De études récentes suggèrent une forte relation entre susceptibilité au paludisme et réponse allergique. Des niveaux élevés d’histamine plasmatique et tissulaire ont été associés à la sévérité de la maladie chez l’homme infecté par P. falciparum et dans de nombreux modèles animaux. Histamine releasing factor (HRF), une protéine pro-inflammatoire libérant l'histamine au cours des processus allergiques, est produite par le parasite au cours des infections palustres modérées et sévères, suggérant l’implication du HRF dans l’altération des réponses immunitaires et dans la pathogenèse. Les objectifs de ce travail consistaient à évaluer le rôle de la protéine parasitaire HRF dans le développement de la réponse immunitaire et à déterminer si son expression est associée à la sévérité de la maladie en étudiant deux parasites murins, PbANKA et PbNK65, déficients pour la protéine HRF (hrfΔ). Les souris infectées avec des sporozoïtes PbANKA-hrfΔ ont montré une diminution de la fréquence du neuropaludisme associée à un déficit du développement des parasites mutants au cours du stade hépatique et à une augmentation précoce systémique d’IL-6. En outre, l'infection par les parasites PbNK65-hrfΔ est caractérisée par l’élimination du parasite conduisant à une protection durable et au développement d’une mémoire immunitaire caractérisée par une augmentation d’IL-6, une diminution de l’expression de PD-1 sur les cellules T et une amélioration de la phagocytose dépendante des anticorps, confirmant l'importance de la protéine HRF dans la virulence du parasite. HRF est le premier gène de parasite Plasmodium dont l’effet direct sur la réponse immunitaire de l’hôte est démontré. / Recent findings have raised the hypothesis that clinical susceptibility to malaria may be related to allergy-type response. In human infection with P. falciparum, as well as in murine models of malaria, increased levels of histamine have been shown to be associated with disease severity. Histamine releasing factor (HRF), shown to be implicated in the release of pro-inflammatory histamine during late-phase allergy, was demonstrated to be produced by the parasite during mild and severe malaria infections suggesting that Plasmodium HRF may affect host immune responses and contributes to the pathogenesis. The objectives of this work were to evaluate the role of Plasmodium HRF in the development of the immune response and to determine whether its expression is associated with the severity of malaria disease by studying two HRF-deficient (hrfΔ) murine parasites (PbANKA and PbNK65). Infection with PbANKA-hrfΔ sporozoites showed a decrease in the frequency of ECM due to the impairment of the development of the mutant parasites in liver stages as a consequence of the up-regulation of IL-6. Infection with PbNK65-hrfΔ parasites confirmed the importance of HRF in enhancing the virulence of the parasite. Indeed, PbNK65-hrfΔ infection results in parasite clearance leading to a long-lasting protection and immune memory as reflected by an up-regulation of IL-6, a down-regulation of PD-1 expression on T cells and in the enhancement of Ab-mediated phagocytosis. HRF is the first parasite gene which directly modulates the host immune response.

Objetivos: Desenvolver, caracterizar e avaliar a eficácia in vivo e o perfil farmacocinético de suspensões de nanocápsulas (NC) poliméricas contendo quinina (QN). Metodologia: As NC-QN foram preparadas através de nanoprecipitação com diferentes concentrações de QN: 2 (NC2-QN), 3 (NC3-QN) e 4 mg/mL (NC4-QN). A NC4-QN também foi revestida com quitosana. Todas as formulações foram caracterizadas através de taxa de encapsulação, teor, diâmetro, índice de polidispersão, potencial zeta e pH, sendo a estabilidade avaliada por 30 dias. A avaliação da eficácia foi realizada com diferentes doses de cada formulação, em modelo de malária experimental em ratos Wistar infectados com Plasmodium berguei. A dose de NC-QN com a qual se obteve 100 % de cura dos animais foi selecionada para a avaliação farmacocinética. Nesses experimentos, os animais sadios ou infectados receberam a QN livre ou a NC2-QN, 25 mg/kg, iv bolus. As amostras de plasma coletadas em tempos pré-determinados foram quantificadas por CLAE com método validado para a determinação da QN. Os protocolos com animais foram aprovados pelo Comitê de Ética em Pesquisa da UFRGS (#2005477). Resultados e discussão: As suspensões coloidais preparadas com diferentes concentrações de QN apresentaram diâmetro adequado, população modispersa, potencial zeta diferente de zero, doseamento e taxa de encapsulação superior a 90 %. Somente a NC2-QN, na dose de 75 mg/kg/dia, q8h, curou todos os animais. Nas doses de 30 e 60 mg/kg/dia, q8h, 28,6 e 85,7% dos animais sobreviveram, respectivamente, com a NC2-QN. Houve uma diminuição significativa no t½ das NC2-QN (32,9 ± 8,9 min) em relação ao fármaco livre (69,8 ± 44,6 min), no grupo de animais infectados (α = 0,05) em função de uma tendência de aumento do CLtotal (7,1 ± 3,3 versus 9,9 ± 2,1 L/h/kg) do fármaco nanoencapsulado. Conclusões: A nanoencapsulação da QN reduziu a dose efetiva do fármaco no modelo animal avaliado em 30 % e aumentou a sobrevivência dos animais infectados em cerca de 60 %, apresentando-se como uma alternativa potencial para o tratamento da malária. / Objectives: The aims of this study were to develop and characterize polymeric