The sulfur containing vitamin, <I>biotin</I> has a cofactorial role for a number of carboxylation enzymes responsible for a variety of essential metabolic processes. Biotin is produced in minute quantities by micro-organisms and plants via a similar biosynthetic route that has few parallels in mammalian biochemistry, making it an attractive target for inhibition studies. This thesis describes the development of synthetic strategies for intermediates of the biotin biosynthetic pathway, and some structurally related compounds. An early step in biotin biogenesis is the decarboxylative condensation of pimeloyl-CoA and L-alanine to give 8-amino-7-oxononanoic acid (AON), catalysed by the <I>bio</I>F gene product, 8-amino-7-oxononanoate synthase (AONS). A novel preparation of AON is described here, along with a description of various alternative strategies which have been employed in the synthesis of this, and related compounds. Dethiobiotin synthetase (DTBS), the penultimate enzyme in the biotin biosynthetic pathway, catalyses the incorporation of a carbonyl moiety between the adjacent amino groups of 7,8-diaminononanoic acid (DAN), in a process utilising ATP and carbon dioxide. A phosphorus containing structural analogue of a postulated intermediate in this process has been synthesised. The study of the interactions between these compounds and the purified enzymes will enable the design and development of mechanism-based enzyme-inhibitors.

The naturally occurring secondary alcohols, known as the sterols, are classified biologically as zoosterols, phytosterols, and mycosterols, according to the source, animal, vegetable, or fungoid, from which they are obtained. The discovery that irradiation with short wave-length ultra-violet light confers powerful anti-rachitic properties on ergosterol, the principal member of the mycosterol sub-division, has given a great. impetus to the study of these compounds and of their derivatives. Mycosterols are mainly derived from the non-saponifiable matter in the fats of yeast and of ergot of rye, both of which contain complex mixtures of the individuals of this group. The separation of these mixtures has frequently been, attempted. and the isolation of many new sterols has been claimed, but the views of the various investigators are in such conflict that an independent and complete examination has become necessary.

Against the background of growing competition in the global marketplace, understanding customers, is a significant aspect of marketing. In the search for competitive advantage, there is a need to measure service quality to better understand its antecedentsa nd consequencesa, nd establish methods for its improvement. In the Libyan economy, the banking sector is one of the most important. Its significance increased after the 2003 lifting of the United Nations sanction. This was followed by entry to the sector of a number of domestic and multinational firms. Despite this increased competition, domestic banks are still widely considered to suffer from low levels of service quality. The main purpose of this study is to evaluate the actual level of service quality provided by Libyan public commercial banks as perceived by their customers. A modified SERVQUAL model was developed to measure service quality in Libyan commercial public banks. The resulting instrument is intended to help these banks to measure their service quality and focus on the service quality dimensions of most importance to their customers. It also aimed to gain an understanding of cultural and environmental influences on service quality in the Libyan banking sector, and their effect on banking management practices. It is also expected that this instrument, and its results, will contribute to future research into service quality. The findings of the present study have produced some important results. Firstly, the level of service quality offered by the Libyan public commercial banks as it was perceived by their customers was relatively high. Secondly, the theoretical five-factor structure of the SERVQUAL model was not confirmed in the Libyan banking context, and the service quality structure in the Libyan context appears to be four-dimensional. Furthermore, the study offers suggestions to banking managers to allocate their resources more efficiently to the most important dimensions, i. e. reliability and tangibles, to improve service quality, since the factor analysis indicates that these are the most important dimensions to customers. Finally, reflections on the methods used to modify SERVQUAL to make it more sensitive to a particular cultural context have implications for future researchersin terms of methodology, method and data analysisbiosynthesis pathway is present and essential for Acanthamoeba growth. We have cloned and sequenced the sixth enzyme in this pathway, imidazole glycerol-phosphate dehydratase (IGPD) (EC 4.2.1.19), from A. castellanii. This enzyme is the molecular target for 3-amino-1, 2, 4-triazole (3AT), a commonly used commercially available herbicide. The growth of A. castellanii was restricted by 3AT and this inhibition was ablated in a dose dependent manner by the addition of histidine to the medium. 3AT was also not toxic to RCE cells. This demonstrates that 3AT acts specifically to inhibit IGPD and that the histidine biosynthesis pathway is present in A. castellanii. This study provides the first evidence of the histidine biosynthesis pathway in a protozoan and demonstrates its potential as a target for therapeutic intervention during Acanthamoeba infection.

