Orientador: Paulo Eduardo de Abreu Machado / Resumo: Com o objetivo de contribuir para o esclarecimento de alguns pontos obscuros nas relações existentes entre a atividade da via das pentoses e a redução das metahemoglobina pela cistamina, em indivíduos normais e doentes com malária, causada pelo Plasmodium falciparum, bem como a relação entre a atividade da glutationa redutase e o efeito do tratamento pela riboflavina, foi proposto novo método de pesquisa pouco dispendioso, simples e que permitisse aplicação em estudo de campo. Com o método padronizado, foram estudados indivíduos normais de Botucatu, Amazônia e Anhembi e doentes com malária causada pelo Plasmodium falciparum, atendidos em Humaitá... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Click electronic access below. / Doutor

The Plasmodium falciparum parasite is the causative agent of the most severe form of malaria. The increase in resistance against the majority of antimalarial compounds underpins the need for the development of new antimalarial compounds, targeting novel biological activities of the parasite. As the P. falciparum parasite develops through its life cycle stages, the parasite is exposed to different environments, resulting in both strategy-specific differences between the asexual (proliferation) and gametocyte (differentiation) stages, as well as stage-specific (i.e. ring – schizont stages; stage I - V gametocytes) differences within each strategy. These strategy- and stage-specific differences might be supported by the presence of different membrane transport proteins (MTPs) in the asexual and gametocyte stages. P. falciparum-encoded MTPs (permeome) are promising novel drug targets because they are specific to P. falciparum and essential for the survival of the P. falciparum parasite as these proteins mediate the uptake and removal of metabolites and waste products. However, to propose parasite-encoded MTPs as potential novel drug targets in the asexual and gametocyte stages, the presence of these MTPs in these stages should be investigated. The P. falciparum-encoded permeome is well characterised in the asexual stages. However, limited knowledge is available about the permeome in the gametocyte stages. Therefore, to address this knowledge gap, the strategy- and stage-specific expression of the entire complement of parasite-encoded MTPs were investigated in the asexual and gametocyte stages to infer the presence of MTP transcripts in the absence of biochemical uptake data. The transcript expression of the permeome revealed strategy-specific expression, with the entire permeome expressed during asexual stages, as expected, given the metabolic adaptations that support the high proliferation rate. By contrast, the gametocyte stages that are undergoing sexual differentiation towards transmission, as opposed to active proliferation, less than half of the permeome were expressed, indicating a reduced range of MTPs active in the gametocyte stages. Subsequently, stage-specific expression of the permeome was investigated by correlating stage-specific metabolic processes that occur within the asexual and gametocyte stages, to the expression profiles of MTP genes involved in these processes. Most of the MTPs involved in these processes showed stage-specific expression, with a few MTP genes showing no stage-specific expression within the asexual and gametocyte stages, respectively. When comparing the stage-specific expression between the asexual and gametocyte stages, it was observed that during the gametocyte stages, there was an absence of some MTPs (decreased expression) that were expressed during the asexual stages, suggesting that the gametocyte stages require only certain metabolites to maintain the investigated metabolic processes. In conclusion, these expression profiles of the permeome in the asexual and gametocyte stages suggest the differential expression of the permeome in these stages. The data presented in this study provides the first complete evaluation of expression of the permeome across P. falciparum asexual and gametocyte stages and serves as a blueprint for future biochemical investigations of transport in these stages, thereby providing a foundation for identifying novel MTP drug targets in future drug development programmes. / Dissertation (MSc)--University of Pretoria, 2018. / NRF / Biochemistry / MSc / Unrestricted