nanocapsules (NC) containing quinine (QN) and to evaluate their efficacy in vivo as well as the pharmacokinetic profile of the nanoencapsulated drug. Methodology: NC-QN were prepared by nanoprecipitation with different drug concentration 2 (NC2- QN), 3 (NC3-QN) and 4 mg/mL (NC4-QN). All formulations were characterized in terms of encapsulation efficacy, drug loading, zeta potential, particle size, polydispersion index, and pH. The stability nanocapsules suspensions were evaluated during 30 days. An experimental malaria model with Plasmodium berghei was used to evaluate NC-QN efficacy in Wistar rats. Different doses were tested for each formulation. The pharmacokinetic evaluation was performed with the dose of NC–QN which presented 100 % efficacy in malaria model. The NC2-QN or QN free were administrated by i.v. route (25 mg/kg) to health and infected rats. Blood samples were collected at pre-determinated time points and quantified by an HPLC validated method. Animal protocols were approved by UFRGS Ethics in Research Committee (# 2005477). Results and discussion: All suspensions presented adequate particle size, monodisperse population, negative zeta potential, drug content and encapsulation efficiency higher than 90 %. The formulation NC2-QN (75 mg/kg/day) administrated q8h daily during 7-9 days post-infection cured all infected rats. For NC2-QN, 30 and 60 mg/kg/day, q8h, 28.6 and 85.7 % of survival were observed, respectively. NC2-QN presented significant decrease in QN t½ compared to the free drug (32.9 ± 8.9 min and 69.8 ± 44.6 min, respectively), in infected rats (α = 0,05). This occurs due to the tendency of increase in CLtotal (7.1 ± 3.3 to 9.9 ± 2.1 L/h/kg, for free QN and NC2-QN, respectively). CLtotal increased in the encapsulated group. Conclusion: Nanoencapsulation reduced QN effective dose in 30 % and increased in 60 % the survival of the infected animals. Theses results indicate that NC-QN is a potential strategy to be investigated for malaria treatment.

L'épigénétique joue un rôle majeur dans le développement érythrocytaire de Plasmodium falciparum, tels que variation antigénique, pathogenèse, différenciation sexuée. Jusqu'à présent, ces éléments n'ont jamais été décrits chez les sporozoïtes. Pour caractériser la régulation épigénétique au niveau des sporozoïtes de P. falciparum, nous avons étudié les principaux régulateurs épigénétiques PfHP1 (P. falciparum hétérochromatine Protein 1) ainsi que PfSET6 et PfSET7 (méthyltransférases histone lysine). J'ai établi une cartographie génomique des marques épigénétiques répressives associées à l'hétérochromatine, et actives associées à l'euchromatine. J'ai identifié un nouveau mécanisme stade-spécifique de contrôle de l'expression génique, qui réprimés plusieurs gènes codant pour des protéines exportées. Ce mécanisme repose sur une expansion d'hétérochromatine. De plus, je démontre qu'un membre de la famille des gènes var, qui code pour le facteur de virulence PfEMP1 des stades sanguins, est exprimé à la surface des sporozoïtes. Cette localisation contraste avec les stades sanguins, où PfEMP1 est transporté à la surface des érythrocytes et participe à cytoadhérence. L'ensemble de ces résultats ouvre de nouvelles questions biologiques: quels sont les facteurs qui régulent la formation d'hétérochromatine chez les sporozoïtes? Quelle est la fonction de PfEMP1 sur la surface d'un sporozoïte? Mes conclusions indiquent un rôle putatif de PfEMP1 lors de la migration des sporozoïtes. En outre, l'expression, à la surface du sporozoïte, d'un antigène polymorphique et spécifique de souche pourrait expliquer la réponse immunitaire souche-spécifique, induite par les sporozoïtes atténués. / Epigenetic mechanisms control key processes during Plasmodium falciparum blood stage development such as antigenic variation, malaria pathogenesis and sexual commitment. However, the epigenetic landscape has not been reported for the sporozoites stage. To characterize epigenetic regulation in sporozoites, we tested the major epigenetic regulators P. falciparum Heterochromatin Protein 1 (PfHP1) and the histone lysine methyltransferases (PfSET6 and PfSET7) in P. falciparum sporozoites. I obtained a reliable genome-wide occupancy data for repressive heterochromatin and active euchromatin marks. Notably, I discovered an unprecedented stage specific mechanism of silencing, which represses several hundreds of genes, encoding parasite surface exported proteins. This is based on an expansion of facultative heterochromatin boundaries in sporozoites. Moreover, I demonstrate that a single member of the polymorphic var gene family, encoding the blood stage virulence factor PfEMP1, is expressed at the surface of sporozoites. This is in contrast to blood stages where PfEMP1 is transported to the erythrocyte surface participating in cytoadhesion. Overall, my findings rise new biological questions including what are the factors that regulate heterochromatin boundaries and what is the function of a virulence-associated surface antigen in sporozoites stage. My findings point to a putative function of this adhesion molecule in sporozoites migration. Moreover, the expression of a highly polymporphic and strain-specific antigen on the surface of sporozoites might provide a molecular explanation for the strain-specific protective immune response induced by attenuated sporozoites.

