La famille des Venus Kinase Receptors (VKR) est une nouvelle famille de Récepteurs Tyrosine Kinase découverte au laboratoire chez le ver parasite Schistosoma mansoni (SmVKR1), puis chez 14 autres organismes invertébrés, principalement des insectes. Les études réalisées au laboratoire ont montré la présence des transcrits vkr dans les organes reproducteurs des organismes adultes mais aussi dans les stades larvaires suggérant un rôle dans le développement et/ou la reproduction de ces organismes. Ma thèse concerne la caractérisation structurale, biochimique et fonctionnelle des VKRs de deux organismes d’intérêt sanitaire majeur : le schistosome, parasite responsable de la bilharziose qui représente la seconde endémie parasitaire mondiale et l’anophèle, vecteur principal du paludisme en Afrique. Dans une première partie nous avons mis en évidence la présence d’un second VKR chez le schistosome, nommé SmVKR2. Nous avons montré que ses transcrits sont exprimés à tous les stades du cycle parasitaire et principalement dans les organes génitaux de la femelle au niveau des ovocytes immatures et de l’ootype. Nous avons exprimé SmVKR1 et SmVKR2 et montré qu’ils sont tous deux des récepteurs à activité tyrosine kinase, activables respectivement par la L-Arginine et par le calcium. Des expériences complémentaires semblent indiquer qu’un ligand naturel de SmVKR1 pourrait se trouver dans le canal gynécophore des vers appariés. Parallèlement, nous avons entrepris l’identification des partenaires cytosoliques de SmVKR1 et SmVKR2 grâce au criblage d’une banque d’ADNc de vers adultes par la technique de double hybride en levure en utilisant les domaines intracellulaires (DIC) des récepteurs comme appâts. L’analyse partielle des résultats obtenus montre que les DIC de SmVKR1 et SmVKR2 interagissent en autres avec des protéines du cytosquelette et avec des protéines cytoplasmiques pourvues de domaines d’interaction protéine-protéine SH2, acteurs de voies de signalisation classique des RTKs. Nous avons montré par ARNi que la diminution des transcrits SmVKR1 et SmVKR2 a un impact majeur sur la morphologie des organes génitaux de la femelle. Les sporocystes interférés pour les deux récepteurs présentent une diminution significative de leur taille comparée aux témoins.La deuxième partie de mes travaux de thèse a été centrée sur l’étude d’AgVKR, le récepteur d’Anopheles gambiae. En utilisant deux systèmes d’expression hétérologue, nous avons pu établir que ce récepteur était lui aussi catalytiquement actif et activable par la L-arg comme SmVKR1. L’étude de la fonction d’AgVKR a été abordée grâce à la découverte récente de son expression constitutive dans une lignée de cellules d’A. gambiae nommées SuA5B et de type hémocytaire. Des expériences d’ARNi ont été mises au point dans le but de visualiser l’impact d’une diminution de transcrits sur la physiologie des cellules. Dans leur ensemble, les résultats de ces travaux participent à la compréhension des mécanismes de régulation et de la fonction des VKRs, des récepteurs qui semblent d’une grande importance pour le développement et la reproduction des organismes. / Venus Kinase Receptors (VKRs) form a new family of Receptor Tyrosine Kinases discovered for the first time in the parasite Schistosoma mansoni (SmVKR1), then in fourteen other invertebrate organisms and mainly in insects. In our laboratory, previous studies have shown that vkr transcripts are present in reproductive organs of adult organisms but also in larval stages, suggesting a role of VKRs in reproductive and development mechanisms. My thesis was concerned with the structural, biochemical, and functional characterization of the VKRs of two invertebrate organisms causing serious public health concerns: the worm S. mansoni responsible for the second human parasitic disease, and the principal malaria vector in Africa, Anopheles gambiae. At first, we have shown the existence of SmVKR2, a second VKR in the worm. Its transcripts are expressed in all the parasitic stages and localized in the immature oocytes and the ootype of the female worm. Recombinant SmVKR1 and SmVKR2 proteins showed a tyrosine kinase activity in vitro. Their catalytic activity could be induced by small molecules such as L-Arginine for SmVKR1 and calcium ions for SmVKR2. Preliminary experiments showed the presence of a potential natural ligand inside of the gynaecophoral duct of paired worms, able to activate SmVKR1 but not SmVKR2. To identify cytoplasmic