A hallmark trait of P. falciparum malaria is sequestration, in which parasite infected erythrocytes (IEs) adhere to the vasculature, causing organ failure and death. Current antimalarials only kill the parasites, necessitating development of anti-adhesion drugs. Using our two-step approach, we can efficiently screen for anti-adhesion small molecules. Screenings of 75libraries using Bio-Plex 200 identified the most active TPI libraries, which were deconvoluted to single compounds. Screenings library TPI 1319 yielded 3 inhibiting non-optimized compounds, each of which inhibits binding between two receptors, CSA and ICAM1, and their binding PfEMP1 domains. Two compounds deconvoluted from TPI 2103 prevent binding between PfEMP1 and ICAM1. Cytoadhesion assays with live IEs support the results seen with Bio-Plex, with best hits showing inhibition below 200 nM. Cytotoxicity testing of active compounds showed minimaltoxicity. Identified hits appear to be amenable to Structure Activity Relationship studies to develop powerful anti-adhesion drugs to treat severe malaria. / Includes bibliography. / Thesis (MS)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Includes bibliographical references / [No subject] Malaria infection caused by the apicomplexa pathogen Plasmodium falciparum has a high rate of resistance to existing anti-malarial drugs. The World Health Organisation recommended interventions are unlikely to eliminate the growth of resistance and it would therefore be prudent to continue the search for new drug targets for the continued combatting of malaria. Plasmodium falciparum is parasitic on the host for its metabolites and therefore inhibiting the transportation of glutamine from the host, has long been considered a potential strategy for combating the spread of infection. The recently sequenced Plasmodium falciparum genome has however shown that pathways for independent survival are also conserved. Therefore, combating the spread of Plasmodium falciparum in the human host, in addition to inhibiting the transportation of glutamine, will also require the inhibition of the de novo expression of essential amino acids within the Plasmodium falciparum cell. This could be achieved by inhibiting the glutamine synthetase gene, which is an essential step in the tri-carboxylic acid cycle.

Introducción: Malaria representa una emergencia médica debido a la posible complicación y muerte del paciente cuando este no fue tratado apropiadamente. Malaria severa y/o complicada (MSC) es causada casi exclusivamente por Plasmodium falciparum. Uno de los factores de riesgo asociado con MSC es el tratamiento inadecuado de los casos de malaria no complicada (MNC). Objetivos: Se genotipificó a los genes dihidrofolato reductasa (Pfdhfr) y dihidropteroato sintasa (Pfdhps) en muestras de 60 pacientes con MSC. La resistencia al tratamiento combinado sulfadoxina-pirimetamina (SP) es causado principalmente por mutaciones puntuales en esos genes Diseño de estudio: Los pacientes con MSC de este estudio fueron enrolados durante el brote de malaria de 1998, cuando SP era la primera línea de tratamiento. Materiales y métodos: Se usó el método de secuenciamiento de Sanger para la identificación de los polimorfismos en el gen Pfdhfr y los métodos PCR-RFLP y PCR alelo-específico para el gen Pfdhps. Resultados: Se encontró que el 84% de las muestras tenían el genotipo del parásito cuádruple mutante N51I/S108N/I164L/inserción repetición Bolivia, y el 16% restante el genotipo mutante simple S108N. Con respecto al gen Pfdhps, encontramos cuatro genotipos, siendo el triple mutante A437G/K540E/A581G el más frecuente (78%). Conclusiones: Observamos que las mutaciones I164L de Pfdhfr y K540E de Pfdhps en los casos de MSC fueron más del doble de frecuente comparado con los reportes publicados en casos de MNC en la misma área y periodo de estudio. / --- Introduction: Malaria represents a medical emergency because it may rapidly progress to complication and death without prompt and appropriate treatment. Severe and/or complicated malaria (SCM) is almost exclusively caused by Plasmodium falciparum. One of the risk factors associated with SCM is an inappropriate treatment of the noncomplicated malaria (NCM). Objectives: We genotyped the dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes from 60 SCM patients. Resistance to SP in P. falciparum is caused mainly by specific mutations at those genes. Study design: SCM patients of this study were enrolled during the malaria outbreak in 1998, when sulfadoxine/pyrimethamine (SP) was the first line of treatment. Material and methods: We used a Sanger sequencing approach for the identification of polymorphisms at Pfdhfr gene codons, and in the case of Pfdhps gene we used a PCRRFLP and PCR allele-specific methodology. Results: We found that 84% of samples harbored a quadruple mutant genotype N51I/S108N/I164L/insertion Bolivia repeat, and the left 16% of the sample contained an infection with a simple mutant genotype (S108N). Regarding the Pfdhps gene, we found four genotypes, the triple mutant genotype A437G/K540E/A581G was the more frequent (78%). Conclusions: We observed that the mutations I164L and K540E, known as highly predictor to SP resistance, in this group of patients with SCM were twice of frequency of the mutations from patients with NCM from published reports, also in the same area and period of study. / Tesis

