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The glycerophosphoinositols (GPIs) are ubiquitous, bioactive metabolites that are produced by the phospholipase A2 IVa activity on the membrane phosphoinositides. Glycerophosphoinositol (GroPIns) and glycerophosphoinositol 4-phosphate (GroPIns4P) are the most active and well studied of the GPIs. When added exogenously, the GPls can enter cells and have multiple effects, such as modulation of actin cytoskeleton organisation in fibroblasts and reduction of the invasive potential of metastatic cells. To gain insights into the molecular mechanisms of the effects of the GPIs, I set out to identify their protein targets. Therefore, a proteomic approach based on high- throughput differential-LC-MS/MS analysis was used with the modified GPIs: with a biotin moiety bound to their glycerol backbone. The targets identified include proteins involved in cell signalling, cytoskeleton organisation, protein folding and metabolic processes. Among these, I focussed my attention on Src-homology phosphatase-l (Shpl), a well-known regulator of Src activation, as it might be related to the reported signalling pathway leading to GroPlns4P-mediated modulation of the actin cytoskeleton, which involves Src. Based on biochemical data, I provide evidence of a direct interaction between Shp 1 and both GroPIns4P .and GroPIns. During my project, Shpl was studied in the context of GroPIns4P-induced membrane ruffle formation in NIH 3T3 fibroblasts, where inhibition of enzymatic activity of Shp 1 completely abolished GroPIns4P-mediated reorganisation of the actin cytoskeleton. In addition, I have also shown that GroPIns4P treatment results in the dephosphorylation of the inhibitory tyrosine residue of Src, as a consequence of increased binding between Shp 1 and Src. A role for Shp 1 is also demonstrated in GroPIns-mediated inhibition of tumour cell invasion. In A375MM melanoma cells, GroPIns treatment results in inhibition of extracellular matrix degradation, and this activity is suppressed when the inactive mutant form of Shp 1 is expressed, while it is essentially unaffected by expression of the native enzyme. In agreement with these results, a lack of effect of GroPIns was observed also in Shpl knock-down cells. In conclusion, my PhD project has led to the definition of Shpl as the first direct GPI target that has been identified to date, and it reveals a positive role for Shpl in the mechanisms of action of both GroPIns4 P and GroPIns.

Cytochrome P450 3A represents the most abundant P450 subfamily in human liver, and is responsible for the metabolism of >50 % of drugs on the market today. An area of intense study has been the xenobiotic regulation of CYP3A gene expression: this phenomenon potentially leads to altered pharmacokinetics/pharmacodynamics of substrates and adverse drug reactions. The use of hepatoma cell lines is common in drug metabolism studies; HuH7 is a human hepatoma cell line increasingly being used in both academia and the pharmaceutical industry. However, little is known regarding the expression profile of these cells. Recent data have made it increasingly clear that the gene expression profile of a cell system, and its alteration in response to external stimuli, is important in controlling expression of cytochrome P450 3As. Using quantitative PCR, transcript levels of CYP3A subfamily members and transcription factors implicated in their regulation were obtained in human adult and foetal RNA samples. These levels were then compared to those obtained from both primary human hepatocytes and HuH7 cells, showing that in general, primary or hepatoma cells show a distinct profile compared to either adult or foetal samples, with the major variables between the cell types being ratios of the transcription factors COUP-TF1 and RXRalpha to PXR. Whereas exposure to the CYP3A transcriptional activators PCN, rifampicin, dexamethasone and phenobarbital produced induction in primary human hepatocytes, no transcriptional activation was observed in HuH7 cells, suggesting that regulatory pathways were disrupted. Alterations in the gene expression profile, particularly the levels of COUP-TFl and PXR, and in the chromatin conformation surrounding the CYP3A gene cluster, were implicated to underlie this disruption. In conclusion, I have demonstrated that there are a number of levels of control of CYP3A gene expression, with both transcription factor expression levels and chromatin status having an impact on transcriptional activation of CYP3A genes in human hepatocytes.

