School of Biology

Research: Recent Theses

School of Biology theses in Research@StAndrews:Full text

• A taxonomic study of Phascum cuspidatum Scheb. ex Hedw. (Pottiaceae) and its allies
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  Abstract: The moss genus Phascum is common in the Northern Hemisphere. Its species may be frequently encountered by botanists studying the moss flora of bare earth in fallow fields and gardens. Species within this genus have been recognised by taxonomists since at least 1741. The present form of the generic name dates from 1753. Yet Phascum still appears to be poorly understood. Even with available modern treatments, in floras such as Nyholm (1960) and Lawton (1971), bryologists find it difficult to recognise taxa and seem hesitant to name species. Why are these taxa difficult to identify? Is it because they are a complex of "good" but difficult to recognise species and varieties or a loose assemblage of intergrading forms and ecotypes? The present work attempts to answer the following questions: - 1. Is Phascum divisible into recognisable entities in the Northern Hemisphere? And, if it is divisible, what should the subunits be correctly called? 2. If the species Phascum cuspidatum Schreb. ex Hedw. is proved to be a valid taxon, is it possible to subdivide it into infraspecific units? And, if it is divisible, at what level should the subunits be recognised and what should they be correctly called? The problem was tackled in three phases. First, in order to cover the nomenclatural questions, using the Index Muscorum edited by Wijk (1967) as a starting point, all the relevant publications which contained citations of putative taxa or new combinations within the genus were assembled. From the information provided in these works a chronological survey of the taxonomy of the genus from 1770 to the present day was compiled (Chapter I). Hopefully this synopsis, of what turned out to be an extremely complex literature, will provide a reference point not only for this study but for any further researches in the genus in Europe and North America. Alterations and amendments to the information provided in the Index Yuscorun, together with other notes and comments, are incorporated in the survey where appropriate. Secondly, from these taxonomic publications, a list of the definitive characters used in the descriptions of the many putative taxa was compiled. This information was used to provide direction in a study of the taxonomic usefulness of these characters and thus the validity of the proposed taxa. This work, which included some growth experiments, was carried out within the eight fields of study suggested by Turrill (1942). Attention was paid to both traditional and more modern taxonomic methods, including computational techniques. An attempt was also made to fill the gaps in areas not covered by this work by reference to the specialised published literature within some of these eight fields (Chapter II). Thirdly, the genus Phascum is discussed in terms of both historical treatment and current practice concerning generic and specific concepts. And, finally, from the results of the studies carried out and with due reference to the literature and specimens seen the genus Phascum is redefined in terms of its component, taxa. These are thereafter typified and named, as far as possible at the present state of knowledge, in accordance with the International Code of Botanical nomenclature (I.C.B.N.) of 1972. An indication is provided of work which still remains to be carried out (Chapter III).
  
  
  
• Palaeobiology, taphonomy and diagenesis of a lower carboniferous fish fauna
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  Abstract: Chapters one and two of this work contain a study of helically-coiled spiral coprolites. Spiral coprolites with complex microstructures have been interpreted as enterospirae – fossilized valvular intestines (Fritsch 1907, Williams 1972). However other evidence seems to indicate that recent dogfish produce spiral faecal pellets with complex microstructures (McAllister 1985). The search for an answer to the problem of whether certain spiral coprolites are fossilized intestines or faecal pellets includes a study of diet and the physiology of digestion in fishes, with particular attention to those with valvular intestines; the sharks, skates and rays. The faunal associations of various coprolite assemblages and the fossil evidence for the presence of the valvular intestine in fishes and other groups are also reviewed. Chapter one includes a revision of the definitions, terminology and taxonomy of coprolites. In the second chapter the various forms of spiral coprolites in the Lower Carboniferous of Scotland are examined. Actualistic research into taphonomic processes in fishes is divided into two areas described in chapters three and five. Chapter three covers various aspects of the physical processes which affect fish carcasses, with particular emphasis on small sharks. A major aspect of this work is concerned with the post mortem build-up of gas in the body cavity and its taphonomic consequences. Chemical aspects of decay and mineralization form the basis of chapter five. The roles of changes in pH and the levels of calcium magnesium and phosphates in early mineralization are investigated. An investigation of the taphonomy and diagenesis of the Wardle Shales fish beds is contained in chapter four. The Lower Carboniferous Wardie Shales fish fauna consists of palaeoniscoids, sharks and acanthodians preserved in siderite concretions. The preservation of uncrushed calcified cartilage is particularly striking. Well-preserved spiral coprolites are exceedingly common. Wide-ranging conclusions help to clarify the processes that have led to the preservation of the remarkable assemblages of coprolites at Anstruther and Wardie and of the Wardie fishes.
  
  
  
• Causes and consequences of variation in the energy expenditure in grey seals (Halichoerus grypus)
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  Abstract: Oxygen consumption of captive grey seals was measured over a period of 2.5 years at the captive facility of the Sea Mammal Research Unit at the University of St Andrews. The conditions under which in-water resting metabolic rate (RMR) was measured had a significant effect on the resulting estimates. RMR was highest when seals were at the surface breathing periodically. Surface apnoea and periodic submergence reduced RMR estimates by 25 and 35% respectively. There was significant seasonal variation in RMR's of adult females, with rates being highest in the spring and declining throughout the summer months. This variation was unrelated to changes in water temperature. Changes in mass explained some of this variation but this seasonality was still evident when rates were expressed mass-specifically. RMR of juvenile grey seals increased with age, as did lean body mass, although there was no relationship between age and total body mass. Diving metabolic rate (DMR) was measured while seals were voluntarily diving in a quasi- natural setting. Overall DMR was lower than RMR measured in the same animals. Mean DMR was 1.6 times predicted BMR. Average DMR decreased with dive duration and increased swimming activity during dives increased DMR. A model was developed that predicts DMR given information on the behaviour during the dive. This model was used to predict the at-sea metabolic rate of 5 wild grey seals from telemetry data. Overall, predicted at-sea metabolic rates were similar to DMR of the captive seals. Travelling dives had a higher energetic cost than foraging dives. Feeding during diving caused DMR to be increased 1.4-1.6 times pre-feeding levels. Metabolic rate data was incorporated into a population energy flux model. Energy requirements of different components of the population were estimated on a quarterly basis and all these were summed to produce an estimate of population annual energy requirements. Error in input parameters were used to calculate confidence limits in these estimates.
  
  
  
• Distance measurements using pulsed EPR : noncovalently bound nitroxide and trityl spin labels
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  Abstract: The function of biomacromolecules is controlled by their structure and conformational flexibility. Investigating the structure of biologically important macromolecules can, therefore, yield information that could explain their complex biological function. In addition to X ray crystallography and nuclear magnetic resonance (NMR) methods, pulsed electron paramagnetic resonance (EPR) methods, in particular the pulsed electron electron double resonance (PELDOR) technique has, during the last decade, become a valuable tool for structural determination of macromolecules. Long range distance constraints obtained from pulsed EPR measurements, make it possible to carry out structural refinements on structures from NMR and X ray methods. In addition, EPR yields distance distributions that give information about structural flexibility. The use of EPR for structural studies of biomacromolecules requires in most cases site specific incorporation of paramagnetic centres known as spin labelling. To date, spin labelling nucleic acids has required complex spin labelling chemistry. The first application of a site directed and noncovalent spin labelling method for distance measurements on DNA is described. It is demonstrated that noncovalent spin labelling with a rigid spin label can afford detailed information on internal DNA dynamics using PELDOR. Furthermore, it is shown that noncovalent spin labelling can be used to study DNA protein complexes. PELDOR can also yield information about spin label orientation. Therefore, spin labels with limited flexibility can be used to measure the relative orientation of the spin labelled sites. Although information on orientation can be obtained from 9.7 GHz PELDOR measurements in selected applications, measurements at 97 GHz or higher, increases orientation selection. It is shown that PELDOR measurements on semi rigid and rigid nitroxide biradicals using a home built high power 97 GHz EPR spectrometer (Hiper) and model based simulations yield quantitative information on spin label orientations and dynamics. The most widely used spin labels for EPR studies on biomacromolecules are the aminoxyl (nitroxide) radicals. The major drawbacks of nitroxide spin labels include low sensitivity for distance measurements, fast spin spin relaxation in solution and limited stability in reducing environments. Carbon centered triarylmethyl (trityl) radicals have properties that could eliminate some of the limitations of nitroxide spin labels. To evaluate the use of trityl spin labels for nanometer distance measurements, models systems with trityl and nitroxide spin labels were measured using PELDOR and Double Quantum Coherence (DQC). This study shows that trityl spin labels yield reliable information on interlabel distances and dynamics, establishing the trityl radical as a viable spin label for structural studies on biomacromolecules.
  
  
  
• Investigating the molecular basis of adaptation and speciation in divergent populations
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  Abstract: The creation of biodiversity involves the evolution of new species. Recent trends in the study of speciation have increased the emphasis on the role of ecology in adaptation and the evolution of reproductive isolation. This includes examining the relative contributions of different types of selection, the role of gene flow and the genomic changes that occur during ecological speciation. The search for speciation genes continues, however our growing knowledge of how the genome translates into phenotypes means we should now consider a broader molecular basis of speciation, which includes genetic, transcriptomic and potentially epigenetic variation that contribute to phenotypic variation. This thesis addresses the molecular basis of speciation by using three different complementary methods to examine the early stages of ecological speciation and the evolution of premating reproductive isolation between two incipient species of the cactophilic fly, Drosophila mojavensis. First, the genetic basis was examined through the sequencing of two candidate genes underlying reproductive isolation (Chapter 2). Second, the historical biogeography of population divergence was uncovered using multiple sequenced loci (Chapter 3). Lastly, gene expression across the whole transcriptome associated with phenotypic plasticity and mating success was assessed (Chapter 4). Further, the role of epigenetic imprinting in the population divergence of a freshwater fish, Girardinichthys multiradiatus, was examined through sequencing of a well known gene involved in sexual conflict (Chapter 5). These studies find that uncovering the genetic variation underlying speciation is difficult, especially when there is extensive phenotypic plasticity. Further, gene expression plasticity may play an important role in the evolution of premating isolation, and this includes a role for epigenetic mechanisms of gene expression. Additionally, it is important to assess the demographic scenario of population divergence to put into context the ecological and functional data on divergent groups. Through these studies this thesis examines the genetic, expression and epigenetic variation associated with on-going population divergence, and emphasises the need to consider the potential role of the full range of gene expression changes during ecological speciation.
  
  
  
• Microphytobenthic diversity and function in estuarine soft sediment
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  Abstract: Corophium volutator (Pallas) fit the criteria of ‘ecosystem engineers’ as defined by Jones and colleagues (1994, 1997): they are widely distributed within and across North Atlantic estuaries, are often present in intertidal soft sediment in vast numbers, and build semi-permanent burrows in the sediment matrix, which they irrigate continuously. Previous studies have demonstrated that C. volutator burrowing and feeding not only modifies the sediment biogeochemistry but can also modify the overlying water biogeochemistry (during immersion). C. volutator activities have also been shown to be detrimental to microphytobenthic (MPB) biofilms in the immediate vicinity of the burrows. As MPB are the stabilizing force in the estuary, the decimation of biofilm destabilizes the habitat for all the organisms colonising it. However, several aspects of C. volutator ecology remain unclear. First, previous studies on the effect of C. volutator on local (within burrow proximity) MPB diversity have not presented a clear signal as to whether they increase or decrease biodiversity or established whether there is preferential survival amongst MPB taxa with certain cell shapes and sizes or lifestyles. Second, as it has been established that C. volutator have the potential to change the water column, it is possible for them to effect MPB populations remotely (outwith burrow proximity). It is therefore of interest to determine the effects they have, whether such an effect can be achieved within a tidal period, and whether these effects can change MPB biomass, behaviour or diversity over time. A series of controlled mesocosm experiments were carried out to quantify those effects of C. volutator on the water column which were likely to impact MPB survival, to determine whether those effects were specific to C. volutator or common to deposit‐feeding bioturbators, to determine to what degree they could be achieved within a single immersion period, and to separate the effects of C. volutator on MPB bulk (chlorophyll-a in top 5 mm) and photosynthesizing (fluorescing) biomass and diversity both ‘locally’ and ‘remotely’. The results of the first 3 experiments consistently showed that C. volutator substantially increased the resuspension of sediment to the overlying water column and that the resulting turbidity could reduce lightpenetration to the sediment by as much as 50% within one immersion period. Results of nutrient fluxes were less consistent and clear within and between experiments but there was some suggestion that increased bioirrigation increased inorganic nitrogen flux to the overlying water column in accordance with previous studies. The effects of C. volutator on local and remote MPB biomass (bulk and surface) and diversity varied between experiments but, broadly speaking: (1) bulk biomass was unaffected, reduced locally, or increased remotely; (2) surface biomass was reduced both locally and remotely; and (3) community diversity (Simpson’s diversity index) was consistently unaffected, both locally and remotely. Because increased water column turbidity is the most distinctive calling card of C. volutator but is only likely to affect the photosynthetically active (surface) MPB biomass during immersion, a controlled laboratory experiment was designed to examine the extent to which turbidity could influence MPB migratory behaviour and photosynthetic activity. MPB bulk migration was shown to be driven by sitespecific, entrained rhythms of light availability and spatial variation in light availability only drove micro‐cycling in the photic zone during the immersion period. So, in the absence of C. volutator, or any other turbidity producing phenomenon (deep water columns, high flow rates, physical disturbance, etc.), MPB will remain at the surface to photosynthesize during immersion and the productivity during this period is determined by total light intensity and exposure hours (or ‘light dose’). Therefore, the proximity and size of C. volutator populations to a site is likely to be influential in determining local productivity patterns of MPB. In addition, differences in MPB assemblage composition were shown to influence the biofilm productivity but what drives changes in MPB assemblage composition is still unclear and requires further investigation.
  
  
  
• Mammalian upstream Hippo signalling pathway proteins activate core pathway kinases and functionally antagonize oncogenic YAP
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  Abstract: The mechanism of body and organ size control is an unsolved puzzle. Initially characterized in Drosophila melanogaster, the Salvador/Warts/Hippo (Hippo) signalling pathway, highly conserved throughout evolution, defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis, and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. Previously Willin/FRMD6 has been proposed as the human orthologue of Expanded and, to date, little is known about the functional role of Willin in mammalian cells. My study elucidated the mechanism by which Willin antagonizes the transcriptional co-activator YAP. In MCF10A cells, Willin ectopic expression antagonizes YAP-induced epithelial-mesenchymal phenotypes via YAP Ser127 phosphorylation site. Loss of Willin expression attenuates MST1/2, LATS1, and YAP phosphorylation promoting YAP’s oncogenic transformation activity in vitro, as analysed by its ability to display epithelial-to-mesenchymal transition (EMT) features. These biological outputs are YAP dependent. These data support the involvement of Willin in the regulation of the mammalian Hippo signalling activity by activating the core Hippo pathway kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but the functional consequences of these biochemical changes have not been previously addressed. I showed that in MCF10A cells, loss of KIBRA expression displays EMT features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the Ser 127 phosphorylation site and I showed that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Willin/FRMD6 was first identified in rat sciatic nerve, which is composed of Schwann cells and fibroblasts. To elucidate the function of Willin in the mammalian sciatic nerve, I showed that Willin is predominantly expressed in fibroblasts and that its expression activates the Hippo signalling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These evidence show that Willin modulates sciatic nerve fibroblast activity, indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.
  
  
  
• Maternal effects on oocyte quality in farmed Atlantic halibut (Hippoglossus hippoglossus L.)
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  Abstract: Atlantic halibut (Hippoglossus hippoglossus) oocyte quality is highly variable and one of the major bottlenecks during fry-production for on-growth in commercial Atlantic halibut farming. In this study, the effect of maternally derived oocyte constituents (i.e. yolk components and mRNAs) on oocyte quality (i.e fertilisation, embryonic hatching and normal blastomere symmetry) in farmed Atlantic halibut has been investigated. Atlantic halibut embryos and larvae depend on nutritional yolk components until larval first feeding. The importance of yolk n-3 fatty acids for oocyte quality was confirmed. However, highest positive correlations with oocyte quality were found for the less studied fatty acids dihomo-γ-linolenic acid (DGLA, 20:3n6) and docosapentaenoic acid (DHA, 20:5n3) that are known to compete with two of the most abundant fatty acids, arachidonic acid (ARA, 20:4n6)and docosahexaenoic acid (DPA, 22:5n3), respectively during fatty acid metabolism. High methionine and aspartic concentrations, amino acids essential to eukaryotic protein synthesis, were found to influence oocyte quality positively while no significant correlations were found between oocyte folate concentrations and oocyte quality. Before activation of zygotic transcription, maternal mRNAs control cell divisions and embryonic patterning. Due to the limited available genomic information on Atlantic halibut maternal transcripts, an expressed sequence tag (EST) maternal library containing 2,341high quality ESTs was created by suppressive subtractive hybridization (SSH). The maternal library constitutes an EST pool to identify suitable Atlantic halibut reference genes and identify differentially expressed maternal genes in high and low quality Atlantic halibut oocytes. To perform reliable quantification of gene expression by qPCR, stable reference genes have to be used to normalize target gene expression. Tubb2/Actb and Tbb2/Fau were identified as the best two-gene normalization factors during Atlantic halibut embryonic and larval development. Either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut. Tubb2/Actb was further used as reference gene during this study. Poor embryonic hatching success was found to not be correlated with a general decrease in oocyte maternal transcript abundance but with low transcript levels of specific maternal transcripts by qPCR. The majority of genes showed either no or very minor correlations between their transcript levels and oocyte quality parameters (Fertilisation: 13-93 %, embryonic hatching: 1-94 %). However, maternal transcript levels of three genes, most likely involved in nuclear protein and mRNA transport, growth factor regulation, and embryonic patterning, correlated with oocyte quality. Further, a new Atlantic halibut 4x44k oligonucleotide microarray was constructed and used to identify 192 strictly maternal genes during Atlantic halibut embryonic development and 20 differentially expressed genes between high and low quality oocytes, involved in immune response, metabolism, RNA transcription, protein degradation, cell signalling and the cytoskeleton. Microarray validation confirmed its suitability for future gene expression studies during Atlantic halibut embryonic development. The identified maternal genes in this study can serve as a pool for future in-depth studies of embryonic gene expression to advance the knowledge of important developmental processes such as germ cell development, growth and immune response in Atlantic halibut. Some of these may serve as possible markers for Atlantic halibut oocyte quality due to their high expression differences between high and low quality oocytes. Future nutritional studies on Atlantic halibut broodstock should focus on the identified yolk constituents acting positively on oocyte quality.
  
  
  
• The integration of phylogenetic and functional trait information into monocot biodiversity conservation
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  Abstract: It is now widely acknowledged that a high proportion of the world’s species are threatened with global extinction in the near future. Conserving all endangered species is impractical due to limited financial resources and manpower. Therefore, there is a real need to understand the mechanisms behind the extinction process in order to develop a proactive rather than reactive conservation strategy. Furthermore, given these limitations there is a need to be selective, prioritising species or areas not just on the basis of extinction risk or the number of species present, but with consideration for their role within the ecosystem. In this thesis, I investigate three aspects of current global extinction: What causes species to become threatened, how we should prioritise those that are, and how such information can be translated into area-based conservation priorities. Firstly, I developed models of plant extinction risk in relation to environmental (anthropogenic, climatic, and physical) variables and used them to show that non- randomness in extinction risk is highly influenced by interactions between the plants traits and their environment. Incorporating such interactions, my models were able to explain ~30% of the variation in plant extinction risk. Results from this large-scale comparative study suggest the biological traits that increase species’ susceptibility are dependent upon local environmental conditions. Secondly, I looked at how much unique evolutionary history, a proxy for species value, will be lost under current extinction patterns, whilst investigating how effective current conservation methods are at prioritising species based on their evolutionary value. The results are not promising; species threatened with extinction had disproportionately high quantities of unique evolutionary history. Furthermore, the IUCN Red List makes no allowance for this, for example, species considered Critically Endangered, the highest threat status under the IUCN Red List, may not necessarily be the most evolutionary unique. Finally, I looked at the relationships among species richness, versity (FD) and phylogenetic diversity (PD) as measures of biodiversity and therefore the conservation value of an area. It was found species richness is generally a good surrogate for FD and PD. However, agricultural development and climatic variation bring out discrepancies between species richness, FD and PD, challenging the claim of interchangeability of different diversity measures, with potential consequences for conservation planning.
  
  
  
• Changes in localisation and dynamics of splicing and alternative splicing factors in human lens epithelial cells of myotonic dystrophy type 1 patients
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  Abstract: Lens growth and development is a life-long process in which epithelial cells from the perimeter of the lens migrate towards the centre of the lens and follow a dynamic programme of differentiation and structured DNA and organelle degradation resulting in the optical clarity of the lens. Myotonic dystrophy type 1 (DM1) is a genetic disease resulting in multiple symptoms including skeletal muscle wasting, cardiac conduction defects, myotonia and endocrine system malfunction. DM1 is caused by an expanded CTG repeat in the 3' untranslated region (UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Mutant DMPK RNA has been demonstrated to form abnormal foci within the cell nucleus in muscle cells from DM1 patients. The link between these foci and the multiple symptoms of DM1 is not fully understood. The alternative pre-mRNA splicing factor muscle blind-like 1 (MBNL1) is found in the foci and sequestration of MBNL1 is a leading hypothesis for pathogenesis. Results shown in this thesis demonstrate that only a small percentage of nuclear MBNL1 is sequestrated into foci. Furthermore MBNL1 is shown to co-localise with splicing speckles in a transcriptionally dependent manner. Interestingly eye lens histological samples suggest a change in MBNL1 distribution during lens growth. The data presented in this thesis shows a strong relationship between MBNL1 and CUGexp pre-mRNA foci and presents data which highlights the sensitivity of lens epitheial cells to changes in MBNL1.
  
  
  
• Diving and depth use in seals : inferences from telemetry data using regression and random walk movement
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  Abstract: This thesis focuses on methods for using telemetry data to make inferences about the diving behaviour of seals, in terms of their use of depth over time. Three species are considered: grey seals (Halichoerus grypus) and elephant seals (Mirounga leonina and Mirounga angustirostris). Data came from Geographic Positioning System phone tags (GPS phone tags) for grey seals, and Conductivity Temperature Depth Satellite Relay Data Loggers (CTD-SRDLs) for southern elephant seals (M.leonina); both are instruments that transmit Information in abstracted form. Data for northern elephant seals (M.angustirostris) came from anarchival prototype SRDL-type instrument that stored tri-axial acceleration information at high resolution and required recovery to obtain the data. The usefulness of maximum dive depth as a measure of depth use in grey seals, known to forage on the seabed, is explored with a logistic regression analysis using a Generalized Additive Model. Often, maximum dive depth will not be a representative measure of the way seals apportion their time in the water column, so a framework for quantifying depth use is developed for abstracted dive data from southern elephant seals and validated with high resolution time-depth data from northern elephant seals. The implications of using a broken-stick model for abstracting dive data on-board CTD-SRDLs are investigated in terms of its performance and uncertainty. A method for obtaining limits on the time-depth area within which these abstracted dives occurred is developed and used as part of a Bayesian state-space random walk model framework to reconstruct dive trajectories and estimate depth use profiles for abstracted dive data.
  
  
  
• Cellular and proteomic studies of the mitochondrial ABAD/Aβ complex : investigating its role in Alzheimer's disease
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  Abstract: The focus of this thesis is to investigate the intracellular protein-peptide complex 3-hydroxyacyl-CoA dehydrogenase (HADH), also known as ABAD (amyloid- binding alcohol dehydrogenase) and amyloid-beta peptide (Aβ). This complex has been identified in the development of Alzheimer's disease (AD), and this study tries to identify if ABAD is a useful biomarker for genetic risk profiling strategies for the early diagnosis of Alzheimer’s disease, or a suitable target for disease-modifying drug development. The major aim of this project is to explore the biochemical and cellular processes activated as a result of the interaction of ABAD and Aβ. Understanding the cellular responses to these interactions could help identify important biomarkers and/or drug targets for the diagnosis or treatment of Alzheimer’s disease. This study assesses the cytotoxic effects of Aβ in tissue culture and in animal models overexpressing ABAD. An in vitro cell system using SK-N-SH cells was developed for investigating the effects of ABAD expression in cells when incubated with synthetic Aβ peptide. In vitro studies confirmed ABAD to be a mitochondrial protein. There were problems with the efficiency of the synthetic Aβ peptide used, which was found to aggregate excessively. Trial of a soluble oligomeric Aβ peptide proved to be more efficient. A cell system was also developed, culturing neurospheres from murine stem cells. This proved to be a reliable system for culturing primary cells and keeping them in culture for up to 8 weeks. Cells were grown from a wild type strain and then differentiated and stained for endogenous expression of proteins. Proteomic studies were carried out with novel transgenic mouse models for AD. Seven proteins were identified with changed expression in the 2x Tg mouse model. Further immunocytochemistry of human AD brain tissue confirmed the upregulation of peroxiredoxin II and endophilin I. Both proteins could be returned to normal expression in the mouse models by peritoneal injection for two weeks of a novel peptide inhibitor to ABAD, confirming the involvement of the ABAD/Aβ complex in the increased expression of these proteins. Finally ABAD/Aβ was investigated as a possible target for AD therapy by screening with a small molecule fragment library. Only an initial screen was carried out, but several small molecule compounds were found to bind to ABAD. Further screening may produce lead compounds for a synthetic drug to inhibit the enhanced Aβ toxicity associated with the ABAD/Aβ complex.
  
  
  
• Using a tryptophan-regulated promoter system to study chromosal DNA replication in archaeal model organism Haloferax volcanii
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  Abstract: Chromosomal DNA replication is an essential process for all forms of cellular life. Archaea, the third domain of life, possess a DNA replication apparatus that shares significant protein sequence similarity with eukaryotic replication factors, making the Archaea a good model system for understanding the biology of chromosome replication in Eukaryotes. The results described in this thesis contribute to the genetic and physiological characterisation of DNA replication in the model organism Haloferax volcanii, a halophilic euryarchaeon. The thesis documents the generation of conditional lethal mutants of replication genes in H.volcanii using the tryptophan- regulated tna promoter and its TATA-box mutant, tnaM3. This system was used to study the cellular function of the triple OB fold containing single-stranded DNA binding protein RpaC and for characterisation of PriS-GINS operon. Deletion analysis of three putative SSB proteins in H.volcanii indicated that RpaA1 and RpaB1 are individually non-essential for cell viability but share an essential function, whereas RpaC protein is essential. Loss of RpaC function can however be rescued by elevated expression of RpaB, indicative of functional overlap between the two classes of haloarchaeal SSB. Down-regulation of RpaC caused growth retardation and a significant reduction of DNA synthesis in vivo suggesting, that RpaC is required for DNA replication. In addition, RpaC overexpression increased resistance to various types of DNA damage implying its role in DNA repair. This function is probably mediated by the N-terminus as deletion of this region makes cells sensitive to DNA damaging agents. Analyses of PriS-GINS operon indicated that GINS could be down-regulated without severe consequences for the cells suggesting that it is non-essential protein in H.volcanii. In addition, bioinformatics studies identified sequence similarity between the C-terminal domain of the catalytic subunit of archaeal primase (PriS CTD) and B-domain of GINS51 and B-domain of GINS23 that offer insights into mechanisms for the evolution of these proteins.
  
  
  
• An investigation into the Trypanosoma brucei CDP-DAG synthase and downstream pathways
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  Abstract: Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host, allowing a plethora of novel drug targets to be discovered and validated. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate produced by the enzyme CDP-DAG synthase (CDS), but nothing was known about CDS in T. brucei. Only one gene encodes CDS in Trypanosoma brucei (Tb927.7.220) and this was shown to encode a functional CDS by overexpression in E. coli and complementation of a yeast CDS null, which was created during this study. Expression and activity of TbCDS was confirmed in T. brucei, and was shown to be essential in both life cycle stages. Disruption of TbCDS altered the lipid profile of T. brucei, confirming a central role for CDP-DAG in phospholipid synthesis. Biochemical and morphological characterisation of mutants in TbCDS expression elucidated at least two separately localised and regulated pools of CDP-DAG and phosphatidylinositol in T. brucei. In bloodstream form these pools are localised to the Golgi and the ER, however in procyclics it is possible that both of these pools are localised to the Golgi, since no phosphatidylinositol synthase protein was detected in the ER of procyclics. Reduction in TbCDS was shown to affect cell cycle regulation and Golgi segregation possibly due to a depletion of phosphorylated phosphatidylinositols (PIPs). These studies also indicate that phosphatidylglycerol may be synthesised by the phosphatidylglycerol-phosphate synthase which may be capable of using phosphatidylserine as a substrate in a headgroup swapping reaction. TbCDS has now been genetically validated as a drug target, and has highlighted novel aspects of lipid biosynthesis in T. brucei. Collectively, these findings highlight the central role played by TbCDS and the new knowledge gained here may lead to the discovery and validation of other novel drug targets against African sleeping sickness.
  
  
  
• Crows' nests on power poles : finding a solution
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  Abstract: Crows (Corvus cornix and C. corone) nest on power-poles throughout northern Scotland causing interruptions to electricity that cost the electricity provider more than £250,000 annually. I aimed to establish what factors influence the nest site selection of crows in the islands of Orkney to help determine ways to reduce pole nesting by these birds. Crows preferred nest sites in trees that were coniferous, tall, mature, densely grouped, and far from occupied houses. However, when such trees were scarce, the number of occupied houses was high, and there were no cliffs within 1km, crows were more likely to nest on power-poles. I found that the fitting of a Firefly diverter at sites where nests were removed was ineffective at deterring rebuilding, but rebuilding was less likely to occur the later in the season that nests were removed. Nests in the middle phase of construction were the most likely to be rebuilt. Making an appropriate decision as to when to remove a crow nest, therefore, would seem more effective for deterring nest rebuilding than is the fitting of Firefly diverters. Nest removal also reduced the level of pole nesting both within years and between years. Furthermore, planned interruptions led to fewer customers losing power and to a twelve-fold reduction in the number of minutes that customers were without power, relative to unplanned power-cuts caused by nests. I also attempted to determine the efficacy of providing alternative nesting platforms and of insulating the live wires on transformer boxes, but I collected too few data to confirm if either of these might reduce the number of power-cuts caused by crows' nests. As both nest platforms and insulation remain in place, however, data collected from these sites in the future may allow confirmation (or not) as to their value.
  
  
  
• Functional, biochemical and structural analyses of two plasmodium membrane proteins
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  Abstract: Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The most severe form of human malaria is caused by P. falciparum, responsible for approximately three quarters of a million deaths each year. One major problem in the treatment of malaria is resistance to existing chemotherapies. Consequently, there is an urgent need to identify and validate novel drug targets. A possible recently identified drug target is the PfNitA protein of P. falciparum which contains orthologues in other Plasmodium species but is absent from humans. The gene is annotated as a putative formate-nitrite transporter and orthologues are found in a range of prokaryotes as well as the lower eukaryotes algae and fungi. To determine the biological function of the protein, pfnita was expressed in Escherichia coli strains lacking the endogenous formate and nitrite transporters. In order to analyse the essentiality of the gene a reverse genetics approach was taken and the data discussed. Results indicate that the PfNitA protein is located in the plasma membrane and digestive vacuole of intraerythrocytic parasites suggesting a role in the uptake or excretion of metabolites. A second complexity with regard to treatment is the lack of a vaccine. A problem in crating a vaccine is antigenic variation. The PIR family of proteins contain a so-called hypervariable domain that has led to the suggestion that the family may play a role in antigenic variation. The objective of the work carried out in this thesis was to investigate the topology and structure of the PcCir2 protein of Plasmodium chabaudi, using E. coli as the expression host. The topology of Cir2 has been examined by means of reporter fusions and overexpression/purification studies undertaken towards crystallisation. As the PcCir2 amino acid sequence does not show significant homology to other proteins, structural data may provide insights into potential functional or binding domains.
  
  
  
• From social learning to culture : mathematical and computational models of cultural evolution
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  Abstract: Humans are unique in the extent and complexity of their cultures. As a species, we generate extensive knowledge and innumerable norms, attitudes, traditions, skills, beliefs and technologies that we share with those around us through teaching, imitation and language. These cultural practices have their roots in our uniquely potent ability for social learning. This thesis sets out to elucidate the process of cultural evolution using a series of mathematical and computational models. These models first investigate the evolution of the capacity for social learning, the rare ability to teach, and the evolution of the smart and strategic use of social learning, in the animal lineage. They go on to investigate the implications of these strategies and mechanisms for culture and find that the form human culture takes is dependant on the amount and nature of social learning as well as on the underlying learning strategies deployed. The thesis also investigates the effect that culture has had on the human evolutionary niche. Cultural practices fundamentally change the selection pressures to which humans are subject and these in turn change both our cultures and our genes through gene-culture coevolution. Finally, a demographic cultural niche construction model is presented, which investigates the application of cultural evolution modelling, cultural niche construction theory and demographic models to the growing problem of sex-ratio imbalance in modern China and considers the implications for policy-making. The analyses presented in this thesis support the argument that the uniquely potent human ability to transmit acquired information through teaching, imitation and other forms of social learning, and through this to shape our cultural and ecological environments, has played and continues to play a central role in human evolution.
  
  
  
• Acoustic behaviour of Risso's dolphins, Grampus griseus, in the Canary Islands, Spain
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  Abstract: The Risso's dolphin (Grampus griseus) is a poorly studied species, particularly with respect to its acoustic behaviour. The little we know about Risso's dolphin acoustics shows that they are an interesting case study given that they combine acoustic features that place them inbetween very different delphinids. I investigated the acoustic repertoire of the species showing that Risso's dolphins produce mainly whistles, burst-pulses and click trains. I discussed the possible functions of each type of vocalization by contextualizing them into group behavioural patterns, size and habitat features. Although capable of whistling, Risso's dolphins seem to favour burst-pulses to communicate and maintain group cohesion following deep dives. Click trains were compared based on their inter-click interval patterns. Those were placed in their behavioural context and used to determine the number of animals echolocating at any given time. Risso's dolphins use a variety of inter-click intervals in each context. My data suggests that they may avoid predators by eavesdropping on the echoes of conspecific clicks while slow travelling and resting. Using 3D acoustic localization, I studied the biosonar performance and on-axis click characteristics of Risso's dolphin clicks. On one hand, Risso's dolphins do not seem to display range locking behaviour which makes them similar to beaked whales; on the other hand, they seem to apply automatic control to the transmitting side of their biosonar, which is similar to other delphinids such as the bottlenose dolphin. I also compared the whistle repertoire from Gran Canaria to that of another four locations. A discriminant function analysis using fundamental frequency parameters showed geographical distinction, most likely due to divergence caused by geographic isolation. To conclude, the Risso's dolphin is an interesting species that combines acoustic features from different cetacean species. This could suggest that Grampus griseus may be misplaced within the Delphininae sub-family.
  
  
  
• Activity-dependent development of the locomotor network in Xenopus laevis larvae
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  Abstract: The impact of activity in Xenopus embryonic and larval development was studied with regards to the locomotor system and developmental rate. The data suggest that pharmacologically suppressing neuronal and muscular activity decreases, and enhancing swimming activity, by raising tadpoles in a rotating water column, increases the rate of development. Moreover the latter adapted their swimming behaviour in accordance to the treatment: their swimming was characterized by longer episode durations and more frequent turning manoeuvres. Patch clamp recordings revealed that spinal neurons of agitated animals receive more synaptic drive, probably deriving from descending INs, enabling the animals to maintain swimming for long time periods. An increase in the incidence of miniature potentials suggests that synaptic connections are strengthened. Probably in response to the increase in synaptic excitation the intrinsic excitability, and thereby the probability of signal transmission, decreased. I argue that this represents a case of homeostatic plasticity and serves to prevent over-excitation and maintain pattern generation in the network. Stronger bursts might be explained by enhanced signal transmission from motoneurons to muscle cells. The amount of extrasynaptic AChRs at the NMJ appeared reduced. In contrast, when activity was suppressed behavioural, ventral root and patch data were comparable to those of control animals, suggesting that the locomotor system can develop normally up to stage 42 in the absence of electrical activity. However, animals appeared less excitable, indicating that the establishment of sensory systems might indeed require extrinsic inputs.
  