Malaria is arguably one of the most overwhelming infectious diseases throughout the world's existence. The most virulent parasite, Plasmodium falciparum, has a redundancy of invasion proteins, allowing it to switch between different receptors on the host red blood cell. These invasion proteins are stored in the apical organelles, the rhoptries and micronemes, but very little is known about how newly synthesized proteins are transported to these organelles. The hypothesis in this study was that a common protein is involved in trafficking invasion proteins from the trans-Golgi network and PfSORTILIN was investigated as a potential escorter protein. The CCys domain of PfMAEBL, a rhoptry protein, and the prodomain of PfAMA-l, a microneme protein, have been implicated in trafficking to the apical organelles. These domains and the VPS 10 domain of PfSORTILIN were cloned into expression vectors encoding a GST- or Histag. Recombinant proteins were expressed in E. coli and purified by affinity chromatography on glutathione- or Ni-particles. In vitro binding assays were performed, which showed that PfSORTILIN VPS 10 bound to PfMAEBL ccys but not to the PfAMA-1 prodomain, suggesting that PfSORTILIN is a rhoptry protein escorter and is not involved in microneme trafficking. To identify novel binding partners of PfSORTILIN VPS 10, the protein was biopanned against a P. falciparum phage display library. No binding partners were identified, most likely because the library is not schizont-stage specific, which is when PfSORTILIN and invasion proteins are predominantly expressed. The results from this study were integrated with other studies and a trafficking model for PfMAEBL was proposed. This study enhances our knowledge of trafficking pathways and suggests that PfSORTILIN may serve as a common rhoptry protein escorter. / MT 2019

Malaria remains a global health problem, despite over a century of efforts towards control and prevention. It is responsible for over 2 million deaths a year. Plasmodium falciparum, the protozoan parasite that causes malaria, presents quite an unexplored field of study, significant both for the purposes of understanding the complex life cycle of the parasite, and for identifying novel and unique targets for anti-malarial therapy. Cyclin-dependent kinases. (CDK.s) play a number of crucial roles in the progression of the cell cycle such as regulating the onset of DNA replication and entry into mitosis. Plasmodium falciparum protein kinase 5, PfPK5, manifests characteristics of eukaryotic CDKs. It is a serine/threonine kinase, has 60% amino acid identity to eukaryotic cyclin-dependent kinase cdc2, and shares the mechanism of activation with CDKs. To establish if PfPK5 indeed is the major cell cycle regulatory kinase, as well as to expand our knowledge about the signaling networks of the parasite, it is necessary to identify proteins that interact with the kinase, such as its putative substrates. Currently, only one Plasmodium falciparum protein is known to interact with PfPK5 - its cyclin partner, Pfcycl. Identifying substrates of PfPKS is a particularly important research endeavor since it would provide insight into the yet unknown downstream signaling pathways of PfPK5. It is likely that pathways unique to Plasmodium falciparum will be found, which may be specifically targeted for anti-malaria therapy. A potential substrate of Plasmodium falciparum cell cycle regulatory kinase PfPK5 has been identified. The new protein, which we call SPOK, was identified by screening a phage display cDNA library. Since SPOK is a large protein of approximately 140kDa, a domain containing a tandem CDK/cdc2 phosphorylation motif of SPEK (single amino acid code, S/TPXK/R) was expressed in E.coli. Our results show that this domain of SPOK is indeed phosphorylated in vitro by PfPK5. This raises the possibility that SPOK could be an in vivo substrate of PfPK5 and may play a role in regulating the cell cycle of the parasite.

