Proneural genes are required for neural competence and subtype specification. I have investigated the nature of this subtype specificity by analysing the number, type and pattern of ectopic sense organ precursors produced by proneural gene misexpression in transformant flies. The bHLH domains of Atonal and Amos share 88% identity, however these proneural proteins have very different wildtype functions. Moreover, I show that they have abundantly distinct misexpression phenotypes. Importantly, I show that an apparent overlap in function is actually due to cross-activation of atonal by amos. I have investigated the functional specificity of amos with regard to atonal and scute by constructing chimeric proteins of Amos and Atonal, and also Amos and Scute. All previously published studies concerning sub-type specificity have concluded that the bHLH domain is the determining factor in all bHLH proteins. Contrary to published studies, I found that Atonal specificity is determined to a large extent by its non-bHLH sequence. Reciprocally, the non-bHLH region of Amos can also facilitate Amos functions. However these results are only valid in the context of an Atonal-like bHLH domain. Phenotypic analysis of Amos and Scute chimeras has reiterated the requirement for the Amos bHLH domain for Amos-like function and correspondingly the Scute bHLH domain for Scute-like function. In synopsis I conclude that subtype information is contained within the bHLH domains of highly divergent proneural proteins such as Amos and Scute; however closely related proneural proteins such as Amos and Atonal require elements outside the bHLH domain for complete subtype specification. I propose a model in which these sequence elements function via interaction with specificity co-factor proteins.

The inability of the VERO cell line to produce interferon suggested that it might represent a regulatory variant which would be useful for somatic cell genetic studies of the regulation of interferon production. It was confirmed that VERO cells are unable to produce interferon in response to virus infection whereas BSC.B cells (similarly derived from the African Green monkey) were able to do so. Treatment of a number of cell lines and embryonic cells with polyriboinosinic. polyribocytidylic acid (poly(rI). poly(rC)) (usually in the presence of diethylaminoethyl (DEAE)-dextran) resulted in the development of an anti-viral state but VERO cells were found to be only weakly responsive. No effective method for significantly enhancing the anti-viral action of poly(rI). poly(rC) or interferon production was found. The cytotoxic effect of interferon and poly(rI). poly(rC) treatment was found to be limited to L929 cells and thus could give no information on the non-inducibility of VERO cells. Mutagenized VERO cells surviving selection by infection with temperature-sensitive Sindbis virus after treatment with poly(rI). poly(rC) were similar to VERO cells in their ability to support the growth of wild-type or temperature-sensitive Sindbis virus or to respond to poly(rI). poly(rC) treatment and they did not release virus or interferon into the medium, VERO cells cannot therefore give rise to inducible revertants. HAT medium containing ouabain was found to be suitable for the selection of hybrids of VERO cells with mouse D1 cells lacking thymidine kinase. Mouse D1 cells are able to produce interferon. Polyethylene glycol treatment effectively replaced treatment with inactivated Sendai virus for the promotion of cell fusion. All VERO-mouse hybrid clones examined were sensitive to the anti-viral action of poly(rI). poly(rC) treatment and many were shown to produce mouse interferon, however, none were able to produce monkey interferon. Certain clones were less sensitive than mouse D1 cells to the anti-viral action of poly(rI). poly(rC) and this may have been an indication of the activity of a VERO cell-coded repressor of interferon production. Thus there is no evidence that VERO cells are able to produce interferon and only slight evidence for an altered regulatory function in these cells.