  
  
• Seasonal mass variation as a life history trait in West African savannah birds
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  Abstract: Seasonality influences life history through its effect on the availability of essential resources, with birds timing breeding to occur during peak food availability. Due to density-dependence, investment in breeding is determined largely by the seasonality of food availability, with an increased investment being traded-off against adult survival. A bird’s mass acts as an index of a species’ foraging environment, because a bird bases its foraging decisions on a trade-off between the risk of predation and the risk of starvation. Under constant predation risk a bird increases its mass as insurance against increased foraging unpredictability. In tropical savannahs day length and temperature remains relatively constant, and there is not a season of increased density-dependent mortality which acts across all species. Thus species have evolved a broad range of life history traits under the same environmental conditions, although how a species experiences seasonality depends largely on its foraging niche. This thesis shows that most savannah species varied their mass across the year, having a reduced mass in the non-breeding season which suggests that foraging remained predictable. Independent of gonad or egg growth they then increased their mass as they started to breed, with the timing of breeding coinciding with peak food availability. Across species in the same foraging niche mass acts as an index of breeding investment, with females increasing their mass more than males. While across species in different foraging niches an increased mass response was associated with higher adult survival, probably because breeding strategy and subsequently adult survival are governed by food limitation. This thesis shows that birds adaptively manage their mass during breeding and that mass is not a result of energetic stress, thus under constant predation risk a bird’s mass is a result of foraging predictability as a function of competition for available food and investment in breeding.
  
  
  
• The distribution and physiological roles of nitric oxide in the locomotor circuitry of the mammalian spinal cord
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  Abstract: The mammalian spinal cord contains the neuronal circuitry necessary to generate rhythmic locomotor activity in the absence of inputs from the higher brain centre or sensory system. This circuitry is regulated by local neuromodulatory inputs, which can adjust the strength and timing of locomotor output. The free radical gas nitric oxide has been shown to act as an important neuromodulator of spinal circuits, which control locomotion in other vertebrate models such as the tadpole and lamprey. Despite this, the involvement of the NO-mediated soluble guanylate cyclase/cyclic guanosine monophosphate secondary messenger-signalling pathway (NO/sGC/cGMP) in mammalian locomotion has largely been under-investigated. The NADPH diaphorase histochemical reaction was used to identify sources of NO in the lumbar spinal cord. The largest population NADPH diaphorase reactive neurons were located in the dorsal horn, followed by the laminae of the ventral horn, particularly around the central canal (lamina X) and lamina VII. NADPH diaphorase reactive neurons were found along a rostrocaudal gradient between lumbar segments L1 to L5. These results show that that discrete neuronal sources of NO are present in the developing mouse spinal cord, and that these cells increase in number during the developmental period postnatal day P1 – P12. NADPH diaphorase was subsequently used to identify NADPH diaphorase reactive neurons at P12 in the mouse model of ALS using the SODG93A transgenic mouse. Physiological recordings of ventral root output were made to assess the contribution of NO to the regulation induced rhythmic fictive locomotion in the in vitro isolated spinal cord preparation. Exogenous NO inhibits central pattern generator (CPG) output while facilitating and inhibiting motor neuron output at low and high concentrations respectively. Removal of endogenous NO increases CPG output while decreasing motor neuron output and these effects are mediated by cGMP. These data suggest that an endogenous tone of NO is involved in the regulation of fictive locomotion and that this involves the NO/sGC/cGMP pathway. Intracellular recordings from presumed motor neurons and a heterogeneous, unidentified sample of interneurons shows that NO modulates the intrinsic properties of spinal neurons. These data suggest that the net effect of NO appears to be a reduction in motor neuron excitability.
  
  
  
• Biodiversity assessment of freshwater fishes: Thailand as a case study
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  Abstract: A key challenge in biodiversity is: How many species are there on earth? This issue is especially acute in poorly surveyed regions with high diversity, particularly Southeast Asia that also experiences many limitations such as lack of funds, documents and experts. To help meet this challenge, I have developed a five-tiered approach for diversity measurement of freshwater fish for use in Thailand. This is: (1) the creation of a newly updated species inventory that uses existing data; (2) exploration of the patterns of species richness, endemism, and uniqueness; (3) estimation of the total species richness; (4) investigation of patterns of rarity; and (5) integration of this knowledge into conservation practice. The system should be applicable to other regions and other taxa where a similar challenge exists. My work shows that eight hundred and seventy-two species in 17 orders, 55 families and 255 genera of freshwater fishes, accounting for roughly 10% of the world’s freshwater fish diversity, have been reported for Thailand to date. This number was derived from information in the museum collections, literature and all other available sources, including reports written in Thai as well as in English. During this work I uncovered many gaps in biodiversity information, in terms of taxonomic and spatial records, though some families and basins are better represented than others. Taxonomic uncertainty also continues to be a challenge for taxonomists and users. The high diversity of freshwater fishes in Thailand is the result of both high alpha (α) diversity (diversity within a particular locality) and beta (β) diversity (diversity differences between localities). I concluded that the substantial beta diversity I detected is associated with the geographical separation of the six river basins in Thailand. For example, the species composition of freshwater fishes in the Salween Basin dramatically differs from all other basins of Thailand. In contrast, the Chao Phraya Basin and the Mekong Basin contain the greatest number of shared species. Approximately 55% of species have a wide distribution range (being reported from more than two basins), whereas 45% are highly restricted within a single basin. Analyses using species richness estimators suggest that the figure of 872 species is an underestimate and that there may be between 1000 and 1300 fish species in Thailand, in other words an increase of between 14.7% and 49.1% over the list I compiled (which is itself an increase of 52.2% over the last report in 1997). Freshwater fish have become increasingly vulnerable to anthropogenic activities. Of the 872 Thai fish species, 6.8% and 15.1% are globally and nationally threatened, respectively. Nonetheless, a striking feature of the database is that the conservation status of the vast majority of species has not so far been assessed, either globally or nationally. Scientists and policy makers will find these results useful in appreciating the magnitude of the tasks involved in surveying, describing and conserving the country’s freshwater fish biota. My work highlights localities and taxa where conservation is a priority and is thus an important resource for policy makers and conservation planners concerned with the management of freshwater fish in Thailand.
  
  
  
• Investigating protein- protein interactions in order to develop novel therapeutics for the treatment of Alzheimer’s disease
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  Abstract: Alzheimer’s disease (AD) accounts for around two thirds of all dementia cases and an increase in life expectancy of the population has resulted in a substantial increase in dementia cases and with that a rise in AD. AD is a debilitating and ultimately fatal neurodegenerative disorder of the elderly, and despite being identified over a century ago, the current treatments do not treat the underlying causes behind the disease, instead they help to mask the symptoms of the disease and prolong the brain’s remaining function. It is therefore vital that an effective, disease modifying treatment for this disease is established as soon as possible. Soluble intracellular forms of amyloid β (peptide Aβ), a hallmark of AD have been identified and intracellular targets of Aβ are being investigated as potential drug targets for the disease. Two key intracellular, mitochondrial proteins investigated as potential drug targets: amyloid binding alcohol dehydrogenase (ABAD) and cyclophilin D (CypD) are the focus of the work reported in this thesis. To begin identifying potential inhibitors of the ABAD-Aβ interaction, a two-pronged approach was taken. Firstly, a series of analogues based on a known inhibitor of the interaction were tested using a variety of biophysical assays, for their therapeutic affect on the interaction, and secondly a fragment based screening approach was used to identify new small molecule binding partners of ABAD which could potentially be modified to produced inhibitors of the ABAD-Aβ interaction. Three different CypD constructs have been successfully expressed and purified, and taken into crystal trials. It is hoped that these constructs can be used to significantly aid the progress of identifying any potential inhibitors and binding partners of CypD that may produce therapeutic effects, and in the future could lead to the identification of an effective disease modifying drug in the treatment of AD. The work reported in this thesis has built upon previously reported findings and the groundwork has also been established for several in vitro biophysical assays, these include for example: measuring ABAD enzyme activity, and the novel morphology specific Aβ aggregation assay, which can be used as screening tools to help identify potential inhibitors of these interactions. Both the ABAD-Aβ interaction, and the blockade of CypD are known to be drug targets in the treatment of AD, and by elucidating the molecular mechanisms behind these interactions, through implementing biophysical assays, this will help in the identification and design of potential new therapeutic agents for the treatment of AD.
  
  
  
• Estimating whale abundance using sparse hydrophone arrays
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  Abstract: Passive acoustic monitoring has been used to investigate many aspects of marine mammal ecology, although methods to estimate absolute abundance and density using acoustic data have only been developed in recent years. The instrument configuration in an acoustic survey determines which abundance estimation methods can be used. Sparsely distributed arrays of instruments are useful because wide geographic areas can be covered. However, instrument spacing in sparse arrays is such that the same vocalisation will not be detected on multiple instruments, excluding the use of some abundance estimation methods. The aim of this thesis was to explore cetacean abundance and density estimation using novel sparse array datasets, applying existing methods where possible, or developing new approaches. The wealth of data collected by sparse arrays was demonstrated by analysing a 10-year dataset collected by the U.S. Navy’s Sound Surveillance System in the north-east Atlantic. Spatial and temporal patterns of blue (Balaenoptera musculus) and fin whale (Balaenoptera physalus) vocal activity were investigated using generalised additive models. Distance sampling-based methods were applied to fin whale calls recorded by an array of Ocean Bottom Seismometers in the north-east Atlantic. Estimated call density was 993 calls/1000 km².hr⁻¹ (CV: 0.39). Animal density could not be estimated because the call rate was unknown. Further development of the call localisation method is required so the current density estimate may be biased. Furthermore, analysing a single day of data resulted in a high variance estimate. Finally, a new simulation-based method developed to estimate density from single hydrophones was applied to blue whale calls recorded in the northern Indian Ocean. Estimated call density was 3 calls/1000 km².hr⁻¹ (CV: 0.17). Again, density of whales could not be estimated as the vocalisation rate was unknown. Lack of biological knowledge poses the greatest limitation to abundance and density estimation using acoustic data.
  
  
  
• Male mating tactics in the rose bitterling (Rhodeus ocellatus) and European bitterling (Rhodeus amarus)
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  Abstract: The aim of this study was to investigate the basis to male mating decisions in two related species of bitterling: Rhodeus ocellatus and R. amarus. Bitterling have a resource-based mating system; females lay eggs in the gills of live freshwater mussels and males fertilize the eggs by releasing sperm into the inhalant syphon of the mussel. Male bitterling perform courtship behaviour and aggressively defend mussels in a territory from which they exclude other males. Using laboratory and field experiments it was shown that male aggressive behaviour is inherited through additive maternal genes. Male aggression is also influenced by the number of conspecific males encountered in competition for a mussel, and by the degree of clustering of mussels. Limited availability of mussels results in stronger selection on traits males use in mating context: hence they are more aggressive, larger and more colourful. The differences in mating behaviours in different environments may indicate a conflict between male dominance and female choice, but have not led to reproductive isolation. Resource availability during ontogenesis and male density during embryogenesis, however, do not exert an effect on male aggressive behaviour. Red carotenoid-based nuptial coloration functions as an inter- and intra-sexual signal and undergoes rapid variation in response to changes in mating context. Male bitterling do not modulate their courtship and aggressive behaviour in response to variation in female size, and their choice of mussel species is influenced by, and consistent with, female oviposition choice.
  
  
  
• The effect of interferon on the transcription pattern of parainfluenza virus 5
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  Abstract: Interferon (IFN) is activated in response to virus infections and upregulates interferon-stimulated genes (ISGs) resulting in the expression of hundreds of proteins, many of which have direct or indirect antiviral activity. Parainfluenza virus 5 (PIV5) of the Paramyxoviridae family is a non-segmented negative sense single-stranded RNA virus with seven genes encoding eight proteins. Here we present that IFN induces alterations in the pattern of both virus transcription and translation and that ISG56 is primarily responsible for these effects. We report that when cells were treated with IFN post-infection, virus protein synthesis was inhibited while virus transcription levels were increased. These results suggest that ISG56 selectively inhibits the translation of viral mRNAs. In addition, the relationship of various PIV5 isolates was analysed by next generation sequencing. Four areas with a high degree of single nucleotide polymorphisms (SNPs) were identified and mapped to the intergenic regions of NP-V/P, M-F and HN-L, as well as the entire SH gene. Three of the isolates, the porcine strain SER and the canine strains CPI+ and CPI-, did not express an SH protein due to the lack of a start codon. A low degree of variation was found in the amino acid sequence of the HN glycoprotein suggesting that PIV5 may be less pressured to evolve in order to evade immune responses, such as neutralising antibodies.
  
  
  
• A computational approach to discovering p53 binding sites in the human genome
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  Abstract: The tumour suppressor p53 protein plays a central role in the DNA damage response/checkpoint pathways leading to DNA repair, cell cycle arrest, apoptosis and senescence. The activation of p53-mediated pathways is primarily facilitated by the binding of tetrameric p53 to two 'half-sites', each consisting of a decameric p53 response element (RE). Functional REs are directly adjacent or separated by a small number of 1-13 'spacer' base pairs (bp). The p53 RE is detected by exact or inexact matches to the palindromic sequence represented by the regular expression [AG][AG][AG]C[AT][TA]G[TC][TC][TC] or a position weight matrix (PWM). The use of matrix-based and regular expression pattern-matching techniques, however, leads to an overwhelming number of false positives. A more specific model, which combines multiple factors known to influence p53-dependent transcription, is required for accurate detection of the binding sites. In this thesis, we present a logistic regression based model which integrates sequence information and epigenetic information to predict human p53 binding sites. Sequence information includes the PWM score and the spacer length between the two half-sites of the observed binding site. To integrate epigenetic information, we analyzed the surrounding region of the binding site for the presence of mono- and trimethylation patterns of histone H3 lysine 4 (H3K4). Our model showed a high level of performance on both a high-resolution data set of functional p53 binding sites from the experimental literature (ChIP data) and the whole human genome. Comparing our model with a simpler sequence-only model, we demonstrated that the prediction accuracy of the sequence-only model could be improved by incorporating epigenetic information, such as the two histone modification marks H3K4me1 and H3K4me3.
  
  
  
• The development of free-swimming in Xenopus laevis larvae
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  Abstract: In Xenopus laevis frog tadpoles, highly self-motive, free-swimming behaviour emerges at the onset of feeding. This is in contrast to the earlier post-hatched larval form, which is capable of escape swimming when stimulated, but normally lies dormant. This developmental transition in behaviour has been documented here and studied in a semi-intact preparation developed to examine the motor output from larvae at the onset of filter feeding. There is a progressive increase in spontaneous motor activity during this period, where spontaneous fictive swimming occurs in episodes of variable duration but with significantly larger burst durations. This spontaneous activity persists after removal of both the fore- and midbrain, but is absent in spinalised preparations. The spontaneous activity is similar to NMDA (100µM) induced rhythm but shows greater periodic variability in the frequency and occurrence of swimming activity. The activity is not dependent on inhibitory synaptic transmission, but is under the control of central GABAergic restraint, as blocking this inhibition with bicuculine (10µM) increased spontaneous locomotor activity. This is distinct to the role of glycinergic inhibition which influences the character of the ventral root bursts, as strychnine (5µM) caused an initial increase in frequency before bursts were synchronised on both the left and right sides, but not the presence of spontaneous activity. However, activity is abolished by the persistent sodium current blocker riluzole (5µM) and enhanced by veratridine (90nM) which potentiates the current, which may suggest that it originates in neurons with pacemaker-like properties - possibly within the hindbrain. Evidence is also provided which shows that the neuromodulatory gas nitric oxide becomes an excitatory modulator of the Xenopus swimming network at the onset of a free-swimming existence, switching from having a global inhibitory role on locomotion in early larval life. The nitric oxide donor DEA/NO (200µM) increased spontaneous fictive swimming in the semi-intact preparation. In contrast, the nitric oxide synthase inhibitor L-NAME combined with PTIO, which sequesters nitric oxide, decreased spontaneous fictive swimming. It is proposed that the emergence of this rhythmic, spontaneous motor activity parallels the increase in swimming at the onset of feeding, suggesting a direct behavioural role for spontaneous network activity in the developing animal.
  
  
  
• Attenuation of bunyavirus replication by modification of genomic untranslated regions
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  Abstract: Bunyamwera orthobunyavirus (BUNV) is the prototype for the family Bunyaviridae. BUNV has a tripartite RNA genome of negative polarity composed of the large(L),medium (M)and small(S)segments. Each segment contains an open reading frame (ORF) flanked by untranslated regions (UTRs). The eleven terminal nucleotides are conserved between the three segments while the internal regions are unique. The UTRs play an important role in the virus life cycle by promoting transcription, replication and encapsidation of the viral genome.The work presented in this thesis explores UTRs plasticity and examines ways to engineere attenuated viruses by modifying only their UTRs. Using reverse genetics, mainly two ways of attenuation were explored: rescue of viruses either carrying deletions within their 3’ and/or 5’ UTRs in all three segments, or of viruses carrying one segment bearing heterologous UTRs. Both approaches resulted in virus attenuation in tissue culture, with viruses producing smaller plaques or even no plaques, and growing to lower titres than wild-type BUNV. Through serial passage, viruses were shown to regain some level of fitness while the mutations introduced in the UTRs proved to be stable. Thus, to investigate the mechanism behind fitness recovery, the nucleotide sequence of the entire genome of viruses with deletions in their UTRs was determined. Amino acid changes were observed in the viral polymerase (L protein) of most mutant viruses and the vast majority of the amino acid changes occured in the C-terminal region. The function of this domain is unclear to date, however data obtained using a mini-replicon assay suggest that the C-terminal domain of the L protein might be involved in UTR recognition. Full genome sequencing also allowed the identification of an amino acid mutation within the polymerase that resulted in a temperature sensitive phenotype when introduced in an otherwise wild-type BUNV. Thus, it was shown that mutations introduced within the UTR regions of the genome were stable through serial passage and resulted in attenuation. Such a strategy could be used in combination with mutations of the ORF to design live-attenuated vaccines against serious pathogens within the family Bunyaviridae.
  
  
  
• The influence of mid-ocean ridges on euphausiid and pelagic ecology
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  Abstract: 1 & 2. Euphausiids comprise a major component of ecosystems in the pelagic realm, the world’s largest habitat, but basin scale drivers of euphausiids diversity and abundance are poorly understood. Mid-Ocean Ridges are the largest topographical feature in the pelagic realm and their benthic and pelagic fauna have only just recently become the focus of research. This thesis present new analyses on the drivers of euphausiids species richness in the Atlantic and the Pacific, giving specific attention to the influence of Mid- Ocean Ridges. New information is given on the biogeography of euphausiids and pelagic food-web trophology of the Mid-Atlantic Ridge, and on the biogeography of pelagic decapods on the South-West Indian Ocean ridge. 3. A Generalized Additive Model framework was used to explore spatial patterns of variability in euphausiid species richness (from recognized areas of occurrence) and in numerical abundance (from the Continuous Plankton Recorder survey) in conjunction with variability in a suite of biological, physical and environmental parameters on, and at either side of, the Mid-Atlantic Ridge. Euphausiid species richness peaked in midlatitudes and was significantly higher on the ridge than in adjacent waters, but the ridge did not influence numerical abundance in the top 10 m significantly. Sea surface temperature (SST) was the most important single factor influencing both euphausiid numerical abundance (-76.7%) and species richness (34.44%). Dissolved silicate concentration, a proxy for diatom abundance, significantly increased species richness (29.46%). Increases in sea surface height variance, a proxy for mixing, increased the numerical abundance of euphausiids. GAM predictions of variability in species richness as a function of SST and depth of the mixed layer were consistent with present theories, which suggest that pelagic niche-availability is related to the thermal structure of the near surface water. 4. Using a Generalized Additive Model in the Pacific, the main drivers of species richness, in order of decreasing importance, were found to be sea surface temperature (explaining 29.53% in species variability), salinity (20.29%), longitude (-15.01%, species richness decreased from West to East), distance to coast (10.99%), and dissolved silicate concentration (9.03%). An additional linear model poorly predicted numerical abundance. The practical differences in drivers of species richness in the Atlantic and Pacific Ocean were compared. Predictions of future species richness changes in the Pacific and Atlantic were made using projected environmental change from the IPCC A1B climate scenario, suggesting an increase in species richness in temperature latitudes (30° to 60° N and S) and little to no change in low latitudes (20° N to 20° S). 5. New baseline information is presented on biogeography, abundance and vertical distribution of euphausiids along the Mid-Atlantic Ridge (40° to 62° N). 18 species were recorded, with Euphausia krohni and Thysanoessa longicaudata being most abundant. Eight species had not been recorded in the area previously. The Subpolar Front is a northern boundary to some southern species, but not a southern boundary to northern ubiquitous species that show submergence. Four major species assemblages were identified and characterised in terms of spatial distribution and species composition. Numerical abundance was highly variable but decreased by orders of magnitude with depth. The Mid-Atlantic Ridge showed only a marginal effect on euphausiid distribution and abundance patterns. 6. Zooplankton and micronektic invertebrate epi- and mesopelagic (0-200 and 200- 800m) vertical distribution (e.g. Euphausiacea, Decapoda, Amphipoda, Thecosomata, Lophogastrida) on either side of the Subpolar Front of the Mid-Atlantic Ridge is described. Dietary relationships are explored, using stable isotope ratios and fatty acid trophic marker (FATM) composition. An increase in trophic level with size was observed. Individuals from southern stations were higher in dinoflagellate Fatty Acid Trophic Markers (FATM) (22:6(n-3)) and individuals from northern stations were higher in Calanus spp and storage FATMs (20:1(n-9) and 22:1(n-9)) reflecting primary production patterns in the two survey sectors. Observations on the geographical and vertical variability in trophodynamics are discussed. 7. New baseline information is presented on the biogeography, abundance, and vertical distribution of mesopelagic (200-1000 m), crustacean micronekton on- and offseamounts of the South-West Indian Ocean Ridge (26° to 42° S). Species richness and numerical abundance were typically higher near seamounts and lower over the abyssal plains, with several species being caught uniquely on seamounts. Observations suggest that the ‘oasis effect’ of seamounts conventionally associated with higher trophic levels is also applicable to pelagic micronektic crustaceans at lower trophic levels. Biophysical coupling of micronekton to seamounts may be an important factor controlling benthopelagic coupling in seamount food-webs. 8. Euphausiid and pelagic diversity is driven primarily by geographical variability in temperature, by longitudinal patterns in upwellings, and by variability in nutrient concentration. Mid-Ocean Ridges modify pelagic ecology, by raising the seafloor and by bringing in proximity true pelagic and bathypelagic predators associated with the seabed. The increase in specialized fauna and biomass associated with ridges and seamounts serves to deplete zooplankton in the near bottom layer (0-200 m) and affect systems in and above the benthic boundary layer (<200 m from the seafloor), and the benthopelagic faunal layer. Mid-Ocean Ridges may serve to structure pelagic faunal distribution, and increase the overall diversity of the world ocean. The influence of ridges in the ocean basin may be comparable to that of hedges in a farmland; whilst delimiting the extent of crops (or zooplankton assemblages), hedges serve as local hotspots of mammal and avian diversity.
  
  
  
• Evolutionary genetics and genomics of the female side of sexual interactions in Drosophila
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  Abstract: Sexual interactions play an important role in generating sexual selection and antagonistic co-evolution. These forces can shape differences between the sexes, but also have the potential to generate population divergence and contribute to speciation. The aim in this thesis was to provide new insights into the genes involved in different stages of female sexual interactions, using Drosophila as a model system. In chapter 2 I tested whether a candidate gene (period) that influences species-specific rhythmic characteristics in male courtship song in D. melanogaster also has a pleiotropic effect on female song preference. Using mutant and transgenic strains I found support for this. In chapter 3 I examined further how females respond to the song at the level of gene expression, using microarrays. Expression profiles revealed modest changes in transcripts abundance overall, which were dominated by antennal olfactory genes, neuropeptide encoding genes and immunity genes. Many of these have previously been found to respond to mating. In chapter 4 I therefore studied further two of these genes, TurandotM and TurandotC and their role in female post-mating fitness. Using RNA interference I found that knocking down these genes influenced immediate fecundity. In chapter 5 I focused on analysing post-mating gene expression patterns in relation to sexual selection in D. pseudoobscura using microarrays. I explored the consequences of experimental variation in female promiscuity on gene expression divergence as a whole, and in response to mating. I found large-scale expression divergence between monandrous and polyandrous females after 100 generations of experimental evolution. Experimental polyandry increased the expression of genes that show female-biased expression in wild-type individuals and decreased male- biased gene expression. Females experiencing no sexual selection showed the opposite pattern. Out of the genes affected by mating, the majority showed increased expression in polyandrous compared to monandrous females, with enrichment e.g. in oogenesis-related genes.
  
  
  
• Examining the response of top marine predators to ecological change using stable isotope proxies
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  Abstract: Monitoring the response of upper trophic level animals to ecological change is important to understanding the state and stability of ecosystems. Marine predators integrate information over large geographical scales and are relatively long-lived; furthermore, many of these organisms are restricted to terrestrial or freshwater habitats at certain times during their life history and are accessible to researchers. This thesis investigated the response of marine predators to ecological change at a variety of spatial and temporal scales using stable isotope ratio methods with the aims of developing meaningful proxies, or indices, of variability in marine ecosystems. The first study explored the intrinsic (i.e. ontogenetic) and extrinsic (i.e. environmental) factors important to modulating variation in the stable isotope ratios of C and N in tooth dentin of male Antarctic fur seals (Arctocephalus gazella) in the Southern Ocean. In the second study, long-term records of variation in δ15N δ13C values of Atlantic salmon (Salmo salar) scales and grey seal (Halichoerus grypus) tooth dentin provided evidence for large-scale climate forcing across the eastern North Atlantic. In the following study, a more detailed examination of intra- and inter-individual stable isotope variation in Atlantic salmon within a single year was undertaken in an attempt to better understand recent declines in somatic condition of these fish. The last two studies were concerned with the development of high resolution sampling of fish otoliths using secondary mass spectrometry (SIMS) and the application of this technique to reconstructing the thermal and metabolic histories of individual Atlantic salmon from intra-otolith δ13C and δ18O values. Stable isotope proxies can be used to document shifts in trophic dynamics and animal movement that may be associated with ecological change. Using multiple tissues, elements and species, such studies provide unique monitoring tools at a range of spatial and temporal scales.
  
  
  
• Putting pollination quality into analyses of floral ecology: testing syndromes through pollinator performance
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  Abstract: Over recent years, the extent of specialised and generalised plant-pollinator relationships, and the predictive powers of floral traits (often grouped into “pollination syndromes”) as indicators of the most effective pollinators of plant species, have been questioned. Such studies, however, have used proxies such as visitation frequency rather than direct measurements of pollinator effectiveness (PE). The main objective of this thesis was to test the predictive powers of various pollination syndromes using a specific measure of PE: single-visit stigmatic pollen deposition (SVSPD). Six different classical pollination syndromes were tested, using 13 different plant species from tropical and temperate habitats, and in the case of flowers typical of the hummingbird, hoverfly, bee, oil flower and long-tongued insect syndromes, the expected pollinators were the most effective at a single-visit scale. For generalist pollination syndrome flowers, not all observed visitors were significant pollinators, and the species studied were not as broadly generalised as their visitor assemblages would suggest. In all 13 plant species, pollinator performance could appear consistent within functional visitor groups but was variable between visitor species, and in almost all cases not all of the observed visitors were effective pollinators. The pollinator performance proxies of visit duration and feeding behaviour were neither significantly, nor consistently, related to PE. Visit duration was not an accurate indicator of pollinator performance on its own, though it was useful when combined with SVSPD to define pollinator performance at a given time scale, for example per hour, per day or per season. My findings suggest that the results of recent “pollination” networks and webs, based on visitors but not necessarily pollinators, should be treated with caution. SVSPD therefore proved to be an effective and relatively simple direct measure of PE, confirming the predictive powers of pollination syndromes, and giving further insight into the extent of specialisation and generalisation.
  
  
  
• An investigation into the effects of a simulated human gastro-intestinal tract has on Bacillus cereus and Bacillus weihenstephanensis viability and pathogenicity
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  Abstract: Bacillus cereus is one of the known causes of diarrhoeal food poisoning. In their natural environment of soil surviving as spores facilitates their colonisation of raw food ingredients enabling their access to the food chain. Recently psychrotrophic strains of B. cereus have been reclassified based on divergent cold shock gene (cspA) sequences and renamed B. weihenstephanensis. It is the modified cspA gene that is thought to confer the psychrotolerant phenotype witnessed by these strains. Aside from cspA, B. cereus and B. weihenstephanensis are closely related, leading to questions about its pathogenicity and ability to mediate diarrhoeal food poisoning outbreaks. Food producers use a variety of processes to limit microbial contamination within food products. Although effective against vegetative cells, spores are often resistant and as such can persist within this environment. Chilled temperatures (4°C) are often used to limit the growth of any contaminating microbes. Under such conditions B. cereus spores would remain dormant however B. weihenstephanensis spores have been shown to germinate and outgrow under refrigerated conditions. This could result in the consumption of both B. cereus and B. weihenstephanensis spores and vegetative cells. The effect that the human gastro-intestinal tract (GI) has on B. cereus and B. weihenstephanensis vegetative cells and spores is unclear. This study showed no difference in the viability of B. cereus or B. weihenstephanensis strains to survive and grow within a simulated human GI tract. Vegetative cells were revealed to die quickly in the stomach. Spore viability was shown to reduce in the stomach environment by approximately 10⁴-fold. With a larger initial inoculum, 10⁷ spore/ml, viable spores were still recorded after 4 hours. These spores subsequently germinated within the small intestinal simulation and the resulting vegetative cells rapidly proliferated. Mass spectrometry illustrated the ability of vegetative cells from both B. cereus and B. weihenstephanensis to produce an array of secreted proteins whose function were predominately related to virulence and pathogenesis. B.weihenstephanensis strain 10202 was shown to produce the potent cytotoxin, CytK-1, while other B. weihenstephanensis and B. cereus tested strains possessed either or both Nhe and Hbl toxins. The primary diarrhoeal virulence factor/haemolysin BL was shown to be present in the supernatant of each strain through western blotting. Significantly smaller concentrations of each protein were detected, however, under simulated human GI tract conditions when compared to optimal conditions. The effects of the simulated human GI tract on virulence gene expression were monitored through real time PCR. No pattern between B. cereus and B. weihenstephanensis strains was found confirming that virulence gene expression is strain specific. Some genes were shown to be significantly upregulated such as fur, (the ferric iron uptake regulator and groEL, encoding a molecular chaperone. The expression of others however was reduced such as haemolysin BL components, hblA and hblC. Overall there were no significant differences detected between B. cereus and B. weihenstephanensis strains in their ability to survive the human GI tract and express virulence factors associated with diarrhoeal food poisoning.
  
  
  
• The effects of sound propagation and avoidance behaviour on naval sonar levels received by cetaceans
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  Abstract: The use of active sonar is deemed to be essential for naval operations, but its potential impact on marine life has raised concerns worldwide. In a risk-assessment framework, characterisation of risk of harm is accomplished by combining exposure assessment and dose−response relationships. The overall topic of this thesis is an evaluation of factors that influence exposure assessment, including analysis of how sound levels received by cetaceans are affected by in-situ sound propagation and the influence of diving, movement and possible avoidance behaviour of the whales themselves. Data from an international research programme based on controlled exposure experiments (CEEs) were available for this study. During these experiments, low-frequency active sonar (LFAS: 1-2 kHz band) and mid-frequency active sonar (MFAS: 6-7 kHz band) signals were recorded by suction-cup tags attached to killer whales, long-finned pilot whales and sperm whales, and by a hydrophone array towed near the whales. Chapter two describes how the sonar signals recorded by these systems were quantified, and investigates the influences of range, depth and propagation conditions on the received sound levels. Chapter three focuses upon the effect of simulated vertical and horizontal exposure-avoidance strategies of whales in response to an approaching source on the received sound levels. A total of 7,091 sonar signals were analysed from the towed-array (2,794) and tag (4,297) recordings. Transmission loss (TL) and excess attenuation (EA) from a simple 20log(range) model were compared among species, signal types and acoustic receivers. TLs followed expected geometric spreading versus range and TL coefficients were 15.5−20.1 for LFAS and 18.8−23.6 for MFAS. One experiment where levels on the animal-attached tag were attenuated due to ‘body shielding’ (when the animal’s body is interposed between the sound recording tag and the sound source) was documented, and other sources of variation in received level dataset were discussed. Variations in EA with depth were consistent with TL patterns predicted using the acoustic propagation model Bellhop with the highest EAs occurring near the sea surface. The effect of depth on EA was clearest in killer and pilot whale experiments which occurred at locations with stronger gradients in the sound-speed profile, while sperm whale experiments in deeper homogenous offshore waters showed little influence of depth on EA. The results indicate that a simple TL model like 20log(range)+absorption does not accurately predict attenuation levels over the distances (0.1−11.1 km) from a sonar source to a freely-diving animal, but that the overall patterns of TL can be fairly well explained using sound propagation models that take into account local environmental conditions. A consistent different in TL between LFAS and MFAS signals was not explained by the Bellhop model, however, indicating that unidentified sources of variation do influence the sonar signals recorded on freely-diving whales. To evaluate the potential effect of avoidance strategies of whales on received sound levels, whale positions were simulated with a Monte Carlo method in which a simulated source vessel directly approached the whale. The cumulative sound exposure level (SELcum) received by the whales was estimated using the Bellhop model. Horizontally-stationary animals received the highest levels. The optimal course in terms of reducing SELcum for animals moving in a straight line was 100° from the heading of the source vessel, while 120−130° was optimal for animals dynamically moving relative to the position of the source. Moving horizontally in the optimal direction away from the vessel path yielded 9−17 dB reduction of SELcum and vertical avoidance led to reductions of up to 10 dB in certain circumstances. Actual observations of the whales during the sonar experiments indicated that animals often move sideways out of the path of the approaching vessel, close to the optimal angle predicted. The simulation approach is therefore potentially useful to predict how whales react to an approaching sound source. This type of analysis may also be useful to understand the patterns of cetacean strandings relative to the movement of sonar-transmitting military vessels.
  
  
  
• The vocal imitation of bottlenose dolphin (Tursiops truncatus) signature whistles: their use in vocal matching interactions and their role as vocal labels
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  Abstract: The bottlenose dolphin uses vocal learning to develop its own unique acoustic signal. This signal encodes the identity of the signaller, and is known as the animal’s signature whistle. The dolphin’s ability for vocal learning means that the signature whistle of one animal may be found in the vocal repertoire of other animals. This copying of signature whistle types may allow conspecifics to label or address one another. This thesis investigated the use of signature whistle copying in both captive and wild animals. Dolphins have been known to rapidly imitate each other’s signature whistle in vocal matching exchanges. This matching of sounds has an aggressive connotation in songbirds, yet the function in bottlenose dolphins remains unclear. I have shown, through playback experiments with captive animals, that signature whistle matching in dolphins is not aggressive but appears to be affiliative, and may be used as a tool to initiate contact with the signature whistle owner. In addition, the rapid matching of whistle types in wild dolphins appears to play a specific role in their foraging behaviour, indicating the function of vocal matching may indeed be multi-faceted. This thesis also uses a unique dataset to extensively describe the occurrence of signature whistle copying in wild animals. The imitation of individual signature whistles occurred almost exclusively between close associates during separations. All copies were accurate representations of the original signature whistle, but were clearly recognisable due to fine-scale differences in selected acoustic parameters, and are therefore unlikely to be used deceptively. Finally, I provide evidence that wild bottlenose dolphins respond to hearing a copy of their own signature whistle by calling back. This offers support to the notion that signature whistles function as distinctive labels that are used to address individuals, and perhaps even label them referentially.
  