Plasmodium falciparum, l'agent étiologique du paludisme, est un parasite intracellulaire obligatoire du phylum des Apicomplexa, responsable de 445 000 décès par an. Le développement de Plasmodium dans les globules rouges (GRs) humains correspond à la phase symptomatique de la maladie. Il commence par la pénétration active de la cellule hôte par la forme invasive nommée mérozoïte, suivie par la multiplication du parasite dans un processus appelé schizogonie pour former 16 à 32 nouveaux mérozoïtes qui sont alors libérés des GRs (étape de sortie) et peuvent alors initier un nouveau cycle. Au cours de son développement intra-érythrocytaire de 48h, ce parasite utilise la phosphorylation réversible de protéines pour réguler les étapes d‘invasion, de schizogonie et de sortie du GR, mais nos connaissances actuelles sur la contribution des phosphatases parasitaires dans ces mécanismes demeurent très incomplètes.L'objectif de ma thèse était d’identifier et de caractériser des phosphatases potentiellement impliquées dans la sortie ou l'invasion des GRs par P. falciparum. J'ai centré mon travail sur 4 d'entre elles, à savoir PP1, PP4, PP7 et Shelph2, sur la base de leur profil d'expression transcriptionnelle tardive au cours du cycle intra-érythrocytaire, qui correspond à ces deux évènements cellulaires. La première partie de cette étude est consacrée à la caractérisation fonctionnelle de Shelph2, une phosphatase d'origine bactérienne. Par génétique inverse utilisant la stratégie CRISPR-Cas9, nous avons étiqueté le gène au locus endogène et montré que Shelph2 est stockée dans des vésicules apicales des mérozoïtes en formation. Nous avons également démontré que cette phoshpatase n’est pas essentielle pour le développement intra-érytrocytaire du parasite dans les GRs car la délétion du gène n'affecte pas les étapes d'invasion, de multiplication des parasites ou de leur sortie des GRs, ce qui suggère la possibilité d’une redondance fonctionnelle avec d'autres phosphatases parasitaires.Dans la deuxième partie de ce travail, nous avons cherché à décrire les rôles de PP1, PP4 et PP7. Les gènes codant pour ces enzymes étant décrits comme probablement essentiels, nous avons mis en place au laboratoire une stratégie de knock-down conditionnel (ribozyme glmS), avec l’idée de déstabiliser l’ARNm après auto-clivage du ribozyme lors de l’addition d‘un métabolite, ici la glucosamine. Nous avons introduit avec succès la séquence glmS en 3 'des gènes d’intérêt pour PP4 et PP7, mais nous n’avons pas observé de déplétion protéique significative lors de l’addition de glucosamine, empêchant d’utiliser ces lignées pour étudier les fonctions de PP4 et PP7. Cependant, ces lignées parasitaires modifiées ont été utilisées pour analyser la localisation subcellulaire de ces phosphatases. Comme alternative au ribozyme, nous avons utilisé une approche de knock-out inductible (iKO) basée sur une recombinase Cre dimérisable (système DiCre) qui excise des fragments d'ADN situés entre deux sites loxP. Nous avons établi deux lignées de parasites, iKO-PP7 qui n'a pas encore été caractérisée et la souche iKO-PP1. En utilisant les parasites iKO-PP1, nous avons montré que PP1 était principalement une phosphatase cytosolique majoritairement exprimée au stade schizontes. De plus, l'excision inductible du gène PP1 à deux moments différents du cycle érythrocytaire de P. falciparum nous a permis de révéler que PP1 joue deux rôles essentiels, l'un pendant la schizogonie et l'autre au moment de la sortie du parasite. A notre connaissance, ce travail représente la première description d'une phosphatase parasitaire requise pour ces étapes du développement asexué de P. falciparum. / Plasmodium falciparum, the etiologic agent of malaria, is an obligate intracellular parasite of the Apicomplexa phylum that is responsible for 445000 deaths annually. Plasmodium development in human red blood cells (RBCs) corresponds to the symptomatic phase of the disease. It starts by the active penetration of the host cell by the invasive form named merozoite, followed by the parasite multiplication in a process called schizogony to form 16-32 new merozoites that are released from the RBC (egress step) and start a new cycle. During its 48h intra-erythrocytic development, this parasite uses reversible protein phosphorylation to regulate invasion, schizogony as well as egress, but our current knowledge on the contribution of parasite phosphatases in these cellular events is still very poor. The objective of my thesis was to identify and functionally characterize phosphatases potentially involved in egress or invasion during P. falciparum RBC cycle. I focused my work on 4 of them, namely PP1, PP4, PP7 and Shelph2, on the basis of their late transcriptional expression profile during the intra-erythrocytic cycle, as this profile matches the timing of these two essential events. The first part of this study is dedicated to the functional characterization of Shelph2, a phosphatase of bacterial origin. By reverse genetics using CRISPR-Cas9 strategy, we endogenously tagged the gene, and showed that Shelph2 is stored in apical vesicles in the developing merozoites. We also demonstrated that it is dispensable for parasite RBC development, as the deletion of the gene did not affect invasion, parasite multiplication nor egress, suggesting possible functional redundancy with other parasite phosphatases.In the second part of this work, we aimed to describe the roles of PP1, PP4 and PP7. As they were described as likely essential, we set up in the laboratory a conditional knock-down strategy named the glmS ribozyme, with the idea of destabilizing the mRNA following self-cleavage of the ribozyme upon metabolite addition, here glucosamine. We successfully introduced the glmS sequence in 3’ of the genes of interest for PP4 and PP7 but we did not observe any significant protein depletion upon glucosamine addition, thus preventing us to use these lines to study PP4 and PP7 functions. Yet, these engineered parasite lines were used to analyze the subcellular localization of these phosphatases. As an alternative to the ribozyme, we used an inducible knock-out (iKO) approach based on a dimerizable Cre recombinase (DiCre system) that excises DNA fragments located between two loxP sites. We established two parasite lines, the iKO-PP7 that has not been further characterized and the iKO-PP1 strain. Using the iKO-PP1 parasites, we showed that PP1 is predominantly a cytosolic phosphatase mostly expressed during schizogony. Furthermore, the inducible excision of PP1 gene at two different time points of P. falciparum RBC cycle permitted us to reveal that PP1 plays two essential roles, one during schizogony and the other one at the time of parasite egress. This is to our knowledge the first description of a parasite phosphatase required for these developmental steps.