Photo-oxidation is a light-dependent reaction, catalysed by photo-sensitive dyes, which can result in the oxidation of a large variety of substances. The photo-oxidation of enzymes with the anionic dye rose bengal was shown to follow one of two types of pH dependence; with an increase in pH the rates of photo-inactivation either increased (conventional photo-oxidation pH profiles), or decreased (unconventional photo-oxidation pH profiles). Photo-oxidation of the same enzymes with the cationic dye methylene blue gave rise to conventional pH profiles, irrespective of the type of pH dependence obtained with rose bengal. The production of unconventional photo-oxidation pH profiles was found to be dependent on the anionic nature of the photo-sensitiser. Using visible absorption spectroscopy and enzyme kinetic studies, rose bengal, but not methylene blue, was found to be capable of binding to and inhibiting the activity of enzymes. However, amino acid analysis of pig heart citrate synthase demonstrated that the occurrence of unconventional photo-oxidation pH profiles and dye-enzyme interactions was not accompanied by increased specificity of residue destruction. Some restriction of dye-protein interactions was achieved by the immobilisation of rose bengal on Sepharose. The photo-oxidation of various enzymes with this immobilised form of the dye was found to result in the formation of unconventional pH profiles only, irrespective of the type of photo-oxidation pH profile originally observed with the free form of the dye. The total prevention of all contact between rose bengal and enzymes was attained by compartmentalising the immobilised dye within a dialysis sac, the protein remaining on the outside. Photo-oxidation of enzymes with this compartmentalised dye again resulted only in the formation of unconventional pH profiles. Both the free and immobilised states of rose bengal were shown to photo-oxidise the five known susceptible amino acids---cysteine, histidine, methionine, tryptophan and tyrosine. The cysteine-containing peptide glutathione, and a glutathione-Sepharose complex, were also shown to be sensitive to photo-oxidation by free rose bengal, and the formation of conventional pH profiles only was observed. Although glutathione was shown to be similarly sensitive to photo-oxidation by the immobilised dye, no loss of cysteine occurred when the glutathione-Sepharose complex was irradiated in the presence of either immobilised or compartmentalised dye. Furthermore, the cysteine residues of mercaptalbumin proved insensitive to photo-oxidation by these two states of the dye. This demonstrated the necessity of direct interactions between rose bengal and cysteine residues as a prerequisite for their photo-oxidation. In the case of photo-oxidation of enzymes with free or immobilised rose bengal, the involvement of singlet oxygen was indicated by the enhanced rates of photo-inactivation obtained when H2O was replaced by D2O as solvent and also by the protective effects of sodium azide. It is therefore suggested that the pH profile obtained on photo-oxidation of enzymes by rose bengal consists of two components; a component at high pH which involves the photo-oxidation of cysteine residues by a direct contact mechanism and a component at low pH which involves the photo-oxidation of other susceptible residues by a singlet oxygen mechanism. This suggestion is supported by the demonstration of a correlation between the type of pH profile obtained on the photo-oxidation of various enzymes with free rose bengal and the presence of cysteine residues in their active sites, as indicated by inactivation experiments with the thiol-specific reagents DTNB and pHMB. The combined use of all the three forms of rose bengal may therefore both increase the specificity for residue destruction and provide information on the mechanisms involved in the photo-oxidation of biological macromolecules to a greater extent than is possible with the free dye alone. Immobilised rose bengal was found capable of photo-oxidising the active site of Acinetobacter citrate synthase yet, in contrast to free rose bengal, was incapable of desensitising the enzyme to NADH inhibition. The advantages and potential offered by the use of immobilised and compartmentalised rose bengal are discussed in this thesis and attention is drawn to the difficulties encountered and the caution required in the interpretation of photo-oxidation pH profiles.

CATH is a protein database of structural domains which are assigned to superfamilies through evidence of a common evolutionary ancestor. These superfamilies are further grouped by overall structural similarity into folds. This thesis explores several automated methods for recognising homologous relationships between these domains using the structural data from the Protein Data Bank (PDB). The aim of this work was to aid the manual classification of domains into the database and provide putative functional assignments to structures solved by the structural genomics initiatives. A fast and novel algorithm, CATHEDRAL, was developed to make fold assignments to regions of polypeptide chains. By combining a fast secondary-structure method (GRATH) and a slower residue-based method (SSAP), the algorithm was able to accurately assign boundaries for distant relatives, undetectable by sequence methods. Sequence and structural conservation patterns were combined in a novel algorithm, FLORA, to develop structural templates specific to catalytic function. FLORA was able to predict the correct functional site in 80% of cases and combined with global structure comparison, it was able to assign domains to enzyme families within diverse superfamilies. Techniques in structure comparison were also applied to ab initio models of protein domains, in order to assign them to fold groups within the CATH database. A novel scoring method was developed to pre-select models that were more likely to have adopted the correct fold. A selected sample of models for each target structure was then compared against representatives from the CATH database using the MAMMOTH and SSAP algorithms. Data from these alignments were combined using a Support Vector Machine to assign the target to a fold group within CATH. This work was generously supported by the Engineering and Physical Sciences Research Council.

No description available.