Plasmodium falciparum est un parasite appartenant au phylum Apicomplexa et est à l’origine de la forme la plus sévère de la malaria. Dans les zones endémiques d'Afrique subsaharienne, la plupart des victimes sont des enfants de moins de cinq ans. L’entrée de P. falciparum dans sa cellule cible, le globule rouge, repose sur la sécrétion de protéines par des organites spécialisés : les micronèmes, les rhoptries et les granules denses. Les mécanismes de biogenèse de ces organites et la coordination de la libération de leur contenu lors de l'invasion sont cependant pour la plupart inconnus. Il a été toutefois été démontré que les protéines destinées à ces organites apicaux se concentrent dans des microdomaines de l’appareil de Golgi, dont la composition en lipides et en protéines détermine leur destination finale. À ce jour, les mécanismes de sélection et de transport des protéines apicales vers les organites d'invasion ainsi que leurs mécanismes de sécrétion durant l’invasion sont pour la plupart inconnus. Nous avons donc posé l’hypothèse que les phosphoinositides (PI) et leurs protéines effectrices sont impliqués dans ces processus chez P. falciparum. Les PI sont sept lipides phosphorylés retrouvés de façon minoritaire dans les différentes membranes cellulaires. Chaque membrane subcellulaire contient une espèce caractéristique de PI qui peut être reconnue et liée spécifiquement par des protéines effectrices. Une large gamme de processus biologiques sont régulés par les PI, tels le trafic vésiculaire, les canaux ioniques, les pompes d’efflux et les transporteurs, ainsi que certains processus endocytiques et exocytaires. Des études antérieures ont été en mesure de détecter seulement cinq des sept espèces de PI chez P. falciparum. Dans le cadre d’un premier projet, nous avons étudié la distribution de six PI, à savoir PI3P, PI4P, PI5P, PI (4,5)P2, PI(3,4)P2 et PI(3,4,5)P3, chez P. falciparum. Pour ce faire, nous avons exprimé chez le parasite des rapporteurs spécifiques correspondant à des domaines humains de liaison aux PI, fusionnés à une protéine fluorescente. Cette méthode nous a permis de confirmer des rapports antérieurs sur la localisation du PI3P dans la membrane de la vacuole alimentaire, dans de petites vésicules près ou sur la membrane plasmique du parasite ainsi qu’à l’apicoplaste. De plus, nous avons révélé pour la première fois la présence de PI5P chez P. falciparum et montré qu’il se localisait à la membrane plasmique, au noyau et potentiellement dans le réticulum endoplasmique de transition. Nous avons aussi montré que le PI4P est localisé dans la membrane plasmique ainsi que dans l’appareil de Golgi et que le PI(4,5)P2 est présent dans la membrane plasmique tout au long du cycle érythrocytaire. Cette carte de la distribution subcellulaire des PI constitue un excellent outil pour mieux déchiffrer les rôles de ces lipides chez le parasite P. falciparum. Dans le cadre d’un second projet, nous avons caractérisé une protéine possédant un domaine conservé chez les Apicomplexa, le domain d’homologie de la Pleckstrine, la protéine PfPH2. En utilisant la stratégie de Knock-sideways pour inactiver conditionnellement la protéine d’intérêt, nous avons montré que PfPH2 est impliquée dans l’attachement initial du mérozoite à la surface du globule rouge. Cet effet est directement lié à un défaut de sécrétion d'une population spécifique de micronèmes en l’absence de la protéine PfPH2. Enfin, nous avons mis en évidence que le domaine PH de PfPH2, lorsque exprimé sous forme de protéine recombinante, se lie aux PI avec une grande spécificité. Pris ensemble, nos résultats démontrent le rôle essentiel des PI dans le processus d’invasion et proposent un modèle mécanistique pour l'exocytose des micronèmes. / Plasmodium falciparum belongs to the phylum of Apicomplexa and causes the most severe form of malaria. In endemic areas of sub-Saharan Africa, most of the victims are among children under the age of five. P. falciparum relies on proteins released from sophisticated invasion organelles called micronemes, rhoptries and dense granules to enter human erythrocytes. The mechanism of biogenesis of invasion organelles and the coordinated release of their contents during