  
  
• The role of intertidal seagrass Zostera spp. in sediment deposition and coastal stability in the Tay Estuary, Scotland
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  Abstract: The Tay estuary is situated on the east coast of Scotland. The estuary is dominated by sediment biotopes, including mudflats which support sparse beds of two nationally scarce seagrass species, Zostera marina var. angustfolia (Hornem.) and Z. noltii (Hornem.). Seagrasses have been described as ecosystem engineers, shaping their sediment environment, and this may increase sediment deposition and stability. In this thesis the ecosystem engineering characteristics of seagrass habitats are explored. In 2008, the distribution of Zostera spp. in the Tay estuary was surveyed and mapped for the first time. Sediments within beds of Z. marina and Z. noltii were compared to investigate the influence of seagrasses on sediment characteristics. To explore the role of seagrass in sediment deposition and erosion, and coastal stability, sediment depth measurements were made in patches of Z. noltii, Z. marina and bare sediment over one year. The role of the root/rhizome system on sediment retention over winter was also considered. Sediment deposition in Z. noltii beds, and the influence of the plants on near-bed flow dynamics was further explored in the laboratory, using an 8 m seawater flume. In the field the retention of particles over 2 and 14 tides was measured, and the results of this experiment led to a study of the influence of leaf and sediment biofilms on particle retention, using the novel method of magnetic particle induction (MagPI). The efficacy of artificial seagrass beds and Z. noltii transplantation as habitat restoration techniques were compared over one year. During the trial, sediment deposition and changes in sediment characteristics were determined, and the protection given to saltmarsh cliffs fringing the study plots was assessed. Mechanisms underlying the results are suggested and the findings discussed. This study provides an insight into the ecology of seagrass in the Tay estuary and its role as an ecosystem manager. It may offer valuable data which could be utilised for future conservation policies, habitat restoration schemes and management planning of the area.
  
  
  
• Impact of interferon β and interferon stimulated gene induction on Bunyamwera virus replication
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  Abstract: The first line of defence against viral infection is the interferon (IFN) response, which must be overcome by a virus for successful replication. Pattern recognition receptors detect virus which triggers induction of IFNβ. Secreted IFNβ stimulates the JAK/STAT signal transduction pathway and the upregulation of IFN stimulated genes (ISGs) culminating with expression of hundreds of antiviral proteins. Bunyamwera virus (BUNV) is the prototype virus for the genus Orthobunyavirus and the family Bunyaviridae. BUNV is a trisegmented single stranded negative sense RNA virus whose genome comprises the Large (L), Medium (M) and Small (S) RNA segments. The L segment encodes the RNA polymerase, the M segment the two glycoproteins Gn and Gc and a non-structural protein NSm, and the S segment the nucleoprotein and a non-structural protein NSs in overlapping reading frames. The NSs protein interferes with RNA polymerase II mediated transcription thereby inhibiting cellular mRNA production, including IFN mRNA, and hence it is the primary IFN antagonist. A recombinant virus, rBUNdelNSs, that is unable to express the NSs protein, does not inhibit cellular transcription and is thus a strong IFN inducer. The aim of this thesis was to understand how IFN inhibits BUNV replication. Cells stimulated into the antiviral state by IFN treatment were protected against BUNV infection but addition of IFN 6 hours (or later) post infection had little effect on the replication cycle. However, addition of IFN immediately following infection conferred restriction on BUNV replication by initially increasing viral protein synthesis and then by blocking translation of positive sense viral RNA. To identify ISGs with anti-BUNV activity, I screened a panel of 26 cell lines that inducibly express individual ISGs. To aid screening, recombinant BUNV that expressed green fluorescent protein (GFP) were employed, including an NSs deletion virus with GFP fused to the Gc, rBUNGceGFPdelNSs, that I created and characterised. By a combination of virus yield assays, Western blotting and fluorescence techniques, three cell lines that inducibly express PKR, viperin or MTAP44 were shown to restrict BUNV replication. More detailed studies revealed PKR to restrict BUNV RNA and protein synthesis, but when PKR was knocked-down in IFN competent A549 cells viral replication was not blocked in cells pre-treated with IFN. Viperin inhibited viral protein synthesis and virally-induced host cell protein synthesis shut-off. Additionally, viral RNA synthesis was restricted by viperin and this was dependent on the CX₃CX₂C motif 1 of viperin. Taken together, these data show that the restriction of BUNV replication mediated by IFN is an accumulated effect of several different ISGs acting on different stages of the viral life cycle.
  
  
  
• Statistical developments for understanding anthropogenic impacts on marine ecosystems
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  Abstract: Over the past decades technological developments have both changed and increased human in influence on the marine environment. We now have greater potential than ever before to introduce disturbance and deplete marine resources. Two of the issues currently under public scrutiny are the exploitation of fish stocks worldwide and levels of anthropogenic noise in the marine environment. The aim of this thesis is to investigate and develop novel analyses and simulations to provide additional insight into some of the challenges facing the marine ecosystem today. These methodologies will improve the management of these risks to marine ecosystems. This thesis first addresses the issue of competition between humans and grey seals (Halichoerus grypus) for marine resources, providing compelling evidence that a substantial proportion of the sandeels consumed by grey seals in the North Sea are in fact H. lanceolatus, which is not commercially exploited, rather than the commercially important A. marinus. In addition, we present quantitative results regarding sources of bias when estimating the total biomass of sandeels consumed by grey seals. Secondly, we investigate spatially adaptive 2-dimensional smoothing to improve the prediction of both the presence and density of marine species, information that is often key in the management of marine ecosystems. Particularly, we demonstrate the benefits of such methods in the prediction of sandeel occurrence. Lastly this thesis provides a quantitative assessment of the protocols for real-time monitoring of marine mammal presence, which require that acoustic operations cease when an animal is detected within a certain distance (i.e. the "monitoring zone") of the sound source. We assess monitoring zones of different sizes with regards to their effectiveness in reducing the risks of temporary and permanent damage to the animals' hearing, and demonstrate that a monitoring zone of 2 km is generally recommendable.
  
  
  
• Biological diversity of freshwater fishes in small streams in peninsular Malaysia
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  Abstract: Peninsular Malaysia has a diverse flora and fauna, much of which is yet to be documented. The freshwater fishes are one important group that have received little attention. Accordingly, the overarching goal of my study is to investigate the pattern of species richness and analyse the community composition and assemblage structure of fishes in the small streams in Peninsular Malaysia. Small stream habitats appeared to be particularly important repositories of fish biodiversity in this region thus obtaining a reliable census of species occurring in such habitats is critical for conservation and management of biodiversity. Although samplings were far from completed, these habitats support a great variety of species with more than 100 species were recorded from fifty streams sampled in this study. A few are extremely rare with restricted distribution and can thus be considered important in biodiversity conservation of the Peninsular Malaysian ichthyofauna. Human-influenced modification of lowland, headwater stream habitats in Peninsular Malaysia is common and often exemplified by the creation of pools in stretches of rapids and riffles. However, it was not possible to separate pristine and disturbed sites which contained almost identical for species diversity. These findings suggest that local habitat modification does not necessarily cause a decrease in freshwater fish diversity, with only minor negative consequences for other community variables recorded in this study, and therefore raise interesting issues regarding conservation. That said it remains premature to conclude that small stream fishes are insensitive to disturbance and thus their potential utility as bioindicators of disturbance-influenced community changes remain to be confirmed. The maintenance practises being applied to small streams modified for recreational usage were not imposing detectable negative consequences, at least across the sites sampled in this study. The rich diversity of tropical stream environments is the result of both within-habitat (alpha) diversity and between-habitat (beta) diversity. The results showed that there was substantial beta diversity particularly amongst sites that are geographically separated from one another. On the contrary, the lowest beta diversity values were portrayed by contiguous sites. Many fishes exhibited discontinuous patterns of distribution and were considered to be rare while only a handful were widely distributed and abundant. Ordination based on the relative resemblance of fish communities to one another support the existence of two distinct ichthyogeographic divisions in Peninsular Malaysia. It was possible to assign the species recorded to all seven of Rabinowitz’s categories of rarity, with at least 10 restricted to a single stream and locally scarce, although not all of these could be described as hyper-endemic. It is recommended that a sizeable augmentation of the existing protected areas is needed to safeguard Malaysia’s exceptionally diverse stream-dwelling fauna of which fishes are simply the most well-known inhabitants. Conservation managers should therefore place particular emphasis on small streams since localities in close proximity to one another can exhibit surprisingly high beta diversity, meaning that partial or small-scale habitat protection may prove insufficient.
  
  
  
• The function of roll in foraging behaviour of sperm whales
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  Abstract: Though direct experiments to test echolocation in sperm whales (Physeter macrocephalus) remain impossible, the indirect evidence is overwhelming that one function of their clicks is echolocation used during foraging, as has been shown for other toothed whales. In this thesis, the aim was to test: 1) the hypothesis by Fristrup and Harbison (2002) that the sperm whale might be using downwelling light during the day to spot prey or prey patches as silhouettes against the lighter sea surface. 2) A second hypothesis by Fristrup and Harbison (2002) suggested that the sperm whales use vision to look for bioluminescence in prey at depth, or during the night when no light is available. 3) Indications that the sperm whales changed their roll-behaviour, rolling more extensively when they were producing regular clicks during the descent as well as for the ascent. Data from 51 sperm whales tagged in the Gulf of Mexico, the Atlantic, the Mediterranean and northern Norway, were tested to explore how large a proportion of the bottom-interval the whales spent at angles where they were able to see towards the lighter sea surface at three different pitch-modes while either searching or buzzing, for each dive. The majority of whales were found to be swimming predominantly upside down or on the side during both the day and night time. The whales were not found to change the way they use roll at night compared to the day time, when looking at the bottom-interval when the whales were both searching for prey and buzzing. Looking at another function for roll and a possible association with the echolocation of the sperm whales, it was found that during the descent, the whales had a tendency to have a higher variance of roll when they were producing regular clicks. A very similar trend showed for the ascent-phase as well, that they seemed to roll more when clicking compared to when they were not, before they reached the sea surface. These analyses of the roll-behaviour of sperm whales used to test the hypotheses put forward by Fristrup and Harbison do not seem to support the use of vision to detect prey during foraging at depth, but might indicate that the sperm whale was intentionally changing its roll-behaviour, when they were producing regular clicks during the ascent and descent-phase in the Gulf of Mexico, the Atlantic, the Mediterranean and Norway.
  
  
  
• Sexual selection and sex allocation in the gregarious parasitoid wasp Nasonia vitripennis
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  Abstract: Sex allocation and sexual selection have been heavily studied, but rarely linked. In this thesis I investigated the interface between them in the gregarious parasitoid wasp Nasonia vitripennis, both directly and through their interactions with the mating system and sexual conflict. Chapter 2 investigated sexual selection and mating at the natal site: earlier eclosing males mated more females independently of body size. Nasonia follows Local Mate Competition, which describes how a female laying eggs alone on a patch of resources (a so-called single-foundress) should lay an extremely female-biased brood to minimise competition between her sons, yet ensure all her daughters are fertilised. Based on this I predicted that males with with fewer brothers would be better inseminators. Despite finding significant among-strain variation in (1) single-foundress sex ratio, (2) mate competitiveness when alone and (3) when in competition, (4) sperm resources, but not (5) sperm-depletion (Chapters 3 & 4), I did not find the predicted relationship. Conversely males from strains with more brothers had a higher mating success under competition (Chapter 3) leading to the question: does mating success select on sex ratio or vice versa? Either way it is a result of an interaction between sexual selection and sex allocation. Chapter 5 investigated the role of male post-copulatory courtship on female re-mating, and found that among- strain variation in female re-mating was not associated with variation in the duration of the post-copulatory courtship. Chapter 6 reviewed sexual conflict in the Hymenoptera: their haplodiploid genetics, newly sequenced genomes and varied life- histories provides a base for future research to build on. Finally I highlight the novel links between sexual selection, sex allocation, sexual conflict and the mating system found during my studies that will hopefully prompt future research on this topic.
  
  
  
• An investigation of human protein interactions using the comparative method
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  Abstract: There is currently a large increase in the speed of production of DNA sequence data as next generation sequencing technologies become more widespread. As such there is a need for rapid computational techniques to functionally annotate data as it is generated. One computational method for the functional annotation of protein-coding genes is via detection of interaction partners. If the putative partner has a functional annotation then this annotation can be extended to the initial protein via the established principle of “guilt by association”. This work presents a method for rapid detection of functional interaction partners for proteins through the use of the comparative method. Functional links are sought between proteins through analysis of their patterns of presence and absence amongst a set of 54 eukaryotic organisms. These links can be either direct or indirect protein interactions. These patterns are analysed in the context of a phylogenetic tree. The method used is a heuristic combination of an established accurate methodology involving comparison of models of evolution the parameters of which are estimated using maximum likelihood, with a novel technique involving the reconstruction of ancestral states using Dollo parsimony and analysis of these reconstructions through the use of logistic regression. The methodology achieves comparable specificity to the use of gene coexpression as a means to predict functional linkage between proteins. The application of this method permitted a genome-wide analysis of the human genome, which would have otherwise demanded a potentially prohibitive amount of computational resource. Proteins within the human genome were clustered into orthologous groups. 10 of these proteins, which were ubiquitous across all 54 eukaryotes, were used to reconstruct a phylogeny. An application of the heuristic predicted a set of functional protein interactions in human cells. 1,142 functional interactions were predicted. Of these predictions 1,131 were not present in current protein-protein interaction databases.
  
  
  
• The genetic basis of flesh quality traits in farmed Atlantic salmon
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  Abstract: The aim was to develop new methods for measuring texture of Atlantic salmon (Salmo salar L.) fillets and investigate the genetic basis of flesh quality traits. Firstly, a new tensile strength method was developed to quantify the force required to tear a standardized block of salmon muscle with the aim of identifying those samples more prone to factory downgrading as a result of gaping. The repeatability, sensitivity and predictability of the new technique was evaluated against other common instrumental texture measurement methods. Data from the new method were shown to have the strongest correlations with gaping severity r=-0.514, P<0.001) and the highest level of repeatability of data when analysing cold-smoked samples. The Warner Bratzler shear method gave the most repeatable data from fresh samples and had the highest correlations between fresh and smoked product from the same fish (r=0.811, P<0.001). It is therefore recommended that the new method be adopted for measuring gaping potential and the Warner Bratzler method become the standard for firmness assessment. Genes associated with post mortem softening in mammals were characterised in Atlantic salmon. A previously unknown ancient paralogue of calpastatin (here named CAST2) was identified. Evidence was provided for the existence of highly homologous recent paralogues of CAST2 and CTSD1. Evidence for the ancestral history of these paralogues was provided by phylogenetic analysis. Recent gene duplicates of 6 further genes were identified. In all cases, homology between recent paralogues was greater than 94%. Analysis of synonymous vs non-synonymous nucleotide substitution between the observed paralogue pairs shows a significant purifying selection in most cases. The CTSD1 gene shows significant purifying selection in a pairwise analysis between 12 teleost species (all cases P<0.0001) but a similar analysis of CTSD2 revealed no significant occurrence of purifying selection. The present study provides further support for the idea of asymmetrical selective pressure on paralogues. Genetic markers were developed that can distinguish individuals with above average fillet yield and texture. A database of firmness, tensile strength and fillet yield was made from 254 individuals from 5 batches of farmed salmon and these fish were genotyped at 7 novel SNP loci. Individuals with the combined favourable genotype at CAPN1a and MYOD1b were associated with an average increase in fillet yield of 2.7% above batch average. A combined genotype of CAPN1a, MYOD1b and MYF5 was significantly associated with an average increase in tensile strength of 9.8% above batch average (P=0.015). In both cases individuals with the combined favourable genotype occurred with a frequency of c. 6% across all batches. The favourable genotypes had no unfavourable effects on other traits. Highly polymorphic microsatellite loci were used to perform tests of assignment, which revealed an overall correct assignment rate of 92.7% to batch of origin and a minimum reference sample number of 25 was empirically determined. A phylogenetic analysis supported the results of the assignment tests. Given that 7 microsatellites is a relatively small number for a study of this nature, these results suggest that reliable assignment of unknown fish to the true batch of origin is potentially rapid and cost effective. Overall, the thesis presents molecular markers for broodstock selection, new genes of relevance to flesh quality, a new method of texture analysis and a proposal for an escapee traceability project.
  
  
  
• Nitrate transport and assimilation in Aspergillus nidulans
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  Abstract: In this study, several aspects of nitrate assimilation and transport have been studied using the filamentous fungus Aspergillus nidulans, which has been shown to be safe laboratory organism as judged by it’s pathogenicity towards insect larvae. In silico analysis of the A. nidulans genome sequence, identified two putative genes designated cnxL and cnxK that might be involved in molybdenum cofactor (a component of nitrate reductase) biosynthesis as well as two putative nitrate reductases encoding genes niaB and niaC. All four genes are hitherto unknown. Although many features of these proteins provided clues of functionality, biochemical and genetical approaches employed in this present study failed to elicit expression of any of these four genes. A NrtA protein structure model was developed based on residue homology with the E. coli GlpT a protein, the structure of which has been solved. The results of thiol cross-linking of three double cysteine mutants in four NrtA essential residues, R87, R368, N168 and N459, indicated that the molecular distance between R87 and R368 is ~ 0.4 Å, R368 and N168 ~ 6.2 Å, R87 and N459 is ~ 2.2 Å. Another important observation was the change in the confirmation of Tm 2 and Tm 8 in the presence of nitrate. This shift resulted in an increase of ~ 2 Å gap between the residues R87 and R368. Distances between amino acid residue pairs estimated using such molecular rulers contradicted the NrtA existing model. Cysteine-scanning mutagenesis studies were extended to the generation of a library of single cysteine mutants of NrtA residues spanning Tm 2 and Tm 8. The majority of single cysteine mutants possessed wild type NrtA protein expression levels but unfortunately most were found to be loss-of-function. Consequently, thiol chemistry of this crop of mutants was not perused. Attempts were also made to overexpress and crystallise the bacterial nitrate transporters but none of the transporter tested proved to be a successful candidate for crystallisation. In this regard, bacterial nitrate transporters, NarU (E. coli), Nar (Bacillus cereus), NarK1 and NarK2 (Pseudomonas aeruginosa) and NarK2 (Thermus thermophilus) fused with GFP were expressed in E. coli and used in crystallisation trials. Although this approach has proved successful for a number of membrane proteins, unfortunately was not helpful with regard to the purification of any of the above bacterial nitrate transporters to yield protein expression levels required for successful protein crystallography. Finally, the effects of potential nitrate transport inhibitors were studied on net nitrate transport by NrtA and NrtB proteins of A. nidulans. The results indicated that chlorate had more of an inhibitory effect on NrtA net nitrate transport than that by NrtB. Chlorite and sulphite equally affected net nitrate transport by either NrtA or NrtB proteins while caesium strongly inhibited the net nitrate transport by NrtB transporter.
  
  
  
• New intracellular mechanisms involved in Alzheimer's disease and frontotemporal dementia
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  Abstract: Dementia causes an increasing social and economic burden worldwide, demanding action regarding its diagnosis, treatment and everyday management. Recent years have seen many advances in neurodegeneration research, but the search for new truly disease modifying therapies for Alzheimer's disease (AD) and frontotemporal dementia (FTD) has so far not been successful. This is mainly due to a lack of understanding of the precise intracellular events that lead up to neuronal dysfunction in early and in late stages of the disease. This thesis describes the approaches taken to extend the current knowledge about the intracellular effects of neuronal amyloid-beta and the signalling pathways causing neuronal death or disturbed synaptic function in dementia. Endophilin-1(Ep-1), amyloid-binding alcohol dehydrogenase (ABAD), peroxiredoxin-2 (Prx-2) and the EF-hand domain family, member D2 (EFHD2) have been found to be elevated in the human brain with dementia and in mouse models for frontotemporal lobar degeneration (FTLD) or AD. The expression of these proteins as well as the expression of c-Jun N-terminal kinase (JNK), c-Jun and APP were analysed by western blotting and real-time PCR in human brains affected by AD or FTLD as well as in mouse models for AD. This provided a new insight into the regulation of these proteins in relation to each other in the ageing brain and uncovered a new potential link between elevated levels of EFHD2, Prx-2 and APP in FTLD. By studying the effects of the overexpression of Ep-1 in neurons, this research has led to a better understanding of its role in JNK-activation. It furthermore verified a protective role for Prx-2 against neurotoxicity and pointed towards a new function for Prx-2 in the regulation of JNK-signalling. The analysis of the effect of increased levels of EFHD2 uncovered for the first time its involvement in the PI3K-signalling cascade in neuronal cells. The current work has therefore contributed to the knowledge about the cellular processes that are affected by Ep-1, Prx-2 and EFHD2 in different types of dementia and will greatly benefit future research into their actions in the neuronal network.
  
  
  
• Characterisation of proteins involved in CRISPR-mediated antiviral defence in Sulfolobus solfataricus
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  Abstract: One of the most surprising realisations to emerge from metagenomics studies in the early ‘00s was that the population of viruses and phages in nature is about 10 times larger than the population of prokaryotic organisms. Thus, bacteria and archaea are under constant pressure to develop resistance methods against a population of viruses with extremely high turnover and evolution rates, in what has been described as an evolutionary “arms race”. A novel, adaptive and heritable immune system encoded by prokaryotic genomes is the CRISPR/Cas system. Arrays of clustered regularly interspersed short palindromic repeats (CRISPR) are able to incorporate viral or plasmid sequences which are then used to inactivate the corresponding invader element via an RNA interference mechanism. A number of CRISPR-associated (Cas) protein families are responsible for the maintenance, expansion and function of the CRISPR loci. This system can be classified in a number of types and subtypes that differ widely in their gene composition and mode of action. This thesis describes the biochemical characteristics of CRISPR-mediated defense in the crenarchaeon Sulfolobus solfataricus. The process of CRISPR loci transcription and their subsequent maturation into small guide crRNA units by the processing endonuclease of the system (Cas6) is investigated. After this step, different pathways and effector proteins are involved in the recognition and silencing of DNA or RNA exogenous nucleic acids. This thesis reports the identification and purification of a native multiprotein complex from S. solfataricus P2, the Cmr complex, a homologue of which has been found to recognise and cleave RNA targets in P. furiosus. The recognition and silencing of DNA targets in E. coli has been shown to involve a multiprotein complex termed CASCADE as well as Cas3, a putative helicase-HD nuclease. S. solfataricus encodes orthologues for the core proteins of this complex, and the formation and function of an archaeal CASCADE is investigated in this thesis.
  
  
  
• A modulatory role for the NMDA receptor glycine binding site during fictive locomotion in Xenopus laevis larvae
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  Abstract: One of the most surprising realisations to emerge from metagenomics studies in the early ‘00s was that the population of viruses and phages in nature is about 10 times larger than the population of prokaryotic organisms. Thus, bacteria and archaea are under constant pressure to develop resistance methods against a population of viruses with extremely high turnover and evolution rates, in what has been described as an evolutionary “arms race”. A novel, adaptive and heritable immune system encoded by prokaryotic genomes is the CRISPR/Cas system. Arrays of clustered regularly interspersed short palindromic repeats (CRISPR) are able to incorporate viral or plasmid sequences which are then used to inactivate the corresponding invader element via an RNA interference mechanism. A number of CRISPR-associated (Cas) protein families are responsible for the maintenance, expansion and function of the CRISPR loci. This system can be classified in a number of types and subtypes that differ widely in their gene composition and mode of action. This thesis describes the biochemical characteristics of CRISPR-mediated defense in the crenarchaeon Sulfolobus solfataricus. The process of CRISPR loci transcription and their subsequent maturation into small guide crRNA units by the processing endonuclease of the system (Cas6) is investigated. After this step, different pathways and effector proteins are involved in the recognition and silencing of DNA or RNA exogenous nucleic acids. This thesis reports the identification and purification of a native multiprotein complex from S. solfataricus P2, the Cmr complex, a homologue of which has been found to recognise and cleave RNA targets in P. furiosus. The recognition and silencing of DNA targets in E. coli has been shown to involve a multiprotein complex termed CASCADE as well as Cas3, a putative helicase-HD nuclease. S. solfataricus encodes orthologues for the core proteins of this complex, and the formation and function of an archaeal CASCADE is investigated in this thesis.
  
  
  
• Conservation ecology and phylogenetics of the Indus River dolphin (Platanista gangetica minor)
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  Abstract: The historical range of the Indus River dolphin has declined by 80% since the 19th century and has been fragmented into 17 river sections by construction of irrigation barrages. Dolphin sighting and interview surveys showed that river dolphins persist in six river sections, have been extirpated from ten, and are of unknown status in the remaining section. Logistic regression and survival modelling showed that low dry season river discharge was the primary factor responsible for the Indus dolphins range decline. Abundance of the three largest Indus dolphin subpopulations was estimated using tandem vessel-based direct counts, corrected for missed animals using conditional likelihood capture-recapture models. The entire subspecies was estimated to number between 1550-1750 in 2006. Dolphin encounter rates within the Guddu-Sukkur subpopulation (10.35/km) were the highest reported for any river dolphin and direct counts suggest that this subpopulation may have been increasing in abundance since the 1970s when hunting was banned. The dry season habitat selection of Indus dolphins was explored using Generalised Linear Models of dolphin distribution and abundance in relation to river geomorphology, and channel geometry in cross-section. Channel cross-sectional area was shown to be the most important factor determining dolphin presence. Indus dolphins avoided channels with small cross-sectional area <700m2, presumably due to the risk of entrapment and reduced foraging opportunities. The phylogenetics of Indus and Ganges River dolphins was explored using Mitochondrial control region sequences. Genetic diversity was low, and all 20 Indus River dolphin samples were identical. There were no haplotypes shared by Indus and Ganges River dolphins, phylogenetic trees demonstrated reciprocal monophyletic separation and Bayesian modelling suggested that the two dolphin populations diverged approximately 0.66 million years ago. Declining river flows threaten Indus dolphins especially at the upstream end of their range, and it is important to determine how much water is required to sustain a dolphin population through the dry season. Fisheries interactions are an increasing problem that will be best addressed through localised, community-based conservation activities.
  
  
  
• Modulation of mammalian spinal motor networks by group I metabotropic glutamate receptors : implications for locomotor control and the motor neuron disease amyotrophic lateral sclerosis
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  Abstract: The present study examined the role of group I metabotropic glutamate receptors (mGluRs) in mammalian spinal motor networks and investigated the potential role of mGluRs in the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Group I mGluR activation was found to modulate locomotor-related activity recorded from ventral roots of in vitro mouse spinal cord preparations. Activation of group I mGluRs led to an increase in the frequency of locomotor-related bursts and a decrease in their amplitude. The cellular mechanisms underlying group I mGluR-mediated modulation were investigated using whole-cell patch-clamp recordings from spinal neurons. Recordings from motoneurons revealed a wide range of effects, some of which were expected to increase motoneuron excitability, such as membrane depolarisation and hyperpolarisation of action potential thresholds. However, the net modulatory effect of group I mGluR activation was a reduction in motoneuron excitability, likely reflecting a reduction in the density of fast inactivating Na+ currents. The activation of group I mGluRs also reduced excitatory synaptic input to motoneurons, suggesting that modulation of motoneuron properties and synaptic transmission both contribute to group I mGluR-mediated reductions in locomotor motoneuron output. Recordings from spinal interneurons revealed a smaller range of modulatory effects for group I mGluRs. The clearest effect on interneurons, membrane depolarisation, may underlie group I mGluR-mediated increases in the frequency of locomotor activity. Finally, the potential role of group I mGluRs in the pathogenesis of ALS was investigated using a mouse model of the disease. Although no major perturbations in group I mGluR-mediated modulation were demonstrated in ALS affected spinal cords, there appeared to be a difference in the intrinsic excitability of spinal interneurons between wild type and ALS affected animals. Together these data highlight group I mGluRs as important sources of neuromodulation within the spinal cord and potential targets for the treatment of ALS.
  
  
  
• The passage of electric currents through tissues : (with particular reference to the percutaneous stimulation of human nerve and muscle)
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  Abstract: The physiologist enjoys a great advantage over the clinical neurologist in the electrical stimulation of muscle, in that he is generally able to kill or anaesthetise his experimental animal and to excise or expose the tissues to be stimulated. The electrical impedance of the preparation is predominantly resistive, and the question of the comfort and safety of the animal scarcely arises. The stimulation of human muscle, on the other hand, normally has to be carried out on ac cious subject, whose comfort and safety must be considered, by means of electric currents applied through electrodes placed in contact with the skin, which exhibits very complex electrical properties. Conflict arises between the requirements of technical accuracy and ease of interpretation of results on the one hand, and comfort and convenience on the other. This thesis represents an attempt to assemble as much as possible of the information required for intelligent solution of the problems encountered in the percutaneous stimulation of muscle. Part I of the thesis reviews published information on nerve, muscle and denervated muscle, the electrical impedance of tissues, and diagnostic and therapeutic stimulation of muscle. Part II describes the author's personal research into the electrical impedance of the body, the interplay between body impedance, stimulator impedance and the electrical excitability characteristics of nerve and muscle, and the influence of these various factors on stimulator performance. In view of the variety of topics involved, the material has been arranged in separate more or less self-contained sections, including discussion of the implications of the results in each section. Technical details of the main electronic devices used are given in the Appendix, (Part III, together with details of the various mathematical analyses and calculations. Part of the work on which this thesis is based was carried out during the tenure of a temporary lectureship in Biophysics donated by the late Sir David Russell, to whom the author is deeply indebted. The author is also greatly indebted to Professor A. E. Ritchie for creating the circumstances which made this work possible, for permitting the author access to unpublished material, and for his patient encouragement and support of this protracted project; to Dr J. Crossland for assistance in the translation of German texts, and to Mr E. Carstairs for advice and assistance in devising the photographic techniques used in recording and reproducing the experimental results.
  
  
  
• The development of a system to facilitate the stable expression of mammalian proteins in the filamentous fungus 'Aspergillus oryzae'
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  Abstract: Human interleukin 6 (hIL6) is a multifunctional cytokine effecting the function and proliferation of many cell types. The further understanding of hIL6 and its possible medical applications rely on the availability of this protein. The filamentous fungus Aspergillus oryzae is an important industrial organism and is used for the large scale production of many enzymes. As this fungus has an impressive secretory output it was decided to attempt to produce hIL6 in this organism. The initial work was based on the development of a gene transfer system for A. oryzae in order that the hIL6 gene could be introduced to the organism. A transformation system based on the homologous nitrate reductase gene is described. This system yielded up to 800 transformants per µg plasmid DNA. Additionally, the adaptation of the transformation system based on the A. nidulans anuiS gene and the use of other transformation systems is reported. In order to ensure that the hIL6 gene was efficiently transcribed it was considered important that homologous control regions from highly produced and regulated A. oryzae genes were linked to the hIL6 gene. Therefore the genes encoding glucoamylase and a-amylase were isolated from A. oryzae. A method for the purification of A. oryzae Si nuclease is described and the amino acid sequence of the N terminus is reported. A. oryzae produces large amounts of extracellular proteases, a feature unlikely to be attractive in a heterologous host. Therefore the production of protease production in A. oryzae was studied. A method is described for the selection of protease deficient mutants. Using this method two protease mutants were isolated and these have been characterized. One mutation designated prtA2 protects against the degradation of hJL6 in vitro. The A. oryzae alkaline protease gene was isolated and mutagenised and the attempts made to produce specific protease mutant by reverse genetics are described. A system is described where by A. oryzae can be engineered to produce relatively high levels of hIL6. Using gene fusion constructs transformants producing in the range of 1 mg per Litre have been isolated. This system is heterologous, recommendations for increasing hIL6 production are included.
  
  
  
• Studies on the pollination biology and breeding systems of some genera with sympatric species in the Brazilian cerrados
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  Abstract: Flowering phenology, floral strategies pollinator activity and breeding system were investigated in 14 species of the cerrado vegetation in Braeflia (DF), from April 1985 to April 1986 in Tabebuia caraiba (Mart.) Bur., T. ochracea (Cham.) Standl.; Erythroxylum campestre St. Hil., E. suberosum St. Hil., E. tortuosum Mart.; Diplueodon crulsianue Pohl., D. oblongus Pohl., D. ramosissimus Pohl., D. roomarinifolius St. Hil., D. villosus Pohl.; Kielneyera abdita Saddi, K. coriacea Mart., K. speciosa St. Hil. and K. variabilis Mart. The species occur sympatrically, often only a few meters apart, and conspecifics frequently occur in clumps. The majority of the species flowered during the dry season (May to August) except D. oblongue, D. villosue and K. speciosa which flowered during the wet season (December to March). Most of them flowered for several months except Tabebuia (one month). The most frequent flowering pattern was the "Cornucopian"' type, except for D. crulsianue and D. Villosus which were "Steady-State" types. Most congeners flowered synchronously, except K. speciosa which showed displacement. Flowers of all species are pollen donors (Tabebuia and Erythroxylum also have nectar rewards), but each genus presents specific floral strategies; Tabebuia, Massflowering, Erythroxylum, distyly and cauliflory, Diplueodon, enantiostyly and Kielmeyera, andromonoecy. Flowers were visited by a large spectrum of bees (38spp. ) and Erythroxylum were also visited by wasps (16 app. ). However, each genus had particular pollen vectors. Tabebuia; Centris and Bombus; Erythroxylum: wasps; Diplueodon; Apia and Trigona; and Kielmeyera; Xylocopa, despite the fact that other bees occur all year round. Controlled pollination experiments (self-, intraspecific-, interspecific crosses, tests for apomixis and from untreated bagged flowers), revealed that all species have a high outcrossing level and are selfincompatible. Only E. campeatre (shrub), D. crulsianus and D. villosus (hemixyles), showed some self-compatibility. Fluorescence microscopy indicated that pollen tubes from selfing and from interspecific cross pollinations were blocked in the ovary, suggesting that the selfincompatibility barrier operates late. Only in Erythroxylum the blockage occurred in the stigma (Thrum flowers) or in the style (Pin flowers). Fruit-set number from hand-pollinations and from natural pollination were low, but the former frequently yielded a higher number of fruits than the latter. Low fruit-set was associated with lack of resources allocated by the maternal parent for fruit maturation. In addition, it seemed that, in all species, many flowers were programmed to be pollen donors only, since they dropped without any sign of ovary enlargement even if they were hand-pollinated or were frequently visited by pollinators.
  
  
  
• Isolation and characterisation of chlorate resistant mutants of barley
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  Abstract: The object of this study has been to characterise barley mutants which lack the functional nitrate reductase (NR) enzyme system. In the long term it is hoped that such studies will lead to improved nitrate utilisation and ultimately to improved quality barley protein. The progeny of nine chlorate resistant selections, in the barley cultivars Mavis Mink and Golden Promise, were studied. Four (R9201, R11301, R12202 and R12801) lacked NADH-NR and FMNH2-NR activities, the rest had the NR+ phenotype. None of the four were nitrate uptake mutants since they all possesed wild type or greater levels of nitrate. R9201, R11301 and the previously characterised R9401 (Bright et al, 1983) were not molybdenum uptake or Mo-accumulation mutants. R9201, R11301 and R12202 lacked xanthine dehydrogenase (XDH) (an enzyme which contains the same molybdenum-containing cofactor, MoCo) activity suggesting that these lines have defective MoCo's, whilst R12801 possessed XDH activity indicating that it might have defective apoprotein subunits. These four lines are similar to R9401 since they lack "NR activity and are unlike other previously selected ' barley NR mutants (Kleinhofs ' et al, 1980) which are leaky and possess up to 5% of the wild type (cv. Steptoe) in vitro NR activity. R9201 and R11301, like R9401, were all caused by single recessive nuclear gene mutations. The MoCo mutants, R9201, R9401, R11301 and R12202, could be divided into two groups on the basis of i) allelism, ii) presence or absence of wild type levels of dimeric NR and iii) the ability of their extracted MoCo's to reconstitute NADPH-NR in an extract of N. crassa nitl mutant (which supplies NR monomers) in the presence of excess molybdate. R11301 is not allelic to R9201, whilst R9201 and R9401 are allelic. R9401 is also thought to be allelic to Az 34, a barley MoCo mutant isolated and characterised by Kleinhofs et al (1980). Az 34 has been designated nar2a and it is proposed that the allelic R9401 and R9201 should be classified as nar2b and nar2c respectively. It is possible that R11301 is either allelic with one of the other barley MoCo lines, nar3, nar4 or it is defective in a different MoCo gene. The same is also true for R12202. R11301 was shown to possess inactive NR dimer at wild type concentrations, whilst R9201, R9401 and R12202 had little or no NR (inactive dimer) present under these conditions. R12801 possessed no dimer. The MoCo extracted from R11301 was able to reconstitute the same level of NADPH-NR in the Hill extract as MoCo extracted from-the wild types. R9201, R9401 and R12202 lacked this ability.
  