Introduction: The hBCAT proteins have a unique redox active CXXC motif which governs its transamination activity. Recent studies have identified that specific neuronal proteins with either redox activity or functions in cell signalling form interactions with the hBCAT proteins that are disrupted when the environment becoming oxidising. However, how hBCAT functions as an oxidoreductase is unknown or how it compares to other known cellular repair enzymes such as thioredoxin, glutaredoxin or protein disulphide isomerase. Moreover, leucine, a substrate for hBCAT regulates protein synthesis through the mTOR pathway, yet the importance of hBCAT itself in this mechanism remains undetermined. Aims: This thesis firstly investigated the redox substrates for the hBCAT proteins, and their oxidoreductase activity in comparison to the cellular repair enzymes. The second main aim was to establish if hBCAT can be regulated through phosphorylation both in-vitro and in the neuronal cell line (IMR-32). Finally, in addition to understanding their role as oxidoreductases, the importance of the reactive cysteines in redox binding to neuronal proteins was determined. Results: These studies demonstrated that both hBCAT isoforms have oxidoreductase and isomerase activity, but of lower activity relative to protein disulphide isomerase. The oxidoreductase activity was dependent on a functional CXXC motif, where in particular S-glutathionylation enhanced the ability of hBCAT to catalyse the folding of proteins. It has been demonstrated that hBCATc is regulated through phosphorylation and this is dependent on the redox environment unlike hBCATm which was only affected in the presence of thiol blocker, NEM. Both isoforms required the CXXC motif to be phosphorylated. Although phosphorylation of hBCATc in neuronal cells was observed, the exact mechanism needs to be elucidated. Finally, these studies have identified putative new partners for hBCAT such as inositol polyphosphate multikinase, GRINL1a upstream protein and parvalbumin. These indicate potential new roles for these proteins in cell division, neurotransmitter signal transduction and mTOR pathway, which have previously not been reported. Conclusion: The hBCAT proteins have oxidoreductase activity and are involved in a number of metabolic pathways such as cell division, neurotransmitter signal transduction and the mTOR pathway. This implies that these proteins may have a protective role in the cell, similarly to previous studies which have shown that upregulation of the hBCAT proteins is protective. Future work will elucidate further the role these proteins have under normal and pathological conditions.

Objective: To investigate the hepatic and serum lipid profile, and to examine the gene expression of the START family of intracellular lipid-trafficking proteins in liver tissues derived from lean (Fal?) and obese (jaIJa) Zucker rodents, a model of leptin receptor deficiency and genetic obesity. To investigate the functional role of cholesterol-trafficking StarD proteins on the lipid metabolism of McA-RH7777 cells. Methods: Glycaemia, body weight, serum and hepatic lipids were measured from animals aged three to six months. Gene expressions of the START family (Stardl-dI5) and protein expression of the cholesterol-binding StarD proteins were profiled by quantitative-PCR and immunoblotting, respectively; all values were nOlmalized to GAPDH. Stably transfected McArdle cell lines overexpressing cholesterol-binding StarD proteins were generated, and siRNA-mediated transient gene silencing was employed to study the effects of reduced expression of StarD3. The lipidation of ApoA-I with eH]cholesterol and the synthesis and secretion of radiolabeled cholesterol, fatty acids, triglycerides and cholesteryl esters from radiolabeled precursors (eH]glycerol, [,4C]acetate and ['4C]0Ieate) monitored (2h). The genetic profile of McArdle cells overexpressing StarD3 was determined by RT2-qPCR array. Results: Obese male Zucker rats accumulated greater quantities of hepatic triacylglycerol and phospholipid than obese female rats, while obese female rats exhibited a more profound hyperlipidaemia. Protein expressions of StarD3 and StarD4 were repressed in obese animals compared to lean controls, and the expression of StarD5 was repressed by obesity in female rats. Notably, protein levels of StarD3 and StarD4 were higher in female, compared with male rodents, while the abundance of StarD5 was higher in males than females. Linear regression analyses revealed gendered expression of Stard3, Stard4, Stard5, Stard8, Stardl2 and Stard13 to be inversely correlated with hepatic and/or serum lipid concentrations. Overexpression of StarD3 and StarD4 in McArdle cells increased the lipidation of exogenous ApoA-I and StarD3 increased the incorporation of de novo fatty acids into triglycerides and cholesteryl esters. Knockdown of StarD3 reduced the synthesis of cholesterol and cholesteryl esters, and repressed the incorporation of [,4C]acetate into the triglycerides pool. Overexpression of StarDI and StarD5 increased the incorporation of exogenously supplied preformed fatty acids into the triglycerides pool. No increase in the cellular lipid mass was measured in any of the stable cell lines.