invasion are mostly unknown. It has been shown that proteins targeted to the apical organelles accumulate in microdomains of the Golgi apparatus with specific lipid and protein composition that determine the final destination of their cargo. To date, the mechanisms of transport of the cargo molecules to the invasion organelles and their release mechanism are mostly unknown. We proposed that phosphoinositides (PIPs) and their effector proteins could be involved in these processes in P. falciparum. PIPs are seven minor phosphorylated lipids in cellular membranes. Each subcellular membrane contains a characteristic species of PIPs that are specifically bound by PIPinteracting proteins. A wide range of biological processes regulated by PIPs such as vesicular trafficking, ion channels, pumps, and transporters and control both endocytic and exocytic processes. Based on previous reports five out of seven PIP species have been detected in P. falciparum. In my first project, we have studied the distribution of six PIPs namely PI3P, PI4P, PI5P, PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3 using expression of specific reporters made up of human PIP-binding domains fused to a fluorescent protein. Here, we have confirmed previous reports on PI3P localization to the food vacuole membrane, small vesicles close/on the parasite plasma membrane and the apicoplast. Also, we have reported for the first time the presence of PI5P in P. falciparum and showed that it localizes to the PM, nucleus and potentially transitional ER. PI4P shows localization to the PM and Golgi and PI(4,5)P2 localizes to the PM all over the erythrocytic cycle. The resulting map of the subcellular distribution of PIPs will now be a great tool to further decipher the roles of these lipids in P. falciparum, In the second project, we have characterized a Pleckstrin Homology domain-containing protein (PfPH2) conserved in all apicomplexan parasites. Using the knock sideways strategy to conditionally inactivate the protein, we show that PfPH2 is involved in an early step of the invasion process, when the merozoites initially attach to red blood cells. We further demonstrate that this is due to the abrogated secretion of a specific population of micronemes. Finally, we reveal that recombinantly expressed PfPH2 binds PIPs with a broad specificity. Taken together, our results present evidence for the role of PI in invasion and propose a mechanistic model for the exocytosis of micronemes.

Le paludisme est une des maladies tropicales les plus dévastatrices au monde causée par des parasites protozoaires intracellulaires du genre Plasmodium. Cinq espèces de plasmodies sont responsables du paludisme chez l'homme et causent 600 000 décès par an principalement chez les enfants de moins de 5 ans et les femmes enceintes vivant dans les régions les plus pauvres du globe. Les parasites ont généré une résistance contre les chimiothérapies existantes et aucun vaccin efficace n'est encore disponible. Il est donc impératif d'identifier et de valider de nouvelles cibles qui peuvent être exploitées pour la découverte de nouveaux médicaments.Cette étude a porté sur la caractérisation d'un enzyme, la phosphoénolpyruvate carboxylase (PEPC), produit d'un gène spécifique au parasite et absent chez l'hôte humain, ce qui constitue l'un des pré-requis d'une cible potentielle pour la découverte de médicaments. Le gène avait été montré comme essentiel pour des parasites seulement en absence de malate ou de fumarate, suggérant un rôle de la protéine dans le métabolisme du carbone intermédiaire des parasites.Mes études de thèse avaient pour but de caractériser le rôle de la PEPC en utilisant la métabolomique. J'ai d'abord établi et normalisé une méthodologie d'analyses métabolomiques des globules rouges infectés par Plasmodium et optimisé l'analyse des métabolites hydrophiles présents dans le parasite intracellulaire et sa cellule hôte. Nous nous sommes concentrés sur les métabolites du métabolisme du carbone intermédiaire, où la PEPC pouvait jouer un rôle déterminant par analogie avec les plantes et les bactéries. Des analyses ciblées utilisant un marquage isotopique du métabolome à partir de 13C-U-glucose, 13C-bicarbonate et 