Inositol phosphates (IPs) are important signalling molecules with various biological roles in a cell. One such role it is often associated with is DNA repair. The DNA repair process following DNA insult is considered crucial for the genomic integrity and stability. Failure to perform this task will result in mutations and possibly disease. Thus, it is important that we expand our knowledge on how these repair processes occur and identify the key factors involved in its regulation. The aim of this project was to investigate whether DNA repair was mediated by inositol diphosphates (IDPs). Using a family of yeast knockout mutants with modulated levels of IPs, it was found that IDPs are crucial in repair of DNA following insult with bleomycin and 5-fluorouracil. The observed sensitivity of the mutants was thought to be due to lack of functional repair protein, UDG-like or APE-like, in the absence of essential cofactor such as IDPs. Experiments conducted revealed that the hypersensitive kcs1Δ contain both the repair proteins required to process the DNA lesions. However, extreme extraction methods were required to access these proteins, suggesting that the proteins are mislocalised and unavailable to access the damage site and perform DNA repair. GFP-tagging the proteins Ung1, Apn1 and Rad52 in kcs1Δ proved to be of little use as it failed to show exact localisation, movement and functionality status of these proteins following bleomycin insult. The enzymes accountable for the dephosphorylation of the IDPs in vivo are the diphosphoinositol polyphosphate phosphohydrolases (DIPPs). Little is known regarding the Michaelis-Menten kinetics parameters for Ddp1p/DIPPs. In this study, using improved methods for the enzymatic synthesis and electrophoretic purification of 1-InsP7, 5-InsP7 and InsP8, the DIPP family has been kinetically characterised. Each DIPP was found to ii display similar Km values for every substrate tested (range: 35-148 nM). The rank order of Kcat values (1-InsP7 > 5-InsP7 = InsP8) was identical for each enzyme, although DIPP-1 activity was observed to be 10- to 60-fold more than DIPP-2α/β and DIPP-3α/β, irrespective of the substrate. This study reveals that Ddp1, the yeast DIPP, is capable of hydrolysing not only 5-InsP7 but also 1-InsP7 and InsP8 to a single product, InsP6. The HPLC data found InsP7 accumulation to be relatively little during InsP8 breakdown by DIPPs. Such low build-up was found to be due to rapid conversion of InsP7 to InsP6. Through this study it is also clear that InsP8 prefers to dephosphorylate through 1-InsP7. In contrary, metabolically and functionally significant steady-state route of InsP8 synthesis was observed to be via 5-InsP7. Oesophageal cancer is considered as one of the deadliest cancers worldwide because of its aggressive nature and low survival rate. Epidemiologic studies have shown that low-dose daily intake of aspirin can decrease the incidence of oesophageal cancer. The data presented in this study show the effects of a number of in-house synthesized novel aspirin analogues on oesophageal cancer cell lines, squamous cell carcinoma (SSC) and adenocarcinoma (ADC). The aspirin analogues, fumaryldiaspirin (PN517) and benzoylsalicylates (PN524, PN528 and PN529), were observed to be more potent against the oesophageal cell lines than aspirin itself. Both, quantitative and qualitative apoptosis experiments conducted revealed that these compounds largely induced apoptosis, although some necrosis was evident with PN528 and PN529. Failure to recover following the treatment with these analogues emphasized that these drugs are largely cytotoxic in nature. The SSC cells (oe21) displayed increased sensitivity to the aspirin analogues compared to the ADC cell lines (flo-1 and oe33). The anticancer properties of these novel aspirin compounds appear to not involve the COX-enzymes at the tested concentrations. These initial findings support further studies into the potential of these aspirin analogues as chemotherapeutic agents against oesophageal cancer.