L’un des obstacles majeurs au développement de nouveaux antipaludiques est notre connaissance limitée de la biologie parasitaire et la rareté des cibles thérapeutiques potentielles identifiées. Les interactions protéines-protéines sont impliquées et essentielles dans divers processus biologiques incluant les modifications post-traductionnelles. Les interactions substrats-kinases ou phosphatases sont considérées comme une liaison transitoire et jouent un rôle central et essentiel dans le cycle cellulaire de Plasmodium. La Ser/Thr Protéine Phosphatase de Type 1 (PP1), l’une des phosphatases majeurs des eucaryotes, est essentielle à la survie du parasite Plasmodium falciparum, responsable du paludisme. Elle est régulée par diverses sous-unités régulatrices dont plus de 200 ont été identifiées chez l’Homme, mais seulement 4 chez Plasmodium.Afin d’explorer le réseau de régulation de la P. falciparum PP1 (PfPP1), nous avons utilisé trois approches complémentaires pour caractériser l’interactome de la PfPP1. La purification par co-affinité suivie d'une analyse par spectrométrie de masse a identifié 6 protéines interagissant avec la PfPP1 dont 3 contenaient le motif consensus d’interaction RVxF, 2 autres le motif Fxx[RK]x[RK], également connu pour interagir avec la phosphatase et une protéine avec les deux motifs de liaison. Le criblage par double hybride chez la levure a identifié 134 protéines dont 30 présentent le motif RVxF et 20 ont le motif de liaison Fxx[RK]x[RK]. Le criblage in silico du génome de P. falciparum en utilisant une séquence consensus du motif RVxF a révélé 55 partenaires potentiels de la PfPP1. Afin de confirmer l’interaction de certaines protéines, 35 partenaires candidats ont été validés par un test d’interaction de type ELISA. Les résultats ont permis de détecter aussi bien des partenaires conservés de la PP1 qu'un nombre élevé d'interacteurs spécifiques à la PP1 du parasite et montrent une grande diversité dans les fonctions biologiques impliquant la PP1 chez Plasmodium. Parmi ces candidats, un partenaire appelé Gametocyte EXported Protein 15 (GEXP15) a été confirmé comme un réel partenaire de la PfPP1 par différentes approches. De plus, GEXP15 est surexprimé chez les gamétocytes, stade responsable de la transmission du parasite chez le moustique. Ces résultats ainsi que des études fonctionnelles seront présentés. / A major obstacle in the development of novel anti-malarials is our limited knowledge of basic parasite biology and the paucity of identified potential intervention targets. Protein-protein interactions are involved and essential in broad range of biological processes including the post-translational modifications. Substrate-kinase or phosphatase interactions are considered as transient binding and play a central and essential role in Plasmodium cell cycle. The Ser/Thr Protein Phosphatase Type 1 (PP1), one of the main contributors of eukaryotic phosphatase activity, is essential to malaria parasite Plasmodium falciparum and is highly regulated by many regulatory subunits. In humans, there are about 200 distinct regulators, however, only 4 have been so far reported in Plasmodium.To explore the P. falciparum PP1 (PfPP1) regulatory network as complete as possible, we carried out three complementary approaches to characterize the PfPP1 interactome. Co-affinity purification followed by Mass Spectrometry-based proteomics identified 6 PfPP1 interacting proteins (PIPs) of which 3 contained the RVxF consensus binding motif, 2 PIPs with a Fxx[RK]x[RK] binding motif, one with both binding motifs. The Yeast Two-Hybrid screening identified 134 proteins of which 30 have the RVxF binding motif and 20 contain the Fxx[RK]x[RK] binding motif. The in silico screen of the P. falciparum genome using a consensus RVxF motif as template revealed the presence of 55 potential PfPP1 interacting proteins. As further demonstration, 35 candidate partners were validated in an independent ELISA-based assay using recombinant proteins. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1, indicating a high diversity of biological functions for PP1 in Plasmodium. Among these candidates, one partner assigned as Gametocyte EXported Protein 15 (GEXP15) has been confirmed as a direct interactor of PfPP1 by different approaches. In addition, GEXP15 is over-expressed during gametocyte stage, responsible for the transmission of the parasite in the mosquito. These results as well as functional studies will be presented and discussed.