13C, 15N-glutamine ont aussi été réalisées permettant de mieux appréhender les conséquences d'un KO de l'enzyme PEPC sur le métabolisme du parasite.Les données montrent que l'enzyme PEPC permet une fixation du bicarbonate et catalyse une réaction anaplérotique conduisant à du malate qui est introduit dans le cycle de l'acide tricarboxylique mitochondrial, transférant ainsi des équivalents réducteurs du cytoplasme à la mitochondrie et fournissant aussi un point d'entrée du squelette carboné dans le cycle. Les résultats montrent surtout que les parasites possèdent un cycle complet et de type oxydatif de l'acide tricarboxylique mitochondrial. Il parait y avoir trois points d'entrée: 1. l'acétyl CoA résultant du pyruvate généré par la glycolyse et décarboxylé dans la mitochondrie; 2. l'acide alpha-cétoglutarique provenant du glutamate, qui lui-même résulte de la désamination de la glutamine essentiellement fournie par l'environnement externe; 3. le malate, produit en aval de la malate déshydrogénase qui réduit l'oxaloacétate produit par la PEPC. En aval de la PEPC, la biosynthèse des pyrimidines opère grâce à l'activité de l'aspartate aminotransférase agissant sur oxaloacétate.En dehors du malate, le fumarate est le seul autre métabolite qui permet de s'opposer au défaut de croissance des parasites déficients en PEPC, ce qui a conduit à évaluer le rôle de la fumarase. À cette fin, l'étiquetage du gène endogène fumarase avec une étiquette HA, a permis de montrer que la protéine est exprimée dans les stades intra-érythrocytaires de P. falciparum et de montrer que la protéine se trouve à la fois dans la mitochondrie et le cytoplasme. La protéine recombinante a été exprimée avec succès et partiellement caractérisée biochimiquement. De nombreuses tentatives visant à générer des mutants de délétion génétique de P. falciparum n'ont pas abouti, laissant en suspens la question du caractère essentiel du gène pour les parasites. Cependant, il est possible de cibler le locus du gène via un marquage C-terminal. Ceci suggère que l'enzyme peut être essentielle pour la survie du parasite et donc une cible exploitable pour la découverte d'un type nouveau de médicament antipaludique. / Malaria is one of the world's most devastating tropical diseases caused by obligate intracellular protozoan parasites of the genus Plasmodium. Five species of these parasites cause malaria in humans and infection results in ~600,000 deaths annually primarily in children under the age of 5 and pregnant women living in the poorest areas of the globe. The parasites have an outstanding ability to generate resistance against existing chemotherapies and an efficacious vaccine is not available yet. Therefore it is imperative that attempts are being made to identify and validate new targets that can be exploited for future drug discovery.This study focused on the validation and elucidation of a parasite-specific gene product namely phosphoenolpyruvate carboxylase (PEPC), which is not present in the human host and thus has one of the pre-requisites of a potential drug target. The gene had been previously genetically validated and it was demonstrated that mutant parasites lacking pepc were only viable in the presence of malate or fumarate, suggesting a role of the protein in intermediary carbon metabolism of the parasites.My studies had the goal to assess the role of PEPC using a metabolomics approach. Initially the methodologies to perform metabolomics analyses of Plasmodium-infected RBCs were established and standardised and it was assessed how to best analyse the hydrophilic metabolites present in the intracellular parasites and its host cell. We focused on metabolites of intermediary carbon metabolism, as it is likely that PEPC is important for metabolic functions linked to this in the parasites, in analogy to plants and bacteria. While global metabolomics analyses were appealing, it was decided to apply a targeted metabolomics and comparative approach using stable isotope labelling of the parasite metabolomes with 13C-U-glucose, 13C-bicarbonate and 13C-,15N-glutamine to assess the consequences of the pepc knockout on parasite metabolism.The data demonstrated that PEPC has an anaplerotic function fixing