The ability to digest significant quantities of the dissacharide lactose is dependent upon high expression of the enzyme lactase in the small intestine. Downregulation of lactase occurs in the vast majority of adult mammals, and most humans lose the ability to produce high levels of lactase after weaning (lactase non-persistence) whereas others keep high levels into adult life (lactase persistence). This variation in adult enzyme expression is under genetic control, and frequencies of the two phenotypes vary in throughout the world. A correlation between a longstanding culture of dairying and a high frequency of lactase persistent individuals in a population group has been reported. This has led to the theory that the high levels of lactase persistence seen in some groups could be the result of natural selection. This thesis examines variation in and around the lactase gene in to explore the possible role of natural selection in explaining modern frequencies of lactase persistence. A series of single nucleotide polymorphisms (SNPs) in the vicinity of the lactase gene have alleles known to associate with lactase persistence in Northern Europe. These were investigated in a series of4024 individuals from 73 population groups throughout the world to generate a global distribution that could be compared with anthropological data, and previously reported frequencies of lactase persistence. One of these SNPs, a C-T transition located - 13.9kb upstream of the lactase gene, was reported during the course of this thesis, (Enattah et al 2002), with the T allele proposed as a putative cause of lactase persistence. The haplotypic background of this allele is reported here.

Phosphoinositide 3-kinases (PI3Ks) are a family of signal transduction enzymes which generate lipid second messengers (1) and control a wide variety of cellular processes such as growth, proliferation, survival, differentiation, intracellular traffic, cytoskeletal organisation and cell migration (1-7) Mammals have 8 isoforms of PI3K, most of which have poorly defined individual roles in cells and in the organism. Deregulated PI3K signalling has been implicated in cancer, inflammation and diabetes, and PI3Ks are being pursued as new therapeutic targets by the pharmaceutical industry. Therapeutic intervention with PI3K will almost certainly have to be targeted at individual isoforms, given the risk that global alteration of PI3K signalling will be deleterious to the organism. This work focuses on the pi 108 isoform of PI3K. pi 105 belongs to the class IA subset of PI3Ks which signal downstream of tyrosine kinase receptors and Ras. These PI3Ks are heterodimers that consist of a pi 10 catalytic subunit and a regulatory subunit. Mammals have genes for 3 catalytic subunits, called pi 10a, pi 10(3 and pi 105. Whereas pi 10a and pllOp show a broad tissue distribution, pi 105 is more restricted, with the highest levels in leukocytes (8) There is mounting evidence for a non-redundant function of these pi 10 isoforms (7,9). Studies using mice have implicated pi 105 in immune signalling, making it an interesting new target for anti-inflammatory therapies. More recent work (7) also indicates a role for pi 105 in cancer. This thesis studies the mechanism of tissue specific distribution of pi 105 at the mRNA and protein level. Using Real-Time reverse transcriptase PCR we have demonstrated that regulation of pi 105 expression occurs at the level of transcription. Using two different approaches, 5' RACE (Rapid Amplification of cDNA Ends) and reverse transcriptase PCR, we have also discovered different transcription start sites in the pi 105 mRNA, leading to three discrete mRNAs which contain distinct 5' untranslated exons. In a further stage of the work, we have found evidence that differential usage of these 5' untranslated exons correlates with the distinct tissue distribution of the pi 105 protein. Our findings imply that there are at least 3 distinct promoters for the pi 105 gene. Efforts to delineate the promoter elements using reporter assays and deletion analysis have given ambiguous results, and have not lead to the desired insight into the regulation of the pi 105 promoters and their regulation in different tissues.