First, regulation of its subcellular localisation: the upstream activators of P-Rex1, Gβγs and PI(3,4,5)P₃, as well as its downstream effector Rac, are all intrinsically associated with the plasma membrane. Yet in resting cells P-Rex1 is mainly cytosolic. Here, it is shown that Gβγ and PI3K together synergistically cause a robust increase in membrane-localised P-Rex1. The isolated DH/PH domains of P-Rex1 are sufficient for synergistic Gβγ- and PI3K-driven membrane localisation. Furthermore, membrane-derived purified P-Rex1 has a higher basal activity than cytosol-derived P-Rex1. Second, regulation by, and of, protein binding partners: regulation of GEF activity can be achieved by interactions between GEFs and other cellular proteins. Few protein binding partners (aside from Rac) are currently known for P-Rex family GEFs. Five new potential P-Rex1 binding partners have been identified here. The interaction of P-Rex1 with one of these novel binding partners, neurochondrin, has been confirmed and further characterised. Coexpression of P-Rex1 and neurochondrin affects both their subcellular localisation and causes characteristic changes in membrane ruffling. Third, P-Rex1 GEF activity is known to be inhibited through phosphorylation by PKA. Here, an activating phosphatase for P-Rex1 has been identified for the first time, the serine/threonine phosphatase Protein Phosphatase 1α (PP1α). The interaction between P-Rex1 and PP1α as well as the PP1α-dependent dephosphorylation of P-Rex1 and the activation of P-Rex1 Rac-GEF activity by PP1α has been characterised both <i>in vitro</i> and <i>in vivo.</i>

The cell surface Notch receptor and its ligand, presented on adjacent cells, bind through their extracellular regions and lead to the release of Notch intracellular domain (NICD). NICD then translocates to the nucleus to form the Notch transcription activating complex. ANK domain of NICD plays an essential part in the formation of this complex and the role of ANK domain was analysed both structurally and functionally using site directed mutagenesis. A single residue E2072 on the sixth ANK repeat was found crucial to maintain the stability of the Notch transcription activating complex. It can therefore be considered as a putative “hot-spot” for the purpose of drug discovery. Recently the interaction of Neutralized (an E3 ubiquitin ligase) with Tom (a Bearded protein) was identified. This study was undertaken to understand and structurally characterise the Tom-Neuralized interaction. Expression constructs were designed using the information from sequence analysis of Tom and Neuralized proteins and expressed using bacterial expression system. The NHR1 domain, expressed as an N-terminal His-tag protein, was used to determine the crystal structure of NHR1 domain. The structure was found to be structurally related to SPRY proteins consisting of jelly roll topology. Potential binding sites were predicted that localised on a cluster referred as ‘hydrophobic patch’ in this study. The expression of Tom remained insoluble under most experimental conditions and therefore a 12 amino acid peptide was synthesized. Biophysical analysis showed the binding affinity between NHR1 and Tom was weak but still could form the complex. Comparison of NHR and SPRY domain proteins suggested that the binding interface of the two could be common indicating that the interaction of Delta could require the same binding interface of NHR1 domain as Tom. Lastly, interaction of NHR1 with PI4P was analysed and verified using interfacial studies, which showed that NHR1 protein localised below the lipidic layer.

P-Rex1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac that is primarily expressed in peripheral blood leukocytes and brain. P-Rex1 is directly and synergistically activated by both the phosphoinositide 3-kinase (P13K) product PtdIns(3,4,5)P₃ and Gbg subunits of heterotrimeric G-proteins. Rac is a member of the Rho family of monomeric GTPases and is involved in a variety of cellular processes. P-Rex1 has been shown to provide a major link between P13K heterotrimeric G-proteins and Rac in neutrophils. P-Rex1 is an 185kDa protein containing a typical catalytic DH (or GEF) domain and tandem PH domain, two DEP and two PDZ domains and significant homology over its C-terminal half to Inositol Polyphosphate 4-Phosphatase. A panel of P-Rex1 deletion, truncation and point mutants have been created to investigate the mechanisms of activation of P-Rex1 Rac GEF activity by PtdIns(3,4,5)P₃ and Gbg subunits. A P-Rex1 mutant lacking the PH domain (DPH) cannot be stimulated by PtdIns(3,4,5)P₃ which implies that the PH domain meditates the activation of P-Rex1 Rac GEF activity by PtdIns(3,4,5)P₃. Consistent with this, PtdIns(3,4,5)P₃ binds to the PH domain of P-Rex1 and the isolated DHPH tandem is sufficient for PtdIns(3,4,5)P₃-stimulated P-Rex1 Rac GEF activity. The Rac GEF activities of both the DPH mutant and the isolated DHPH tandem can be stimulated by Gbg subunits which implies that the catalytic DH domain mediates the activation of P-Rex1 Rac GEF activity by Gbg subunits. Deletion of the DEP, PDZ or the homology to Inositol Polyphosphate 4-Phosphatase domains has no major consequences on the abilities of either PtdIns(3,4,5)P₃ or Gbg subunits to stimulate P-Rex1 Rac GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac GEF activity, suggesting that there are important functional interactions between the DHPH tandem domains and the DEP, PDZ and homology to Ionsitol Polyphosphate 4-Phosphatase domains of P-Rex1.

