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Key Research Profiles

• Centre for Biomolecular Sciences
• Centre for Evolution, Genes and Genomics
• Centre for Magnetic Resonance
• Centre for Research into Ecological & Environmental Modelling
• Centre for Social Learning & Cognitive Evolution
• Institute of Behavioural and Neural Sciences
• Scottish Oceans Institute
• Sea Mammal Research Unit

Some Recent Publications

Recent Theses

• Causal pattern inference from neural spike train data
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  Title: Causal pattern inference from neural spike train data
  
  Authors: Echtermeyer, Christoph
  
  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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  Title: Changes in gene expression, lipid class and fatty acid composition associated with diapause in the marine copepod Calanus finmarchicus from Loch Etive, Scotland.
  
  Authors: Hill, Katie A.J.
  
  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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  Title: Variation in sperm whale (Physeter macrocephalus) coda vocalizations and social structure in the North Atlantic Ocean
  
  Authors: Antunes, Ricardo
  
  Abstract: This study aimed at complementing studies of sperm whale social and vocal behaviour thatwere restricted to the Pacific Ocean.The characteristic multi-pulsed structure of sperm whale clicks allows for estimation ofwhales' size from measurements of the inter-pulse intervals (IPI). I have developed two newautomatic methods for IPI estimation from clicks recorded during foraging dives. When comparedto 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 unitmembers as the function for the sperm whale communication signals called codas. I tested withincoda type variation for individual specific patterns and found that, while some coda types do notallow for individual discrimination, one did so. This variation suggests that different coda typesmay 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 thecosts and benefits of group formation. I argue that these are due to differences in terms of predationpressure 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 thatpreviously documented in the Eastern Tropical Pacific. In the North Atlantic, coda repertoirevariation is mostly geographic, which is parsimoniously explained by random drift of culturallytransmitted coda repertoires. No sympatric vocal clans with distinct dialects were found as has beennoted in the Pacific. Drawing upon the differences found in social structure I argue that selection formaximization of differences between units with similar foraging strategies may have led to thePacific vocal clans.The differences between oceans suggest that sperm whales may adaptively adjust theirbehaviour according to experienced ecological conditions.
  
  
  
• Viral interferon antagonists and antiviral drugs
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  Title: Viral interferon antagonists and antiviral drugs
  
  Authors: Nobre, Rita
  
  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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  Title: Structural studies on the sialidases from Streptococcus pneumoniae and Pseudomonas aeruginosa
  
  Authors: Xu, Guogang
  
  Abstract: The sialidases are a group of glycosyl hydrolases that specifically remove terminalsialic acid (Neu5Ac) residues from various glycans. In the two common humanpathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, theseenzymes have been shown to be key virulence factors directly involved in bacterialcolonization and infection. However, little is known about their detailed structural andmechanistic features and lack of this information significantly slows down the progressof new drug discovery targeting these enzymes. Therefore, we embarked structural andkinetic studies towards the three distinct sialidases (designated as NanA, NanB andNanC) 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 disorderedregions. The catalytic domain of NanA (CNanA) was therefore subcloned, which wascrystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer withclose contacts between the two monomers. The second pneumococcal sialidase NanBonly shares 24% sequence identity with NanA. Crystal structure of NanB was alsodetermined to 1.7 Å, which exhibits a multi-domain monomeric architecture. Ingeneral, 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 conservedresidues stacking around the proposed active sites. NanC is a close homologue ofNanB, sharing over 50% sequence identity. However, NanC crystallization is notsuccessful so far. To compare the three sialidases in more detail, a computationalNanC model was made based on the structure of NanB. Mapping of the active sites ofCNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as theprobe. Although sharing many common features, NanA, NanB and NanC presentdifferent topologies around the catalytic centre, give these enzymes a high level ofdiversity in enzymatic kinetics, substrate specificity and catalytic properties. NMRstudies show that NanA acts as a classic hydrolytic sialidase; while NanB is found tobe an intermolecular trans-sialidase like the leech sialidase; NanC, however, handlesmultiple 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, whichmight be essential for pneumococcal pathogenesis at various stages. Based on thecrystal structures of CNanA and NanB, preliminary work towards S. pneumoniaesialidases inhibitor design is under way, in which, a variety of techniques, such as thefluorescence-based thermal shift assay, NMR spectroscopy, computational dockingand X-ray crystallography, are incorporated in.The crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be aunique trimer in crystal that is associated, in part, by the immunoglobulin-liketrimerization domain around a three-fold crystallographic axis. The core catalyticdomain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidaseactivity was detected with this enzyme. In addition, two key catalytic residuesincluding one of the arginine in the arginine triad and the acid/base catalyst asparticacid are missing in PaNA. In silico docking suggests that Phe129 may confer substrateselectivity towards pseudaminic acid, which is a specific carbohydrate superficiallysimilar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directedmutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNAinto a poor sialidases. Moreover, the crystal structure of PaNA also indicates thatHis45, Tyr21 and Glu315 may form a charge relay to compensate the missing asparticacid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315to be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNAcould be a pseudaminidase with structural and mechanistic variations. This enzyme,together some other uncharacterized fellow proteins, might form a novel subclass inthe sialidase superfamily.The various findings in the current projects provide meaningful insights towardsseveral sialidases that have been linked to bacterial virulence, which may contribute toa more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.
  