  
  
• Endocrine control of reproduction of the powan of Loch Lomond, 'Coregonus lavaretus (L.)' (Teleostei): with special reference to the pituitary gonadotrophin-gonadal steroid axis
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  Abstract: In order to provide an accurate basis for the correlation of endocrine changes and phases of the reproductive (and associated) cycles in the powan of Loch Lomond, 'Coregonus- lavaretus (L.), the following experiments were carried out, Cell types in the pituitary were identified using light microscopy and transmission electron microscopy. To locate gonadotropic cells an immunofluorescence technique was employed using antibody prepared in rabbit and an immunodiffusion method to detect the presence of antibody in the rabbit serum. In order to purify pituitary gonadotropin, two purification techniques 'were used. One was alcoholic extraction followed by gel filtration on sephadex; the other was buffer extraction followed by affinity chromatography on Concanavalin A-sepharose. To characterise the fractions from columns, two in vitro bioassays were employed; (i) ovulation of the oocytes of medaka (Oryzias latipes); (ii) Cyclic AMP production in immature trout gonads. The concentration of pituitary gonadotropins was measured using a heterologous radioimmunoassay technique. Using a radioimmunoassay technique, the serum testosterone and oestradiol-17β concentrations were determined. Serum proteins were characterised using SDS gel electrophoresis.
  
  
  
• Measurement of changes in marine benthic ecosystem function following physical disturbance by dredging
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  Abstract: Measuring the impact of physical disturbance on macrofaunal communities and sediment composition is important given the increased demand for the exploitation and disturbance of marine ecosystems. The aim of the present investigation was to provide a comprehensive study about the extent to which the disturbance (especially aggregate dredging) may affect benthic ecosystem function. The first part of the thesis concerns a field investigation of the impacts of dredging on the benthic community and related ecosystem function which was measured by different approaches including traditional methods based on benthic community structure and a more novel approach based on the functional traits of benthic organisms. The assessment was done by comparing dredged sites (Area 222, southeast England) with nearby undisturbed reference sites from the years 2001 to 2004 and in 2007. In general, low dredging intensity did not appear to impose great impacts on the benthic community and related ecosystem function compared to the higher intensity activity. Most of the analyses suggested that the community at the high dredging intensity site had yet to recover at the end of this study period. Among many factors related to the recovery of the benthic community was sediment composition where gravel deposits appeared to support a faster biological recovery. Meanwhile, the recovery of species with specific traits, such as tube-building and filter feeding also indicate a faster recovery for the whole community. The experimental work to determine different impacts of Hediste diversicolor on its surrounding depending on its relative size is discussed in Appendix 1.
  
  
  
• Cetacean studies using platforms of opportunity
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  Abstract: As human impact on marine ecosystems continues to grow, so too does the need for sound conservation and management strategies that are informed by science. Cetaceans, the whales, dolphins and porpoises, epitomise this challenge, because they are hard to study, they have been heavily exploited in the past, and because some of their habitats, behaviours and life-history strategies make them acutely vulnerable to human activities. Unfortunately, research on free-ranging cetaceans in remote areas is costly, and financial resources are limited. The approach used in this thesis to acquire inexpensive quantitative information on cetacean populations and behaviour was to seek out platforms of opportunity. Tourism and environmental education projects provided access to remote areas of importance to cetaceans. The topic was explored in two main areas. First, studies were conducted to investigate the use of ships of opportunity in estimating distribution and abundance, namely of Antarctic baleen whales. The second area of interest was the effect of boats on killer whales in the northeast Pacific. Platforms of opportunity proved valuable for collecting data to model the role of measurement error on abundance estimation. Measurement error was found to be a potential source of bias in four distance estimation experiments. Platforms of opportunity could be used to train observers on protocols, and to learn to use range- finding photogrammetric equipment well before conducting dedicated surveys, which would eliminate this source of bias, as well as estimating abundance in some cases. Abundance and distribution of three whale species were modelled using data collected aboard Antarctic tourist ships. Spatial modelling techniques were used to model distribution of minke, fin and humpback whales using line-transect data collected from a survey that could not be randomised. Strong gradients in animal density were predicted, which could be used to inform future surveys. In the meantime, rough estimates of abundance were obtained, and this approach shows promise for other areas where lack of resources makes systematic surveys prohibitively expensive. A government-funded environmental education project provided logistical support for two studies that dealt with effects of boats on killer whale behaviour. One quantified the extent to which a particular style of whalewatching was disruptive to whale behaviour, and commercial whalewatchers agreed to halt this activity. The other found that a protected area conferred benefit to killer whales, even though it protects only a fraction of the whales' habitat for a fraction of the year. The thesis contains four case studies that illustrate how inexpensive methods may be used to obtain practical quantitative information to aid decision-making about conservation and management of wild cetaceans that interact with (i.e., whalewatching), compete with (i.e., fishing) or are exploited by (i.e., whaling) humans. Description: Chapters 1-5 of this thesis have been updated and subsequently published in multi-authored articles in peer-reviewed journals. If required, citations should be made to these updated articles listed in the full item record below
  
  
  
• Biodiversity of freshwater fishes of Trinidad and Tobago, West Indies
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  Abstract: The proximity of the speciose South American continent to Trinidad and Tobago ensures that these islands have a rich and dynamic fauna. According to the equilibrium theory of island biogeography (MacArthur & Wilson 1967), these islands should be subjected to frequent immigrations from the nearby continent, and these additions will, in response, fuel local extinctions to maintain a balance of species richness. The aquatic habitat is further impacted by man's activities, (Government of Trinidad and Tobago 1962; Government of Trinidad and Tobago 1976b) which have the potential to amplify the natural rates of immigration and extinction. Despite the possible consequences, the effects of this disturbance on the fish fauna have not been studied. A survey of the islands' freshwater fishes was therefore carried out to investigate the natural spatial and temporal dynamics of local fish communities, and the effects of anthropogenic perturbations, on these. These baseline data can be used as a benchmark to address some of the problems that are threatening the fish diversity of Trinidad and Tobago. The fish fauna of Trinidad and Tobago is diverse. Total species richness was estimated at between 37 and 40. Four zoogeographic zones were recognised. A zone of Antillean fishes included Tobago and the north coast of Trinidad. A zone of recent colonising South American fauna existed along the south coast of Trinidad. The rest of Trinidad contained a relict South American fauna dating back to the time when Trinidad was still part of the mainland. Included here was a centrally located zone of unstable fauna at risk of local extinction. The fish fauna was temporally dynamic due to frequent colonisation and extinction events. Examination of the historic record showed that observed species richness varied from 38 to 43 between the mid 1950s and the present. During this time there were 15 introductions and 12 local extinctions. A conservative estimate was that a new species was recorded for Trinidad almost every three years. The geographic distribution of species also showed temporal changes which indicated a natural tendency of the fauna to vary over time. Human interference, particularly the introduction of exotic fish species and long-term habitat alteration, has affected species diversity. Forty seven percent of the new introductions and 43% of the extinctions were human-introduced exotics. About four fifths of sites in Trinidad, and almost one fifth of the sites in Tobago, were either perturbed or polluted. Polluted rivers coincided with areas of high urbanisation and industrial development in the west and southwest of Trinidad. Several effects of human interference on the fauna were recorded. Almost 8% of the sites examined contained one of the three exotic species still extant on the island. At each of these sites, the exotic species accounted for between 1.3% and 80.4%, by number, of the fish caught. Some of the effects of habitat disturbance on individual sites were increased frequency of diseases, extirpation of species, changes in species richness and other diversity measures, and the eventual regression of the fish community to opportunistic species (r-strategists). The potential of two fishes, Poecilia reticulata and Astyanax bimaculatus, as indicator species was examined. Astyanax showed better potential as an indicator of habitat quality as it was not found in depauperate communities, typical of severely disturbed habitats, and its proportional abundance and biomass were negatively affected by pollution. Poecilia populations, on the other hand, were found to be insensitive to habitat quality when the above-mentioned criteria were used. They did, however, have a high frequency of diseased individuals at polluted sites. One of the aims of conservation is to protect that portion of biodiversity most at risk of extinction, the rare species (Rabinowitz 1986). Over 70% of freshwater fish species found in Trinidad and Tobago were classified as rare in these islands. This fact, in addition to the loss of diversity recorded for some sites indicates that the implementation of a management strategy for the conservation of the freshwater fish fauna of Trinidad and Tobago is imperative. The management strategy should focus on the amelioration and protection of aquatic habitats since at least 80% of the rare species had either a restricted geographic distribution or narrow habitat specificity. Additionally, protection from overexploitation should be offered to commercially important species with only small populations. Finally, a minimum sample size of 35 sites, spread over different zoogeographic areas, is recommended for estimating species richness for monitoring, an intrinsic part of any management strategy.
  
  
  
• Molecular systematics of 'Rhododendron ponticum' L. and its close allies
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  Abstract: Rhododendron ponticum, introduced to the British Isles in 1763, has become a noxious invasive pest species, particularly in the west. Material of R. ponticum and its close allies in subsection Pontica were subjected to chloroplast DNA RFLP analysis. A preliminary phylogeny based on all cpDNA variation detected is presented. Suggestions regarding the interrelationships of these species are made, and a cpDNA-type of unknown taxonomic identity is tentatively identified. Chloroplast DNA differences were detected between native material of R. ponticum from Turkey, Spain and Portugal. Based on these differences, it was determined that approximately 90% of material naturalised in the British Isles originates from Spain and 10% from Portugal. These two types of material occur in roughly the same proportion throughout the British Isles. About 10% of naturalised accessions were found to contain an rDNA marker indicating nuclear introgression from R. catawbiense. Introgressed individuals are shown to be most common in the coldest region of the British Isles surveyed, i.e. E. Scotland, and it is suggested that introgression from R. catawbiense may confer increased frost tolerance to R. ponticum. The occurrence of the rDNA marker was not correlated with that of morphological markers indicating introgression. Correlations were found between morphological characteristics, which may result from introgression from cultivated species, such as R. maximum. Molecular evidence for the involvement of R. maximum in the ancestry of British R. ponticum was found in two accessions which resembled R. maximum. in certain morphological characteristics. It was established that another unidentified species was involved in the parentage of naturalised material. Five natural hybrid combinations amongst Turkish Rhododendron species were detected using morphological, cpDNA and nuclear rDNA markers. These were R. ponticum x R. ungernii, with introgression in both directions; R. ponticum x R. smirnovii; R. smirnovii x R. ungernii with introgression at least towards R. ungernii; R. smirnovii x R. caucasicum; and R. ponticum x R. caucasicum. The unusual characteristics of the last combination are discussed in some detail.
  
  
  
• The gaseous messenger molecule, nitric oxide : a modulator of locomotor movements during early amphibian development
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  Abstract: 1. The free radical gas nitric oxide (NO) is now recognised as a ubiquitous and versatile signalling molecule and the investigation of its biological roles has involved a wide range of scientific disciplines in many different species. Yet despite this, its potential roles in the development of rhythmic motor activities in vertebrates have been largely ignored. 2. Physiological experiments recording extracellular ventral root output suggest that NO is playing an inhibitory role in the swimming system of Xenopus laevis larvae, shortening the duration of swim episodes and slowing swim frequency. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry labelled three populations of neurons in the brainstem, which putatively co-localise NO with the aminergic neuromodulators serotonin (5-HT) and noradranaline (NA), and the fast descending inhibitory neurotransmitter, y-aminobutyric acid (GABA). This suggests that the inhibitory role is supraspinal in origin. 3. Intracellular recordings from neurons presumed to be spinal motor neurons provide further evidence for the inhibitory influence of NO. My experiments suggest that NO potentiates both glycinergic and y-aminobutyric acid (GABA)-ergic inhibition onto spinal motor neurons. The facilitation of the release of these inhibitory transmitters is consistent with the observed effects on swim frequency and swim episode duration, respectively. Additionally, NO appears to affect membrane properties, causing a pronounced membrane potential depolarisation and a decrease in membrane conductance. This suggests that NO shuts off a resting membrane conductance. 4. NADPH-diaphorase histochemistry was subsequently applied to determine the four dimensional expression of putative nitrergic neurons in the central nervous system and related structures. The developmental sequence of staining identifies groups and subgroups of interconnected intemeurons, and provides further clues to their identity. NADPH-diaphorase labelling was also located in the eyes, skin and blood vessels, further confirming the validity of this staining technique for identirying nitric oxide synthase. 5. In the related anuran species, Rana temporaria nitric oxide donor drugs appear to have no affect on swimming, but instead reliably initiates a non-rhythmic "lashing" motor pattern similar to that elicited by dimming of the illumination. Interestingly the NADPH-diaphorase technique labelled three clusters of apparently homologous interneurons in the brainstem and additionally the inner layer of the skin was intensely stained, implicating a species-specific role for NO released from brainstem neurons.
  
  
  
• Aspects of olfaction, social behaviour and ecology of an island population of the European rabbit (Oryctolagus cuniculus)
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  Abstract: Olfactory behaviour in the European rabbit (Oryctolagus cuniculus) has been studied extensively under laboratory and semi-natural conditions. Results of observations on aspects of the olfactory behaviour of a free-living population of rabbits are presented. To facilitate interpretation of these results, considerable preliminary information about the population was collected. The study was conducted over a three year period on the Isle of May off the east coast of Scotland. A total of 326 rabbits were trapped and marked to permit identification in the field, and data on sex, age and social status of these individuals was collated. Data on the overall structure and fluctuations in the population are presented. The social organisation and home ranges of rabbits at four study sites throughout the three years are described. Observations indicate that the social organisation of free-living populations is more complex and variable than previous descriptions of semi-natural populations would have led us to expect. The reproductive performance of the population was investigated and intra and interwarren variations are analysed with respect to warren and group size. Results indicate an inverse relationship between warren size and reproductive success. The most frequently observed group composition (2 males, 2 females) was also the most reproductively successful. Daily and seasonal activity patterns of different age, sex and social status classes of rabbits are described. Olfactory communication was investigated by analysis of the frequency, daily and seasonal variation, and behavioural context of odour related activities performed by members of different age, sex and social status classes. The importance of using appropriate methods for the sampling of behaviour in field studies of olfaction is stressed. The present study concentrates on behaviour related to latrines; chin marking of the substrate and of conspecifics; enurination and urine squirting; and pawscraping. The results suggest that different scent products may carry similar information but analysis of variations in the frequency and context of odour deposition suggests that the deposition of scent fulfills a variety of functions.
  
  
  
• Natural history and conservation of pirarucu, Àrapaima gigas', at the Amazonian Várzea : red giants in muddy waters
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  Abstract: The pirarucu, Arapaima gigas (Osteoglossidae, Teleostei), an important natural resource in the Amazonian flooded forest, has been exploited for centuries and is now threatened by overfishing. The aim of this thesis, therefore, is to investigate the biology and ecology of this species so that sustainable management programmes can be put in place and the conservation of this fish ensured. Most of the research was carried out at Mamiraua Reserve, a protected area of Varzea forest in the Central Brazilian Amazon. Additional data were also recorded at the aquarium of the Amazonian Museum of Natural History, at Manaus, Brazil. A study of the main environmental aspects varzea aquatic systems, showed that the lakes at Mamiraua Reserve could be grouped into two main categories based on their limnological morphological features. The investigation of the fisheries management by local fishermen revealed that the use of traditional fishing technology was responsible for the maintenance of local stocks. However, recent technological changes had increased CPUE to alarming level. The analysis of pirarucu feeding habits demonstrated that pirarucus are specialised carnivores, living mainly on fishes. Other invertebrate species are also important in their diet. High degrees of food selectivity and strong seasonal changes in diet proved to be the most important factor affecting the seasonal growth of pirarucus. Pirarucu growth was examined by analysing the growth marks deposited on scales twice a year, at times of high ingestion of food. L was circa 2.5.m and k was about 0.18. No significant differences in growth patterns between the sexes were found. Ten cohorts or annual age classes were identified. These ranged from 0.80m to 2.25m in total length. Very high annual growth rates during the first years are replaced by moderate rates after the age of first sexual maturation. Maturity is reached when pirarucus are around 5 years of age, or about 1.65m in total length. Fecundity increases with age of females, and so does oocyte diameter. However, fecundity is divided between 4 to 6 clutches of gametes that can be used throughout the breeding season. Each clutch is spawned separately, in small batches with less than 500 eggs in average. This strategy probably protects pirarucu against the unpredictability of the environment and avoids reproductive failures - total loss of fertilised eggs - due to catastrophic events. By spawning eggs in separate batches pairs may breed again if the first attempt was not successful. Males perform parental care, protecting the nest and guarding the offspring for about 6 months. The typical red coloration of pirarucus is particularly evident in males after sexual maturation. Almost 70% of male body surface may be covered in bright red pigmentation, brighter than among females. It is proposed that red coloration signals the size and/or age of pirarucus, and is used in the context of sexual selection. Male-male competition and female choice of males are likely to be operating in this species. The information in this thesis leads to several recommendations for the conservation of pirarucus. Criteria for selection of lakes in a rotational system of fisheries management, and age-specific impact of fishing and natural mortality rates on the stock are major topics discussed. The aim is the maximisation of breeding stock and local natural recruitment. Pirarucu farming and large scale conservation actions are also considered. Suggestions for future research on the biology of conservation of pirarucus are made.
  
  
  
• The physiology of the reproductive cycle of the powan of Loch Lomond, Coregonus lavaretus (L) (Euteleostei, Salmonidae) in relation to the deposition and mobilization of storage products
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  Abstract: There have been numerous studies in which the reproductive cycles of teleosts have been correlated with either environmental cycles or associated physiological cycles, or both. Such correlation is seldom accurately achieved; usually because the reproductive cycle of the species concerned is lax, sometimes because only one or two factors of an integrated whole were examined. The powan of Loch Lomond, Coregonus lavaretus (L. ) (Teleostei, Salmoniformes) is the subject of a long-term study investigating its growth, in particular reproduction. This race is a freshwater glacial relict form of a boreal group, and thus has an exceptionally strictly times reproductive cycle. It thus represents an ideal subject for cyclical studies. This thesis investigates the relationship between lipid storage and the reproductive cycle and the role of thyroid gland.
  
  
  
• The food and feeding of powan Coregonus lavaretus (L.) (Salmonidae: Coregoninae) in two Scottish lochs
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  Abstract: The present study is mainly a descriptive account of the food and feeding of the two Scottish powan populations from Loch Lomond and Loch Eck. Quantitative and qualitative comparisons of powan diet are made. Seasonal variations of diet are determined for the two populations and some aspects of the planktivorous feeding of adult pawan in Loch Lomond are examined. The partitioning of the ingested energy into somatic and non-somatic growth is considered. Factors affecting the feeding of the two populations are discussed.
  
  
  
• Growth processes in the two Scottish populations of powan, Coregonus lavaretus (L.) (Eateleosteia, Salmonidae)
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  Abstract: The powan, Coregonus lavaretus (L.) is endemic to only two British waters, Loch Lomond and Loch Eck, Scotland. This thesis describes the seasonal and longer term growth processes of the two populations, concentrating on growth in length back-calculated from scales, factors affecting recruitment and mortality, reproductive cycles, and seasonal deposition and mobilisation of storage products, particularly lipid. The interrelationships of these cycles is discussed. The populations differ in their diet and duration of feeding, and it is shown that most of the inter-population differences in seasonal cycles of growth relate to these feeding differences. The Loch Eck population is the more variable. In addition to adult and immature powan, a third category is identified, termed adolescents. These are fish which are entering their first reproductive cycle. Immature and adolescent fish are analysed separately and compared with the adults. There are some differences in seasonal cycles between the juveniles and adults, mainly in relation to the presence or absence of the reproductive cycle. A preliminary histological study of the ovaries of adolescent females is carried out. Comparison of historical data with the results of the present study shows that there has been little change in the Loch Lomond powan in the past 200 years. Both lochs are coming under increasing human pressure, and conservational measures urgently need to be taken if the powan populations are to survive.
  
  
  
• Foraging strategies, diet and competition in olive baboons
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  Abstract: Savannah baboons are amongst the most intensively studied taxa of primates, but our understanding of their foraging strategies and diet selection, and the relationship of these to social processes is still rudimentary. These issues were addressed in a 12-month field study of olive baboons (Papio anubis) on the Laikipia plateau in Kenya. Seasonal fluctuations in food availability were closely related to rainfall patterns, with the end of the dry season representing a significant energy bottleneck. The distribution of water and of sleeping sites were the predominant influences on home range use, but certain vegetation zones were occupied preferentially in seasons when food availability within them was high. The influence of rainfall on monthly variation in dietary composition generally mirrored inter-population variation. Phytochemical analysis revealed that simplistic dietary taxonomies can be misleading in the evaluation of diet quality. Food preferences were correlated with nutrient and secondary compound content. The differences between males and females in daily nutrient intakes were smaller than expected on the basis of the great difference in body size; this was partly attributable to the energetic costs of reproduction, and possibly also to greater energetic costs of thermoregulation and lower digestive efficiency in females. A strongly linear dominance hierarchy was found amongst the adult females. Dominance rank was positively correlated with food ingestion rates and daily intakes, but not with time spent feeding or with dietary quality or diversity. In a provisioned group, high-ranking females occupied central positions, while low-ranking females were more peripheral and were supplanted more frequently. In the naturally-foraging group, the intensity of competition was related to the pattern of food distribution, but not to food quality, and was greater in the dry season than in the wet season. The number of neighbours and rates of supplanting were correlated with rank, and evidence was presented that high-rankers monopolised arboreal feeding sites.
  
  
  
• The control of mitochondrial morphology and dynamics in Arabidopis thaliana
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  Abstract: Mitochondria are ubiquitous eukaryotic organelles which carry out a range of essential functions, most notably the production of ATP through the process of oxidative phosphorylation. While the main biochemical function of mitochondria was established over 50 years ago, the processes which control mitochondrial morphology are, at present, poorly understood. The thesis aims to add to our knowledge of the factors that control mitochondrial morphology and dynamics in the model plant species, Arabidopsis thaliana. The phenotypic characteristics of two novel mitochondrial morphology mutants, motley mitochondria I (mmtl) and network mitochondria (nmt), were examined and quantified. mmtl has an increased heterogeneity of mitochondrial plan area relative to wild-type, which is matched by a similar chloroplast phenotype. nmt exhibits a reticular mitochondrial morphology, similar to the mitochondria found in yeast and animals. Genetic mapping of the two mutant loci has established that mmtl resides on a short region of chromosome 11w, hile nmt was mapped to a small area of chromosome V. This thesis describes the identification and functional analysis of two novel orthologs of yeast and animal mitochondrial division genes. Using TDNA reverse genetics, it is shown that disruption of the dynamin-like DRP3A or BIGYIN (an Arabidopsis orthologue of yeast FISI) led to an increase in mitochondrial plan area, which is coupled with a decrease in the number of physically discrete mitochondria per cell. Finally, the morphology and behaviour of Arabiclopsis mitochondria is investigated upon the induction of cell death. Abiotic stress treatments that induce cell death led to fast and irreversible changes in mitochondrial morphology. The role of these changes, as possible early indicators of cell death, are discussed.
  
  
  
• Transcriptional regulation mediated through the conjugation and deconjugation of the small ubiquitin-like modifiers SUMO-1, SUMO-2, and SUMO-3
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  Abstract: SUMO-1/2/3 are members of the ubiquitin-like family of protein modifiers. These proteins are covalently attached to numerous proteins in a directed and controlled manner. SUMO conjugation primarily occurs to proteins containing an exposed SUMO conjugation motif, (I, V, L, F)KxE, where x represents any amino acid. SUMO conjugation is controlled by key enzymes, a SUMO activating enzyme, SAE1/2 and a SUMO conjugating enzyme, Ubc9, which is responsible for substrate recognition, and the efficiency of this pathway can be increased by one of many SUMO ligase enzymes. This modification alters the substrate's characteristics and results in a change of state, such as stability, localisation, or activity. p300, a transcriptional co-activator, contains an evolutionary conserved tandem SUMO modification motif, located in a transcriptional repression domain. p300 was efficiently conjugated, both in vitro and in vivo, by SUMO-1/2/3, within this repression domain to both SUMO conjugation motifs. The SUMO conjugation to p300 correlated with p300 ability to repress transcription, requiring both SUMO conjugation motifs for full transcription repression activity. This repression activity was mediated through SUMO recruitment of histone deacetylase 6. Repression could be alleviated through co-expression of a SUMO-specific protease thereby suggesting a potential regulatory mechanism for transcription control of SUMO modified substrates. Despite utilising the same conjugation machinery, there remained the potential for distinct roles for the SUMO isoforms. SUMO -2/3, which form a distinct group from SUMO-1, were shown to preferentially mediate the transcription repression abilities of a number of known SUMO modifiable substrates: p300, Elk-1, and SP3. Further differences were observed in the ability of SUMO-1 and SUMO-2/3 to influence the nuclear organisation of p80 coilin.
  
  
  
• Role of SUMO-1 modification in transcriptional activation
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  Abstract: In unstimulated cells, the transcription factor NF-κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Activation of NF--KB is mediated by signal induced degradation of IκBα via the ubiquitin proteasome-dependent pathway. Targeting the proteins for ubiquitin-mediated proteolysis is an irrevocable decision, and as such, the process needs to be highly specific and tightly regulated. This task is achieved by conjugation and deconjugation enzymes that act in a dynamic and coordinated mechanism. In a yeast two hybrid screen designed to identify proteins involved in IκBα signalling Ubch9 was found to interact with the N-terminal regulatory region of IκBα. Although Ubch9 is an enzyme homologous to E2 ubiquitin conjugating enzymes we have shown that is unable to form a thioester with ubiquitin but it is capable to form a thioester with the small ubiquitin-like protein SUMO- 1. To fully characterise the SUMO-1 modification reaction we have purified the proteins and cloned the genes encoding the SUMO-1 activating enzyme (SAEl/SAE2) and shown that it is homologous to enzymes involved in the activation of ubiquitin, Smt3p, the yeast SUMO-1 homologue, and Rublp/Nedd8, another ubiquitin-like protein. SUMO-1 is conjugated to target proteins by a pathway that is distinct from, but analogous to, ubiquitin conjugation. SUMO-1 was efficiently conjugated, both in vivo and in vitro, to IκBα on lysine 21, which is also utilised for ubiquitin modification. Thus, by blocking ubiquitination SUMO-1 modification acts antagonistically to generate a pool of IκBα resistant to proteasome-mediated degradation which consequently inhibits NF-κB dependent transcription activation. In view of several lines of similarity between NF-kB and p53, the involvement of SUMO-1 modification in the metabolism of the tumour supressor p53 was investigated. We have shown that p53 is modified by SUMO-1 at a single site, lysine 386 in the C-terminus of p53. Although p53 is regulated by ubiquitination, SUMO-1 and ubiquitin modification do not compete for the same lysine in p53. However, overexpression of SUMO-1 activates the transcriptional activity of wild type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. A consensus sequence was obtained by comparison of the sequences surrounding the SUMO-1 acceptor lysine in proteins that have been shown to be modified by SUMO-1 and revealed a possible recognition site for SUMO-1 conjugation machinery. Tagging of proteins with SUMO-1 regulates transcriptional activation, either by interfering with subcellular location or with the ubiquitination pathway. The pathway may represent a novel target for drug development.
  
  
  
• The physiological ecology and life history strategies of the nudibranch molluscs 'Adalaria proxima' (Alder & Hancock) and 'Onchidoris muricata' (Müller) (Gastropoda: Opisthobranchia)
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  Abstract: This study investigated the physiological ecology, larval biology and population genetics of the nudibranch molluscs Adalaria proxima (A & H) and Onchidoris muricata (Müller). These two species are annual, simultaneous hermaphrodites and are ecologically very similar with the exception that A. proxima reproduces by means of pelagic lecithotrophic larvae whereas Omuricata has long-term planktotrophic larvae. The aim of the study was therefore to determine the selective pressures which resulted in the evolution of different larval types in these two species, and to ascertain the ecological and population genetic consequences thereof. Simple energy budgets comprising the major components (consumption, growth, respiration and reproduction) were constructed for laboratory populations of each species. In both A. proxima and O. muricata, feeding rate displayed an asymptotic increase with body size. Mean feeding rates of A. proxima were greater than those of comparable O. muricata individuals, and overall assimilation efficiency was higher in A. proxima than in O. muricata. This difference was reflected in the somatic growth rates which were correspondingly greater in A. proxima than in O. muricata. Net growth efficiencies were broadly comparable between the two species, however, growth of A. proxima was approximately linear over' time whilst that of O. muricata displayed a curvilinear, almost exponential, pattern. This is interpreted as demonstrating that some form of constraint (possibly feeding rate) operated on the growth rates of A. proxima but not on those of O. muricata. Respiration rates were found to be relatively constant within given animals, but significant differences were found between individuals. The allometry of respiration rate was not constant; Omuricata demonstrated a more rapid increase in respiration rate with increasing body size than did A. proxima. Individual variations in respiration rate did not reflect variations in the energy partitioned to either growth or reproduction. Reproductive patterns in the two species were dissimilar. A. proxima laid fewer spawn masses containing fewer, larger ova than those laid by O. muricata individuals. In addition, the spawning period of A. proxima was shorter than that of O. muricata (60 days and 105 days respectively). Both species exhibited a similar (proportional) degree of somatic catabolism over these periods. The consequently more rapid "degrowth" of A. proxima is interpreted as the necessary utilization of an energy resource (i. e. the soma) caused by an inability to meet the energy demands of reproduction through feeding alone. This was not the case in Oanuricata individuals which exhibited a much smaller maximum body size and were able to feed at a sufficiently rapid rate to maintain reproduction. In the latter case, the longer reproductive period served to maximise the total reproductive output. Several different measures of "Reproductive Effort" (RE) were calculated. These generally indicated that the RE of Omuricata was considerably greater than that of A. proxima. Although such differences have been used in the literature to classify the respective costs of different larval types or "reproductive strategies", the variability of the RE's obtained from the different measures used here has led to the suggestion that the general lack of association between RE and reproductive strategy which has been reported elsewhere may (partially) be attributable to the different measures of RE employed in different studies. Studies of the embryonic and larval period showed that the egg-to-juvenile period of O. muricata was approximately 50% longer than that of A. proxima. This difference was primarily attributable to the extended pelagic development of O. muricata larvae. Estimates of the degree of dispersal, and hence gene-flow, between populations of these species were tested by investigating the biochemical genetics of such populations. No data were available for O. muricata, but A. proxima populations proved to be more genetically heterogeneous than had been expected. It is therefore concluded that actual pelagic dispersal may be considerably abbreviated over that expected on the basis of larval culture data alone. A model is developed to explain the possible consequences of different egg-to-juvenile periods (which accrue from different larval types) on both the ecology of the benthic adult, and on overall energy partitioning to reproduction. However, although (probable) proximate causes and effects of the different reproductive traits exhibited by A. proxima and Oanuricata are shown, it has not been possible to determine the exact selective pressures which caused A. proxima to diverge from the ancestral "O. muricata" stock through the evolution of a pelagic lecithotrophic larva.
  
  
  
• Characteristics of breeding passerine communities at Earlshallmuir and Tentsmuir, North-East Fife
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  Abstract: The literature pertaining to a variety of methods for estimating the abundances of songbirds is reviewed, and the use of two of the main methods - mapping and transects - is described and discussed. The mapping method is found to be the most efficient and reliable in the context of the present study, and work elsewhere in the British Isles suggests that the method has wide applicability in the region. Various methods for describing the habitats in which birds occur are discussed. In the present study the method chosen involved measuring various parameters of the vegetation within a limited area at each of several sampling points in five study plots in North-east Fife. It proved a useful method in the habitats studied - dune grassland, commercial and semi-natural coniferous woodlands, and deciduous scrub and mature woodland - and results of the vegetation sampling are discussed in terms of habitat structure and succession, and in the relationship of this structure to the breeding bird communities. The composition of these communities in the study plots is described, as are the changes over the three breeding seasons 1979-1981, which followed on from a meteorologically "hard" winter. Over the study period there was an overall increase in the populations of all five study plots, and this increase was greatest in the poorest habitat - the dune grassland - and least in the richest habitat - the mature deciduous woodland. Using indices to compare the study plots a major difference was found between the grassland plot and all four woodland plots, amongst which the two coniferous ones were most similar. Of the more abundant bird species, Robin, Song Thrush and Coal Tit were more prevalent in coniferous habitats; Willow Warbler and Blue Tit were more prevalent in the deciduous habitats. In relation to habitat structure, the more diverse bird communities were found in the more complex habitats, and there was evidence of succession in the bird communities concomitant with habitat succession. The present study is one of very few in Europe involving woodland habitats especially of a commercial nature near sea-level, and it was found that the four woodland study plots were rich in terms of the diversities and densities of their passerine breeding populations when compared with other European studies.
  
  
  
• Studies on the preservation of flowers
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  Abstract: A known method for the preservation of green foliage was adapted in order to preserve floral tissues, retaining the colour and texture, thereby providing a method suitable for the preservation of whole flowers. Initially, the effects of the existing foliage preservation process on floral tissues were studied and the resulting problems of limp sticky petals and colour loss were identified. Subsequently, with a knowledge of basic plant anatomy and of the properties of the main floral pigments, the anthocyanins, a series of experiments on petals and whole flowers were carried out in an attempt to rectify these problems and to incorporate the remedies into a method for preserving whole flowers. The problem of improving the texture and firmness of flower heads was tackled by investigating the effects of adding bulking or setting ingredients to the process fluid and establishing their optimum concentrations. In the case of flower colour, the addition of acid was required in order to maintain the bright anthocyanin colours and a range of acids was investigated. Furthermore, since it is known that in nature the anthocyanin pigments are stabilised by metal ions and copigments, the use of these agents in the preservation process was also considered. This empirical work was then validated by confirming the identity of the main pigments involved and by studying various aspects of the new preservation process. Factors examined included acid concentration, temperature, solvent composition and the addition of metal ions and copigments to solutions of petal extracts containing anthocyanin pigments. Physical changes resulting from processing, including process fluid content and the moisture absorption properties of processed petals were also measured. Finally, the application of a selection of coating materials was assessed in an attempt to increase the life span of the processed flowers by providing extra protection against environmental stresses.
  
  
  
• Systematics and biogeography of Myricaceae
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  Abstract: Two molecular phylogenetic studies were undertaken to examine relationships within Myricaceae. Analyses of DNA sequences of the plastid rbcL gene, trnL-Fregion and nuclear ITS region showed the family to be monophyletic. In all analyses Canacomyrica, a monotypic genus endemic to New Caledonian that bears several distinctive features such as staminodes in the female flowers, fell into a well- supported clade sister to the rest of Myricaceae. Phylogenetic analyses of ITS and trnL-F sequence data, representing all genera and subgeneric groups, were undertaken using maximum parsimony and Bayesian methods. The following relationships were strongly supported: (Canacomyrica (Comptonia (Myrica, Morella))). The clade containing all species formerly considered to comprise Myrica s.l. was split into two strongly supported clades corresponding to Myrica s.s. and Morella; this finding strengthens the argument for recognition of these as separate genera. Within Morella, two clades corresponded to previously recognized subgenera. Molecular dating analyses were performed using Penalized Likelihood. Close correlations between lineage-specific diversification and major orogenic or climatic events were inferred. This study suggests that much of the diversity in Morella arose during the Neogene and seed-dispersal by birds has been a significant factor in determining the modem distribution. A study of the conservation status of Canacomyrica was conducted using field observations and data from herbarium specimens. This species was found to occur in just eleven fragmented localities: six outside protected areas and three threatened by mining or bush fires. IUCN Red List status of Endangered was recommended. The morphology and ecology of Canacomyrica was studied to enhance knowledge of this poorly known species and provide comparative data for use in a study of the morphology of the entire family. A new classification scheme with keys was presented including, for the first time, Canacomyrica. New combinations in Morella were recommended.
  
