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Caractérisation moléculaire des Récepteurs Venus Kinase : étude fonctionnelle chez le parasite Schistosoma mansoni

Abstract : 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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Nadège Gouignard. Caractérisation moléculaire des Récepteurs Venus Kinase : étude fonctionnelle chez le parasite Schistosoma mansoni. Médecine humaine et pathologie. Université du Droit et de la Santé - Lille II, 2011. Français. ⟨NNT : 2011LIL2S034⟩. ⟨tel-00769951⟩

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