P. falciparum est le principal responsable des formes graves du paludisme. Le parasiteévolue entre deux hôtes (homme et moustique) qui lui imposent différents environnements; ettout particulièrement, des modifications des pressions partielles d’O2 nécessitant des capacitésd’adaptation surprenantes pour un parasite microaérophile. Chez l’hôte vertébré, lesphénomènes de cytoadhésion, ralentissant la progression du parasite notamment au niveau despoumons, augmentent la durée d’exposition aux conditions hyperoxiques.La dynamique de la réponse parasitaire à l’hyperoxie a été étudiée par une approchecombinée de transcriptomique et de protéomique. Certains mécanismes de défense contre lesespèces réactives d’oxygène ont été appréciés, dont une éventuelle fonction oxydasealternative.L’exposition du parasite à 21% d’O2 induit un retard de croissance au niveau de laschizogonie. Le stress oxydatif induit par l’hyperoxie entraîne des perturbations métaboliquescomme une inhibition de la glycolyse en faveur de la respiration et un ralentissement dumétabolisme de la vacuole digestive. Cette action combinée sur le métabolisme mitochondrialet vacuolaire permet au parasite de s’adapter à un environnement hyperoxydant, en régulant laproduction d’espèces réactives d’oxygène. Nos travaux ont montré qu’un inhibiteur de lafonction oxydase alternative, l’acide salicylhydroxamique ou SHAM, avec un effet mineur surla croissance parasitaire en microaérophilie, avait un effet létal sur les parasites en hyperoxie.Une meilleure compréhension de la biologie parasitaire pourrait contribuer audéveloppement de nouveaux traitements antipaludiques associés à une thérapie hyperbarique. / P. falciparum is the main species responsible for severe case of malaria. The parasiteevolves between two hosts (human and mosquito), imposing to it different environments;especially changes in the O2 pressure, demanding astonishing adaptation skills for amicroaerophilic parasite. In the vertebrate host, the phenomena of cytoadhesion, which slowdown the spread of the parasite among others in the lungs, increase the timing of exposure tohyperoxic conditions.The parasitic response dynamic to hyperoxia has been analysed by a combinedtranscriptomic and proteomic approach. Some of the defense mechanisms against reactiveoxygen species have been evaluated, among which a potential alternative oxidase function.The exposure of the parasite to 21%O2 atmosphere leads to a growth delay atschizogony level. The oxidative stress resulting from the hyperoxia conducts to metabolicalterations, as an inhibition of the glycolysis in favour of respiration and as a slowdown of themetabolism of the digestive vacuole. This combined action on the mitochondrial and vacuolarmetabolisms allows the parasite to adapt itself to hyperoxic environment, by regulatingreactive oxygen species. Our works have shown that an inhibitor of the alternative oxidasefunction, the salicylhydroxamic acid or SHAM, with a minor effect on the parasite growth inmicroaerophily, had letal effect on parasites in hyperoxia.A better understanding of the parasitic biology could contribute to the development ofnew antimalarial treatments, associated with a hyperbaric oxygen therapy.

Le paludisme, responsable du décès d’un million de personnes chaque année, reste un problème majeur de santé publique. Des études récentes ont évoqué des mécanismes immunopathologiques communs entre les manifestations allergiques et le paludisme, soutenus par l’existence d’un homologue parasitaire du facteur de relargage d’histamine humain (PfTCTP). Notre objectif était de déterminer l’implication des acteurs de la réponse allergique dans la gravité de l’accès palustre, et la capacité de la PfTCTP à moduler ces réponses.Trois groupes de sujets ont été recrutés dans des structures de santé à Dakar. Les patients infectés présentaient des taux élevés d’IgE totales et spécifiques, par rapport aux sujets sains, mais sans corrélation avec la gravité. A l’aide d’un test d’activation des basophiles basé sur l’expression du CD203c, nous avons montré que les basophiles de patients en accès simple présentaient un niveau d’activation basal significativement réduit. Ces mêmes patients présentaient des concentrations plasmatiques élevées en IL-10, suggérant un meilleur contrôle de leur réponse inflammatoire. Les basophiles de patients en accès grave présentaient une hyperréactivité à l’hémozoïne, ainsi qu’à des stimulations IgE-dépendantes (anti-IgE). Enfin, la présence de PfTCTP circulante a été associée à une plus grande réactivité des basophiles, tandis que l’acquisition d’anticorps contre cette protéine est associée à une protection envers une trop grande réactivité cellulaire.En conclusion, nos travaux soutiennent l’hypothèse d’une activation allergique excessive au cours du paludisme grave. Ce travail ouvre la voie à de nouvelles approches thérapeutiques. / Malaria is responsible of the death of a million person each year and remains a major public health problem. Recent studies have suggested the existence of common immunopathologic mechanisms between allergic manifestations and malaria, supported by the existence of a parasite homolog of the human histamine releasing factor (PfTCTP). Our objective was to determine i) the implication of allergic response actors in the severity of malaria, and ii) the ability of PfTCTP to modulate these responses.Three groups of subjects were recruited in health structures in Dakar. Infected patients presented high levels of total and specific IgE, compared to