  
  
• The structure and function of microphytobenthic biofilms
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  Abstract: Microphytobenthos are the dominant primary producers on estuarine mudflats playing a key role in the functioning of the ecosystem. Studies into microphytobenthic ecology have previously been limited by scale but the advent of fine scale analysis techniques (gm) as well as non-destructive sampling has enabled the system to be examined at a level not previously possible. This study examined the formation, structure and function of microphytobenthic biofilms using non-destructive (remote sensing by PAM fluorescence; fibreoptic light microprofiling) and destructive (cryo-freezing and Low temperature scanning electron microscopy) sampling. Many microphytobenthic organisms are motile and have evolved complex migratory strategies. Microphytobenthic migratory patterns are widely described but much remains to be elucidated about the controlling factors. The fluorescence parameter F015 (minimum fluorescence yield after 15 minutes dark adaptation) was used to monitor short-term changes in biomass at the sediment surface. Light, tidal state, endogeny and combinations thereof were all shown to control migration, demonstrating that predictable migratory rhythms cannot be assumed. Microscale sectioning showed that chlorophyll a was always concentrated in the top 400 gm (the photosynthetically active biomass). Clear migratory patterns were not detected using microscale sectioning therefore indicating that migration occurs over a scale < 400gm. Despite no changes in the chlorophyll a content in the surface layers, LTSEM analysis demonstrated diurnal taxonomic shifts providing circumstantial evidence that microphytobenthic cells sub-cycle at the sediment surface to optimise fitness. The light extinction co-efficient (k) of microphytobenthic biofilms significantly vaned with site, assemblage and also over time. 90% of the surface PPFD had always been attenuated by 400, and in many cases before 200. Traditional microphytobenthic primary productivity models do not account for changes in the spatial and temporal distribution of biomass or light attenuation and therefore their applicability to the real situation may be limited.
  
  
  
• Environmental timing and control of reproduction in the powan of Loch Lomond 'Coregonus lavaretus (L) (Teleostei)' in relation to its pineal organ
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  Abstract: The reproductive cycle of Coregonus lavaretus (L. ) in Loch Lomond was investigated by monthly sampling. The stages of the cycle occurred at the same time each year and were precisely timed. The environment followed a regular pattern which varied seasonally and was repeated annually. Spawning synchrony within the population during the short breeding period may be in response to lunar phases. A qualitative echosounding survey was made. The spatial distribution of the fish is probably related to their feeding behaviour and was mainly pelagic in summer and benthic in winter. Diel vertical migrations were recorded at dawn and dusk and appeared to be related to negative solar altitudes. The fish occurred at the surface during the night and persisted with their diel vertical migrations when feeding behaviour was benthic. The regulation of the reproductive cycle in the common sole Solea solea was investigated. The timing of spawning is ultimately determined by sea temperature, and spawning synchrony within the population is probably achieved in the initiation of exogenous vitellogenesis by a unified response within the breeding population to a stimulatory photoperiod. In both Solea solea and Coregonus lavaretus, initiation of exogenous vitellogenesis occurred during a rapid rise in the condition of the fish. The photosensitivity of the reproductive system may possibly be linked to a threshold condition. The pineal organ of Coregonus lavaretus is typically salmonid and the convoluted epithelium contained photoreceptors, interstitial cells, and neurones. The interstitial cells gave rise to processes which extended into the perivascular space. Photoreceptor cells synapsed with neurones, photoreceptor cells (lateral processes) and possibly other cell types. The results suggest that the pineal organ functions as a photoreceptor.
  
  
  
• Some aspects of the anatomy of the alimentary canal of the lesser octopus, Eledone cirrhosa (Lamarck 1817), with special reference to the nervous system
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  Abstract: The modern cephalopod molluscs, particularly the octopods, are highly developed macrophageous predators which have a pronounced ability to learn. Striking resemblances are found between these animals and the vertebrates, particularly the fish. The study of cephalopods therefore provides an opportunity to examine systems which are composed of structural units common to molluscs but which may be similar in function and performance to those of vertebrates. This investigation concerns the alimentary canal and its innervation in the lesser octopus Eledone cirrhosa. Detailed anatomical investigation shows the alimentary canal of E. cirrhosa to be very similar to that of the better known Octopus vulgaris. Current concepts of the functions of the alimentary organs are discussed utilizing data from both E. cirrhosa and O. vulgaris. Silver staining reveals a basic pattern of innervation in the alimentary organs. Large nerve trunks running in the external collagenous layer give rise to a nerve plexus within the circular muscle. The longitudinal muscle plexus arises from branches of the circular muscle plexus or direct from the nerves of the external layer, Nerves of both plexuses contact muscle fibres in an 'en passant' manner. Fibres run out from the longitudinal muscle to the subepithelium, where they are observed associated with muscle fibres and beneath the epithelial basal lamina. Good evidence for fibres crossing into the epithelium was observed only in the cuticularized regions of the digestive tract. The digestive gland ducts differ from this pattern in the very large numbers of major nerves seen in their external and muscular regions. Outwith the alimentary ganglia nerve cells are only regularly observed within the major intestinal nerves. Receptor like cells were also repeatedly observed only in the posterior intestine. These results axe compared with data from O. vulgaris and the physiological evidence for the presence of receptors. The blood vessels of the alimentary canal are innervated at all levels. The distribution of other densely staining cells is reported. Evidence for the presence of particular neurotransmitters within the alimentary canal and alimentary nerve centres is reviewed. Fluorescence histochemistry shows that at least two types of nerves are present in the alimentary wall. The majority axe aminergic (including those associated with blood vessels and some sphincters), as the pattern of fluorescent nerves is predominantly that shown by silver studies. However, fluorescent nerves decrease and then disappear anteriorly from the crop/oesophageal sphincter and posteriorly from the mid-intestine. The stomach has fluorescent nerves, other than those associated with blood vessels. Fluorescent fibres enter the gut via the sympathetic and possibly the digestive gland duct nerves. Non-fluorescent fibres enter via the atrio-rectal nerves and from the gastric ganglion. The fluorescence is ascribed to catecholamines as no evidence of 5HT was obtained, Specific fluorescence was also observed in some cells of the subepithelium and the external region. These results are discussed with reference to available physiological data. The anterior intestine was the representative region chosen for fine structural studies. These show the alimentary muscles to be the same basic type (cross or pseudo-striated) as that found in cephalopod somatic or heart muscle. Three types of myomuscular and neuromuscular junctions axe described. The presence of mineralized concretions (spherites) in the external layer of the intestine, together with the complex relations of its epithelial cells and heavy vascularization suggest a secondary function of mineral and/or water balance for this organ. These results are discussed. Finally, a pathological condition affecting the octopuses during this study is described.
  
  
  
• Parasitological studies : on the structure, biology and taxonomy of Nucellicola kilrymontis gen. et. sp., nov., (Crustacea : Copepoda) parasitic in Nucella lapillus (L.), (Gastropoda : Prosobranchia)
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  Abstract: The modern cephalopod molluscs, particularly the octopods, are highly developed macrophageous predators which have a pronounced ability to learn. Striking resemblances are found between these animals and the vertebrates, particularly the fish. The study of cephalopods therefore provides an opportunity to examine systems which are composed of structural units common to molluscs but which may be similar in function and performance to those of vertebrates. This investigation concerns the alimentary canal and its innervation in the lesser octopus Eledone cirrhosa. Detailed anatomical investigation shows the alimentary canal of E. cirrhosa to be very similar to that of the better known Octopus vulgaris. Current concepts of the functions of the alimentary organs are discussed utilizing data from both E. cirrhosa and O. vulgaris. Silver staining reveals a basic pattern of innervation in the alimentary organs. Large nerve trunks running in the external collagenous layer give rise to a nerve plexus within the circular muscle. The longitudinal muscle plexus arises from branches of the circular muscle plexus or direct from the nerves of the external layer, Nerves of both plexuses contact muscle fibres in an 'en passant' manner. Fibres run out from the longitudinal muscle to the subepithelium, where they are observed associated with muscle fibres and beneath the epithelial basal lamina. Good evidence for fibres crossing into the epithelium was observed only in the cuticularized regions of the digestive tract. The digestive gland ducts differ from this pattern in the very large numbers of major nerves seen in their external and muscular regions. Outwith the alimentary ganglia nerve cells are only regularly observed within the major intestinal nerves. Receptor like cells were also repeatedly observed only in the posterior intestine. These results axe compared with data from O. vulgaris and the physiological evidence for the presence of receptors. The blood vessels of the alimentary canal are innervated at all levels. The distribution of other densely staining cells is reported. Evidence for the presence of particular neurotransmitters within the alimentary canal and alimentary nerve centres is reviewed. Fluorescence histochemistry shows that at least two types of nerves are present in the alimentary wall. The majority axe aminergic (including those associated with blood vessels and some sphincters), as the pattern of fluorescent nerves is predominantly that shown by silver studies. However, fluorescent nerves decrease and then disappear anteriorly from the crop/oesophageal sphincter and posteriorly from the mid-intestine. The stomach has fluorescent nerves, other than those associated with blood vessels. Fluorescent fibres enter the gut via the sympathetic and possibly the digestive gland duct nerves. Non-fluorescent fibres enter via the atrio-rectal nerves and from the gastric ganglion. The fluorescence is ascribed to catecholamines as no evidence of 5HT was obtained, Specific fluorescence was also observed in some cells of the subepithelium and the external region. These results are discussed with reference to available physiological data. The anterior intestine was the representative region chosen for fine structural studies. These show the alimentary muscles to be the same basic type (cross or pseudo-striated) as that found in cephalopod somatic or heart muscle. Three types of myomuscular and neuromuscular junctions axe described. The presence of mineralized concretions (spherites) in the external layer of the intestine, together with the complex relations of its epithelial cells and heavy vascularization suggest a secondary function of mineral and/or water balance for this organ. These results are discussed. Finally, a pathological condition affecting the octopuses during this study is described.
  
  
  
• Transcriptional regulation in skeletal muscle of zebrafish in response to nutritional status, photoperiod and experimental selection for body size
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  Abstract: In the present study, the ease of rearing, short generation time and molecular research tools available for the zebrafish model (Danio rerio, Hamilton) were exploited to investigate transcriptional regulation in relation to feeding, photoperiod and experimental selection. Chapter 2 describes transcriptional regulation in fast skeletal muscle following fasting and a single satiating meal of bloodworms. Changes in transcript abundance were investigated in relation to the food content in the gut. Using qPCR, the transcription patterns of 16 genes comprising the insulin-like growth factor (IGF) system were characterized, and differential regulation between some of the paralogues was recorded. For example, feeding was associated with upregulation of igf1a and igf2b at 3 and 6h after the single-meal was offered, respectively, whereas igf1b was not detected in skeletal muscle. On the other hand, fasting triggered the upregulation of the igf1 receptors and igfbp1a/b, the only binding proteins whose transcription was responsive to a single-satiating meal. In addition to the investigation of the IGF-axis, an agnostic approach was used to discover other genes involved in transcriptional response to nutritional status, by employing a whole-genome microarray containing 44K probes. This resulted in the discovery of 147 genes in skeletal muscle that were differentially expressed between fasting and satiation. Ubiquitin-ligases involved in proteasome-mediated protein degradation, and antiproliferative and pro-apoptotic genes were among the genes upregulated during fasting, whereas satiation resulted in an upregulation of genes involved in protein synthesis and folding, and a gene highly correlated with growth in mice and fish, the enzyme ornithine decarboxylase 1. Zebrafish exhibit circadian rhythms of breeding, locomotor activity and feeding that are controlled by molecular clock mechanisms in central and peripheral organs. In chapter 3 the transcription of 17 known clock genes was investigated in skeletal muscle in relation to the photoperiod and food content in the gut. The hypothesis that myogenic regulatory factors and components of the IGF-pathway were clock-controlled was also tested. Positive (clock1 and bmal1 paralogues) and negative oscillators (cry1a and per genes) showed a strong circadian pattern in skeletal muscle in anti-phase with each other. MyoD was not clock-controlled in zebrafish in contrast to findings in mice, whereas myf6 showed a circadian pattern of expression in phase with clock and bmal. Similarly, the expression of two IGF binding proteins (igfbp3 and 5b) was circadian and in phase with the positive oscillators clock and bmal. It was also found that some paralogues responded differently to photoperiod. For example, clock1a was 3-fold more responsive than clock1b. Cry1b did not show a circadian pattern of expression. These patterns of expression provide evidence that the molecular clock mechanisms in skeletal muscle are synchronized with the molecular clock in central pacemaker organs such as eyes and the pineal gland. Using the short generation time of zebrafish the effects of selective breeding for body size at age were investigated and are described in chapter 4. Three rounds of artificial selection for small (S-lineage) and large body size (L-lineage) resulted in zebrafish populations whose average standard length were, respectively, 2% lower and 10% higher than an unselected control lineage (U-lineage). Fish from the L-lineage showed an increased egg production and bigger egg size with more yolk, possibly contributing to the larger body size observed in the early larval stage (6dpf) of fish from this lineage. Fish from S- and L-lineage exposed to fasting and refeeding showed very similar feed intake, providing evidence that experimental selection did not cause significant changes in appetite control. Investigation of the expression of the IGF-axis and nutritionally-response in skeletal muscle after fasting and refeeding revealed that the pattern of expression was not different between the selected lineages, but that a differential responsiveness was observed in a limited number of genes, providing evidence that experimental selection might have changed the way fish allocate the energy acquired through feeding. For example, a constitutive higher expression of igf1a was recorded in skeletal muscle of fish from the L-lineage whereas igfbp1a/b transcripts were higher in muscle of fish from the S-lineage. These findings demonstrate the rapid changes in growth and transcriptional response in skeletal muscle of zebrafish after only three rounds of selection. Furthermore, it provides evidences that differences in growth during embryonic and larval stages might be related to higher levels of energy deposited during oogenesis, whereas differences in adult fish were better explained by changes in energy allocation instead of energy acquisition. In chapter 5 the main findings made during this study and their impact on the literature are discussed.
  
  
  
• What does it mean to experience paediatric depression?: the ethics of mental health diagnosis and treatment in children and adolescents
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  Abstract: This thesis explores the ethics of mental health diagnosis and treatment in children and adolescents through a collection of different perspectives based on literature analysis and interviews with practising clinicians. Compared to other medical fields, paediatric psychiatry is considerably novel; it has only recently become a medical specialty and differs from other medicine by involving both biological and psychological aspects of care. Recently, human brain development has become better understood, but the effect of mental illness on the trajectory of development is only just beginning to be studied. Despite this limited understanding, children who may not have been diagnosed for psychiatric disorders in the past are increasingly receiving off-label prescription treatments. This trend may be due to improved sensitivity of diagnostic practices as well as the medicalisation of normal, classifying previously healthy borderline behaviours as pathological. What are the implications of increasing medicalisation? How are child-specific diagnoses and treatment plans determined? I focus on identity development in young people with mental disorders, specifically depression, and try to explain the effects of labelling and medication on that development. Also the physiological impact of psychopathology and psychotropic treatment on neurodevelopment is examined, as well as the lack of long-term clinical data for drug treatment in children. Though the key source material has been from literature in the US and UK, the thesis broadens to an international perspective with a global look at the relationship between culture and paediatric psychiatry practices. Finally, mental health care professionals were interviewed to provide a clinical perspective to supplement the literature analysis. By drawing historical, philosophical, psychological, biological, cultural, and professional perspectives together in this novel way, ethical considerations necessary in the psychiatric care of paediatric patients are given a more thorough understanding, and a framework for assessing these considerations is presented through a focused interdisciplinary lens.
  
  
  
• Structural studies of Vibrio cholerae quorum sensing proteins
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  Abstract: The spread of cholera is always associated with contaminated food or water and this is the reason this disease has been endemic in developing countries for centuries due to their lack of proper sanitation facilities and poor or no infrastructure for sewage systems. Cholera can spread quickly and sporadically after any natural disaster that destroys the sewage system or safe drinking water supply of both developed and undeveloped countries. In Southeast Asia in December 2004 and in Pakistan and Haiti 2010, cholera outbreaks followed the natural disasters; with most of the cholera victims being children. Although it is known that the best way to prevent cholera outbreak is the development of the infrastructure, provision of a safe drinking water supply and proper sanitation, this is a very long-term process, and most of the developing countries cannot afford such improvements. These situations can be made worse by natural disasters. Therefore there is a pressing need for the development of a cholera vaccine and there have been numerous research projects working towards this end for several decades. A few of them have been successful to date but because of the severe side effects and narrow range of protection, more effective and wider range vaccine development is still ongoing. In this study, crystallographic and enzymatic studies have been carried out on several novel proteins involved in the control of the production of the factors required for quorum sensing. Quorum sensing is a process in which bacterial cells communicate among themselves by the synthesis, release and detection of small chemical compounds called autoinducers. In this work, structural analysis was carried out on proteins involved in the synthesis and detection of the major autoinducer of Vibrio cholerae, named CAI-1. The crystal structure of CqsA involved in CAI-1 synthesis has been successfully solved and its enzymatic properties have been characterized. The structure of one domain of the cytoplasmic region of the CAI-1 receptor CqsS was also elucidated, and other domains were expressed. The crystal structure of another enzyme (VCA0859, an aldo-keto reductase) thought to have been involved in the synthesis of CAI-1 was also determined. Another protein named VCA0939 was also studied, due to its importance in biofilm development, and its ability to control quorum-sensing in an alternative pathway in the mutated version of pathogenic strains of V. cholerae that were responsible for the seventh cholera pandemic. The aim of this project was to understand the three dimensional structure of some proteins that are involved in quorum sensing and control of the expression of virulence genes for the pathogenesis of V. cholerae. Understanding the three dimensional structure of the proteins and the mode of autoinducer binding to its specific receptor could be highly valuable in the development of a chemical compound that could lead to the discovery of a novel drug with the ability to target cross species specification.
  
  
  
• Functional design and use of acoustic signals produced by killer whales (Orcinus orca)
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  Abstract: This study aimed to investigate possible functions of the sounds produced by herring-eating killer whales in the Northeast Atlantic. In this study, I investigated the whistle repertoire of killer whales, which had previously only been studied in British Columbia, where it appeared to be restricted to the audible range. However, I show that high frequency whistles (> 17 kHz) were detected in Northeast Atlantic populations but not in Northeast Pacific populations. These results indicated substantial intraspecific variation in whistle production in killer whales. Little variation was observed in high frequency whistles recorded from three different sites in the Northeast Atlantic, suggesting this signal has a similar function across locations. The estimated active space of high frequency whistles and burst-pulse calls suggested that these are short-range signals used for within-group communication. Source levels of burst-pulse calls were lower than what was previously described in British Columbia, which possibly reflected the fact that these sounds do not need to propagate far because distances between group members are generally short. Calls, high frequency whistles and herding calls produced at different depths did not appear to suffer effects due to increased pressure, such as changing frequency or duration characteristics. Feeding appeared to take place below 10 m of depth, as suggested by the localisation of depth of production of feeding-related sounds. These depths were consistent with those at which tailslaps were produced in Dtags attached to individual whales. Feeding periods were characterised by deep diving, increased sound production and highly non-directional movement. These findings suggested that killer whales in a herring spawning ground use a feeding strategy different from carousel feeding used in herring overwintering grounds. These findings showed that Northeast Atlantic killer whales have a different sound repertoire to other populations, and suggested that they may employ different feeding strategies depending on prey behaviour.
  
  
  
• Ants as flower visitors : floral ant-repellence and the impact of ant scent-marks on pollinator behaviour
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  Abstract: As flower visitors, ants rarely benefit a plant, commonly disrupting pollination by deterring other flower visitors, or stealing nectar. This thesis examines three aspects of ant-flower interactions, focusing on the occurrence of floral traits that prevent disruption of pollination and a novel means by which ants may influence pollinator behaviour. To assess which types of plant species possess ant-repelling floral traits I carried out a survey of 49 Neotropical plant species. Around a third of these species were repellent to the common generalist ant Camponotus novograndensis (Formicinae). This repellence was positively correlated with large nectar volumes within individual flowers. It appears that there has been selection for floral ant-repellence as a defence against ant thieves in plant species that invest in large volumes of nectar. In some cases these repellent traits were effective against a wide range of ant species. However, in no plant species were predacious ants particularly repelled, indicating that there may be little selective pressure on non-ant-plants to defend potential pollinators from aggressive ants. To investigate the importance of coevolution in determining the effectiveness of ant-repellents, a small but diverse range of Mediterranean plant species were tested with the invasive nectar thieving ant Linepithema humile (Dolichoderinae) and the native but non-nectar thieving ant Messor bouvieri (Myrmecinae). Responses of both ant species to floral traits were very similar. The ability of some plants to restrict access to ant species with which they have no evolutionary history may help to reduce the impact invasive species, as nectar thieves, have on plant-pollinator interactions. It is reported that flowers recently visited by bees and hoverflies may be rejected for a period of time by subsequent bee visitors through the detection of scent-marks. Nectar-thieving ants could potentially influence the foraging decisions of bees in a similar way if they come to associate ant trail pheromones or footprint hydrocarbons with poor reward levels. However, my empirical work found no differences were found in bee visitation behaviour between flowers of Digitalis pupurea (Plantaginaceae), Bupleurum fruticosum (Apiaceae) or Brassica juncea (Brassicaceae) that had been in contact with ants and control flowers. Ant-attendance at flowers of these species may not reduce reward levels sufficiently to make it worthwhile for bees to incorporate ant scent-marks into foraging decisions. Investigations like these into the interactions between ants, flowers and other flower visitors are essential if we hope to understand the part ants play in pollination ecology, and determine how ants have helped shape floral evolution.
  
  
  
• Willin as a novel 4.1 ezrin radixin moesin (FERM) domain protein in the mammalian hippo signalling pathway
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  Abstract: The Salvador/Warts/Hippo (Hippo) pathway defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Hippo pathway was initially utilised in D. melanogaster, where the Expanded protein acts in the Hippo signalling cascade to control organ size. Willin is the proposed human orthologue of Expanded and the aim of this thesis is to investigate whether willin can activate the mammalian Hippo signalling pathway. Ectopic willin expression causes an increase in phosphorylation of the core Hippo signalling pathway components MST1/2, LATS1 and YAP, an effect which can be antagonised by ezrin. In MCF10A cells, willin over-expression antagonises a YAP-induced epithelial-to-mesenchymal transition via the N- terminal FERM (Four-point-one Ezrin Radixin Moesin) domain of willin. Preliminary results show that willin is expressed within the sciatic nerve of rat and mice, and within the neuromast cells in the zebrafish; suggesting that willin and the Hippo pathway may play a vital role in the developmental regulation within the peripheral nervous system. To conclude, willin influences Hippo signalling activity by activating the core Hippo pathway kinase cassette in mammalian cells. Description: Electronic version excludes material for which permission has not been granted by the rights holder
  
  
  
• The desaturase gene family : an evolutionary study of putative speciation genes in 12 species of Drosophila
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  Abstract: The formation and persistence of species are the subject of much debate among biologists. Many species of Drosophila are behaviourally isolated, meaning that heterospecific individuals are not attracted to one another and do not interbreed. Often, this behavioural isolation is at least in part due to differences in pheromonal preference. Drosophila pheromones are long-chain cuticular hydrocarbons (CHCs). Desaturases are enzymes that are important for the production of CHCs. This thesis investigates the evolution of the gene family across 12 species of Drosophila. Desaturase genes were located in all species. Some genes, those that have previously been shown to have important roles in pheromonal communication, have experienced duplication and loss in several species. Two previously undiscovered duplicates were identified. Generally the desaturase gene family is governed by purifying selection, although following duplication these constraints are relaxed and in some cases duplicated genes show compelling evidence of positive selection. One of the loci under positive selection, the novel duplicate desat1b of the obscura group, was found to have a sex-biased expression pattern and alternative splicing in its 5′ UTR. In RNAi knock-down experiments of desaturase gene function in D. melanogaster, several desaturases were shown to affect CHC profiles of males and females, including some that were previously unlinked to CHC production.
  
  
  
• An investigation of the ABAD-Aβ interaction as a potential therapeutic target for the treatment of Alzheimer’s disease
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  Abstract: Alzheimer’s disease (AD) is the leading cause of dementia but despite being identified over a century ago, current treatments remain limited. To date, no disease-modifying therapies are available. Soluble, intracellular forms of β-amyloid (Aβ), a protein associated with AD, have been identified and intracellular targets of Aβ are being investigated as potential targets for new drugs. Amyloid binding alcohol dehydrogenase (ABAD) was previously identified as a mitochondrial target of Aβ and is known to be up-regulated in AD. This interaction results in production of reactive oxygen species and cell death. Using a small peptide, known as the “decoy peptide”, disruption of this interaction has been shown to reverse biochemical and behavioural symptoms in an AD mouse model. The work reported in this thesis describes the approaches taken to develop methods for in vitro and ex vivo study of the interaction between ABAD and Aβ. A fluorogenic assay for measuring the intracellular activity of ABAD in living cells was developed and using this technique, the intracellular inhibition of ABAD by Aβ was observed for the first time. Surface plasmon resonance was used to measure binding between ABAD and Aβ and also showed the first quantitative analysis of direct binding of the decoy peptide to Aβ42. In order to synthesise small molecule inhibitors of ABAD activity with the aim of developing a molecular probe of the enzyme’s activity, compounds were identified by screening a fragment-based library. Subsequent optimisation of the compound structure led to a 10-fold improvement in the IC50 and has resulted in a lead compound for future development. A similar screening strategy was employed to identify potential small molecule inhibitors of the ABAD-Aβ interaction. This research has resulted in a range of tools and methods for studying ABAD activity and interactions, which will greatly benefit future work on developing compounds that inhibit the ABAD-Aβ interaction to provide a novel method for treating Alzheimer’s disease. Description: Electronic version does not contain associated previously published material
  
  
  
• A comparative investigation of the cognitive and social factors underlying a capacity for cumulative culture
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  Abstract: Human culture has been proposed to uniquely exhibit a ‘ratchet effect’, with beneficial modifications being made to cultural traits over many generations. This is widely thought to have allowed an accumulation of technology and knowledge over time, and to be of central importance to the remarkable ecological and demographic success of humans. Whilst many researchers argue that the roots of human culture lie in social learning, a process widespread in nature, the exact cognitive capacities that set humans apart are not known. To provide a comparative assessment of nine separate hypotheses regarding different social and cognitive factors that may underlie a capacity for cumulative culture, in this thesis a cumulative puzzlebox was presented to three species. Groups of capuchins, chimpanzees and children were provided with the opportunity to solve the puzzlebox to three sequential levels to retrieve rewards of increasing desirability. Higher level solutions spread only in the children. Evidence was found for the occurrence of teaching, imitation, complex communication and prosociality in groups of children, but not in groups of capuchins and chimpanzees. Furthermore, these processes were positively correlated with the performance of individuals within the groups of children which was the only species to show evidence of cumulative cultural learning. Five further hypotheses focussed on alternative social and cognitive factors were not supported by the evidence from this experiment.
  
  
  
• Female mating decisions in the rose bitterling (Rhodeus ocellatus)
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  Abstract: The aim of this study was to obtain an understanding of the basis to female mating decisions in the Chinese rose bitterling (Rhodeus ocellatus). Bitterling have a resource-based mating system that involves the female laying her eggs inside the gills of a freshwater mussel. Male bitterling perform elaborate courtship behaviour and are territorial and aggressively guard mussels in their territory from other territory holders and non-territorial males. Using a series of laboratory experiments it was shown in this study that females were choosy over the males they mated with, but females were not congruent in their preferences. Female mate preferences correlated positively with offspring growth rates and survival during early development. Female mate choice did not correspond with male dominance, and there may be an intersexual conflict between female mate preferences and male dominance as a result. Females tended to prefer males with functionally dissimilar MHC alleles. MHC alleles may influence male odour cues, and females showed a preference for mussels in which the sperm of multiple males had been released, possibly indicating that females use odour cues associated with sperm release in mating decisions. Bitterling show an innate preference for the colour red in a foraging context and there may be a receiver bias for red nuptial colouration in female mating preferences. Despite a significant role for mate preferences, direct (oviposition) mating preferences were shown to be more important in the mating system. Choice of oviposition sites has both immediate (survival) consequences for offspring, as well as longer-term fitness effects.
  
  
  
• The impact of microbial extracellular polymeric substances on sediment stability
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  Abstract: The main objective of this thesis is to investigate the impact of microbial extracellular polymeric substances (EPS) on sediment stability and the related factors which influence “biogenic stabilisation” as a basis to the prediction of sediment erosion and transport. The ability to make direct and sensitive measurements of the physical properties of the biofilm is a critical demand to further understanding of the overall biostabilisation processes.Therefore, attention has been focused on developing a new technique, Magnetic Particle Induction (MagPI) for measuring the adhesive properties of the biofilm. MagPI determines the relative adhesive properties or “stickiness” of the test surface, whether a biofilm, a sediment or other submerged material. The technique may have future applications in physical, environmental and biomedical research. Newly developed Magnetic Particle Induction(MagPI) and traditional techniques Cohesive Strength Meter (CSM) for the determination of the adhesion/cohesion of the substratum were used to assess the biostabilisation capacity of aquatic microorganisms. Whilst these devices determine slightly different surface properties of the bed, they were found to complement each other, increasing the range of measurements that could be made and presented a strong correlation in the overlapping portion of the data. It is recognized that microorganisms inhabiting natural sediments significantly mediate the erosive response of the bed (“ecosystem engineers”) through the secretion of naturally adhesive organic material (EPS: extracellular polymeric substances). Interactions between main biofilm consortia microalgae, cyanobacteria and bacteria in terms of their individual contribution to the EPS pool and their relative functional contribution to substratum stabilisation were investigated. The overall stabilisation potential of the various assemblages was impressive, as compared to controls. The substratum stabilisation by estuarine microbial assemblages was due to the secreted EPS matrix, and both EPS quality (carbohydrates and proteins) and quantity (concentration) were important in determining stabilisation. Stabilisation was significantly higher for the bacterial assemblages than for axenic microalgal assemblages. The peak of engineering effect was significantly greater in the mixed assemblage as compared to the bacterial and axenic diatom culture. This work confirmed the important role of heterotrophic bacteria in “biostabilisation” and highlighted the interactions between autotrophic and heterotrophic biofilm components of the consortia. An additional approach, to investigate the impact of toxins on biostabilisation capacity of aquatic organism was performed on cultured bacterial and natural freshwater biofilm.The data suggest a different mode of triclosan (TCS) action ranging from suppressing metabolisms to bactericidal effects depending on the TCS concentration. The inhibitory effect of triclosanon bacterial and freshwater biofilms was confirmed. This information contributes to the conceptual understanding of the microbial sediment engineering that represents an important ecosystem function and service in aquatic habitats.
  
  
  
• Range-wide analysis of the spatial distribution and genetic diversity of Delonix s.l. (Leguminosae) in Madagascar : enhancing herbarium-based conservation assessments
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  Abstract: Despite their ecological and economic importance, the majority of plant species and their conservation status are poorly known. Only 4% of plants have been assessed globally and listed on the IUCN Red List of Threatened Species; and without plant conservation assessments, many plant species will not feature in conservation planning. Herbarium collection information can significantly increase the number of plant conservation assessments. Thus, the aims of this thesis were: (1) to investigate how the quality of herbarium-based conservation assessments can be optimised; (2) to assess the extent to which herbarium-based conservation assessments reflect the reality on the ground; and (3) to scientifically validate genetic and spatial underpinning of IUCN criteria. Preliminary range-based assessments of the Leguminosae of Madagascar achieved a result consistent with the final conservation rating for over 95% of species when using up to fifteen herbarium specimens. Bioclimatic modelling of range shifts based on future climate change predicted that, in the worst case scenario, up to one third of endemic Leguminosae in Madagascar will be threatened with extinction over the next 100 years. An analysis of the population structure of species of Delonix s.l. (Leguminosae) showed that combining spatial analysis with population genetic data provides a more complete picture of landscape-level population dynamics and the impacts on conservation status. Moreover, range-wide genetic analysis of AFLP markers for four species of Delonix demonstrated a genetic basis for IUCN categories distinguishing between threatened and non-threatened species. Although genetic data are currently not often incorporated in conservation assessments, they are crucial in making accurate management decisions and creating effective action plans for conservation. Only by using all available scientific resources can informed conservation decisions be made and the survival of plants and their associated ecosystems be ensured.
  
  
  
• Studies on influenza A virus PB1-F2 protein
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  Abstract: The influenza A virus genome codes for up to 12 proteins. Segment 2 encodes three proteins, the polymerase subunit PB1, a small protein PB1-F2 and an N-terminally truncated version of PB1 called N40. Different functions have been reported for PB1-F2 such as induction of apoptosis, regulation of the viral polymerase activity, enhancement of secondary bacterial infections and modulation of the innate immune system. So far, no function has been ascribed to N40. To study PB1-F2 in more detail, its coding sequence was deleted from its original position and inserted downstream of the PB1 (segment 2), NA (segment 6) or M (segment 7) open reading frames (ORF) employing different strategies, including the use of an overlapping Stop-Start cassette, a duplicated promoter sequence and the self-cleaving 2A peptide derived from foot-and-mouth disease virus. Viruses with bicistronic segments were rescued and tested for their ability to express PB1-F2. Whereas no expression of PB1-F2 was detected from bicistronic segments 2 and 7, expression of PB1-F2 from segment 6 was observed in high levels. However, the phenotype of all these viruses was similar to that of viruses lacking PB1-F2 which made mutational analysis of PB1-F2 not worthwhile. Previously, the function of PB1-F2 was mainly studied using a virus deficient in PB1-F2 production but showing increased N40 expression. In the present study, recombinant WSN viruses lacking either PB1-F2 or N40, or both proteins were engineered and the effects of these mutations on the viral life cycle were examined. Viruses deficient for PB1-F2 that overexpressed N40 showed the most attenuated phenotype, whereas the loss of PB1-F2 alone did not obviously affect virus replication. Reduced viral polymerase activity was observed for viruses lacking N40, however attenuation in vivo was only seen in combination with the loss of PB1-F2. Neither the loss of PB1-F2 nor N40 alone had a great impact, but changes in the expression level of both proteins were disadvantageous for the virus. Increased levels of N40 shifted the polymerase activity towards replication, suggesting a new function for N40. Thus, it was shown that the segment 2 gene products and their expression level influence viral replication and pathogenicity, and a careful design of mutant recombinant viruses is vital for determining the experimental outcome.
  
  
  
• Virus and interferon : a fight for supremacy : comparison of the mechanisms of influenza A viruses and parainfluenza virus 5 in combatting a pre-existing IFN-induced antiviral state
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  Abstract: The Interferon (IFN) family of cytokines are produced in direct response to virus infection and they constitute the first line of defence against virus infection by inducing hundreds of interferon stimulated genes (ISGs) which act in concert to establish the so-called “antiviral state”. Influenza A viruses and parainfluenza virus type 5 (PIV5) are both small negative strand RNA viruses that must circumvent their hosts’ interferon (IFN) response for replication. However, the ways in which these viruses interact with the IFN system are very different. Although PIV5 replication is initially severely impaired in cells in a pre-existing IFN-induced antiviral state, it manages to overcome the antiviral state by targeting an essential component of type I IFN signalling, STAT1, for degradation. Thus the cells cannot maintain the antiviral state indefinitely without continuous signalling. Consequently, the virus resumes its normal replication pattern after 24-48 hours post-infection. In clear contrast, influenza virus fails to establish its replication in the majority of infected cells (90-95%) with a pre-existing IFN-induced antiviral state, although a few cells are still able to produce viral antigens. To further investigate how influenza virus interacts with cells in a pre-existing IFN-induced antiviral state, I have used in situ hybridization to follow the fate of input and progeny genomes in cells that have, or have not, been treated with IFN prior to infection. Here I show for the first time that IFN pre-treatment blocks the nuclear import of influenza A virus genome, which prevents the establishment of virus replication, but this can be overcome by increasing multiplicities of infection. Of those IFN-induced antiviral molecules, human MxA is an essential component of the IFN-induced antiviral state in blocking influenza virus genome import, as this block can be abolished by lentivirus-mediated knockdown of MxA. I also show that in cells constitutively expressing MxA the viral genome still manages to be transported into the nucleus, indicating that MxA might require an unidentified IFN-induced factor to block nuclear import of the influenza virus genome. These results reveal that IFN exerts its action at an early stage of virus infection by inducing MxA to interfere with the transport of viral genome into the nucleus, which is the factory for viral RNA production.
  
