Le paludisme est causé par cinq espèces du genre Plasmodium, P. falciparum étant le plus mortel. Des résistances de certaines souches du parasite ont été rapportées pour tous les médicaments mis sur le marché. Les moustiques vecteurs du parasite sont résistants aux insecticides et aucun vaccin n'est disponible. Cette maladie est un problème économique et de santé publique pour les pays en voie de développement. Mes travaux de thèses visent à identifier de nouveaux traitements contre le paludisme, en ciblant deux nouvelles protéines. Les Apicomplexes ont développé un mécanisme unique d'invasion, impliquant une interaction forte entre la cellule hôte et la surface du parasite, appelée jonction mobile. La caractérisation structurale et fonctionnelle du complexe AMA1-RON2 a ouvert la voie à la découverte de petites molécules capables d'empêcher l'interaction AMA1-RON2 et de ce fait, l'invasion. Le parasite a aussi besoin de phospholipides pour construire sa membrane durant le cycle érythrocytaire. Il y a six fois plus de phospholipides dans les érythrocytes infectés que dans les érythrocytes sains. Notre stratégie est d'inhiber la voie de synthèse de novo Kennedy et plus précisément, son étape limitante catalysée par la PfCCT. Des filtres basés sur le ligand (LBVS) et sur la structure (SBVS) ont été utilisés pour tester virtuellement les chimiothèques commerciales que j'ai préparées. Pour chaque projet, des molécules ont été sélectionnées pour leurs scores de docking et les interactions qu'elles établissent avec les résidus clés de la protéine. En combinant la bioinformatique structurale et la chémoinformatique, nous avons identifié des inhibiteurs potentiels des deux cibles protéiques. / Human malaria is caused by five parasitic species of the genus Plasmodium, P. falciparum being the most deadly. Drug resistance of some parasite strains has been reported for commercial drugs. Vector mosquitoes are resistant to perythroid insecticides and no successful vaccine is available. This disease is a public and economic health issue for developing countries. My PhD projects investigate new treatments for malaria, by targeting two new proteins. Apicomplexa parasites have developed a unique invasion mechanism involving a tight interaction formed between the host cell and the parasite surfaces called Moving Junction. The structural and functional characterization of the AMA1-RON2 complex pave the way for the design of low molecular weight compounds capable of disrupting the AMA1-RON2 assembly and thereby invasion. The parasite also needs phospholipids to build its membrane during the erythrocytic cycle. There are six times more phospholipids in infected erythrocytes compared to healthy ones. Our strategy is to inhibit the de novo Kennedy pathway and more precisely its rate-limiting step catalysed by the enzyme PfCCT. Filters were used for ligand-based (LBVS) and structure-based virtual screening (SBVS) of commercial chemical databases that I have prepared. For each project, molecules were selected in terms of their docking scores and their interactions with key active site residues. By combining structural bioinformatics and cheminformatics, we identified potential inhibitors of the two protein targets.

T.gondii is a model organism of the phylum Apicomplexa that infects one third of the human population. While the majority of infections are asymptomatic or manifest with mild flu-like symptoms, toxoplasmosis can be fatal in immunocompromised individuals and in the developing fetus. The lytic cycle of tachyzoite-stage parasites causes damage to the host by repeated rounds of host cell invasion, intracellular replication and lysis of the host cell upon egress. Invasion is a key step for the parasite to maintain its intracellular lifestyle. Apical Membrane Antigen 1 (AMA1) is an adhesin released from a unique set of secretory organelles called micronemes. AMA1 plays a central role in the initial stages of host cell invasion. Although parasites without AMA1 are viable in culture, virulence in an animal model of infection is completely attenuated, highlighting AMA1's functional importance. AMA1 is a type I transmembrane protein with a large ectodomain and a short cytoplasmic tail. The ectodomain of AMA1 interacts with domain 3 (D3) of rhoptry neck protein 2 (RON2), which in turn complexes with RONs 4, 5, and 8 in the host cell. Together, this complex of proteins forms the moving junction, through which the parasite pushes itself during invasion. Rhomboid proteases on the parasite surface cleave AMA1 within its transmembrane domain and parasites expressing a non-cleavable form of AMA1 show reduced invasion of host cells and a growth defect. While much is known about the ectodomain of T. gondii AMA1 (TgAMA1), the fate of the TgAMA1 cytoplasmic tail after cleavage remains unclear, its interacting partners remain unidentified, and its role in invasion or thereafter remains a mystery. To address these questions, we: (a) explored the consequences of TgAMA1-TgRON2 interaction during invasion and (b) generated allelic replacement (AR) parasites with point mutations across the tail of TgAMA1 to determine the effect of these mutations on the parasite's ability to invade host cells. Quantitative proteomic techniques were used to analyze the proteins that bind to the tail of TgAMA1 under these different experimental conditions. The results from this work highlight the importance of TgAMA1 post-translational modifications, and potentially TgAMA1-binding proteins, in regulating invasion-related processes in T. gondii.

Pre-mRNA splicing is a vital process of gene expression important for all eukaryotic organisms. For the proper function of this very complex and dynamic event the presence of few specialized RNA and many proteins that hold a variety of tasks is necessary, not only inside the splicing complex itself, but also beyond this complex. The Prp45 is one of the proteins involved in pre-mRNA splicing in yeast Saccharomyces cerevisiae. Its human homologue, SNW1/SKIP, is involved in splicing but also in other crucial cell processes. The Prp45 protein was reliably reported only to participate in the second transesterification reaction of splicing. But there are also data suggesting its possible involvement in the first transesterification reaction. This work provides further evidences linking protein Prp45 with the first splicing reaction, obtained by the research of cells carrying the mutant allele prp45(1-169). Cells carrying this allele show dropped splicing and accumulation of pre-mRNAs. This thesis therefore also investigated the possible influence of Prp45 protein on the RNA export from the nucleus to the cytoplasm. But no connection between this protein and RNA transport was discovered. Keywords pre-mRNA splicing; Saccharomyces cerevisiae; Prp45; Mer1; Mud2; Prp22; Rrp6; AMA1; SNW1/SKIP