healthy controls, although not correlated with severity. With a basophil activation test based on CD203c expression, we demonstrated that basophils from mild malaria patients presented a significantly reduced basal level of activation which paralleled higher levels of IL-10, thus suggesting a better control of their inflammatory responses. Basophils from severe malaria patients showed an hyperreactivity to haemozoin and to IgE-dependent stimulations (anti-IgE). Finally, presence of circulating PfTCTP was associated with a higher basophil reactivity, whereas acquisition of anti-PfTCTP antibodies was associated with protection towards excessive cellular reactivity.In conclusion, our results support the hypothesis of an excessive allergic activation during severe malaria. This work might open the way to new therapeutic approaches.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-30T21:16:53Z No. of bitstreams: 1 Matheus Santos de Sá Avaliacao da atividade antimalarica de substancias obtidas de especies vegetais....pdf: 9210254 bytes, checksum: 6b1ee3754729e2a51460a41ce1709423 (MD5) / Made available in DSpace on 2012-07-30T21:16:53Z (GMT). No. of bitstreams: 1 Matheus Santos de Sá Avaliacao da atividade antimalarica de substancias obtidas de especies vegetais....pdf: 9210254 bytes, checksum: 6b1ee3754729e2a51460a41ce1709423 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A malária é uma das mais importantes infecções parasitárias de seres humanos devido à alta morbidade e mortalidade atribuídas a esta doença, que constitui uma ameaça para mais de dois bilhões de pessoas vivendo nas áreas de alta incidência. O Plasmodium falciparum, um dos agentes causadores da malária, apresenta alta capacidade de adaptação por mutação e pode ser resistente a vários tipos de drogas antimaláricas já disponíveis, como a cloroquina, o que torna importante a busca de novos antimaláricos. A região do semi-árido brasileiro abrange cerca de 11,5% do território nacional, e possui o bioma menos estudado em relação à flora e fauna, e um dos que tem sofrido maior degradação pelo uso desordenado e predatório nos últimos 400 anos. Tendo em vista o potencial farmacológico dos produtos naturais, o objetivo desse trabalho foi avaliar a atividade antimalárica de substâncias puras extraídas de espécies vegetais nativas ou endêmicas do semi-árido brasileiro e derivados sintéticos. A partir de uma biblioteca de 160 substâncias triadas para atividade antimalárica, foram selecionadas duas classes de compostos para avaliações in vitro e in vivo: o ácido betulínico e derivados, bem como o lapachol e derivados. Foi selecionada ainda uma terceira classe de moléculas, as fisalinas, utilizando o método do Similarity Ensemble Approach (SEA), que previu a ação antimalárica dessas substâncias. Dentre os derivados do ácido betulínico testados, o acetato do ácido betulínico apresentou a maior potência farmacológica in vitro quando comparado com os outros derivados, e foi ativo in vivo. A atividade antimalárica das fisalinas foi confirmada em ensaios in vitro. Ao serem analisadas in vivo, as fisalinas F e D apresentaram resultados opostos (exacerbação e proteção contra a infecção, respectivamente), possivelmente devido à atividade imunossupressora da fisalina F e ausente na fisalina D. A análise do lapachol e seus derivados iniciou-se através de estudos in silico por Quantitative Structure-Activity Relationship (QSAR), que indicaram ser o isolacet o derivado com maior atividade, o que foi confirmado por ensaios in vitro. A atividade antimalárica do isolacet foi confirmada in vivo, sendo ainda realizados estudos de Docking desta molécula com a falcipaína 2 de P. falciparum, que indicaram ser esta cisteíno-protease um possível alvo do isolacet. Nossos resultados indicam o potencial antimalárico de compostos isolados a partir de plantas do semi-árido e demonstram a importância da associação de várias abordagens para entendimento dos mecanismos de ação de moléculas com atividade farmacológica. / Malaria is one of the most important parasitic infections of humans due to the high morbidity and mortality attributed to this disease, which threatens to over two billion people living in areas with high incidence. Plasmodium falciparum, a causative agent of malaria, has a high capacity to adapt by mutation and may be resistant to various antimalarial drugs already available, such as chloroquine, which makes it important to search for new antimalarials. The Brazilian semi-arid region cover about 11.5% of the country, and the biome has been less studied in relation to flora and fauna, and one who has suffered further degradation and predation by the inordinate use in the last 400 years. Given the pharmacological potential of natural products, the aim of this study was to evaluate the antimalarial activity of pure compounds extracted from native or endemic plant species of arid and semi-synthetic derivatives. From a library of 160 substances screened for antimalarial activity, we selected two classes of compounds for evaluation in vitro and in vivo: The betulinic acid and derivatives, as well as lapachol and derivatives. It was also selected a third class of molecules, physalins using the method of Similarity Ensemble Approach (SEA), who predicted the antimalarial action of these substances. Among the tested derivatives of betulinic acid, betulinic acid acetate showed the highest pharmacological potency in vitro when compared with other derivatives, and was active in vivo. The antimalarial activity of physalins