  
  
• Geographic and species variation in bottlenose dolphin (Tursiops spp.) signature whistle types
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  Abstract: Geographic variation in the whistle vocalisations of dolphins has previously been reported. However, most studies have focused on the whole whistle repertoire, with little attempt to classify sounds into biologically relevant categories. Common bottlenose dolphins (Tursiops truncatus) use individually distinctive signature whistles which are thought to help maintain contact between conspecifics at sea. These whistles may show a different kind of variation between populations than non-signature whistles. Here I investigate signature whistle use and variation in the two recognised species of bottlenose dolphins (T. truncatus and T. aduncus) from populations inhabiting the coastal waters of the North America, Scotland, South Africa, Tanzania, Japan, Australia and New Zealand, and one captive colony. I identified likely signature whistles (signature whistles types, SWTs) from acoustic recordings by combining two novel techniques: automated contour categorisation in ARTwarp (Deecke and Janik, 2006) and a specific bout analysis based on the timing of signature whistle production in T. truncatus termed SIGID (Janik et al. in press). Three ways of categorising the contours were tested and between 87 and 111 SWTs were identified in total. Repeated emissions of stereotyped contours were apparent in the repertoire of all T. aduncus populations using both automated and human observer categorisation, providing good evidence for signature whistle use in this species. There was significant inter-specific variation in the frequency parameters, looping patterns and duration of SWTs. Inflection points, duration and measures of SWT complexity showed high variation within populations, suggesting inter- and intra-individual modification of these parameters, perhaps to enhance identity encoding or convey motivational information. Using 328 bases of the mtDNA control region, I found high levels of population differentiation (FST and φST) within the genus Tursiops. These data do not support a link between mtDNA population differentiation and variability in call type. Instead, morphological variations at the species level, and learned differences at the population level, better explain the variation found.
  
  
  
• Local development planning and bats in the UK : “an impenetrable fog” ?
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  Abstract: Despite national and international protection, and the focus of conservation biology research, UK bat populations remain under threat from development. Cumulative impacts from development threaten bat populations with the “death of a thousand cuts”: direct mortality; loss of roost spaces; increasing road traffic and wind turbines killing more bats; disturbance, fragmentation and degradation of habitat may increase mortality by reducing fitness. It is timely to investigate the efficacy of conservation measures targeted at protecting bats in local development planning (“development management”), in particular the process of screening planning applications for potential impacts upon bats, acting in resolution of this Human-Wildlife Conflict. Examination of published guidance was combined with review of practitioner experience through Local Planning Authority (LPA) development management staff and bat survey consultants, with the aim of determining weaknesses in principles, mechanisms and resources. Key issues were the lack of political and managerial support, poor enforcement, a lack of expert natural heritage skills, inconsistent interpretation and application of guidelines, and significant gaps in guidance. For one LPA, 22% of sampled planning applications showed a high risk of potential impacts upon bats, yet only 1% had a bat survey undertaken; and 32% of known roost sites had been subject to one or more planning applications in 10 years. Only a few LPAs enjoy excellent access to expert natural heritage skills, biological data and advice from SNCOs. I present a multi-disciplinary synthesis to derive recommendations for process enhancement including seven good design principles, and seven key resources. The foundation to comprehensive adoption and enforcement is clear nationally consistent political support; thus engendering good practice e.g. all UK development applications should declare how biodiversity issues are addressed; research should address bat population dynamics, and responses to development impacts. Description: This study was supported by RPS Group plc (Planning & Development)
  
  
  
• An investigation of factors related to the bycatch of small cetaceans in fishing gear
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  Abstract: The bycatch of cetaceans in fishing gear is considered to be one of the biggest conservation threats to these species. Gear modifications have the potential to reduce these bycatches in global fisheries but there is little available information on how such modifications may change the fishing performance of gear, or indeed the behavior of cetaceans interacting with fishing gear. Generalized linear models (GLMs) were used to identify factors related to cetacean bycatches in UK bottom set gillnets. Rigged net height had a significant positive relationship with harbour porpoise (Phocoena phocoena) bycatch in ICES Area VII suggesting that lowering the profile of gillnets may have the potential to reduce bycatch rates. Modifications to gillnets, such as changing the amount of floatation or increasing the density of the meshes, were found to have significant effects on the active fishing heights of these nets. However, results from a bycatch mitigation trial in Argentina showed that the reduced fishing profile of one experimental net did not result in a concurrent reduction in the bycatch rate of Franciscana dolphins (Pontoporia blainvillei). While there was no significant difference in the rate, length or intensity of harbour porpoise encounters in the presence or absence of gillnets, the proportion of fast echolocation click trains were significantly higher when a net was present, indicating that porpoises either increased acoustic inspection of the net or foraging in the vicinity of the net. An analysis of underwater video footage collected inside trawl nets in an Australia fishery showed that bottlenose dolphins (Tursiops sp.) were present inside nets more frequently than they were caught and were actively foraging inside these nets. The orientation of dolphins inside these nets indicates that the current design of excluder devices used in this fishery could be improved to further reduce bycatch rates.
  
  
  
• Health, disease, mortality and survival in wild and rehabilitated harbor seals (Phoca vitulina) in San Francisco Bay and along the central California coast
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  Abstract: Conventional methods for health assessment of wild-caught and stranded seals were used to describe the disease status of harbor seals in California. Clinical chemistry, infectious disease prevalence, immune function, and contaminant data were collected to evaluate harbor seal health with data collected from three groups of seals. Wild-caught seals of all ages were sampled at two locations: San Francisco Bay (a heavily urbanized estuary) and Tomales Bay (a less developed control site). Stranded seals entered rehabilitation from a more extensive portion of the California coast which included the locations where seals were caught. Hematology reference intervals were generated to provide a baseline for health assessment among the seals. Individual variability in blood variables among seals was affected by age, sex, location, and girth. Disease surveillance focused on pathogens known to cause lesions in harbor seals, zoonoses, and those with terrestrial sources. Specific pathogens of interest were E coli, Clostridium perfringens, Vibrio spp, Campylobacter spp, Salmonella, Giardia, Cryptosporidium, avian influenza virus, Brucella, Leptospira spp., Toxoplasma gondii, Sarcocystis neurona, and Neospora caninum, Leptospira spp, and phocine and canine distemper virus. There was evidence of exposure to all pathogens except for phocine distemper virus. Simple measures of immune response were used to evaluate the immune function of harbor seal pups in rehabilitation that had evidence of previous bacterial infection. The swelling response to a subcutaneous injection of phytohemagglutinin (PHA) was positively associated with growth rate, possibly illustrating the energetic trade-offs between growth and immunity. Blubber contaminant concentrations (PCBs, DDTs, PBDEs, CHLDs, and HCHs) in harbor seal pups were grouped by extent of suckling and strand location. The ratio of PCB:DDT was increased in San Francisco Bay and decreased in Monterey Bay compared with other locations along the coast. Pups that weaned in the wild, lost weight and then stranded had the highest contaminant levels, equivalent to the concentrations detected in stranded adult harbor seals. Dispersal and survival were monitored by satellite telemetry in harbor seal pups released from rehabilitation and recently weaned wild-caught pups to assess the effect of condition, health, and contaminant levels on survival probability. Increased contaminant levels and decreased thyroxine (T4) were associated with decreased survival probability. Increased mass, particularly among the rehabilitated pups, was associated with increased survival probability. This study demonstrates that health and survival of harbor seals pups along the central California coast are impacted by human activities such as contaminant disposal, pathogen pollution and boat traffic, although the variability in individual health measures requires carefully designed studies to detect these effects.
  
  
  
• Enemy within the gates : reasons for the invasive success of a guppy population (Poecilia reticulata) in Trinidad
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  Abstract: The invasion of individuals into new habitats can pose a major threat to native species and to biodiversity itself. However, the consequences of invasions for native populations that are not fully reproductively isolated from their invaders are not yet well explored. Here I chose the Trinidadian guppy, Poecilia reticulata, to investigate how different population traits shaped the outcome of Haskins's introduction, a well-documented invasion of Guanapo river guppies into the Turure river. I especially concentrated on the importance of behaviour for invasive success. I investigated if the spread of Guanapo guppies is due to superiority in behaviour, life-history and/or genetics, or if the outcome of this translocation is due to chance. Despite the fact that by today the invasive front has passed the Turure's confluence with the River Quare many kilometres downstream of the introduction site, and the original genotype only survives in small percentages, as was revealed by genetic analysis in this and other studies, no obvious differences between invasive and native populations could be detected in any of the tested behavioural, life-history and genetic traits. When tested for mate choice, neither Guanapo nor Oropuche (Turure) males seemed to be able to distinguish between the population origin of females, but courted and mated at random. At the same time, females did not prefer to school with individuals of the same population over schooling with more distantly related females. The formation of mixed schools after an invasive event is therefore likely. Because female guppies showed a very low willingness to mate, even after having been separated from males for up to six months, sperm transfer through forced copulations will become more important. Taken together, these behaviours could increase the speed of population mixing after an invasion without the need for behavioural superiority of the invasive population. When tested for their schooling abilities, offspring of mixed parentage, in contrast to pure breds, displayed a large amount of variety in the time they spent schooling, a circumstance that can potentially influence survival rates and therefore the direction of gene pool mixing. Guanapo fish did not show reproductive superiority in a mesocosm experiment, where both populations were mixed in different proportions. On the contrary, in two out of three mixed treatments, the amount of Oropuche (Turure) alleles was significantly higher than expected from the proportion of initially stocked fish. The almost complete absence of distinguishable traits other than genetic variation between the examined populations that belong to different drainage systems, opposes the recent split of the guppy into two different species following drainage system borders, as is argued in this thesis. However, the successful invasion of the Turure by Guanapo guppies and the nearly entire disappearance of the original population can be explained in absence of differing population traits. Here I demonstrate how behavioural and genetic interactions between subspecies influence the outcome of biological invasions and second, how factors other than population traits, such as the geographic situation, can produce an advantageous situation for the invader even in the absence of population differences.
  
  
  
• Life history trade-offs between survival, moult and breeding in a tropical season environment
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  Abstract: The trade-off between current and future investment in reproduction lies at the heart of life history theory. The need to differentially allocate resources between these two options arises generally as a result of environmental pressures. Higher risk of mortality in adults is linked with increased investment in current reproduction, whereas the opposite is true where adults are long-lived (the r- K selection paradigm). Perhaps the most obvious factors influencing the environment stem from seasonality of the climate, since rainfall and temperature affect food availability, resulting in a higher risk of mortality. The available trade-offs that an organism can make will therefore be constrained by environmental variability potentially resulting in general adaptation and so ultimately influencing evolution of biome-specific life-history traits. In this thesis, I examine how the seasonality of a West African tropical savannah environment influences moult and breeding timing and duration, and survival in West African tropical savannah bird species. I show that moult in tropical birds follows the same basic descendant pattern through the wing feathers, but is a much lengthier process than for temperate species (mean = 131 ± 11 days, N = 29 species), and that it frequently overlaps with breeding activities. This suggests either that either the feathers of tropical species take longer to grow; that it is a relatively low-cost activity and has little influence on life history trade-offs; or that individuals further aim to reduce mortality risk by attempting to maintain high flight capability at all times. Breeding also occurred over a longer season than for temperate species, although an obvious peak in occurrence was identified to coincide with the food-abundant period of the late rains and early dry season. Lengthy breeding seasons may indicate an increased tendency to re-nest (possibly as a result of higher nest predation levels), and we also identified a prolonged immature plumage phase – potentially indicating an extended duration of parental care. Survival rates were calculated from mark-recapture models based on mist-netting data. Previous work has focussed on the use of incorporating mark-resighting data alongside that obtained by standard mark-recapture techniques. Here, I assess the models applied in those methods, identify problems associated with over-paramaterisation, goodness of fit and the generation of biologically unrealistic estimates, and so provide suggestions on how to improve the protocol. Average survival from my study (40 species: 0.63 ± 0.02) was higher than previous estimates obtained from this site and were comparable with estimates from other Afrotropical and Neotropical areas, although rates varied greatly between species. Juvenile survival (13 species) was similar or possibly lower than adult survival. I then used my empirically derived estimates of moult, breeding and survival life history traits to identify potential trade-offs between traits. Overall I was unable to identify significant relationships between any of the life history trait estimates, other than between adult survival and clutch size. In this, the results followed those of previous researchers in identifying a pattern of lower investment in current reproduction (clutch size) and maximisation of adult survival in tropical species. My study, however, demonstrates for the first time how moult and breeding duration are likely to be less constrained in tropical environments.
  
  
  
• Target strength variability in Atlantic herring (Clupea harengus) and its effect on acoustic abundance estimates
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  Abstract: Acoustic survey techniques are widely used to quantify abundance and distribution of a variety of pelagic fish such as herring (Clupea harengus). The information provided is becoming increasingly important for stock assessment and ecosystem studies, however, the data collected are used as relative indices rather than absolute measures, due to the uncertainty of target strength (TS) estimates. A fish’s TS is a measure of its capacity to reflect sound and, therefore, the TS value will directly influence the estimate of abundance from an acoustic survey. The TS is a stochastic variable, dependent on a range of factors such as fish size, orientation, shape, physiology, and acoustic frequency. However, estimates of mean TS, used to convert echo energy data from acoustic surveys into numbers of fish, are conveniently derived from a single metric - the fish length (L). The TS used for herring is based on TS-L relationships derived from a variety of experiments on dead and caged fish, conducted 25-30 years ago. Recently, theoretical models for fish backscatter have been proposed to provide an alternative basis for exploring fish TS. Another problem encountered during acoustic surveys is the identification of insonified organisms. Trawl samples are commonly collected for identification purposes, however, there are several selectivity issues associated with this method that may translate directly into biased acoustic abundance estimates. The use of different acoustic frequencies has been recognised as a useful tool to distinguish between different species, based on their sound reflection properties at low and high frequencies. In this study I developed theoretical models to describe the backscatter of herring at multiple frequencies. Data collected at four frequencies (18, 38, 120 and 200 kHz) during standard acoustic surveys for herring in the North Sea were examined and compared to model results. Multifrequency backscattering characteristics of herring were described and compared to those of Norway pout, a species also present in the survey area. Species discrimination was attempted based on differences in backscatter at the different frequencies. I examined swimbladder morphology data of Baltic and Atlantic herring and sprat from the Baltic Sea. Based on these data, I modelled the acoustic backscatter of both herring stocks and attempted to explain differences previously observed in empirical data. I investigated the change in swimbladder shape of herring, when exposed to increased water pressures at deeper depths, by producing true shapes of swimbladders from MRI scans of herring under pressure. The swimbladder morphology representations in 3-D were used to model the acoustic backscatter at a range of frequencies and water pressures. I developed a probabilistic TS model of herring in a Bayesian framework to account for uncertainty associated with TS. Most likely distributions of model parameters were determined by fitting the model to in situ data. The resulting probabilistic TS was used to produce distributions of absolute abundance and biomass estimates, which were compared to official results from ICES North Sea herring stock assessment. Modelled backscatter levels of herring from the Baltic Sea were on average 2.3 dB higher than those from herring living in northeast Atlantic waters. This was attributed to differences in swimbladder sizes between the two herring stocks due to the lower salinity Baltic Sea compared to Atlantic waters. Swimbladders of Baltic herring need to be bigger to achieve a certain degree of buoyancy. Morphological swimbladder dimensions of Baltic herring and sprat were found to be different. Herring had a significantly larger swimbladder height at a given length compared to sprat, resulting in a modelled TS that was on average 1.2 dB stronger. Water depth, and therefore the increase in ambient pressure, was found to have a considerable effect on the size and shape of the herring swimbladder. Modelled TS values were found to be around 3 dB weaker at a depth of 50 m compared to surface waters. At 200 m, this difference was estimated to be about 5 dB. The Bayesian model predicted mean abundances and biomass were 23 and 55% higher, respectively, than the ICES estimates. The discrepancy was linked to the depth-dependency of the TS model and the particular size-dependent bathymetric distribution of herring in the survey area.
  
  
  
• Variation in habitat preference and distribution of harbour porpoises west of Scotland
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  Abstract: The waters off the west coast of Scotland have one of the highest densities of harbour porpoise (Phocoena phocoena) in Europe. Harbour porpoise are listed under Annex II of the EU Habitats Directive, requiring the designation of Special Areas of Conservation (SACs) for the species’ protection and conservation. The main aim of this thesis is to identify habitat preferences for harbour porpoise, and key regions that embody these preferences, which could therefore be suitable as SACs; and to determine how harbour porpoise use these regions over time and space. Designed visual and acoustic line-transect surveys were conducted between 2003 and 2008. Generalised Estimating Equations (GEEs) were used to determine relationships between the relative density of harbour porpoise and temporally and spatially variable oceanographic covariates. Predictive models showed that depth, slope, distance to land and spring tidal range were all important in explaining porpoise distribution. There were also significant temporal variations in habitat use. However, whilst some variation was observed among years and months, consistent preferences for water depths between 50 and 150 m and highly sloped regions were observed across the temporal models. Predicted surfaces revealed a consistent inshore distribution for the species throughout the west coast of Scotland. Regional models revealed similar habitat preferences to the full-extent models, and indicated that the Small Isles and Sound of Jura were the most consistently important regions for harbour porpoise, and that these regions could be suitable as SACs. The impacts of seal scarers on distribution and habitat use were also investigated, and there were indications that these devices have the potential to displace harbour porpoise. These results should be considered in the assessment of sites for SAC designation, and in implementing appropriate conservation measures for harbour porpoise.
  
  
  
• Revealing the past : the potential of a novel small nucleolar RNA (snoRNA) marker system for studying plant evolution
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  Abstract: Despite the existence of various molecular marker systems there are still limitations in distinguishing between closely related species based on molecular divergence, especially when hybridization events have occurred in the past. The characterisation of plant small nucleolar RNA (snoRNA) genes and their organisation into multigene clusters provides a potential nuclear marker system which could help in resolving the phylogenetic history of plants and might be applicable in DNA barcoding. Using closely and distantly related Senecio species, I investigated a combination of fragment length and sequence variation of snoRNA genes/snoRNA gene clusters to assess the utility of this marker system for barcoding and resolving species relationships. SnoRNA gene and gene cluster sequences identified in Arabidopsis thaliana were used to find homologues in other species and subsequently used for the design of universal primers. Most of the universal primer pairs designed were successful in amplifying snoRNA fragments in most Senecio species and fragment length variation between and within species could be detected. Furthermore, the combination of some fragment length datasets produced by different primer pairs enabled the separation of species and the detection of reticulate evolution indicating a high potential of snoRNA gene/gene cluster fragment length polymorphisms (SRFLPs) for phylogenetic reconstructions in Senecio and other plant genera. Most of the examined gene clusters showed a similar gene order in Senecio and Arabidopsis. However, the majority of these clusters appeared to exhibit more copies in Senecio, some of which were distinguishable by a combined sequencing/fragment profiling approach, and shown to be putative single copy regions with the potential to be used as co-dominant markers. However, a high number of paralogues and possible differences in copy number between species excludes these regions from being used in DNA barcoding. This is because specific primers would have to be developed for specific copies which would preclude development of a universal application for barcoding. None of the regions showed enough sequence variation to delimit distinctly closely related Senecio species and were therefore also considered to be unsuitable for DNA barcoding. Although most snoRNA genes and gene clusters might be inapplicable for DNA barcoding, they are likely to be valuable for phylogenetic studies of species groups, genera and families. On this scale, specific primers might act universally and the number of paralogous copies is likely to be equal across the species group of interest.
  
  
  
• The behavioural ecology of the Trinidadian guppy, Poecilia reticulata, as an invasive species
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  Abstract: This thesis focuses on the guppy, Poecilia reticulata, as an invasive species. Its non-native distribution, the biology behind its success and the reasons for its introduction are examined. A worldwide email survey revealed that the guppy is established in at least 73 countries outside of its native range and that mosquito control schemes and the release of unwanted aquarium fish are the two primary routes of introduction. Knowledge gaps were identified; primarily the scarcity of scientific evidence for negative impacts of guppy introductions and similarly for mosquito control efficacy. Replicated mesocosm experiments demonstrated that female guppies are capable of routinely establishing populations, and that these retain behavioural viability over several generations. The first mesocosm study suggested that founders with very different evolutionary histories were equally good at establishing populations. The second mesocosm study suggested that monandrous females were extremely successful at establishing behaviourally viable populations, with no decline in behavioural variation. The effectiveness of guppies as mosquito control agents was examined in two related foraging experiments. The first study found little evidence for the presence of ‘prey switching’ in guppies, questioning the validity of previous work advocating their introduction to stabilise prey populations. The second study revealed a preference for non-vector mosquito larvae in a two-prey system. However, both mosquito species were consumed equally readily when habitat complexity increased. The presence of conspecifics affected female foraging behaviour. The presence of males reduced the strength of prey preference in the first study, and the presence of conspecifics of either sex removed prey preference in the second. Both demonstrate that multi-prey systems have important implications for the efficacy of poeciliids in biological control. Despite severe demographic bottlenecks, their adaptability and ability to rapidly increase in numbers enable guppies to establish and persist when introduced. Such bottlenecks are typical of introduction scenarios, warning that particular caution should be exerted when introducing this species, or other livebearing fish, to natural water bodies.
  
  
  
• Studies on interferon (IFN) induction and isolation of IFN-inducing mutant viruses
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  Abstract: The interferon (IFN) system is a powerful antiviral defense system. Host cell pattern recognition receptors (PRRs) recognise pathogen-associated molecule patterns (PAMPs) which when activated, lead to the transcription of the IFN-β gene. As a consequence IFN is secreted from the cell and activates the JAK-STAT pathway to up-regulate the transcription of IFN-stimulated genes (ISGs). The products of many ISGs inhibit viral replication and cell proliferation. Viruses encode IFN antagonists that dampen down the IFN response, making it less effective. However, within a virus population, there are always likely to be naturally occurring mutant viruses that have lost the ability to circumvent the host IFN response, and if isolated, these viruses would be unlikely to cause severe disease in the host and may therefore be developed as live attenuated virus vaccine candidates. To develop a methodology to rapidly isolate IFN-inducing mutant viruses, we generated an A549 reporter cell-line in which expression of GFP was driven by the IFN-β promoter. Using this cell-line, we show that the number of cells that became positive for GFP correlated with the amount of IFN secreted by the infected cells and the number of defective interfering (DI) particles within the virus preparations. However, we were unable to isolate IFN-inducing mutant viruses using the A549/pr(IFN-β).GFP cell-line(s). Possible reasons for this may be either that, in cells infected by IFN-inducing mutant viruses, an antiviral state was established independent of IFN that prevented virus replication in the reporter cells in which the IFN-β promoter was activated; or the viruses that activated the IFN-β promoter were DIs only which were not be able to replicate without non-defective helper viruses. A549/pr(IFN-β).GFP cells are also being used for high throughput assays to screen chemical libraries for compounds that block IFN induction. Such compounds may be potential candidates for anti-inflammatory drugs.
  
  
  
• Genetic characterisation and social structure of the East Scotland population of bottlenose dolphins (Tursiops truncatus)
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  Abstract: The Eastern Scottish population of bottlenose dolphins (Tursiops truncatus) is the northernmost population of this species. The resident core of this population consists of 120 to 150 different individuals. This small size and its geographical isolation from other populations raises questions about its viability and whether the population has behavioural patterns that differ from those common to other populations of the same species. Microsatellite genetic diversity was low and mitochondrial DNA genetic diversity values were lowest in East Scotland compared to other populations worldwide and to neighbouring populations around UK waters. It has been well documented, from four different field sites worldwide, that male bottlenose dolphins form alliances with preferred male associates. These alliances can last for several years and the males involved show association coefficients similar to those of mothers and calves (0.8-1.0). These alliances appear to be of great importance in obtaining matings for the males. In the Eastern Scottish population males do not form alliances. No evidence of strong associations between individuals of either sex was found and there was no correlation between association and relatedness patterns. I suggest that the isolation and small size of the population together with reduced genetic diversity affects the pressure of kin selection for altruistic behaviours. There is no gain in competing or associating with close relatives for access to mates and it might be more important to avoid inbreeding by dispersing. Although evidence of gene flow between East Scotland and its neighbouring populations was not confirmed with Bayesian clustering analysis, a small set of individuals from Wales were found to be closely related to individuals from the East Coast of Scotland. In general the dynamics found in UK water populations resemble those of the Western North Atlantic with sympatric populations of coastal as well as pelagic individuals.
  
  
  
• Splitting, joining and cutting : mechanistic studies of enzymes that manipulate DNA
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  Abstract: DNA is a reactive and dynamic molecule that is continually damaged by both exogenous and endogenous agents. Various DNA repair pathways have evolved to ensure the faithful replication of the genome. One such pathway, nucleotide excision repair (NER), involves the concerted action of several proteins to repair helix-distorting lesions that arise following exposure to UV light. Mutation of NER proteins is associated with several genetic diseases, including xeroderma pigmentosum that can arise upon mutation of the DNA helicase, XPD. The consequences of introducing human mutations into the gene encoding XPD from Sulfolobus acidocaldarius (SacXPD) were investigated to shed light on the molecular basis of XPD-related diseases. XPD is a 5’-3’ DNA helicase that requires an iron-sulphur (FeS) cluster for activity (Rudolf et al., 2006). Several proteins related to SacXPD, including human XPD, human FancJ and E. coli DinG, also rely on an FeS cluster for DNA unwinding (Rudolf et al., 2006; Pugh et al., 2008; Ren et al., 2009). Sequence analysis of the homologous protein, DinG, from Staphylococcus aureus (SarDinG) suggests that this protein does not encode a FeS cluster. In addition, SarDinG comprises an N-terminal extension with homology to the epsilon domain of polymerase III from E. coli. This thesis describes the purification and characterisation of SarDinG. During replication, DNA lesions or other ‘roadblocks’, such as DNA-bound proteins, can lead to replication fork stalling or collapse. To maintain genomic integrity, the fork must be restored and replication restarted. In archaea, the DNA helicase Hel308 is thought to play a role in this process by removing the lagging strands of stalled forks, thereby promoting fork repair by homologous recombination. Potential roles of Hel308 during replication fork repair are discussed in this thesis. The mechanism by which Hel308 moves along and unwinds DNA was also investigated using a combined structural and biophysical approach. The exchange of DNA between homologous strands, catalysed by a RecA family protein (RecA in bacteria, RAD51 in eukaryotes, and RadA in archaea), defines homologous recombination. While bacteria encode a single RecA protein, both eukaryotes and archaea encode multiple paralogues that have implications in the regulation of RAD51 and RadA activity, respectively. This thesis describes the purification and characterisation of one of the RadA paralogues (Sso2452) in archaea. Description: Electronic version does not contain associated previously published material
  
  
  
• Social learning in fish
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  Abstract: Social learning is known to be a common phenomenon in fish, which they utilise under many different contexts, including foraging, mate-choice and migration. Here I review the literature on social learning in fish and present two studies. The first examines the ability of threespined sticklebacks to use social learning in the enhancement of food preferences. The second study examines the ability of both threespined sticklebacks and ninespined sticklebacks to use social learning in the avoidance of predators.
  
  
  
• Predicting food consumption and production in fish populations : allometric scaling and size-structured models
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  Abstract: Life-history traits in fish populations are highly correlated. A subset of these correlations are called allometric scaling, they refer to biological processes which can be described using body size as independent variable. Particularly, allometric scaling related with food consumption (Q) and biomass production (P) has gained the attention of ecologists for several decades. This thesis proposes a quantitative framework for food consumption, which allows both the identification of the mechanisms underlying the allometric scaling for Q and the development of a predictive model for consumption to biomass ratio (Q/B) in fish populations. This thesis is based on the fact that food consumption can be inferred from first principles underlying the von Bertalanffy growth model. In addition, it has been noticed in the literature that biomass production and food consumption show similar allometric scaling dependence, therefore, both can be derived from these first principles. Thus, a similar quantitative framework was used to produce models for P/B in fish populations. Once functional forms for production and food consumption were identified, a third model was developed for the ratio between production and consumption (P/Q). This ratio is usually named ecological efficiency because it determines how efficiently a population can transform ingested food into biomass. Several authors have noticed that P/Q remains invariant (independent of body size) across species. From a theoretical point of view, the results presented here allow the first quantitative explanation for the existence of the allometric scaling for Q/B and the invariance of P/Q across fish species. These results, together with the explanation for allometry in P/B reported in the literature, suggest that the regular across-species pattern for the trio {P/B,Q/B,P/Q} can be explained by basic principles that underpin life-history in fish populations. This quantitative framework for the trio {P/B,Q/B,P/Q} is based on an explicit dependence with body size, which simplifies the estimation of these quantities. Model complexity depends, in part, on which data are available. Models were applied to real data from commercially important species fished in Chile. Statistical properties of the new models were evaluated by an intensive resampling approach. The simplest possible model for the trio {P/B,Q/B,P/Q} rests on the assumption of a stable age distribution. These quantities have a key importance in ecosystem modelling because they determine population energetics in terms of food intake by predation and the transformation of this energy into population biomass of predators. Application of the new models produces results which were comparable to those given by standard methods. This thesis is a result of multidisciplinary research which attempts to make a contribution to the understanding of the mechanisms underlying the allometric scaling of food consumption and production in fish populations. It proposes models for the trio {P/B,Q/B,P/Q} and thus, has the potential to be widely applicable in fisheries science.
  
  
  
• The biology of South African Bryde's whales
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  Abstract: The biology of South African Bryde’s whales (Balaenoptera brydei/edeni), with a focus on the inshore form, was investigated through estimates of abundance and survival rate, seasonality of occurrence and variation in mitochondrial and nuclear DNA. Photographs, sightings data and biopsy samples were collected in Plettenberg Bay, on the south-east coast of South Africa. Additional genetic material was obtained from the Iziko South African Museum, Marine and Coastal Management, and the Port Elizabeth Museum. Mark-recapture methods applied to photo-identification data were used to estimate abundance and survival rate. Estimates of abundance ranged from 130 to 250 (CV = 0.07 - 0.38) and the estimated annual survival rate was 0.93 (CV = 0.047, 95% CI = 0.852 - 1.0). Seasonal increases in the encounter rate and number of individual whales were observed during summer and autumn, with a peak in April, which corresponded to increased feeding activity and larger average aggregation sizes. Chlorophyll-a, sea surface temperature and wind speed were all significant factors in explaining the variability in the occurrence of whales. No seasonality in the occurrence of calves was detected. Mitochondrial DNA control region sequences (685bp) were compared to published sequences. This confirmed the offshore form as Balaenoptera brydei and the inshore form as closely related to B.brydei, possibly at the sub-specific level, but excluded it as B.edeni. Phylogenetic analyses support complete separation between the two forms. The use of 10 polymorphic microsatellite loci revealed no population structure among the inshore samples (FST = 0.006). Pairwise estimates of relatedness found most individuals to be unrelated, with only a few distant relatives detected.
  
  
  
• Cellular and molecular studies of postembryonic muscle fibre recruitment in zebrafish (Danio rerio L.)
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  Abstract: Cellular and molecular mechanisms of postembryonic muscle fibre recruitment were investigated in zebrafish (Danio rerio L.), a standard animal model for developmental and genetic studies. Distinct cellular mechanisms of postembryonic muscle fibre recruitment in fast and slow myotomal muscles were found. In slow muscle, three overlapping waves of stratified hyperplasia (SH) from distinct germinal zones sequentially contributed to a slow and steady increase in fibre number (FN) through the life span. In fast muscle, SH only contributed to an initial increase of FN in early larvae. Strikingly, mosaic hyperplasia (MH) appeared in late larvae and early juveniles and remained active until early adult stages, accounting for >70% of the final fibre number (FFN). The molecular regulation of postembryonic muscle fibre recruitment was then studied by characterising myospryn and cee, two strong candidate genes previously identified from a large scale screen for genes differentially expressed during the transition from hyperplastic to hypertrophic muscle phenotypes. Zebrafish myospryn contained very similar functional domains to its mammalian orthologues, which function to bind to other proteins known to regulate muscle dystrophy. Zebrafish myospryn also shared a highly conserved syntenic genomic neighbourhood with other vertebrate orthologues. As in mammals, zebrafish myospryn were specifically expressed in striated muscles. Zebrafish cee was a single-copy gene, highly conserved among metazoans, ubiquitously expressed across tissues, and did not form part of any wider gene family. Its protein encompassed a single conserved domain (DUF410) of unknown function although knock-down of cee in C. elegans and yeast have suggested a role in regulating growth patterns. Both myospyrn and cee transcripts were up-regulated concomitant with the cessation of postembryonic muscle fibre recruitment in zebrafish, indicating a potential role in regulating muscle growth. Furthermore, a genome-wide screen of genes involved in the regulation of postembryonic muscle fibre recruitment was performed using microarray. 85 genes were found to be consistently and differentially expressed between growth stages where muscle hyperplasia was active or inactive, including genes associated with muscle contraction, metabolism, and immunity. Further bioinformatic annotation indicated these genes comprised a complex transcriptional network with molecular functions, including catalytic activity and protein binding as well as pathways associated with metabolism, tight junctions, and human diseases. Finally, developmental plasticity of postembryonic muscle fibre recruitment to embryonic temperature was characterised. It involved transient effects including the relative timing and contribution of SH and MH, plus the rate and duration of fibre production, as well as a persistent alteration to FFN. Further investigation of FFN of fish over a broader range of embryonic temperature treatments (22, 26, 28, 31, 35°C) indicated that 26°C produced the highest FFN that was approximately 17% greater than at other temperatures. This finding implies the existence of an optimal embryonic temperature range for maximising FFN across a reaction norm. Additionally, a small but significant effect of parental temperature on FFN (up to 6% greater at 24 and 26°C than at 31°C) was evident, suggesting some parental mechanisms can affect muscle fibre recruitment patterns of progeny. This work provides a comprehensive investigation of mechanisms underlying postembryonic muscle fibre recruitment and demonstrates the power of zebrafish as an ideal teleost model for addressing mechanistic and practical aspects of postembryonic muscle recruitment, especially the presence of all major phases of muscle fibre production in larger commercially important teleost species.
  