partners of SmVKR1 and SmVKR2, we used intracellular domains (ICD) of each receptor as baits to screen an adult worm cDNA yeast library. We could show that SmVKR1 and SmVKR2 ICDs interact with various proteins, including cytoskeleton components and proteins containing SH2 protein-protein interaction domains, known to participate in classical signalling pathways of RTKs. We have also shown by RNAi that the diminution of smvkr1 and smvkr2 transcripts results in major changes in the morphology of genital organs of female worms. In the sporocyst larvae, RNAi of both SmVKR1 and SmVKR2 led to a significant decrease of the size of the parasites, as compared to the controls.The second part of my thesis work concerned the study of AgVKR from A. gambiae. Using two different expression systems, we have established that AgVKR was also catalytically active and activable by L-Arg as was SmVKR1. Functional studies of AgVKR could be facilitated by the recent discovery that the hemocyte-like SuA5B cell line of A. gambiae are constitutively expressing AgVKR. RNAi procedures have been designed to analyse the impact of a diminution of agvkr transcripts on the physiology of SuA5B cells.Taken together, these results already participate in a better knowledge of the mechanisms of VKR regulation and of their function, confirming their potential importance in growth and reproduction of invertebrate organisms.

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Housekeeping enzymes are vital to the metabolism of all plant cells. Two such enzymes adenosine kinase (ADK) and S-adenosylhomocysteine (SAH) hydrolase share a similar function: both sustain S-adenosylmethionine-dependent methylation reactions by removing inhibitory by-products. SAH hydrolase breaks down SAH which is a competitive inhibitor of all methyltransferase activities. ADK phosphorylates the Ado produced by SAH hydrolase and in doing so drives this reversible reaction in the hydrolysis direction. By catalyzing the phosphorylation of Ado into adenosine monophosphate, ADK not only prevents SAH from re-forming but also initiates the recycling of Ado into adenylate nucleotides and cofactors. This thesis documents two distinct research topics related to methyl recycling in plants. The first goal was to identify ADK-deficient mutants to establish the contribution of this enzyme activity to adenosine salvage. The second goal was to determine the subcellular localization of ADK and SAH hydrolase in Arabidopsis thaliana (Columbia). T-DNA insertion lines for highly similar ADK isoforms (ADK1 and ADK2) and silencing lines of overall ADK activity (sADK) were compared to WT Arabidopsis to identify phenotypic abnormalities associated with ADK deficiency. In addition to following their growth, microscopic analysis was performed on the sADK lines. While removal of either ADK1 or ADK2 had no phenotypic effect, lowering ADK levels to 6-20% that of WT lead to several changes including small, wavy rosette leaves, delayed leaf senescence, decreased internode length, reduced branching, clustered inflorescences and lack of petal abscission and silique dehiscence. Further analysis linked the abnormal phenotype to increased levels of hypomethylation throughout the plant (K Engel unpublished data), as was expected; however higher levels of active cytokinin were also observed. Thus ADK appears to be integral in regulating cytokinin levels as well as recycling methylation intermediates. To investigate the relationship between the subcellular localization of SAH production and its metabolism, immunogold labelling was performed on leaf and meristematic tissues of Arabidopsis using antibodies specific for either ADK or SAH hydrolase. As well, ???-glucuronidase and green fluorescent protein translational fusions of each enzyme were examined (S. Lee unpublished data). Results of both the immunogold labelling and fusion lines revealed that all ADK and SAH hydrolase isoforms localize to the cytosol, chloroplast and nucleus. Further analysis of purified chloroplasts has given varying results regarding the targeting of these enzymes to the organelle, and further research will be required before ADK and SAH hydrolase can be conclusively localized to the chloroplast.

Yung, Yuk Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 65-73). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Sou, Weng Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 96-106). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.