was confirmed in vitro assays. When analyzed in vivo the physalins F and D had the opposite results (exacerbation and protection against infection, respectively), possibly due to the immunosuppressive activity of physalin F and absent in physalin D. The analysis of lapachol and its derivatives was initiated through studies in silico by Quantitative Structure-Activity Relationship (QSAR), which indicated that the isolacet the derivative with greater activity, which was confirmed by in vitro assays. The antimalarial activity of isolacet was confirmed in vivo, and further studies of this molecule by Docking with falcipain 2 P. falciparum, which indicated that this cysteine protease is a possible isolacet target. Our results indicate the potential antimalarial compounds isolated from plants of the semi-arid and demonstrate the importance of the combination of various approaches to understanding the mechanisms of action of molecules with pharmacological activity.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-10T19:15:34Z No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-10T19:15:44Z (GMT) No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-10T19:15:54Z (GMT) No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) / Made available in DSpace on 2015-04-10T19:15:54Z (GMT). No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / O diagnóstico adequado de malária permanece como um dos pilares dos programas de controle da doença no mundo, já que um diagnóstico eficiente permite a identificação precoce de casos e definição do esquema terapêutico, contribuindo para interrupção do ciclo biológico do parasito. A microscopia óptica (MO), atual diagnóstico de referência para malária, tem apresentado limitações, principalmente em casos de co-infecções e baixas parasitemias. Assim sendo, busca-se por técnicas mais sensíveis e específicas para auxiliar a MO, sendo os métodos baseados na reação em cadeia da polimerase (PCR) considerados mais adequados para identificação de indivíduos com infecção submicroscópica. Entretanto, os vários protocolos de PCR apresentados até o momento tem se baseado no gene da subunidade menor do RNA ribossomal 18S dos plasmódios (18S rRNA), que se encontra em poucas cópias no genoma destes parasitos. Recentemente, foram descritas sequências não ribossomais para identificação de Plasmodium vivax (Pvr47) e Plasmodium falciparum (Pfr364), sendo estas sequências promissoras para o diagnóstico molecular de malária. Baseando-se nestes achados, este trabalho propôs o desenvolvimento de um protocolo de Real-Time PCR para validar os alvos Pvr47 e Pfr364 para o diagnóstico de malária vivax e falciparum, respectivamente, com ênfase em infecções mistas e baixas parasitemias. Após padronização com sucesso da técnica de Real-Time PCR (RT-LAMAL), a mesma foi comparada a três outros protocolos moleculares, sendo duas técnicas baseadas no gene 18S rRNA – Nested-PCR (Snounou et al., 1993) e Real-Time PCR (Mangold et al., 2005) – e uma PCR convencional baseada nos alvos Pvr47/Pfr364 (Demas et al., 2011). Para avaliar os protocolos quanto aos seus limites de detecção de infecções únicas e mistas por P. vivax e P. falciparum, foram realizadas titulações de misturas artificiais com diferentes concentrações dos parasitos. Os resultados obtidos nesta etapa revelaram que a PCR-Demas e RT-LAMAL apresentaram os menores limites de detecção para P. vivax e P. falciparum, tanto em infecções únicas quanto mistas, e que o protocolo de RT-Mangold foi incapaz de detectar coinfecções. Posteriormente, os protocolos foram avaliados para o diagnóstico de malária em amostras de campo, incluindo área não endêmica (n=117) e área endêmica para malária (n=163). Os resultados revelaram que os protocolos de RTMangold, PCR-Demas e RT-LAMAL foram mais eficientes na detecção de parasitemias submicroscópicas, porém, o protocolo de RT-Mangold novamente mostrou ser incapaz de detectar infecções mistas. Em conjunto, os dados obtidos demonstraram que os alvos Pvr47/Pfr364 foram mais adequados para o diagnóstico molecular de malária vivax e falciparum do que o gene 18S rRNA. Contudo, o protocolo aqui desenvolvido (RT-LAMAL) se mostra em vantagem à PCR-Demas, com maior rapidez na obtenção de resultados, dispensando a revelação em gel de agarose e uso de brometo de etídeo, além de diminuir a possibilidade de contaminação de reagentes e amostras. Portanto, conclui-se que a RT-LAMAL possui grande potencial para o diagnóstico molecular de certeza de pacientes com infecções mistas e baixas parasitemias. / Accurate diagnosis of malaria remains as one of the pillars for prevention and control of the disease. An efficient diagnostic test may allow early case detection and appropriate treatment, therefore contributing to the interruption of malaria transmission cycle. Because the optical microscopy (OM) – the gold standard of malaria diagnosis – presents significant limitations, mainly in cases of co-infections and low parasitemias, it seems to be essential to develop a more sensitive and specific diagnostic tool. In this context, methods based on the polymerase chain reaction (PCR) seem to be more suitable for detecting individuals with submicroscopic malaria infections. Unfortunately, the majority of PCR-based methods still rely on the 18S rRNA gene targets, present in few copies in the parasite genome. Recently, new target DNA sequences were described for the identification of Plasmodium vivax (Pvr47) and Plasmodium falciparum (Pfr364). Due to the importance of these findings, the goal of