  
  
• The trade-off between starvation and predation risk in overwintering redshanks (Tringa totanus)
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  Abstract: In order to meet their energy budget animals must often increase their risk of predation, either through their choice of foraging location or by decreasing anti-predation behaviours, which are incompatible with foraging. I investigated the starvation-predation risk trade-off in redshanks overwintering in the area of the Firth of Forth in Scotland over different spatial scales. On a small spatial scale, where redshanks foraged in an area where risk of attack was high I investigated the role of competition for food and decreased individual vigilance within groups and how this related to predation risk, additionally I looked at the relative roles of individual variation in time spent exposed to risk and variation in anti-predation behaviours on individual survival time. On larger spatial scales of 100s of meters and over several kilometres, I considered how choice of overwintering site was affected by predation risk, profitability and population density. Time available to feed increased with increased group size, allowing redshanks to compensate for increased competition and allowing large groups to form, thus decreasing individual predation risk. Individuals that spent less time exposed to attacking predators survived for longer, however individuals constrained by cold weather to spend long periods exposed to risk could increase their survival through increased intake rates and vigilance. On an intermediate spatial scale redshanks selected overwintering sites based on profitability rather than risk, and only used less profitable site when population density was high. On a large spatial scale redshanks increased their use of less profitable sites in warmer weather, but did this without increasing their risk of predation. Overall this suggests that across most spatial scales redshanks can minimise their predation risk by their choice of foraging location, but when forced by weather conditions or competition to be exposed to attack, capture reducing behaviours also reduce predation risk
  
  
  
• Two-pore channels and NAADP-dependent calcium signalling
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  Abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺ mobilising messenger in mammalian and non-mammalian cells. Studies on a variety of cell types suggest that NAADP evokes Ca²⁺ release from a lysosome-related store and via activation of a receptor distinct from either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R). However, the identity of the NAADP receptor has, until now, remained elusive. In this thesis I have shown that NAADP-evoked Ca²⁺ release from lysosomes is underpinned by two-pore channels (TPCs), of which there are 3 subtypes, TPC1, TPC2 and TPC3. When stably over-expressed in HEK293 cells, TPC2 was found to be specifically targeted to lysosomes, while TPC1 and TPC3 were targeted to endosomes. Initial Ca²⁺ signals via TPC2, but not those via TPC1, were amplified into global Ca²⁺ waves by Ca²⁺-induced Ca²⁺ release (CICR) from the endoplasmic reticulum (ER) via IP₃Rs. I have shown that, consistent with a role for TPCs in NAADP-mediated Ca²⁺ release, TPC2 is expressed in pulmonary arterial smooth muscle cells (PASMCs), is likely targeted to lysosomal membranes, and that TPCs also underpin NAADP-evoked Ca²⁺ signalling in this cell type. However, and in contrast to HEK293 cells, in PASMCs NAADP evokes spatially restricted Ca²⁺ bursts that are amplified into global Ca²⁺ waves by CICR from the sarcoplasmic reticulum (SR) via a subpopulation of RyRs, but not via IP₃Rs. I have demonstrated that lysosomes preferentially co-localise with RyR subtype 3 (RyR3) in the perinuclear region of PASMCs to comprise a “trigger zone” for Ca²⁺ signalling by NAADP, away from which a propagating Ca²⁺ wave may be carried by subsequent recruitment of RyR2. The identification of TPCs as a family of NAADP receptors may further our understanding of the mechanisms that confer the versatility of Ca²⁺ signalling which is required to regulate such diverse cellular functions as gene expression, fertilization, cell growth, and ultimately cell death.
  
  
  
• Wild at heart? : differential maternal investment in wild and domesticated zebra finches (Taeniopygia guttata)
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  Abstract: Over the past twenty years there has been an exponential increase in the investigation of maternal effects. Understanding the adaptive function of maternal allocation strategies is integral to interpreting the evolutionary outcomes of sexual selection. Thus, model animal systems that facilitate experimental manipulation and controlled investigation of the physiological and behavioural mechanisms underlying maternal effects are important to evolutionary biologists. The zebra finch (Taeniopygia guttata) has been used as a model to investigate avian life-history, signalling behaviour, neurophysiology, mate choice, and more recently, maternal effects. However, a potentially influential and rarely addressed problem with this species is the process of domestication. Within this thesis we aimed to both test current predominant maternal allocation hypotheses, but for the first time in both domesticated and wild zebra finches. Chapter 2 develops on earlier work using domesticated zebra finches that has demonstrated differential allocation of maternally derived yolk androgens and antioxidants in eggs dependent on paternal attractiveness. This chapter specifically tests the ratio of these two yolk resources within individual eggs and shows that the balance of androgens to antioxidants varies by offspring sex and paternal attractiveness. Specifically, we found that mothers allocated a smaller androgen to antioxidant ratio to daughters when paired to green ringed (unattractive) males compared to red ringed (attractive) males. This pattern was reversed for sons, where mothers allocated a larger ratio of androgen to antioxidant when paired to red ringed (attractive) compared to green ringed (unattractive) males. We also show that brood sex ratio depended on both female condition and male attractiveness. It is concluded that investigating female allocation of individual resources within egg yolks may lead to incorrect assumptions on offspring fitness consequences, and that individual female state is an important consideration when predicting a resource allocation strategy. Throughout this thesis colour bands are used as a method to manipulate male attractiveness. In chapter 3 the influence of these bands was further tested to elucidate whether they affect male behaviour or quality. Wild birds were used for this chapter as preferences for bands based on colour have only once been demonstrated in wild birds and it was felt this should also be replicated. We confirmed a female preference for males based on colour bands worn in mate choice trials, with red bands preferred over green. Interestingly, we also found that colour of bands worn by males for an extended period in the single sex aviary influenced both their song rate and condition. Males that had worn red bands sang more in mate choice trials than both green banded or un-banded males. In addition red banded males were found to be in significantly better physical condition. These data suggest that earlier experiments in which it has been assumed that colour bands do not manipulate any form of intrinsic male quality should be re-evaluated. The final two data chapters, 4 and 5, return to investigating maternal allocation in response to male attractiveness, but for the first time in wild birds. Chapter 4 presents an experiment that was conducted on a wild, nest box breeding population of birds. Maternal resources allocation was investigated in both an experimental manipulation of male attractiveness, and also by correlating resource allocation with paternal phenotypic traits. A limited sample size meant few conclusions could be drawn from the experimental study, but significant positive correlations were found between both egg size and yolk testosterone (T) concentration and male phenotypic traits. This suggested that wild zebra finches may follow a positive investment strategy but requires further investigation. In chapter 5 experiments were repeated on wild birds that had been brought into captivity, to allow both an improved sample size and further control of influential environmental features. Again, female allocation strategies are tested using colour bands to manipulate male attractiveness, to allow direct comparisons with work on domesticated zebra finches. We found that females laid significantly heavier eggs for attractive compared to unattractive males, supporting the positive investment hypothesis. In addition we found an interaction between offspring size and paternal attractiveness treatment, with daughters of red banded (attractive) males being smaller than sons. This experiment is the first to demonstrate the influence of colour bands on maternal allocation in wild zebra finches and also provides further support for the positive investment hypothesis in this species. The final chapter discusses how overall patterns of female allocation were shown to be similar among wild and domesticated populations. It is concluded that demonstrated variations between populations and/or contexts reported in these studies cannot be explained by inherent differences between wild and domesticated individuals. Thus, the zebra finch remains a robust and reliable model for testing the evolution of avian maternal allocation strategies.
  
  
  
• Environmental constraints on the foraging behaviour, spatial usage and population sizes of albatrosses
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  Abstract: Satellite-tracking of wide-ranging, apex marine predators, combined with remote-sensing, can be used to test ecological hypotheses and to estimate spatial abundance. I used this approach to quantify the habitat usage of central place foraging black-browed albatrosses (BBA) from nine colonies, modelling population-level distribution as a function of habitat accessibility, habitat preference and conspecific competition. Throughout breeding, BBA preferred neritic waters, steeper bathymetry, and, during incubation, warmer sea surface temperatures. BBA from South Georgia also preferred highly dynamic oceanic waters. Foraging areas were partially spatially segregated with respect to colony and region, presumably to reduce intraspecific competition. Although such competition is often invoked to explain observed colony sizes, by accounting for travel costs, I demonstrate a strong relationship between the sizes of regional populations and the availability, accessibility and productivity of neritic waters, supporting the hypothesis that seabird populations are constrained by breeding season food availability. In response to this constraint, albatrosses have evolved to exploit energetically efficient gliding flight, allowing them to access prey 100-1000s of km from their colonies. Hence, I used satellite tracking and activity data to quantify the effects of relative wind speed on the flight speed of four albatross species. Groundspeed was linearly related to the wind speed in the direction of flight, its effect being greatest on wandering albatrosses, followed by BBA, light-mantled and grey- headed albatrosses, and airspeeds were higher in males than females. Commuting birds tended to encounter headwinds during outward trips and tailwinds on their return, such that return trips were faster. This supports the hypothesis that foraging upwind of the colony is more efficient but could also result from wind climate and the relative location of prey. The ability to use tracking data to estimate spatial usage is timely given the acute threat currently posed to albatrosses by incidental fisheries mortality.
  
  
  
• Measuring and understanding biogenic influences upon cohesive sediment stability in intertidal systems
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  Abstract: Intertidal cohesive sediment systems are found throughout the world in areas of low hydrodynamic energy. These systems are ecologically and economically important but are under pressure from global warming, sea level rise and other anthropogenic influences. To protect and conserve these systems it is important to understand the sediment dynamics, especially the erosional properties of the sediment. The study of sediment erosion and transport is complex, encompassing biological, chemical and physical properties of the ecosystem. This thesis contributes towards this area of research, firstly in regard to the methods used to measure sediment erosion on exposed and submerged sediments and secondly with respect to assessing influences upon sediment stability through changes in the ecosystem, comprising of both the sediment environment and the macrofaunal community. Chapter 3: In partnership with Sediment Service a thorough re-evaluation of the Cohesive Strength Meter (CSM), a commercially available device used to measure surface sediment strength, was performed. New components, deployment method and calibration protocol were devised and tested. The new design was not effective, but the deployment and calibration have improved the ease of use and interpretation CSM data. Chapter 4: The study of intertidal sediment stability was conducted during the submerged period of the tidal cycle. Protocols and methods were devised or modified to sample submerged sediments with the aim to determine how sediment properties are affected by submersion and the resulting effect on sediment stability. Sediment stability increased with submersion. The existence of a fine layer of sediment on the surface, similar to the fluff layer found in submerged sediments, is given as a suggested explanation as it may be removed by the incoming tide. However, no other changes in sediment properties were detected. This may be due to flaws in the methods used in detecting fine scale changes in the sediment surface. In situ and laboratory experiments revealed contrasting effects of submersion on sediment stability with disturbance from the sampling and movement of sediment from the field to the laboratory given as an explanation for this. Chapter 5: The influence of the ecosystem engineering polychaete Arenicola marina on sediment properties was examined with an exclusion experiment. A. marina was excluded from five 20m² plots on an intertidal mudflat on the German island of Sylt. A holistic approach was used to measure the ecosystem, including a range of biotic and abiotic sediment properties as well as the macrofauna community. It was hypothesised that A. marina’s exclusion would alter the macrofaunal community and increase sediment stability. However, there was no consistent change in the macrofauna community or sediment environment with the exclusion of A. marina and subsequently no change in sediment stability. Chapter 6: The impact of bait digging for A. marina was examined with six 5m² plots dug up and A. marina removed, the plots were then monitored over a three month period. Bait digging disturbance was expected to have an impact upon the sediment environment and macrofauna community, resulting in a reduction in both sediment stability and microphytobenthic abundance. However, bait digging had minimal impact on the macrofauna community and caused no change in the sediment environment, despite the removal of a large proportion of the A. marina population. No change was recorded in the sediment stability or biomass of the microphytobenthos, indicating that with the exception of removing A. marina, bait digging of this nature was not detrimental to the sediment ecosystem. However, the consequences of larger, longer term digging operations can not be determined from this work and further studies are suggested. The study of intertidal sediment stability was progressed with advances made in methods and protocols. The work highlighted the importance of studying sediment stability as an ecosystem function through a holistic ecosystem approach rather than isolating individual variables.
  
  
  
• Causal pattern inference from neural spike train data
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  Abstract: Electrophysiological recordings are a valuable tool for neuroscience in order to monitor the activity of multiple or even single neurons. Significant insights into the nervous system have been gained by analyses of resulting data; in particular, many findings were gained from spike trains whose correlations can give valuable indications about neural interplay. But detecting, specifying, and representing neural interactions is mathematically challenging. Further, recent advances of recording techniques led to an increase in volume of collected data, which often poses additional computational problems. These developments call for new, improved methods in order to extract crucial information. The matter of this thesis is twofold: It presents a novel method for the analysis of neural spike train data, as well as a generic framework in order to assess the new and related techniques. The new computational method, the Snap Shot Score, can be used to inspect spike trains with respect to temporal dependencies, which are visualised as an information flow network. These networks can specify the relationships in the data, indicate changes in dependencies, and point to causal interactions. The Snap Shot Score is demonstrated to reveal plausible networks both in a variety of simulations and for real data, which indicate its value for understanding neural dynamics. Additional to the Snap Shot Score, a neural simulation framework is suggested, which facilitates the assessment of neural network inference techniques in a highly automated fashion. Due to a new formal concept to rate learned networks, the framework can be used to test techniques under partial observability conditions. In the presence of hidden units quantification of results has been a tedious task that had to be done by hand, but which can now be automated. Thereby high throughput assessments become possible, which facilitate a comprehensive simulation-based characterisation of new methods.
  
  
  
• Changes in gene expression, lipid class and fatty acid composition associated with diapause in the marine copepod Calanus finmarchicus from Loch Etive, Scotland.
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  Abstract: Zooplankton are the major primary consumers in pelagic ecosystems, providing the principal pathway for energy transfer from primary production to higher trophic levels. The marine copepod Calanus finmarchicus is an important component of the pelagic food web in the North Atlantic and peripheral ecosystems, and forms an essential dietary component of a number of commercially important fish. As part of its life cycle, many C. finmarchicus overwinter in a diapause phase (a dormant overwintering phase where development is suppressed in adaptation to the seasonal food supply) at depths of 500 to 2000 m, but little is known about the triggers that initiate and terminate diapause, or the internal processes associated with these triggers. Understanding these processes is important, given that subtle changes in the environmental conditions which may affect diapause could have consequences for the entire Calanus-based ecosystem. In this study I took advantage of relatively easy access to a deep (> 100 m), isolated population of C. finmarchicus in Loch Etive (a sea loch on the west coast of Scotland) to sample Calanus finmarchicus monthly between April 2006 and June 2007 and measure lipid dynamics and gene expression associated with diapause. Chapter 1 of this thesis provides a general introduction to diapause and Calanus finmarchicus, Chapter 2 reports on the population of C. finmarchicus in Loch Etive, Chapter 3 reports changes in the lipid class and fatty acid composition of individual copepods, Chapter 4 reports on differential gene expression between diapausing and active C. finmarchicus and Chapter 5 provides a general discussion and puts this research into context. This study provides some initial insight into possible gene expression patterns, but further work is needed to attribute specific gene expression patterns with initiation and termination of diapause.
  
  
  
• Variation in sperm whale (Physeter macrocephalus) coda vocalizations and social structure in the North Atlantic Ocean
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  Abstract: This study aimed at complementing studies of sperm whale social and vocal behaviour that were restricted to the Pacific Ocean. The characteristic multi-pulsed structure of sperm whale clicks allows for estimation of whales' size from measurements of the inter-pulse intervals (IPI). I have developed two new automatic methods for IPI estimation from clicks recorded during foraging dives. When compared to other previously developed methods, the newly developed method that averages several clicks' autocorrelation function showed the best performance amongst the automatic methods. Previous studies did not support individual identity advertisement among social unit members as the function for the sperm whale communication signals called codas. I tested within coda type variation for individual specific patterns and found that, while some coda types do not allow for individual discrimination, one did so. This variation suggests that different coda types may have distinct functions. Analysis of social structure in the Azores found that, similar to the Eastern Tropical Pacific, sperm whales form long term social units of about 12 individuals. Unlike the Pacific Ocean, Azorean social units do not form temporary groups with other units, suggesting differences in the costs and benefits of group formation. I argue that these are due to differences in terms of predation pressure and intraspecific competition between the Azores and the Pacific study sites. The variation of coda repertoires in the Atlantic also showed a pattern dissimilar to that previously documented in the Eastern Tropical Pacific. In the North Atlantic, coda repertoire variation is mostly geographic, which is parsimoniously explained by random drift of culturally transmitted coda repertoires. No sympatric vocal clans with distinct dialects were found as has been noted in the Pacific. Drawing upon the differences found in social structure I argue that selection for maximization of differences between units with similar foraging strategies may have led to the Pacific vocal clans. The differences between oceans suggest that sperm whales may adaptively adjust their behaviour according to experienced ecological conditions.
  
  
  
• Viral interferon antagonists and antiviral drugs
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  Abstract: For this project, we developed reporter cell lines that express viral proteins with the potential to be used in cell-based screening assays to select chemical candidates for antiviral drugs. The viral proteins expressed in these reporter cell lines (Hepatitis B core and precore, Hepatitis C core (1a and 4a), and Rabies P (BH and SADL16)) were presented in the literature as responsible for interfering with the IFN signaling pathway, specifically for blocking the expression of its key protein STAT1. We cloned the viral genes into the pdl’SurvpkIB reporter plasmid and, through a lentiviral delivery system, infected the Hep2Mx1TIPSE cells and the A549Luc cells resulting in the Hep2Mx1TIPSEHBVprecore, Hep2Mx1TIPSEHBVcore, Hep2Mx1TIPSEHCVcore (1a and 4a), and A549lucRabiesP (SADL16 and BH) reporter cell lines. We assessed the obtained viral cell lines according to their ability to block the IFN signaling pathway by using three different assays: an immunoblot targeting the protein STAT1, a phenotypic assay for survival in the presence of puromycin (in viral Hep2Mx1TIPSE cells), and a quantitative measure of luciferase expression (in viral A549Luc cells). Concerning the immunoblot targeting STAT1, the results showed that only cell lines expressing the Rabies P protein (namely the A549lucRabiesPSADL16 cell line) were able to decrease the level of expression of STAT1. The phenotypic assay conducted on the Hep2Mx1TIPSE viral cell lines were intended to show impairment of the IFN signaling pathway through the down-regulation of the IFN stimulated gene Mx1. Normal Hep2Mx1TIPSE cells contain a puromycin resistance gene controlled by the Mx1 promoter. Therefore, when puromycin is added to these cells in the presence of IFN, the signaling pathway is activated and Mx1 as well as the puromycin resistance gene are expressed resulting in cell survival. Results showed that the cell lines expressing the HCV core and HBV precore proteins also survived puromycin addition. However, the Hep2Mx1TIPSEHBVcore cells died in the presence of puromycin suggesting that in these cells the HBVcore protein affects Mx1 protein expression. Since it was expected that all viral cell lines would be able to down- regulate Mx1 by impairing the IFN signaling pathway, it was assumed that the level of viral expression may not have been enough to be detected by this kind of assay and therefore a quantitative study would be crucial for the continuation of this project. The cell lines expressing the Rabies P protein demonstrated their ability to block STAT1 and contained a luciferase gene under the control of an IFN regulated promoter. These cells were therefore considered the best candidates for the quantitative assay. We compared the difference between luciferase expression in the viral cells in the presence and absence of IFN with A549Luc cells (control cells) and verified that in both cases there was an increase in the amount of luciferase expression upon the addition of IFN, which is concordant with the up-regulation of the IFN signaling pathway. However, this increase was considerably less in cells expressing the viral protein. This result confirms a partial blockage of the IFN signaling pathway in these cells. This experiment demonstrates a new alternative step in the creation of cell lines that express the Rabies P protein and that can be applied to the manufacturing process of antiviral drugs. However, in order to achieve the successful production of cell lines, it would be essential to improve viral protein expression.
  
  
  
• Structural studies on the sialidases from Streptococcus pneumoniae and Pseudomonas aeruginosa
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  Abstract: The sialidases are a group of glycosyl hydrolases that specifically remove terminal sialic acid (Neu5Ac) residues from various glycans. In the two common human pathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, these enzymes have been shown to be key virulence factors directly involved in bacterial colonization and infection. However, little is known about their detailed structural and mechanistic features and lack of this information significantly slows down the progress of new drug discovery targeting these enzymes. Therefore, we embarked structural and kinetic studies towards the three distinct sialidases (designated as NanA, NanB and NanC) from S. pneumoniae, as well as the putative sialidase (designated as PaNA) from P. aeruginosa. Full-length NanA failed to crystallize due to the presence of some natively disordered regions. The catalytic domain of NanA (CNanA) was therefore subcloned, which was crystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer with close contacts between the two monomers. The second pneumococcal sialidase NanB only shares 24% sequence identity with NanA. Crystal structure of NanB was also determined to 1.7 Å, which exhibits a multi-domain monomeric architecture. In general, the core catalytic domain of both CNanA and NanB adopts the classic six- bladed β-propeller fold (or called sialidase fold), with a set of highly conserved residues stacking around the proposed active sites. NanC is a close homologue of NanB, sharing over 50% sequence identity. However, NanC crystallization is not successful so far. To compare the three sialidases in more detail, a computational NanC model was made based on the structure of NanB. Mapping of the active sites of CNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as the probe. Although sharing many common features, NanA, NanB and NanC present different topologies around the catalytic centre, give these enzymes a high level of diversity in enzymatic kinetics, substrate specificity and catalytic properties. NMR studies show that NanA acts as a classic hydrolytic sialidase; while NanB is found to be an intermolecular trans-sialidase like the leech sialidase; NanC, however, handles multiple catalytic roles efficiently, which include releasing Neu5Ac2en from α2,3- sialyllactose and hydration of Neu5Ac2en to Neu5Ac with high efficiency. S. pneumoniae thus expresses NanA, NanB and NanC for disparate but cooperative roles. Such a working pattern of three sialidases in one microbe is unusual in nature, which might be essential for pneumococcal pathogenesis at various stages. Based on the crystal structures of CNanA and NanB, preliminary work towards S. pneumoniae sialidases inhibitor design is under way, in which, a variety of techniques, such as the fluorescence-based thermal shift assay, NMR spectroscopy, computational docking and X-ray crystallography, are incorporated in. The crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be a unique trimer in crystal that is associated, in part, by the immunoglobulin-like trimerization domain around a three-fold crystallographic axis. The core catalytic domain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidase activity was detected with this enzyme. In addition, two key catalytic residues including one of the arginine in the arginine triad and the acid/base catalyst aspartic acid are missing in PaNA. In silico docking suggests that Phe129 may confer substrate selectivity towards pseudaminic acid, which is a specific carbohydrate superficially similar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directed mutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNA into a poor sialidases. Moreover, the crystal structure of PaNA also indicates that His45, Tyr21 and Glu315 may form a charge relay to compensate the missing aspartic acid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315 to be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNA could be a pseudaminidase with structural and mechanistic variations. This enzyme, together some other uncharacterized fellow proteins, might form a novel subclass in the sialidase superfamily. The various findings in the current projects provide meaningful insights towards several sialidases that have been linked to bacterial virulence, which may contribute to a more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.
  
  
  
• RNA virus modulation of IFN, PI3K and apoptosis
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  Abstract: Interferon (IFN) and phosphatidylinositol 3-kinase (PI3K) are apoptosis regulators that are targeted by viruses to promote survival of infected cells. Significant crosstalk exists between IFN and PI3K, and this study sought to investigate the relationships between IFN, PI3K and apoptosis during virus infection. Parainfluenza virus 5 (PIV5) and influenza A virus (IAV) are both negative-sense single-stranded RNA viruses that encode multifunctional proteins in order to maximise their genome coding capacity. The PIV5 V and IAV NS1 proteins are well-studied as IFN antagonists and, in addition, both are reported to modulate PI3K signalling. Less well-studied is the role of these proteins in apoptosis regulation; the ability of V and NS1 to inhibit apoptosis was therefore investigated. PIV5/V was found to limit cell death in response to a number of apoptosis inducers in a manner that required its STAT1- degradative activity and also inhibited activation of the PI3K downstream target, Akt. IAV/NS1 binds directly to PI3K to stimulate its activity, and this is reported to mediate anti-apoptotic signalling during IAV infection. However, a virus expressing an NS1 unable to bind PI3K did not induce more apoptosis than wt virus. NS1 expression, either in a stable cell-line or during virus infection, was also unable to protect cells from pro-apoptotic stimuli. NS1-mediated PI3K activation similarly had no effect on IFN production or ISG expression in infected cells. In contrast, other NS1 mutant viruses induced large amounts of apoptosis. These viruses also induced significant levels of IFN and were unable to cause apoptosis in IFN-deficient cells, indicating that NS1 limits apoptosis induction through its IFN antagonist functions. The implications of this work for anti-cancer and anti-viral therapies are discussed.
  
  
  
• Female mating decisions in the Trinidadian guppy, Poecilia reticulata
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  Abstract: Contrary to traditional belief, the decisions that females make before, during and after mating shape its outcome and ultimately fitness. The aim of this thesis was to examine how females modify and adjust their mating decisions in line with social and environmental variability and how these directly and indirectly affect mating benefits. To address this aim I have formulated four main questions that correspond to chapters 2 to 6 in this thesis. Firstly I asked whether there was evidence for female choice being driven by mating benefits. More precisely, in chapter 2, I reviewed the literature in search of evidence for direct and indirect benefits in female choice among freshwater fish species. Direct mating benefits were defined as an increase in female’s reproductive success (number of offspring). Conversely, increases in offspring reproductive success were considered to be indirect benefits. The results showed that despite the multiple suggestions and the great amount of information available, to date there is still no evidence for both direct (increase of F1) or indirect (increase in F2) mating benefits, nor their influence in female mating decisions if freshwater fishes species. Furthermore, although polyandry occurred in more than 60% of the species reviewed, I was unable to confirm that polyandry was maintained because of indirect benefits. These findings justified the need to experimentally investigate the drivers of female mating decisions in freshwater fish species and lead to the questions addressed on chapters 5 and 6. For the experimental chapters 3-6 I used the Trinidadian guppy, Poecilia reticulata, as a model species. Their unique and remarkable ecological and biological characteristics coupled with easy maintenance in laboratory conditions make guppies an ideal species for behavioural studies. But in particular, the fact that guppies live in a promiscuous mating system where females despite being constantly harassed by males may be able to control paternity, makes guppies the ideal species to address my aims. The second question was how much were females in control of their mating decisions, and how social environment could limit these decisions. In particular, in chapter 3 I looked at how females adapt and adjust their mating decisions in line with extreme differences in population sex ratio. In chapter 4, I examined to what extent male sexual harassment affects female reproductive behaviours. Results from these two chapters indicated that female guppies have a remarkable reproductive plasticity that enable them to control their mating decisions. When faced with extreme differences in population sex ratio, female mating decisions were made in an optimal way that maximized the ratio of female reproductive benefits per investment. This translated into producing bigger offspring when in a strong female biased environment, than when in a male biased environment. Further, chapter 4 illustrated that female guppies can, despite high levels of male sexual harassment, be in control of their mating decisions. These two chapters demonstrated and have reinforced previous findings of the remarkable reproductive adaptation of female guppies to differences in the social environment. The third question I addressed was: do multiply mated females have greater direct or indirect benefits than single mated females? To answer this question I followed for the first time reproductive success of females over two generations. I measured fitness directly (number of F1 and F2) and took as well as multiple indirect measures of fitness components for two generations in search for evidence of direct and indirect mating benefits in explaining the maintenance of female multiple mating. The results of chapter 5 revealed that female guppies do not have a higher number of F1 and F2 from polyandry or either from mating with males possessing allegedly good quality traits. I, therefore, stressed the idea that potential differences in sexual selection pressure between laboratory and wild populations may influence the expression and intensity of mating benefits between thus explaining the difficulty of finding mating benefits. In my fourth and last question, I used a novel statistical approach based on the analysis of the dispersion in phenotypes, to look for potential alternative explanations for the prevalence of polyandry. The results of this analysis show offspring from multiple mated mothers were phenotypically more diverse than offspring from single mated mothers. Given the direct relationship between phenotypic diversity and potential fitness gains in stochastic systems, female guppies are likely to get greater benefits from mating with males with different phenotypes than with males with a particular sexual trait. This result provides an alternative explanation for the maintenance of polyandry in resource free systems. Overall the results of this thesis reinforce previous suggestions that female guppies are active participants in the mating process, and not necessarily limited to post-copulatory mechanisms of selection of sperm. It also showed the remarkable ability of females to adjust their reproductive investment in line with changes in the social conditions. Interestingly, my results contradict the commonly accepted assertion that females’ mating preference converges towards unique male sexual traits. This result stresses the need to look at alternative explanations to justify female mating decisions.
  
  
  
• Structure and function of nitrate and nitrite transporters, NrtA and NitA, from Aspergillus nidulans
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  Abstract: Membrane proteins play an integral role in the control of ion transport across the cell membrane in biological systems. However, due to experimental constraints, structural and functional data available for these proteins is limited, especially considering their importance. In this study, two membrane proteins which transport nitrate and nitrate into the model filamentous ascomycete Aspergillus nidulans were investigated. Work on the twelve trans-membrane domain nitrate transport protein NrtA is well established. As a member of the major facilitator super family (MFS) the role of signature sequences characteristic of this family have previously been studied. Here, a series of point mutations were made to facilitate an understanding of key residues in the nitrate binding domain, the first nitrate signature motif and residues of the unique fungal central-loop domain. Using an expanded alignment package, the proposed secondary structure of NrtA was enhanced and used as a starting point for mutagenesis. Alanine scanning mutagenesis showed that glycine residues in the conserved nitrate nitrite porter (NNP) motif were critical for NrtA function. Two asparagines in the NNP were investigated; N160 and N168. N168 was found to be critical for NrtA function as all mutants were devoid of growth on nitrate solid agar medium though they expressed in the membrane to varying degrees. The nitrate binding site has been studied previously, revealing the interaction of conserved arginine residues with the anion as it traverses the bilayer. Though it was thought that mutations of residue T83 to a small, charge neutral, amino acid would substitute for no alteration to enzyme kinetics in mutant T83S was found when using ¹³NO₃⁻. Another major part of this thesis examined NitA which is part of a distinct nitrite transport family to NrtA (the Formate Nitrite Transporters, FNT). A mutagenesis approach targeted NitA residues conserved amongst homologous proteins. Residues in position D88 in an alignment of homologues were conserved in terms of charge. Mutagenesis of D88 revealed that maintaining charge at this position was essential for NitA function, likely due to a role in salt-bridge formation during conformational changes. Mutations to asparagine, glutamine, serine and valine showed reduced growth on agar though the protein was expressed to approximately wild-type levels. Nitrite uptake assays using a ¹³NO₂⁻ tracer were performed on D88N, D88E and D88Q and all showed wild-type Km and Vmax. Finally, the role of conserved asparagine residues found throughout NitA was investigated by mutagenesis. Expression studies revealed that mutants created in N122 and N246, changed to aspartic acid, lysine, glutamine and serine were generally not present in the membrane and thus did not grow on nitrite agar. However, mutations in N173 (in Tm 4) and N214 (in Tm 5), which are conserved in > 95 % of NitA homologues, showed varying degrees of growth and expression. Both of these residues are located in FNT signature motifs, so it is likely that they are involved with conformational changes or protein dynamics.
  
  
  
• Investigation of the transcriptional response of Sulfolobus solfataricus to damaging agents
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  Abstract: It is vital for the survival of an organism that it can repair damage to its DNA. Exogenous and endogenous sources of damage are dealt with by a variety of repair pathways that have evolved to repair specific types of damage. Organisms in the archaeal domain, the third domain of life, contain homologues of many of the eukaryotic repair proteins, however little is known about how damage is detected in the archaeal domain. Microarray studies in the archaeal species Sulfolobus solfataricus determined a number of genes whose expression was effected by UV radiation (work by Dr D Götz). The change in expression of nine of these genes was confirmed by RT real time PCR. The expression of these genes was then investigated after exposure to different damaging agents, Mitomycin C, Methyl methane sulfonate, Phleomycin and Hydrogen peroxide. The expression of two genes, transcription factor tfb-3 and cell division control gene cdc6-2, was up regulated in all damage conditions. There was a huge induction of the dps-like gene (sso2079) after hydrogen peroxide damage. Transcription from this genes promoter was shown to be strong in vitro (work by Dr S Paytubi) suggesting a repressor was controlling the gene in vivo. A palindromic repeat in the promoter of the dps-like gene was used to ‘fish’ for a transcriptional repressor and the Sso2273 protein, a homologue of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheria, was identified as a possible repressor. Sso2273 was expressed and purified, and its crystal structure solved, its paralogue, Sso0669, was also expressed and purified. Electrophoretic mobility shift assays showed that the Sso2273 protein does not bind DNA, and had no effect on transcription from any promoter used in in vitro transcription assays. However Sso0669 appeared to inhibit transcription, although the inhibition was not sequence specific. A knockout strain of S. solfataricus PBL2025 missing the sso2273 gene was produced and used in microarray experiments in an attempt to determine the role of Sso2273 within the cell. The absence of Sso2273 appeared to have no effect on the expression of the dps-like gene, however strong repression of an operon containing genes involved in Sulphur assimilation was observed.
  
  
  
• Determination of the molecular and physiological basis of citric acid tolerance in spoilage yeast
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  Abstract: The ability of yeasts to grow and adapt under extreme environmental conditions including within the presence of weak organic acid preservatives has led to substantial economic losses through manufactured food and beverage spoilage. The food industry has employed the use of various weak organic acids such as sorbic, benzoic and acetic acid as preservatives to help prevent spoilage by yeasts and moulds. The mechanisms by which S. cerevisiae is able to adapt to these weak organic acids have been extensively studied. A lesser studied weak organic acid preservative is citric acid. The aim of this study was to gain further information on the mechanisms of citric acid adaptation and through this identify potential targets for new preservation strategies. Current knowledge indicates the involvement of the HOG pathway in citric acid adaptation. A citric acid sensitivity screen from a previous study also isolated a SR protein kinase Sky1p, involved in polyamine metabolism, which has been connected with other crucial cellular processes including modulation of ion homeostasis and osmotic shock. In this study we have undertaken a systematic screen for genes that confer increased sensitivity to citric acid paying particular attention to those involved in polyamine metabolism and those known to encode proteins which have evidence of interactions with Sky1p. Many of the deletion strains tested exhibited hypersensitivity to citric acid including Δsky1. Protein-protein interaction maps for Sky1p highlighted an interesting secondary interacting protein Nmd5p, an importin crucial for the nuclear localization of Hog1p. This information suggested there may be the possibility of linkage between Sky1p and Hog1p and their roles in citric acid tolerance, perhaps through Nmd5p. This provided an incentive to perform a range of experiments to test this theory. Proteomic and phosphoproteomic analyses were carried out to study protein expression and phosphorylation changes in response to citric acid stress. Comparative proteomic analyses for Δsky1, Δhog1 and BY4741a with and without citric acid identified four instances of analogous protein expression responses in both Δsky1 and Δhog1, suggesting functional overlap upon exposure to citric acid. Epistasis studies of Δhog1Δsky1 suggested that the two protein kinases do not function on the same pathway. However, overexpression analyses did suggest some functional interaction between Hog1p and Sky1p in mediating citric acid resistance since overexpression of Sky1p in Δhog1 resulted in partial rescue of growth. Further supporting evidence for some functional interaction or linkage was provided by Hog1p phosphorylation and localisation studies. Δsky1 exhibited dual phosphorylation of Hog1p in the absence of citric acid stress; implying that loss of SKY1 results in dual phosphorylation of Hog1p by either prompting phosphorylation or perhaps by interfering with dephosphorylation of Hog1p. Localisation studies of Hog1p proved that like osmotic stress, citric acid stress results in nuclear translocation of Hog1p and deletion of SKY1 seemed to interfere with this localisation to some extent. In light of the results attained in this study we believe we have evidence to propose a novel role for Sky1p in mediating resistance to citric acid and that there is also substantial evidence to suggest that Sky1p shares some functional redundancy and perhaps functional linkage with Hog1p in citric acid adaptation.
  
  
  
• Reproductive success and male traits in the spotless starling, Sturnus unicolor
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  Abstract: Selection operates when the variability among individuals in heritable traits translates to differences in the number of offspring that survive to breed, which is a close estimate of fitness. Consequently, the outcome of sexual-selection should be higher reproductive success for individuals with a greater expression of the selected traits. In this thesis, the relationship between some male spotless starling (Sturnus unicolor) traits and reproductive success was assessed. A particular focus was given to the role of throat feathers (TF) as sexually selected trait. The study was conducted in a wild population using a correlative approach in 2004, while in 2005 and 2006 the TF of males were experimentally shortened. The genetic parentage of the offspring was required for determining the reproductive success of males. Nine highly polymorphic microsatellites (with 11.7± 3.2 alleles per locus) were developed and optimised for this species. Parentage analyses were conducted in NEWPAT XL and CERVUS 3.0.3 and confirmed using observational data. Eighty-five percent of the offspring had at least one parent assigned. The levels of intra-specific brood parasitism, extra-pair paternity and quasi-parasitism were 7%, 7% and 1% of the offspring, respectively. Polygamy levels decreased with year, as the study population matured. The correlative study showed that males with longer TF and with better condition had a higher probability of reproducing and sired more offspring, but their offspring were not of higher quality as measured by their weight and immune response to phytohaemagglutinin. Polygynous males were also in better condition. In the experimental study, males in better condition had a higher chance of reproducing and sired more fledglings. Conversely, males with reduced TF sired significantly fewer eggs and lighter fledglings than control males. Body condition and TF length are shown to be good predictors of reproductive success and TF length is shown to be under sexual selection.
  