Activation of muscle contraction has been a topic of interest for over 60 years, During this period researchers have used various approaches such as electron microscopy, X-ray diffraction and solution kinetics to understand the structural and molecular basis of cooperative activation of muscle contraction. Muscle contraction is a process which occurs because of the interaction of a globular protein, myosin, with the thin filament which consists of filamentous F -actin. However this interaction is regulated by the presence of tropomyosin and troponin on the thin filament, where tropomyosin spans across seven actin monomers and troponin (which is bound to tropomyosin and also interacts with actin) acts as a calcium switch. Tropomyosin is positioned such that it blocks myosin binding sites on actin. The binding of calcium to troponin causes a positional change in the adjacent tropomyosin molecule; as a result the tropomyosin moves away exposing myosin binding sites on the thin filament, facilitating muscle contraction. However, calcium is not the only regulator of thin filament activation, strong myosin-binding also plays a crucial role in activating thin filaments (Rosenfeld and Taylor, 1985).

The ability of microorganisms to adapt to low oxygen concentrations confers a considerable growth advantage. Furthermore, the colonization of microaerobic environments by pathogenic bacteria, such as Pseudomonas aeruginosa is a significant clinical issue. The cbb3 cytochrome c oxidases are members of the heme copper oxidase superfamily that regulate microaerobic respiration in diverse Proteobacteria. Cytochrome cbb3 oxidases are composed of four non-identical subunits encoded by one or two ccoNOQP operons. Surprisingly, the CcoP subunit contains a low potential hexacoordinate heme that binds CO in the reduced state following displacement of the distal endogenous ligand. The biochemical significance of CcoP is poorly understood but the cbb3 complex reports the redox status of the cell leading to transcriptional activation of genes involved in energy metabolism via the sensory kinase RegB/RoxS. By expressing the diheme subunit CcoP from the non-pathogenic Pseudomonad, Pseudomonas stutzeri in Escherichia coli, we have now examined the biochemical properties of the CO binding c-type heme. We characterized wild-type and mutant CcoP using mediated redox potentiometry, UVVisible, magnetic circular dichroism, and electron paramagnetic spectroscopies and have clearly identified two low-spin His/His coordinated c-type hemes, with redox potentials of + 185 mV and -15 mV. Examination of the spectral characteristics and oxidase activity of both cbb3 oxidase isoforms from the clinically relevant P. aeruginosa suggested that the cbb3 -1 oxidase has an important metabolic function at high oxygen tensions and the cbb3-2 oxidase has a more significant role under oxygen limiting conditions. In conclusion, our data suggests a prominent function for the CcoP subunit of cytochrome cbb3 oxidases in the adaptive ability of Pseudomonads to colonize diverse environments. Further understanding of this adaptive biochemistry may reveal drugable targets for P. eeruginosa.

The Saccharomyces cerevisiae Rnq 1 p prion protein plays an integral role in the dynamics of other aggregation-prone proteins, be they other prion proteins or glutamine-rich proteins. The conformational conversion of a prion protein to its prion state is often associated with changes to cellular physiology and two interesting questions arise from this. One, what impact do these physiological changes have on our ability to interpret experimental findings in this model organism? Two, are these changes non-random, representing a novel means of altering cellular physiology? An understanding of the cellular function of Rnq 1 P is important in addressing these questions. Further, the role of Rnq 1 P in its [PIN+] prion state as a universal catalyst for amyloid-formation provides a useful model for dissecting mechanisms of amyloid toxicity, and once again, cellular function is one parameter of multiple that determine the [mal toxicity profile of a protein. To identify a cellular function for Rnqlp both phenotypic assays and a mass spectrometry-based label-free quantitative proteomics analysis were performed. A role for Rnq 1 P as a negative regulator of translation termination was characterised and eo-localisation of Rnq 1 p with P-bodies, tightly packed clusters of untranslating rnRNA, was observed indicating that Rnqlp is intimately associated with mRNA dynamics within the cell. Additionally, evidence for a role in the maintenance of mitochondrial respiratory capacity pertaining to ATP-generation is presented, along with indications that this latter role may be through transcriptional regulation. To further understand the mechanisms of toxic protein aggregation, two analyses were performed. One, a study on the impact of natural Rnqlp polymorphisms identified fifty-three novel RNQI alleles and sequence features affecting Rnq 1 P toxicity. Two, a screen for genetic modifiers of both Rnqlp and mutant huntingtin demonstrated the role of P-bodies and mRNA degradation pathways in modulating amyloid or glutamine-based toxicity, along with a possible role for energy. homeostasis. The results presented in this thesis provided new insight into the functional roles of Rnq 1 P within the cell, and consequently the possible impact on cellular physiology associated with Rnqlp's [pin-] to [PIN+] conversion, and also identified novel modulators of toxic aggregation events in the cell.