Les 3-mercaptopyruvate sulfurtransférases (3-MST) et les thiosulfate sulfurtransférases (TST) sont des enzymes ubiquitaires de la famille des thioltransférases à domaine rhodanèse qui catalysent le transfert d’un atome de soufre d’un substrat donneur vers un substrat accepteur via un intermédiaire Cys-persulfure. Les 3-MST sont impliquées dans la formation de sulfure d’hydrogène (H2S), un gazotransmetteur toxique à forte concentration, alors que les TST interviendraient dans son élimination. L’objectif de mon projet était de décrypter les mécanismes moléculaires impliquant ces thioltransférases afin de mieux comprendre leurs rôles physiologiques. Pour cela, le mécanisme catalytique et les spécificités de substrats des enzymes humaines (3-MST, TSTD1 et Rhodanèse) et d’Escherichia coli (3-MST et GlpE) ont été caractérisés grâce à la mise au point de méthodes spécifiques permettant l’étude de chacune des étapes du mécanisme (fluorescence, stopped-flow, sonde H2S) et par une étude des relations structure-fonction menée en collaboration pour les aspects chimie théorique et cristallographie RX. J’ai montré que le site actif de ces enzymes est adapté à la catalyse d’un transfert de S0 à partir de composés soufrés non activés. De plus, le mécanisme de formation de l’intermédiaire persulfure ne dépend pas de l’enzyme mais du substrat donneur. En effet, la rupture de la liaison C-S du 3-mercaptopyruvate requiert la déprotonation des fonctions thiols du substrat et de la Cys essentielle, fonction assurée par la boucle catalytique CysX5 qui constitue un véritable site de reconnaissance thiolate, et l’intervention concomitante d’une molécule d’eau comme catalyseur acide. En présence de thiosulfate, hormis l’activation de la Cys seule la neutralisation des charges négatives du substrat est indispensable à la réaction de transfert de soufre. Enfin, et de façon inattendue, la 3-MST humaine pourrait être impliquée dans l’élimination cytosolique du sulfite, un composé toxique pour les cellules. Quant aux deux TST mitochondriales humaines, elles pourraient intervenir à la fois dans la signalisation cellulaire H2S-dépendante, via la production d’espèces polysulfure, et dans l’élimination d’H2S / 3-mercaptopyruvate sulfurtransferases (3-MSTs) and thiosulfate sulfurtransferases (TSTs) are ubiquitous enzymes that belong to the rhodanese sulfurtransferase family and catalyze the transfer of a sulfur atom from a donor to an acceptor substrate via a cysteine-persulfide intermediate. While 3-MSTs are involved in the biogenesis of hydrogen sulfide (H2S), a gasotransmitter known to be toxic at high concentration, TSTs are likely responsible of its degradation. My project mainly focused on deciphering the sulfurtransferase-dependent molecular mechanisms to better define their physiological functions. To address these questions, their catalytic mechanisms and substrate specificities were investigated. This was achieved through the development of kinetic approaches (fluorescence, stopped-flow, H2S specific probe) to study each step of the reaction catalyzed by human (3-MST, TSTD1 and Rhodanese) and Escherichia coli (3-MST, GlpE) enzymes and structure-function relationship studies performed in collaboration for the theoretical chemistry and the X-ray crystallography parts. Here, I show that the active site of these enzymes is optimized to perform an efficient S0 transfer from non-activated sulfur compounds. Moreover, the mechanisms leading to formation of the persulfide intermediate do not depend on the enzyme but rather on the donor substrate. Indeed, the cleavage of the carbon-sulfur bond of 3-mercaptopyruvate critically depends on the CysX5 catalytic loop acting as a thiolate hole to favor the deprotonation of the essential Cys and the substrate, and on a water-mediated protonation step. In the presence of thiosulfate, the Cys activation mode remains unchanged and the reaction of sulfur transfer is only driven by the neutralization of the negative charges of the substrate. In addition, we propose a new physiological function for the human 3-MST in the cytoplasmic elimination of sulfite, a toxic compound for the cells. Finally, the two human mitochondrial TSTs are likely to be involved in the H2S-mediated cellular signaling, through the formation of polysulfide entities, but also in H2S catabolism