  
  
• Estimating the impact of bycatch and calculating bycatch limits to achieve conservation objectives as applied to harbour porpoise in the North Sea
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  Abstract: Incidental catch, or bycatch, of harbour porpoise (Phocoena phocoena) in fishing operations is an international conservation issue. The main objective of this thesis was to develop methods for determining the impact of bycatch on the state and dynamics of porpoise populations and for calculating bycatch limits that will achieve conservation objectives in the future. I applied these methods to the North Sea as a case study. First, I analysed sighting rates of harbour porpoise on seabird surveys in the North Sea during 1980-2003 to determine whether these data could provide informative time-series of relative abundance. Some general patterns and trends in sighting rates were consistent with previous studies. However, the standardised indices of abundance were relatively imprecise and thus have limited value for a monitoring framework that relies on statistical detection of trends. Second, I used a population model to integrate available data on harbour porpoise in the North Sea and to assess the dynamics of the population during 1987-2005. There was a high probability that bycatch resulted in a decrease in abundance. The estimated life history parameters suggested a limited scope for population growth even in the absence of bycatch. The model and data were not informative about maximum population growth rate or carrying capacity. The model suggested that dispersal was the most plausible explanation for observed changes in distribution within the North Sea. Third, I considered management procedures for calculating bycatch limits. I performed simulations to compare the behaviour of the procedures, to tune the procedures to specific conservation objectives and to test the robustness of the procedures to a range of uncertainties regarding population dynamics and structure, the environment, observation and implementation. Preliminary annual bycatch limits for harbour porpoise in the North Sea ranged from 187-1685 depending on the procedure, tuning and management areas used.
  
  
  
• Phylogenetic analyses and taxonomic studies of Senecioninae : southern African Senecio section Senecio
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  Abstract: Molecular phylogenetic analyses of subtribe Senecioninae, based on combining sequenced ITS and trnL-F fragments from specimens collected in the field with sequences collected from GenBank, suggest the subtribe is monophyletic, as is Senecio s.str. (including Robinsonia), and suggest an expanded monophyletic section Senecio. Many Senecio species should be removed from the genus, as they are only distantly related to it, emphasising the para- or polyphyletic nature of Senecio as it is currently circumscribed. Area optimisation suggests southern Africa as a possible geographical origin for the genus and section. Harvey’s (1865) sectional classification of South African Senecio species (the only attempt to date to impose infrageneric groupings on these taxa), was tested for monophyly which, however, was not seen in the sections tested. A number of southern African species from Harvey’s sections are suggested for inclusion in an expanded section Senecio. A clade suggested as basal to sect. Senecio, consisting of Senecio engleranus and Senecio flavus, was found to be only distantly related to the section. Resolution of the two species within the clade was not evident; a comparative study was therefore made employing RAPDs, morphometrics and breeding experiments. The two proved to be distinct entities, both genetically and morphologically, although they remain interfertile, suggesting that intrinsic postzygotic barriers between them are weak, and that hybridisation – not found in the wild - is mainly prevented by prezygotic barriers. F1 hybrids created between the two were seen to have intermediate morphologies and RAPD profiles. A single F1 individual self-pollinated to produce a vigorous F2 generation, allowing preliminary surveys of pollen number, pollen fertility and pappus type. Pappus type is seen to be under the control of allelic variations in a single major gene, while pollen numbers and pollen fertility are seen to be under more complex genetic control.
  
  
  
• Biodiversity and ecosystem processes in heterogeneous environments
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  Abstract: The decline in biodiversity over the last decade has motivated researchers to investigate the relationship between species richness (biodiversity) and ecosystem function. Empirical approaches are becoming more realistic as more factors have been included. Spatial heterogeneity is an example. Heterogeneity is an inherent part of the environment and apparent in all habitat types creating a patchy, mosaic of natural landscape. Researchers have reported the extent of heterogeneity in the landscape, but surprisingly not yet included heterogeneity into biodiversity and ecosystem function (BEF) studies. In recent years, empirical studies of marine systems have enhanced the BEF debate. Depauperate estuarine systems are ideal candidates for establishing model systems. In this study, estuarine microphytobenthos (MPB) were used as a response variable since the relationship between MPB and primary productivity is well-known. This relationship was exploited to employ MPB biomass as a proxy for primary productivity. Benthic chambers were used to assess the effect of macrofauna in single species and multi-species treatments on both ecosystem function and net macrofaunal movement. Heterogeneity was created through enriching sediment ‘patches’ with Enteromorpha intestinalis, providing areas of high and low nutrient. Heterogeneity, macrofaunal biomass, species richness, species diversity and flow were all varied in order to assess combined effects on the functioning of the system. Heterogeneity was found to have a significant influence on ecosystem functioning and on macrofaunal movement, however, patch arrangement did not. MPB biomass was highest in patches containing organic enrichment suggesting that nutrients were obtained locally from the sediment/water interface rather than the water column. There was variation in MPB biomass with macrofaunal species, probably resulting from differences in behavioural traits. It was also evident that flow altered species behaviour, as there was a significant difference between static and flow treatments. This work shows the importance of heterogeneity for BEF relationships.
  
  
  
• Structural biology of Vibrio cholerae pathogenicity factors
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  Abstract: The World Health Organization (WHO) states that 30,000 children under the age of five die each day worldwide. Around a quarter of these die from diarrheal disease caused by microbial infection. In addition to this high mortality rate, there are data emerging on the morbidity effects of diarrheal disease, for example a few episodes of diarrhea in the first two years of life can remove 10 IQ points and lead to growth deficiency. Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a serious problem in third world countries, where sanitary and hygiene infrastructure is very poor, and claims several thousand lives every year. In order to better understand the pathogenicity regulation in V. cholerae, structural and functional investigations of a hypothetical protein family present in pathogenicity islands and a transcriptional regulator protein for DNA-binding were investigated. Two adjacent genes, vc1804 and vc1805, encode hypothetical proteins within the Vibrio pathogenicity island-2 (VPI-2) of Vibrio cholerae, and are part of a cluster of genes only present in pathogenic strains of the bacterium. Paralogous adjacent genes, vc0508 and vc0509, are also present within a second pathogenicity island, the Vibrio seventh pandemic island-2 (VSP-2), of V. cholerae O1 El Tor and O139 serogroup isolates. Sequence similarity suggests that the VC0508, VC0509, VC1804 and VC1805 proteins will share a similar fold. The crystal structures of VC0508, VC0509 and VC1805 have been determined to a resolution of 1.9, 2.4 and 2.1 Å, respectively. Several recombinant constructs of vc1804 were made, but no soluble proteins were expressed. This hypothetical protein family reveals structural homology to human mitochondrial protein p32. Human p32 is a promiscuous protein known to bind to a variety of partners including the globular head component of C1q. We have shown that VC1805 binds to C1q. One possibility is that VC1805 is involved in adherence of the bacterium to membrane-bound C1q in the gut. To explore the roles of VC0508, VC0509, VC1804 and VC1805 in vivo, gene knockout and animal model studies of those proteins are underway. The ferric uptake regulator (Fur), a metal-dependent DNA-binding protein, acts as both a repressor and activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, and this opens up the potential of Fur as a drug target for cholera. The first crystal structure of a Fur protein, from Pseudomonas aeruginosa, revealed a dimeric molecule with each monomer containing a dimerization domain, a helical DNA-binding domain and two metal binding sites: Zn1 is proposed to be a regulatory Fe-binding site, and Zn2 is proposed to be a structural Zn-binding site. Here we present the crystal structure of V. cholerae Fur (VcFur) that reveals a very different orientation of the DNA-binding domains. Accompanying these structural changes are alterations in the amino acids coordinating the zinc at the Zn2 site, and this lends support to this being the site regulated by iron. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including the much-studied E. coli Fur. An analysis of the metal binding properties shows that like other Fur proteins, VcFur can be activated by a range of divalent metals. EPR spectroscopy measurements of the movements of the DNA-binding domain, in the presence of DNA and different metals, are underway.
  
  
  
• Tools for probing 2A sequence space
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  Abstract: Foot-and-mouth disease virus (FMDV) 2A is an oligopeptide composed of only 18 amino acids that can mediate a co-translational cleavage at its own C- terminus. It has been observed that 2A sequences do not show cleavage activity within bacterial organisms. Why 2A lacks activity in a prokaryotic organism such as E.coli is unclear. A series of plasmids designed to provide a phenotypic screen for 2A-mediated cleavage (in prokaryotes) were developed. Even though no active 2A sequences were found in bacteria, this system can easily be adapted to eukaryotic cells and will also be very useful in mutagenic studies on 2A sequences. Furthermore, 2A[subscript(FMDV)] has been used in the construction of a reporter of stress in the cell. This may allow us to open a new approach in the use of 2A oligopeptide, which had already been widely used to co-express genes of interest with reporter proteins, in biotechnology and gene therapy. Theiler’s murine encephalomyelitis cardiovirus (TMEV) 2A has the same role as in FMDV but is 150 aa in length instead of the 18 aa in FMDV. It also presents the same C-terminal motif but what is the function of the remaining ~85% of the cardiovirus 2A sequence remains a mystery. To this end we have produced antibodies against TMEV-2A, to study the role of 2A[subscript(TMEV)] within the cell. Database searches probing for 2A’s C-terminal conserved motif (- DxExNPGP-) has identified many 2A-like sequences, not only within picornaviruses but also in trypanosomes, insect and cellular genes. These remarkable findings indicate that the control of protein synthesis by 2A is not solely confined to the Picornaviridae. Bioinformatics analyses of all the known 2A-like sequences, comparing all the different upstream sequences, show a clear pattern on the organization of residues in the upstream region. The discovery of this 2A oligopeptide has led to a breakthrough in protein co- expression technology. It has been used as a highly effective new tool for the co- expression of multiple proteins from a single ORF in plant biotechnology and also gene therapy applications. Although we have gained substantial insights into the general features and biological significance of this process, a great deal still needs to be uncovered about the structural and mechanistic details of this unique mechanism of action.
  
  
  
• Muscle growth and flesh quality of farmed Atlantic halibut (Hippoglossus hippoglossus) in relation to season of harvest
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  Abstract: In the present study, muscle growth and flesh quality have been investigated from both commercially farmed Atlantic halibut (Hippoglossus hippoglossus) (Aga marine AS, Norway)and halibut obtained from small-scale trials at Mørkvedbukta Research Station (Bodø University College, Norway). Morphometric techniques have been utilized to investigate fast muscle growth in halibut ranging from circa 2 g to 100 kg, and it was established that fast muscle fibre recruitment ceases when the fish attain approximately 81 and 177 cm, in the case of males and females, respectively. Different muscle fibre types were distinguished using histochemical (myosin ATPase and succinic dehydrogenase) and immunohistochemical (S-58, an antibody against slow muscle myosin) staining techniques. Females recruit twice as many fast muscle fibres compared to males, which allows them to reach a larger final size. Furthermore, the seasonal growth patterns during a one year production cycle in commercial farmed halibut revealed a winter depression in growth leading to loss of biomass, which was attributed to the maturation of males. Commercial farmed fish of equal size (~1.5 kg) showed sexual dimorphism of fast muscle fibre number, caused by a significantly higher rate of fast muscle fibre recruitment in females. During the winter season fast muscle fibres shrunk significantly, especially in male fish, as a consequence of loss of appetite, low water temperatures and sexual maturation. None of the female fish matured during the trial. Flesh quality of halibut deteriorated during winter and spring, since it had a softer appearance and significantly lower myotomal protein content, particularly in males. Cathepsin activity was measured using spectroscopy and showed a strong negative correlation to protein content, displaying a seasonal variation. The proteolytic depletion of fast muscle proteins affected the water holding capacity of the muscle (determined by centrifugation), which showed concomitant changes with the increase in cathepsin activity and drop in protein content. Despite the soft appearance, the firmness (shear force) of the flesh increased during the winter. The hydroxylysyl pyridinoline cross-link content of the collagen matrix, determined by HPLC, showed a strong correlation to the fillet texture. The increased firmness during the winter, a period of little (female) or negative growth (males), was probably due to an increased cross-linking of the collagen compartment. Partial sequences of IGF-I and IGF-II were cloned from fast muscle of Atlantic halibut, and their relative gene expression levels were determined along with those of cathepsin B, cathepsin D and IGF-IRa in male halibut using qPCR during a fasting and refeeding trial. Transcript levels of cathepsin B and to some extent cathepsin D were significantly higher during fasting than refeeding, suggesting an increased enzyme production during periods of food deprivation. A temporary increase in IGF-I transcripts was observed after 7 days refeeding suggesting that this growth factor is involved in muscle growth control. Both IGF- IRa and IGF-II were down-regulated during refeeding.
  
  
  
• Predictive models of cetacean distributions off the west coast of Scotland
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  Abstract: The main purpose of this study was to produce and test the reliability of predictive models of cetacean distributions off the west coast of Scotland. Passive acoustic and visual surveys were carried out from platforms of opportunity between 2003 and 2005. Acoustic identifications were made primarily of harbour porpoises (Phocoena phocoena), delphinids, and sperm whales (Physeter macrocephalus). Generalised Additive Models (GAMs) were used to relate species’ distributions to a range of environmental variables over a range of temporal and spatial scales. Predictive models of delphinid distributions showed both inter-annual and inter-month variations. Combining all data for all months and years resulted in a model that combined the environmental influences from each monthly and yearly model. Overall, delphinids were found to associate with the deep (> 400m) warm water (10.5°C-12.5°C), and in areas of deep thermocline. Relationships between sperm whales and environmental variables were consistent over changes in grain size (9 km or 18 km), but not between areas. Although sperm whales were distributed in deep water characterised by weak thermoclines and strong haloclines in the most northerly area (Faroe-Shetland Channel), they were found in deep productive areas with cold surface temperature in the more southerly waters (Rockall Trough). Within the southern Inner Hebrides, high use areas for harbour porpoises were consistently predicted over time (in years) and with differing survey techniques (acoustic versus visual), but not over space (southern Inner Hebrides versus whole of the Inner Hebrides). Harbour porpoises were mainly distributed in areas with low tidal currents and with higher detection rates during spring tides. The use of prey as a predictor variable within models of delphinid distribution shows some promise: there were correlations between delphinid and herring (Clupea harengus) in shelf-waters in 2005 but not in 2004. These models can be used in mitigating acoustic threats to cetaceans in predicted high use areas off the west coast of Scotland.
  
  
  
• Acoustic and ecological investigations into predator-prey interactions between Antarctic krill (Euphausia superba) and seal and bird predators
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  Abstract: 1. Antarctic krill (Euphausia superba) form aggregations known as swarms that vary greatly in size and density. Six acoustic surveys were conducted as part of multidisciplinary studies at two study sites, the western and eastern core boxes (WCB and ECB), during the 1997, 1998 and 1999 austral summers, at South Georgia. A quantitative, automated, image processing algorithm was used to identify swarms, and calculate swarm descriptors, or metrics. In contrast to acoustic surveys of aggregations of other pelagic species, a strong correlation (r = 0.88, p = 0.02, 95% C.I.= 0.24 to 0.99) between the number of krill swarms and the mean areal krill density [rho.hat] was found. Multivariate analysis was used to partition swarms into three types, based on contrasting morphological and internal krill density parameters. Swarm types were distributed differently between inter-surveys and between on and off-shelf regions. This swarm type variation has implications for krill predators, by causing spatial heterogeneity in swarm detectability, suggesting that for optimal foraging to occur, predators must engage in some sort of adaptive foraging strategy. 2. Krill predator-prey interactions were found to occur at multiple spatial and temporal scales, in a nested, or hierarchical structure. At the largest inter-survey scale, an index of variability, I, was developed to compare variation in survey-scale predator sightings, sea temperature and [rho.hat]. Using I and a two-way ANOVA, core box, rather than year, was found to be a more important factor in determining species distribution. The absence of Blue-petrels (Halobaena caerulea) and the elevated number of Antarctic fur seals (Arctocephalus gazella) suggest that 1998 was a characterised by colder than average water surrounding South Georgia, and a high [rho.hat] in the ECB. At the smaller, intra-survey scales (<80 km, <5 day), the characteristic scale (distances in which predator group size, or krill density were similar, L_s) were determined. For krill and predators L_s varied by survey and the L_s of krill also varied by depth within a survey. Overlap in L_s were stronger between predator species than between a predator species and krill, indicating predators were taking foraging cues from the activity of predators, rather than from the underlying krill distribution. No relationship was found between swarm characteristics and predator activity, suggesting either there is no relationship between krill swarms and predators, or that the predator and acoustic observation techniques may not be appropriate to detect such a relationship. 3. To overcome the 2-D sampling limitations of conventional echosounders, a multibeam echosounder (MBE) observed entire swarms in three-dimensions. Swarms found in the nearshore environment of Livingston Island situated in the South Shetland Islands, exhibited only a narrow range of surface area to volume ratios or roughnesses (R = 3.3, CV = 0.23), suggesting that krill adopt a consistent group behaviour to maintain swarm shape. Generalized additive models (GAM) suggested that the presence of air-breathing predators influenced the shape of a krill swarm (R decreased in the presence of predators: the swarm became more spherical). A 2D distance sampling framework was used to estimate the abundance, N, and associated variance of krill swarms. This technique took into account angular and range detectability (half-normal, [sigma_r.hat] = 365.00 m, CV = 0.16) and determined the vertical distribution of krill swarms to be best approximated by a beta-distribution ([alpha.hat] = 2.62, [CV.hat] = 0.19; [beta.hat] = 2.41, [CV.hat] = 0.15), giving the abundance of swarms in survey region as [N.hat] = 5,062 ([CV.hat] = 0.35). This research represents a substantial contribution to developing estimation of pelagic biomass using MBEs. 4. When using a single- or split-beam missing pings occur when the transmit or receive cycles are interrupted, often by aeration of the water column, under the echosounder transducer during rough weather. A thin-plate regression spline based approach was used to model the missing krill data, with knots chosen using a branch and bound algorithm. This method performs well for acoustic observations of krill swarms where data are tightly clustered and change rapidly. For these data the technique outperformed the standard MGCV GAM, and the technique is applicable for estimating acoustically derived biomass from line transect surveys.
  
  
  
• Antibacterial free fatty acids from the marine diatom, Phaeodactylum tricornutum
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  Abstract: The aim of this thesis was to isolate the compounds responsible for the antibacterial activity of cell extracts of the marine diatom, Phaeodactylum tricornutum. Marine microalgae are not only important primary producers but, due to their phylogenetic diversity, they are also a potential source of novel bioactive compounds. The marine diatom, P. tricornutum, was selected for study because its cell extracts are known to be antibacterial but the compounds responsible have not been isolated. In this thesis, the compounds responsible for the antibacterial activity are isolated from aqueous methanol P. tricornutum cell extracts by column chromatography and reverse phase high-performance liquid chromatography using a bioassay-guided approach. The compounds in three active fractions were identified by mass spectrometry and nuclear magnetic resonance spectroscopy as the unsaturated fatty acids (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, (9Z)-hexadecenoic acid and (6Z, 9Z, 12Z)-hexadecatrienoic acid. The fatty acids were found to be antibacterial against Staphylococcus aureus at micromolar concentrations. P. tricornutum exists in different cell morphs and, interestingly, extracts prepared from cultures in the fusiform morph were found to have greater antibacterial activity than extracts from oval cultures. This is explained by greater levels of the three antibacterial fatty acids in the fusiform cell extracts. The antibacterial fatty acids are proposed to be released by enzyme action when the diatom cells lose their integrity. The release of free fatty acids by diatoms is suggested to be a simple, very low cost population-level activated defence mechanism against potential pathogenic bacteria triggered when the cell loses its integrity. Further, this pathway may act against multiple threats to the microalga, including grazers, as fatty acids exhibit activity in diverse biological assays. Finally, whilst two of the fatty acids, (9Z)-hexadecenoic acid and (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, inhibited the growth of MRSA their usefulness as therapeutic compounds may be limited due to their instability and their broad biological activity.
  
  
  
• The genetics of sexually dimorphic traits implicated in sexual isolation in Drosophila : QTLs and candidate genes
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  Abstract: This study is primarily concerned with assessing the influence of the sex determination genes, transformer (tra), doublesex (dsx) and fruitless (fru) on three sexually dimorphic traits within Drosophila; pheromone blend, courtship song and sex comb tooth number. The sex determination loci have all been implicated as possible candidate genes affecting these important traits that contribute to sexual isolation, which is a major cause of speciation. Quantitative Trait Loci (QTL) analysis is used to assess the effects of these known candidate genes on the naturally occurring variation of mean interpulse interval (IPI) of courtship song and the differing pheromone blend profiles between Drosophila simulans and D. sechellia. The QTL analysis for both song and pheromone blend variation incorporated Multiple Interval Mapping (MIM), which enables the detection for epistasis. The desaturase loci desat1, desat2 and desatF were also included in the assessment on pheromone blends (cuticular hydrocarbon compounds), since they facilitate ecological adaptation and are also candidate genes, which are likely to exert a large affect on this particular trait. The sex determination genes were not significantly influential on the interspecific variation of the cuticular hydrocarbon compounds between these two sibling species. However significant effects were detected from two of the desaturase loci. desat1 was associated with a strong effect on the interspecific variation of a saturated hydrocarbon chain compound (unbranched-23). Additionally the candidate gene desatF potentially exerts an influence on the variation of 7,11-heptacosadiene, through a large epistatic effect with unidentified loci, situated between the markers pros and Mtn. The candidate gene eloF is situated in this region, and is known to affect the elongation of unsaturated hydrocarbon chains. The QTL associated with the marker desatF influenced the variation of both diene compounds (7,11-heptacosadiene and 7,11-pentacosadiene). Intriguingly epistasis was only detected for the variation of these two diene compounds. The MIM analysis assessing the affects of the sex determination genes on interspecific variation of mean IPI detected the candidate gene fru as the closest marker associated with a significant QTL on the third chromosome. The MIM also found a significant QTL associated with the marker Dgα situated on the second chromosome. Moreover significant epistatic interactions were detected between a further QTL situated nearest the marker forked on the X-chromosome with both of the other significant QTL situated on the third and second chromosomes. The analysis of a number of Recombinant Inbred (RI) lines was also carried out to test for the affects of the sex determination genes on both mean IPI and sex comb tooth number. The fru locus was associated with a significant increase in mean IPI, whereas the opposite was true for the dsx locus. In the analysis of sex comb tooth variation, it appears that all RI lines homozygous for D. sechellia alleles at the sex determination loci had significantly higher numbers of sex comb teeth. The final data chapter involves the sequence analysis of the fruitless locus, including all 13 fru proteins between ten recently sequenced Drosophilid genomes. The PAML program was used to detect the possible influence of natural selection on sequence divergence. There was no significant positive selection detected at the BTB functional domain and the sequences encoding for this domain were extremely conserved. Positive selection was found to be acting on the exon encoding for the Zinc-finger C domain. This domain is present in two protein isoforms including the male sex-specific isoform FRUMC, and the common non-sex-specific isoform FRUComC. Interestingly positive selection was also found at the non sex-specific Zinc-finger D domain.
  
  
  
• Functional analysis of the orthobunyavirus nucleocapsid (N) protein
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  Abstract: Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has a tripartite genome consisting of negative sense RNA segments called large (L), medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of 233 amino acids. The N protein encapsidates all three segments to form transcriptionally active ribonucleoproteins (RNPs). The aim of this project was to determine the domain map of BUNV N protein. To investigate residues in BUNV N crucial for its functionality, random and site- specific mutagenesis were performed on a cDNA clone encoding the BUNV N protein. In total, 102 single amino acid substitutions were generated in the BUNV N protein sequence. All mutant N proteins were used in a BUNV minigenome system to compare their activity to wt BUNV N. The mutant proteins displayed a wide-range of activity, from parental-like to essentially inactive. The most disruptive mutations were R94A, I118N, W134A, Y141C, L177A, K179I and W193A. Sixty-four clones carrying single substitutions in the BUNV N protein were used in the BUNV rescue system in an attempt to recover viable mutant viruses. Fifty recombinant mutant viruses were rescued and 14 N genes were nonrescuable. The 50 mutant viruses were characterized by: titration, protein labelling, western blotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³ -10⁸ pfu/ml, and three different plaque sizes large, medium and small. Protein labelling and western blotting showed that mutations in the N gene did not affect expression of the other viral genes as much as affecting N protein expression. It was demonstrated that single amino acid substitutions could alter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F). Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228T and G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74S and S96G were shown to have a ts defect in genome-synthesis (late replication step), while mutant viruses K228T and G230R had a ts defect in antigenome- synthesis (early replication step). Host-restriction experiments were performed using 5 different cell lines (Vero-E6, BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly in all cell lines. Likewise, the majority of mutant viruses follow this pattern except mutant virus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing the ratios of intracellular and extracellular particles revealed that only 15% of the total virus particles of mutant Y23A was released as extracellular particles compared to 30% of the parental virus. Fourteen N genes were nonrescuable. They were characterized by (i) their activity in the BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) their ability to form multimers, (iv) their ability to interact with L protein, and (v) their impact on RNA synthesis. In summary, BUNV N protein was shown to be multi-functional and involved in the regulation of virus transcription and replication, RNA synthesis and assembly, via interactions with the viral L polymerase, RNA backbone, itself or the viral glycoproteins.
  
  
  
• Theiler's murine encephalomyelitis protein 2C and its effect on membrane trafficking
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  Abstract: Picornaviruses replicate in association with cytoplasmic membranes of infected cells. Poliovirus 2C and 2BC play an important role in the formation of membranous vesicles, and induce dramatic changes in membrane trafficking. Theiler’s murine encephalomyelitis virus protein 2C was localized in infected cells using an anti-TMEV-2C antibody. Early upon infection, TMEV 2C was localized in the cytoplasm in an ER-like pattern. At later stages, 2C redistributed to a juxtanuclear site, which represents the viral replication site. Co-localization with the Golgi complex could not be observed. TMEV 2C seems to interact in vitro with reticulon 3, a highly conserved ER-associated protein. It was not possible to confirm a previously identified interaction with AKAP10, a protein kinase anchoring protein, presumably reflecting conformational constraints of the interaction. Two mutations in the AKAP10 binding site of TMEV 2C were identified, which inhibit the completion of the infectious cycle of TMEV. The intracellular changes that occur during TMEV infection were observed. Both actin filaments and microtubules may be used at early stages of infection; however both cytoskeleton components accumulate at the periphery of the cell during late stages of infection. A computer- based analysis has demonstrated that TMEV 2C is highly similar to katanin, a microtubule- severing protein, and may play a similar role in the reorganization of microtubules during infection. The Golgi complex turns from a solid, crescent-shaped organelle, into a series of punctuate fluorescent points forming an expanding balloon-like structure surrounding the concomitantly expanding site of virus replication. The remnants of the Golgi complex are finally dispersed throughout the cytoplasm. Live imaging confirmed these findings. It was observed that PKA also undergoes displacement to the cell periphery during infection. However, BIG1 seems to locate to the viral replication site during infection, suggesting it may play a role during viral replication. The localization of PKA and BIG1 in the infected cell may in part explain the observed dispersion of the Golgi complex.
  
  
  
• Embryonic temperature and the genes regulating myogenesis in teleosts
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  Abstract: In this study, full coding sequences of Atlantic salmon (Salmo salar L.) muscle genes were cloned, including myogenic regulatory factors (MRFs) (myod1c, myog, mrf4, myf5), inhibitors of Myostatin (fst, decorin), markers of myogenic progenitor cell (MPC) proliferation (sox8) and fusion (calpastatin), a marker of slow muscle fibre differentiation (smlc1) and a novel eukaryotic gene involved in regulating growth (cee). Several of these genes were then characterised using a range of experimental and computational analyses with the aim to better understand their role in myogenesis and their evolution in teleosts. A series of experiments supported previous findings that teleosts have extra copies of many genes relative to tetrapods as a result of a whole genome duplication (WGD) event that occurred some 320-350 Mya. For example, it was shown that genes for myod and fst have duplicated in a common teleost ancestor, but were then specifically lost or retained in different lineages. Furthermore, several characterised Atlantic salmon genes were conserved as paralogues, likely from a later WGD event specific to the salmonid lineage. Phylogenetic reconstruction and comparative genomic approaches were used to characterise the evolution of teleost paralogues within a framework of vertebrate evolution. As a consequence of one experiment, a revised nomenclature for myod genes was proposed that is relevant to all diploid and polyploid vertebrates. The expression patterns of multiple myogenic genes were also established in Atlantic salmon embryos using specific complementary RNA probes and in situ hybridization. For example, co-ordinated embryonic expression patterns were revealed for six salmon MRFs (myod1a, myod1b, myod1c, myog, mrf4, myf5), as well as markers of distinct MPC populations (pax7, smlc1), providing insight into the regulatory networks governing myogenesis in a tetraploid teleost. Furthermore, it was shown that Atlantic salmon fst1 was expressed concurrently to pax7 in a recently characterised MPC population originating from the anterior domain of the epithelial somite, which is functionally analogous to the amniote dermomyotome. In another experiment, the individual expression domains of three Atlantic salmon myod1 paralogues were shown to together recapitulate the expression of the single myod1 gene in zebrafish, consistent with the partitioning of ancestral cis-acting regulatory elements among salmonid myod1 duplicates. Additionally, the in situ expression of cee a novel and highly conserved eukaryotic gene was revealed for the first time in a vertebrate and was consistent with an important role in development including myogenesis. Additionally, Atlantic salmon were reared at 2, 5, 8 or 10 ºC solely to a defined embryonic stage, which was just subsequent to the complete pigmentation of the eye. After this time, animals were provided an equal growth opportunity. Remarkably, changing temperature during this short developmental window programmed the growth trajectory throughout larval and adult stages. While 10 and 8 ºC fish were larger than those reared at 2 and 5 ºC at the point of smoltification, strong compensatory growth was subsequently observed. Consequently, after 18 months of on growing, size differences among 5, 8 and 10 ºC fish were not significant, although each group was heavier than 2 ºC fish. Furthermore, significant embryonic-temperature induced differences were observed in the final muscle fibre phenotype, including the number, size distribution and myonuclear density of muscle fibres. A clear optimum for the final muscle fibre number was observed in 5 ºC fish, which was up to 17% greater than other treatments. In a sub-sample of embryos, temperature induced heterochonies were recorded in the expression of some MRFs (myf5, mrf4) but not others (myod1a, myog). These results allowed the proposition of a potential mechanism explaining how temperature can program the muscle phenotype of adult teleosts through modification of the somitic external cell layer, a source of MPCs throughout teleost ontogeny.
  
  
  
• Consequences of the interaction of amyloid beta with amyloid binding alcohol dehydrogenase and the receptor for advanced glycation end products
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  Abstract: Amyloid beta (Aβ) has been postulated to be the principle initiator of the pathogenesis of Alzheimer’s disease (AD). Therefore, understanding the underlying mechanisms of Aβ induced neurotoxicity in the early stages of AD would be essential for finding potential therapeutic targets of AD. Aβ-binding alcohol dehydrogenase (ABAD) has been shown to be a mitochondrial binding site for Aβ. Expression of ABAD has been found to be increased in brains of AD sufferers. Two dimensional electrophoresis studies have revealed that endophilin 1 was upregulated in Tg mAPP/ABAD mice brains as compared to Tg mAPP, Tg ABAD and non-Tg mice brains. Increased expression of endophilin 1 has also been found in brains of AD patients as compared to non-demented control brain tissues. Endophilin1 has been reported to regulate c-Jun N-terminal kinase (JNK) activation. In this study, expression of dominant negative forms of endophilin 1 (DN-endophilin 1) in mouse cortical neurons exhibited a significant reduction of Aβ induced JNK activation. Furthermore, using cell counting methods, it was shown that the transfection of DN-endophilin 1 increased neuron survival after Aβ treatment. Aβ has also been proposed to disrupt the interaction of ABAD and Cyclophilin D (CypD), which would trigger mitochondrial permeable transition, thereby leading to neurotoxicity. For fluorescence resonance energy transfer (FRET) analysis of the interaction of ABAD and CypD, a mitochondria targeted, EYFP tagged ABAD plasmid (pMito-ABAD-EYFP) and an ECFP tagged CypD (pCypD-ECFP) plasmid were developed. Positive FRET signals in SK-N-SH cells co-expressing pMito-ABAD-EYFP and pCypD-ECFP indicated that ABAD interacts with CypD in the mitochondria of mammalian cells. RAGE (receptor for advanced glycation end products) has been reported to bind to Aβ and mediate the toxic effects of Aβ peptides on neurons and microglia. It has been shown previously that Tg mAPP/DN-RAGE mice display preserved cognitive function as compared to Tg mAPP mice. To investigate possible mechanisms involved in rescuing cognitive function by RAGE blockage, two dimensional electrophoresis was used to analyze differential protein expression between Tg mAPP and Tg mAPP/DN-RAGE mice cortex. Altered expression of four proteins, including NADH dehydrogenase flavoprotein 2 (NDUFV2), glyoxalase 1 (GLO1), proteasome subunit beta type 4 (PSMB4, or β7 subunit of proteasome) and nitrilase family, member 2 (Nit2) have been observed between Tg mAPP/DN-RAGE mice cortex and Tg mAPP mice cortex. NDUFV2 is a 24kDa subunit of complex 1 which is involved in ATP synthesis. GLO1 is a cytosolic enzyme that plays a role the glutathione-dependent detoxification of α-oxoaldehydes, such as methylglyoxal. PSMB4 is a subunit of the 26s proteosome which is in the degradation of ubiquitinylated proteins. The function of Nit2 is still unclear.
  
  
  
• Development of microarray techniques for the study of gene expression in the European eel (Anguilla anguilla) during silvering and migration to seawater
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  Abstract: The European eel, Anguilla anguilla, has a complex life-cycle involving migrations between the Sargasso Sea and the river systems of Europe and North Africa. The requirement to move across large salinity gradients presents a significant physiological challenge and the developmental stages of the eel are closely linked to these migrations. Microarrays were created to elucidate gene expression changes occurring during; i. The transition from juvenile yellow to the adult sexually maturing, migrating silver eel and; ii. Salinity adaptation during the migration from freshwater to seawater. Groups (n = 6) of freshwater-acclimated yellow or silver eels were transferred to seawater for between 6 hours and 5 months and complementary control groups were transferred to freshwater. Brain, kidney, intestine and gill cDNA libraries were constructed using suppression subtractive hybridisation (SSH) techniques and a novel protocol based on Invitrogen's Gateway cloning system. The latter technique produced a low redundancy (~4 %) EST bank with a wide range of insert sizes (0.5 – 10 kb). Two microarray types were produced; one comprised 5760 clones from the two brain libraries whilst the other was a multi-tissue microarray incorporating 6144 clones from the SSH libraries. Pooled RNA samples were probed against the microarrays to highlight differentially expressed genes. Real-time quantitative PCR (QPCR) was used to validate the observed expression changes of selected genes in the tissues of individual fish. Following yellow to silver transformation of freshwater-adapted eels, the expression of tyrosine 3-mono-oxygenase/tryptophan 5-mono-oxygenase activation protein (14-3-3) and vaccinia related kinase 3 was shown to be consistently elevated. Prolactin expression increased in the brains of silver eels following two-day seawater-acclimation but QPCR analysis revealed high variation amongst freshwater-adapted eels. This is the first eel microarray study and the expression profiles highlighted herein will provide new avenues for research into the sexual development and salinity acclimation of A. anguilla Description: Electronic version does not contain associated previously published material
  
  
  
• Investigation into a prominent 38 kHz scattering layer in the North Sea
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  Abstract: The aim of this study was to investigate the composition of an acoustic scattering layer in the North Sea that is particularly strong at 38 kHz. A full definition of the biological composition of the layer, along with its acoustic properties, would allow for it to be confidently removed from data collected during acoustic fish surveys, where it presents a potential source of bias. The layer, traditionally and informally referred to as consisting of zooplankton, appears similar to others observed internationally. The metho