  
  
• RNA virus modulation of IFN, PI3K and apoptosis
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  Title: RNA virus modulation of IFN, PI3K and apoptosis
  
  Authors: Killip, Marian J.
  
  Abstract: Interferon (IFN) and phosphatidylinositol 3-kinase (PI3K) are apoptosis regulatorsthat are targeted by viruses to promote survival of infected cells. Significant crosstalkexists between IFN and PI3K, and this study sought to investigate the relationshipsbetween IFN, PI3K and apoptosis during virus infection. Parainfluenza virus 5 (PIV5)and influenza A virus (IAV) are both negative-sense single-stranded RNA virusesthat encode multifunctional proteins in order to maximise their genome codingcapacity. 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 therole of these proteins in apoptosis regulation; the ability of V and NS1 to inhibitapoptosis was therefore investigated. PIV5/V was found to limit cell death inresponse 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 mediateanti-apoptotic signalling during IAV infection. However, a virus expressing an NS1unable 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 cellsfrom pro-apoptotic stimuli. NS1-mediated PI3K activation similarly had no effect onIFN production or ISG expression in infected cells. In contrast, other NS1 mutantviruses induced large amounts of apoptosis. These viruses also induced significantlevels of IFN and were unable to cause apoptosis in IFN-deficient cells, indicatingthat NS1 limits apoptosis induction through its IFN antagonist functions. Theimplications 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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  Title: Female mating decisions in the Trinidadian guppy, Poecilia reticulata
  
  Authors: Barbosa, Miguel
  
  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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  Title: Structure and function of nitrate and nitrite transporters, NrtA and NitA, from Aspergillus nidulans
  
  Authors: Symington, Vicki F.
  
  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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  Title: Investigation of the transcriptional response of Sulfolobus solfataricus to damaging agents
  
  Authors: Munro, Stacey
  
  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 repairpathways that have evolved to repair specific types of damage. Organisms in thearchaeal domain, the third domain of life, contain homologues of many of theeukaryotic repair proteins, however little is known about how damage is detected inthe archaeal domain.Microarray studies in the archaeal species Sulfolobus solfataricus determineda number of genes whose expression was effected by UV radiation (work by Dr DGötz). The change in expression of nine of these genes was confirmed by RT realtime PCR. The expression of these genes was then investigated after exposure todifferent damaging agents, Mitomycin C, Methyl methane sulfonate, Phleomycin andHydrogen peroxide. The expression of two genes, transcription factor tfb-3 and celldivision control gene cdc6-2, was up regulated in all damage conditions.There was a huge induction of the dps-like gene (sso2079) after hydrogenperoxide damage. Transcription from this genes promoter was shown to be strong invitro (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 atranscriptional repressor and the Sso2273 protein, a homologue of the diphtheria toxinrepressor (DtxR) from Corynebacterium diphtheria, was identified as a possiblerepressor.Sso2273 was expressed and purified, and its crystal structure solved, itsparalogue, Sso0669, was also expressed and purified. Electrophoretic mobility shiftassays showed that the Sso2273 protein does not bind DNA, and had no effect ontranscription from any promoter used in in vitro transcription assays. HoweverSso0669 appeared to inhibit transcription, although the inhibition was not sequencespecific.A knockout strain of S. solfataricus PBL2025 missing the sso2273 gene wasproduced and used in microarray experiments in an attempt to determine the role ofSso2273 within the cell. The absence of Sso2273 appeared to have no effect on theexpression of the dps-like gene, however strong repression of an operon containinggenes involved in Sulphur assimilation was observed.
  
  
  
• Determination of the molecular and physiological basis of citric acid tolerance in spoilage yeast
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  Title: Determination of the molecular and physiological basis of citric acid tolerance in spoilage yeast
  
  Authors: McGuire, Lynne I.
  
  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.
  
  
  

School of Biology Theses in the Digital Repository