bicarbonate and leading to generation of malate that is fed into the mitochondrial tricarboxylic acid cycle and so transfers reducing equivalents from cytoplasm to mitochondrion as well as providing an entry point of carbon skeleton into the cycle. The most important findings with respect to parasite mitochondrial metabolism were that the parasites possess a complete and oxidative tricarboxylic acid cycle, which appears to have three entry points: 1. Acetyl CoA resulting from glycolytically generated pyruvate that is decarboxylated in the mitochondrion; 2. α-ketoglutarate from the reaction of glutamate dehydrogenase and 3. malate, which is a downstream product of malate dehydrogenase that reduces oxaloacetate the reaction product of PEPC. Other downstream reactions supported by PEPC activity are pyrimidine biosynthesis through the activity of aspartate aminotransferase also acting on the PEPC-derived oxaloacetate.Apart from malate, fumarate was the only other metabolite that reversed the growth defect of pepc mutant parasites. Hence the role of fumarase in the parasites was also assessed. To this end the endogenous fumarase gene of P. falciparum was tagged with an HA-tag, which showed that the protein is expressed in the intra-erythrocytic stages of P. falciparum and demonstrated that the protein is located in both mitochondrion and cytoplasm. In addition, the recombinant protein was produced and partially biochemically characterised. Numerous independent attempts to generate genetic deletion mutants of P. falciparum were unsuccessful, leaving the question whether the gene is essential for the parasites unanswered. However, it was possible to manipulate the locus by C-terminal tagging of the fumarase gene suggesting that fumarase might be indeed essential for parasite survival and therefore possibly suitable for future drug design and discovery.

La pompe ATPase Ca2+ du réticulum sarco-endoplasmique Plasmodium falciparum (PfATP6) est une protéine de dix transmembranes, impliqué dans la régulation de l'homéostasie du calcium dans le parasite. L'importance d'étudier cette protéine repose sur l'hypothèse d'être engagé dans le mécanisme d'action et de résistance des artémisinines. Des travaux précédents, fondé sur l'expression hétérologue dans des ovocytes de Xenopus laevis et Saccharomyces cerevisiae, ont montré des résultats opposés, générant de nombreux corollaires vérifiables. Par conséquent, des travaux supplémentaires sont nécessaires pour mieux comprendre la nature des interactions entre les artémisinines et transporteurs de type SERCA.Afin d'évaluer le caractère essentiel du gène de Plasmodium spp., une approche de génétique inverse a été utilisée. Knockout du gène, soit P. falciparum et berghei, ne pouvant pas être obtenu. La complémentation de sauvetage épisomique a été jugée impossible. Marquage à la fin 3' de PfATP6 et PbATP6 a été, également, tenté pour étudier la localisation et l'expression de la protéine chez les parasites. La manipulation des gènes à cette place n'a pas permis la survie du parasite. Nos résultats, pris ensemble, montrent que ATP6 est essentiel dans Plasmodium spp..Au cours de nos études génétiques, un phénotype stable et particulier de parasites du genre Plasmodium falciparum 3D7 a été distingué. Les étranges parasites “monstres" contiennent une vacuole digestive inhabituelle gonflées à travers toutes les étapes du développement du parasite. Caractérisation de l'insolite Plasmodium a été réalisée, montrant une sensibilité accrue à la chloroquine, mais pas à l'artémisinine ou de la méfloquine. Tenant compte de la similitude du PfATP6 avec la pompe SERCA orthologue mammifère, de nouvelles molécules, connu et synthétisé pour cibler spécifiquement la protéine chez les mammifères, ont été testés sur P. falciparum. Quatre classes différentes de composés (sHA 14-1, BHQ, chalcone et des analogues de l'ACP) a montré le blocage de la croissance in vitro du P. falciparum 3D7 et Dd2 à des concentrations inférieure au range micromolaire. En outre, une nouvelle classe de molécules (thaperoxides), conçu comme un hybride entre l'artémisinine et thapsigargine, a été testé contre le type sauvage 7G8 et la ligne muté L263E. Ce dernier porte