the present study was to validate the Pvr47 and Pfr364 targets for the diagnosis of vivax and falciparum malaria, focusing on the development of a real-time PCR for detection of mixed infection and sub-microscopic parasitemia. After successful standardization of the Real-Time PCR (RT-LAMAL), this-PCR protocol was compared with three well-established PCR protocols, two of them relied on the gene 18S rRNA – Nested-PCR (Snounou et al., 1993) and Real-Time PCR (Mangold et al., 2005) -- and a third protocol based on the Pvr47/Pfr364 as target for a conventional PCR assay (Demas et al., 2011). In order to evaluate these different PCR-protocols in terms of their limit of detection, titrations of artificial mixtures of DNA plasmodial were performed. The results revealed that the PCRDemas and RT-LAMAL presented the lowest limit of detection for either single or mixed P. vivax and P. falciparum infections. In addition, the RT-Mangold protocol was unable to detect co-infections. To further evaluate the performance of these four PCR protocols for diagnosis of malaria in the field, we analyzed samples from malaria-endemic areas (n=163) as well as from non-endemic area (n = 117). While the results confirmed that PCR-Demas and RT-LAMAL protocols as being more appropriate for the diagnosis of submicroscopic infection, the data demonstrated again that the RT-Mangold protocol was unable to detect mixed infections. Together, the data confirmed Pvr47/Pfr364 targets as more suitable for molecular diagnosis of P. vivax and P. falciparum. Of interest, the Real-time PCR developed here (RTLAMAL) offers several advantages over the traditional PCR-Demas, including faster processing time and decreased risk of contamination. In conclusion, that RT-LAMAL has great potential for molecular diagnosis of patients with mixed infections and low levels of parasitemia.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:19:42Z No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:19:53Z (GMT) No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) / Made available in DSpace on 2016-02-19T13:19:53Z (GMT). No. of bitstreams: 1 Clarissa Cunha Santana Avaliação... 2015.pdf: 1937824 bytes, checksum: 1d212794cc3bec0caa6b8414a186531b (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A malária é uma doença causada por cinco espécies de parasitos do gênero Plasmodium que causa anualmente a morte de milhares de pessoas, principalmente em países pobres da África. Muito antiga, uma diversidade de fármacos já foram empregados na tentativa de erradicação da doença, entretanto o aparecimento de cepas resistentes, bem como efeitos adversos gerados pelo tratamento impossibilitou tal ação. Os quinolínicos configuram uma grande parte destes tratamentos, apresentando uma notável atividade antimalárica. Neste trabalho nós avaliamos o potencial antimalárico de três novos derivados quinolínicos BS 260, BS 318 e BS 373 em culturas de Plasmodium falciparum, cepa w2, cloroquina resistente. BS 373 apresentou melhor atividade contra culturas de Plasmodium falciparum e, assim como o BS 318, foi capaz de inibir a biocristalização de hemozoína pelos parasitos. A microscopia eletrônica de transmissão revelou uma desorganização celular, diminuição do tamanho e quantidade de cristais de hemozoína no vacúolo digestivo, bem como vacuolizações citoplasmáticas e presença de estruturas membranares no vacúolo digestivo, o que indica a ocorrência de um processo autofágico nas células tratadas com 10 LM e 20 LM do BS 373. A presença de cristais citoplasmáticos indica a ocorrência de autólise pela ruptura da membrana do vacúolo digestivo. Por fim, o efeito dos tratamentos se mostrou irreversível nos parasitos com 24 horas de tratamento para BS 318 e BS 373, enquanto que para BS 260 essa irreversibilidade só foi observada após 48 horas. Nossos dados mostram que os derivados quinolínicos testados são efetivos contra culturas de P. falciparum, configurando bons candidatos à novas moléculas antimaláricas. / Malaria is a disease caused by five Plasmodium species that cause deaths of thousands of people annually, mostly in poor countries of Africa. Very anccient, a variety of drugs have been used in an attempt to eradicate the disease, however the emergence of resistant strains, as well as adverse effects caused by treatment prevented such action. The quinoline are a large part of these treatments, presenting a remarkable antimalarial activity. In this paper we evaluate the antimalarial potential of three new quinoline derivative BS 260, BS 318 and BS 373 in Plasmodium falciparum chloroquine resistant, w2 strain, cultures. BS 373 showed the best activity against Plasmodium falciparum cultures, while and analogously to BS 318 was able to inhibit the hemozoin formation by parasites. The transmission electron microscopy revealed a cell disorganization, decreased size and amount of hemozoin crystals in the digestive vacuole, cytoplasmic vacuolization and presence of membrane structures in the digestive vacuole, which indicates an autophagic process in cells treated with 10 LM and 20 LM BS 373. Cytoplasmic being crystals indicate parasite cell autolusis caused by digestive vacuole membrane disrupture. Finally, the effect of treatment proved irreversible on parasites at 24 hours of treatment for BS 318 and BS 373, whereas for BS 260 this irreversibility was only observed after 48 hours. Our data show that the quinoline derivatives tested are effective against P. falciparum cultures, setting good candidates for new antimalarial molecules.