L'infection des hépatocytes par les sporozoïtes constitue une première étape obligatoire du cycle de Plasmodium chez l'hôte vertébré. Les mécanismes impliqués dans l'invasion des hépatocytes restent mal caractérisés. Nous avons découvert que CD81, une protéine transmembranaire appartenant à la famille des tétraspanines, est nécessaire à l'infection des hépatocytes par les sporozoïtes de Plasmodium falciparum, de Plasmodium yoelii, et dans certains cas de Plasmodium berghei. L'expression de CD81 suffit à rendre les cellules hépatocytaires HepG2 sensibles à l'infection par les sporozoïtes de P. yoelii mais pas P. falciparum, suggérant l'intervention d'autres molécules pour ce parasite. Une caractéristique majeure des tétraspanines est leur capacité de s'associer entre elles et avec d'autres molécules de surface pour former des microdomaines membranaires distincts des radeaux lipidiques classiques (« rafts »). Nos résultats montrent que le cholestérol membranaire contribue non seulement à la localisation de CD81 dans les microdomaines enrichis en tétraspanines mais aussi à l'infection par les sporozoïtes de Plasmodium lorsqu'elle implique une voie dépendante de CD81. Nous n'avons pas pu mettre en évidence d'interaction directe entre CD81 et les sporozoïtes, ce qui suggère que CD81 pourrait ne pas jouer un rôle de récepteur pour le parasite mais intervenir de manière indirecte. EWI-F et EWI-2, deux protéines transmembranaires associées à CD81 dans les hépatocytes, ne sont pas directement impliquées dans l'infection par les sporozoïtes. Au contraire, EWI-F a un effet inhibiteur sur l'infection, peut-être lié à une réorganisation des microdomaines à tétraspanines ou à une compétition avec un autre partenaire de CD81. Une autre molécule associée à CD81 pourrait donc jouer un rôle essentiel dans l'infection par les sporozoïtes, éventuellement comme récepteur, mais sa nature reste à déterminer.

Découper, a priori et de façon précise, les séquences en domaines est d'une grande importance dans le champ de la biologie, notamment pour optimiser les études de génomique structurale et de génomique fonctionnelle. Différentes approches basées sur la composition en acides aminés, la complexité de la séquence ou la construction de modèles 3D ab initio, ont été développées par le passé. Nous proposons, dans le cadre de ce travail, une approche nouvelle et originale pour le découpage automatique et sensible des séquences protéiques en domaines structurés distincts par exploitation de leur texture. Cette approche bénéficie de l'information de voisinage 2D apportée par la méthodologie « Hydrophobic Cluster Analysis » (HCA). La distribution des différentes catégories d'amas hydrophobes, tels que définis par l'intermédiaire de HCA, ainsi que l'analyse de leurs caractéristiques en termes de structures secondaires, permettent d'appréhender de façon différenciée les textures des régions globulaires, non globulaires et/ou désordonnées, répétitives, passages membranaires isolés ou multiples.... L'approche développée, DomHCA, permet in fine de segmenter une séquence protéique en une série de régions et sous-régions caractérisées par des textures précises, segmentation qui, appliquée à l'échelle des génomes, autorise une comparaison rapide et originale de l'ensemble des séquences. Une des applications concerne les séquences du génome de Plasmodium falciparum qui, par leurs fortes proportions en acides aminés N et K, rendent les méthodes classiques de détection de similarité peu efficaces.

<p>Protective immunity against <i>Plasmodium falciparum</i> may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the <i>P. Falciparum</i> infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to <i>P. falciparum</i> infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure.</p><p>Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an <i>in vitro</i> assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes.</p><p>The <i>P. falciparum</i> parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated<i> P. falciparum </i>parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition <i>in vitro</i> by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition <i>in vitro</i>, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses.</p><p>Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area.</p><p>There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against<i> P. falciparum</i> asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.</p>