une mutation ponctuelle unique de nucléotides dans PfATP6, déjà connu d'être impliqué dans la résistance du l'artémisinine.Compte tenu de la difficulté à manipuler les gènes du parasite, et afin de mieux caractériser PfATP6, un gène synthétique a été optimisé pour l'expression hétérologue chez S. cerevisiae. De cette façon, la complémentation d'une ligne de levure mutée (K616) sans les pompes endogènes Ca2+ de type P a été permis avec succès, montrant le sauvetage de la croissance de la levure en présence de forte concentration de calcium libre. Différents inhibiteurs de SERCA, comme la thapsigargine et l'acide cyclopiazonique, ont été testés sur la levure complémenté K616 PfATP6, afin de vérifier l'inhibition de la croissance. Tous les composés ont bloqué la croissance de levure sélectivement ciblant le PfATP6. En outre, le test a été développé comme un criblage de haute performance, afin de tester de nouvelles molécules pour leur activité. La méthode s'est révélée être un outil rapide et très fiable et reproductible pour l'identification de nouveaux composés actifs. / The Plasmodium falciparum sarco-endoplasmic reticulum ATPase Ca2+ pump (PfATP6) is a ten transmembrane protein involved in the regulation of the calcium homeostasis in the parasite. The importance of studying this protein relies on the fact that it has been hypothesized to be involved in the mechanism of action and resistance of artemisinins. Previous works, based on heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae, have shown contrasting results, generating many testable corollaries. Therefore, further work is needed to better understand the nature of interactions between artemisinins and SERCA-type transporters.In order to assess the essentiality of the gene in Plasmodium spp., a reverse genetics approach has been used. Knockout of the gene, in either P. falciparum and berghei, could not be achieved. Complementation for episomal rescue was found to be not possible. Tagging at the 3' end of PfATP6 and PbATP6 has been, also, attempted to study localization and expression of the protein in parasites. Manipulation of the gene at this position did not permit parasite survival. Our results, taken together, show that ATP6 is essential in Plasmodium spp..During our genetic studies, a stable and peculiar phenotype of Plasmodium falciparum 3D7 parasites has been noticed. The odd “monster” parasites contain an unusual swollen food vacuole throughout all stages of parasite development. Characterization of the unusual Plasmodium has been carried out, showing an increased sensitivity to chloroquine, but not to artemisinin or mefloquine. Taking into account the similarity of PfATP6 with the mammalian orthologue SERCA pump, new molecules, designed and synthesized to specifically target the mammalian protein, were tested on P. falciparum parasites. Four different classes of compounds (sHA 14-1, BHQ, chalcone and CPA analogues) showed to inhibit P. falciparum 3D7 and Dd2 growth in vitro at concentrations in the lower micromolar range. In addition, a novel class of molecules (thaperoxides), designed as an hybrid between artemisinin and thapsigargin, has been tested against 7G8 wild type and mutated L263E line. The latter carries a single nucleotide point mutation in PfATP6 that has been previously shown to be involved in artemisinin resistance. Considering the difficulty in manipulating the gene in the parasite and in order to better characterize PfATP6, a synthetic gene was optimized for heterologous expression in S. cerevisiae. This enabled successful complementation of a mutated yeast line (K616) lacking the endogenous P-type Ca2+ pumps, showing rescue of the yeast growth in presence of high concentration of free calcium. Different SERCA inhibitors, such as thapsigargin and cyclopiazonic acid, have been tested on K616 PfATP6 complemented yeast, in order to check for growth inhibition. All compounds showed to inhibit yeast growth selectively targeting PfATP6. In addition, the assay has been developed as a high throughput screening, in order to test new molecules for their activity. The method has proved to be a fast, highly reliable and reproducible tool for identification of new active compounds.