No description available.

NMR spectroscopy and mass spectrometry based metabolomics has been used to investigate the function of PPARs in order to better define the role these receptors play in nutritional sensing, diabetes, obesity and the metabolic syndrome. Examining systemic metabolism in the PPAR-α null mouse and the interaction of phenotype with age, tissues from both fed and fasted mutant mice were characterised by a reduced concentration of glucose and impaired fatty acid oxidation (FAO). Both of these effects were most notable in the liver and increased with age. Additional changes in the PPAR-α null mouse were indicative of increased amino acid metabolism, impaired glucose metabolism regulation, myocardial damage, and substrate switching to maintain ATP homeostasis, the latter being particularly evident in muscle tissue. After a 24 hours fast, the PPAR-α null mouse demonstrated further abnormalities consistent with impaired ketogenesis, as well as increased lipogenesis and fatty acid accumulation in the liver and heart. PGC-1β is a transcriptional co-activator found primarily in highly oxidative tissues, and modulates the activity of many nuclear receptors, including PPAR-α and PPAR-γ. Surprisingly, the PGC-1β null mouse exhibited few signs of perturbed metabolism, except in the gastrocnemius where a perturbation in phosphocreatine concentration was observed in male mice aged 3 months. Finally, as deletion of nuclear hormone receptor (NHR)-49 in <i>C. elegans</i> causes disrupted lipid metabolism, a metabolomics functional genomic comparison was made between <i>nhr-49</i>and PPAR-α. The results demonstrate that <i>nhr-49</i> has a similar role to mammalian PPAR-α with both receptors regulating pathways of lipid synthesis, FAO, glycolysis and gluconeogenesis.

Phosphatidylinositol 4,5 bisphosphate (PI(4,5)P₂) plays a crucial role in cellular signalling. Functions include the generation of the key second messengers, inositol 1, 4, 5 triphosphate and diacylglycerol, as well as direct regulation of the activity of proteins that mediate cytoskeletal attachment, vesicular transport, ion-channel activation and enzyme activation. Despite multiple cellular functions, the total level of PI(4,5)P₂ shows minimal changes. This thesis aims at investigating the mechanisms by which the PI(4,5)P₂ levels are regulated in <i>Drosophila </i>photoreceptors. Two distinct aspects of PI(4,5)P₂ signalling were addressed. (i) Microvillar membrane extracts were used for <i>in vitro </i>binding assays to identify PI(4,5)P₂ binding proteins. The results define an experimental approach by which the identity of PI(4,5)P₂ binding proteins in photoreceptors can be revealed. (ii) We also studied the principal route by which PI(4,5)P₂ might be synthesised in <i>Drosophila </i>photoreceptors. To test the hypothesis that CG3682, the <i>Drosophila </i>putative Type I PI4P 5-kinase, performs its function in <i>Drosophila </i>photoreceptors, we generated a loss-of-function mutant of CG3682 by homologous recombination. Analysis by mass spectrometry showed that the pool of phosphatidylinositol monophosphates (PIP) was elevated in the mutant retinae, suggesting that this gene product regulates the conversion of PI4P to PI(4,5)P₂. This mutant showed a profound defect in the light response with a ca. 500 fold reduction in sensitivity to light compared to the wild type but minimal defects in photoreceptor ultrastructure. These findings together suggest that CG3682 contributes to the Type I PIPkin activity that underpins PI(4,5)P₂ synthesis during phototransduction.

No description available.