<p>Malaria is the most common parasitic disease of humans worldwide. A factor that aggravates the many attempts to control the epidemiologic malaria situation is the spreading of resistance against anti-malarial drugs. In this project the point mutation at position 86 of the <em>Plasmodium. </em><em>falciparum</em><em> </em>multidrug resistance gene (<em>pfmdr1</em>), which is thought to contribute to Chloroquine resistance, was analysed in 188 samples from a low transmission area in eastern Sudan, where malaria endemicity is seasonal. The patient group studied had asymptomatic and sub patent parasitemia that persisted during the transmission-free dry season, after being treated with Chloroquine. To differentiate between wild type and mutant genotypes, nested PCR and restriction fragment length polymorphism with the enzyme Apo1 was used. Out of 188 samples 79 (42%) were successfully analysed. Of those, 72% had parasites with mutant genotypes or where mixed infection. No conclusions on the relevance of the <em>pfmd</em><em>r</em><em>1</em> gene in the studied samples are made due to the many remaining gaps. However, eventual sources of error and previous findings in the study area are discussed.</p>

<p>Malaria is a widespread disease caused by parasites of the genus <i>Plasmodium</i>. Each year 500 million clinical cases are reported resulting in over one million casualties. The most lethal species, <i>P. falciparum</i>, accounts for ~90% of the fatal cases and has developed resistance to chloroquine. The resistant strains are a major problem and calls for novel drugs.</p><p>In this thesis, the process of computational inhibitor design is illustrated through the development of <i>P. falciparum</i> aspartic protease inhibitors. These proteases, called plasmepsins, are part of the hemoglobin degradation chain. The hemoglobin is degraded during the intraerythrocytic cycle and serves as the major food source. By inhibiting plasmepsins the parasites can be killed by starvation.</p><p>Novel inhibitors with very high affinity were found by using a combination of computational and synthetic chemistry. These inhibitors were selective and did not display any activity on human cathepsin D. The linear interaction energy (LIE) method was utilized in combination with molecular dynamics (MD) simulations to estimate free energies of binding. The MD simulations were also used to characterize the enzyme–inhibitor interactions and explain the binding on a molecular level.</p><p>The influence of the partial charge model on binding free energy calculations with the LIE method was assessed. Two semiempirical and six <i>ab initio</i> quantum chemical charge derivation schemes were evaluated. It was found that the fast semiempirical charge models are equally useful in free energy calculations with the LIE method as the rigorous <i>ab initio</i> charge models.</p>

Protective immunity against Plasmodium falciparum may be obtained after repeated exposure to infection. Several studies indicate that immunity against the blood stages of the P. Falciparum infection is mainly antibody mediated. Protective antibodies may act either on their own, mediate antibody-dependent phagocytosis and/or cell-mediated neutralization of parasites. This thesis describes several aspects of humoral immune responses to P. falciparum infection in individuals of different age groups, different genetic background and with different degrees of malaria exposure. Several target antigens for antibody-mediated inhibition of parasite growth or invasion have been identified. One such antigen is Pf332, which appears on the surface of parasitized erythrocytes at late trophozoite and schizont stage. This surface exposure makes the antigen a possible target for opsonizing antibodies. We optimized an in vitro assay for studying cellmediated parasite neutralization in the presence of Pf332-reactive antibodies. Our data demonstrate that, Pf332 specific antibodies are able to inhibit parasite growth on their own and in cooperation with human monocytes. The P. falciparum parasites have evolved several mechanisms to evade the host neutralizing immune responses. In this thesis, we show that freshly isolated P. falciparum parasites from children living in a malaria endemic area of Burkina Faso were less sensitive for growth inhibition in vitro by autologous immunoglobulins (Ig) compared with heterologous ones. Analyses of two consecutive isolates taken 14 days apart, with regard to genotypes and sensitivity to growth inhibition in vitro, did not give any clear-cut indications on possible mechanisms leading to a reduced inhibitory activity in autologous parasite/antibody combinations. The frequent presence of persisting parasite clones in asymptomatic children indicates that the parasite possesses as yet undefined mechanisms to evade neutralizing immune responses. Transmission reducing measures such insecticide treated nets (ITNs) have been shown to be effective in reducing morbidity and mortality from malaria. However, concerns have been raised that ITNs usage could affect the acquisition of malaria immunity. We studied the effect of the use of insecticide treated curtains (ITC) on anti-malarial immune responses of children living in villages with ITC since birth. The use of ITC did neither affect the levels of parasite neutralizing immune responses nor the multiplicity of infection. These results indicate that the use of ITC does not interfere with the acquisition of anti-malarial immunity in children living in a malaria hyperendemic area. There is substantial evidence that the African Fulani tribe is markedly less susceptible to malaria infection compared to other sympatrically living ethnic tribes. We investigated the isotypic humoral responses against P. falciparum asexual blood stages in different ethnic groups living in sympatry in two countries exhibiting different malaria transmission intensities, Burkina Faso and Mali. We observed higher levels of the total malaria-specific-IgG and its cytophilic subclasses in individuals of the Fulani tribe as compared to non-Fulani individuals. Fulani individuals also showed higher levels of antibodies to measles antigen, indicating that the intertribal differences are not specific for malaria and might reflect a generally activated immune system in the Fulani.