L’étude des inositols hautement phosphorylés est un domaine en pleine expansion. Leurs essors ne datent que d’une dizaine d’années, mais de nombreuses fonctions y sont déjà associées bien qu’ils en restent sans doute encore à découvrir. Les inositols phosphates (incluant les inositols hautement phosphorylés) s’inscrivent dans un cycle dont le représentant le plus connu est inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). De ce fait, chaque inositol phosphate influence directement ou indirectement les autres membres de ce cycle.<p>Au cours de la thèse, nous avons pu éclaircir une controverse de la littérature sur la voie de synthèse des inositols hautement phosphorylés. Grâce à un modèle de cellules MEF (mouse embryonic fibroblast) n’exprimant aucune des trois isoformes de l’inositol 1,4,5-trisphosphate 3-kinase (ITPK) et à l’aide des cellules souches déficientes pour l’inositol polyphosphate multikinase (IPMK), nous avons pu révéler le rôle majeur de cette dernière dans la génération de l’InsP5 et l’InsP6.<p>Dans un second temps, nous avons comparé la neurogenèse de ces cellules souches IPMK+/+ et IPMK-/- mises dans un milieu de différenciation par défaut (DDM). Les cellules dépourvues de l’IPMK entrent en apoptose et se différencient très difficilement en progéniteurs neuronaux et en neurones. Afin de comprendre le mécanisme sous-jacent pouvant expliquer ce phénomène, nous avons réalisé des PCRs quantitatives qui ont montré une sous expression des gènes du neuroectoderme ainsi qu’une augmentation de l’expression de gènes du mésoderme dans les cellules IPMK-/- par rapport aux cellules IPMK+/+. De plus, nous avons découvert que le phénomène d’apoptose observé au cours de la différenciation en DDM était spécifique à ce milieu. En effet, nous n’avons pas pu mettre en évidence un tel phénomène au cours de la différenciation en corps embryoïdes.<p>Durant la thèse, nous avons également développé des anticorps dirigés contre l’isoforme B de l’inositol 1,4,5-trisphosphate 3-kinase et contre la forme native de l’IPMK. Ceci nous a permis de mener à bien nos expériences et d’ouvrir de futures perspectives de recherche.<p>En conclusion, nous avons démontré le rôle majeur de l’IPMK dans la voie de synthèse des inositols hautement phosphorylés. Nous avons également découvert que l’IPMK est très importante pour la survie de ces cellules souches en cours de différenciation et nous avons également introduit une nouvelle fonction pour l’IPMK dans la neurogenèse.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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The ability for an organism to sense and respond appropriately to its environment is often critical for survival. One mechanism for this is the inositol phosphate (InsP) signaling pathway. This work focuses on the role of InsP signaling in maintaining energy homeostasis in the plant. InsP signaling is connected to energy sensing in plants via a protein complex containing both the inositol polyphosphate 5-phosphatases (5PTase13) and the Sucrose non-Fermenting Related Kinase 1 (SnRK1). SnRK1 is considered a fuel gauge for the plant cell that senses energy status and reprograms growth appropriately. While the SnRK1.1 gene has been well studied, the role other SnRK1 isoforms play in energy or stress signaling is less well understood. This work examined the role of 3 SnRK1 isoforms in energy signaling, finding that SnRK1.1 and SnRK1.2 are regulated and function differently in Arabidopsis. The second part of this work focuses on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e. PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals. Even though plants produce huge amounts of InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. This work details the presence of PPx-InsPs in plants and delineates two Arabidopsis gene products (AtVip1 and AtVip2) capable of PP-InsP5 synthesis. We further examined the subcellular location of enzymes connected to PPx-InsP synthesis as well as the developmental and tissue specific patterns of expression of the genes that encode these enzymes. We localized the enzymes involved in InsP6 and PPx-InsP production to the nucleus and endoplasmic reticulum (ER). The subcellular compartmentalization of PPx-InsP signaling may be unique to plants. An increased understanding in the pathways involved in energy sensing and metabolic response may reveal novel strategies to improve crops for yield and viability in the future. / Ph. D.