Malaria is a widespread disease caused by parasites of the genus Plasmodium. Each year 500 million clinical cases are reported resulting in over one million casualties. The most lethal species, P. falciparum, accounts for ~90% of the fatal cases and has developed resistance to chloroquine. The resistant strains are a major problem and calls for novel drugs. In this thesis, the process of computational inhibitor design is illustrated through the development of P. falciparum aspartic protease inhibitors. These proteases, called plasmepsins, are part of the hemoglobin degradation chain. The hemoglobin is degraded during the intraerythrocytic cycle and serves as the major food source. By inhibiting plasmepsins the parasites can be killed by starvation. Novel inhibitors with very high affinity were found by using a combination of computational and synthetic chemistry. These inhibitors were selective and did not display any activity on human cathepsin D. The linear interaction energy (LIE) method was utilized in combination with molecular dynamics (MD) simulations to estimate free energies of binding. The MD simulations were also used to characterize the enzyme–inhibitor interactions and explain the binding on a molecular level. The influence of the partial charge model on binding free energy calculations with the LIE method was assessed. Two semiempirical and six ab initio quantum chemical charge derivation schemes were evaluated. It was found that the fast semiempirical charge models are equally useful in free energy calculations with the LIE method as the rigorous ab initio charge models.

Ce travail avait pour objectif : 1) évaluer le portage de gamétocytes et leur génotype avant et après le traitement d'une part, et d'étudier leur infectivité ; 2) exprimer le domaine catalytique (PfMCA1-cd-Sc) de la métacaspase de Plasmodium falciparum (PfMCA1) chez la levure et 3) tester in vitro l'activité antiplasmodiale de nouvelles molécules synthétiques dérivées des pyrano et ferro-quinoléines sur des clones de laboratoire 3D7 et Dd2. Pour cela, le test in vivo de 28 jours de l'OMS, les marqueurs moléculaires de résistance et le " direct feeding " ont été utilisés pour le premier objectif. La culture des levures, l'expression des protéines de la métacaspase 1 de Plasmodium falciparum, le western blot, le test de prolifération et de survie, et les marqueurs de mort cellulaire ont servi pour le second objectif et enfin, la culture parasitaire et tests in vitro par la méthode de fluorimétrie au Sybr Green I ont permis l'évaluation de l'activité antiplasmodiale de nouvelles molécules. Nous avons démontré que les gamétocytes post-traitement étaient porteurs de mutations ponctuelles et plus infectants dans le groupe chloroquine ; que l'expression hétérologue du domaine catalytique de la métacaspase de Plasmodium falciparum (PfMCA1) dans la levure Saccharomyces cerevisiae entraînait une mort clonale de type apoptotique et un retard de croissance dépendant de l'activité VAD-Protéase et enfin, que les substitues aromatiques à base de pyrimidine ou de benzylméthylamine ferrocène révèlent une activité satisfaisante par rapport à la méthoxyéthylidene sur les clones 3D7 et Dd2.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.