Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.). / Written for the Institute of Parasitology. Title from title page of PDF (viewed 2008/02/12). Includes bibliographical references.

L'aggravation actuelle du risque lié au paludisme résulte du développement du phénomène de résistance de souches de Plasmodium falciparum aux molécules antipaludiques. Une telle situation et l’absence de vaccin efficace nécessitent le développement de nouvelles stratégies antiparasitaires. Jusqu’à présent, les mécanismes moléculaires qui contrôlent le cycle parasitaire sont méconnus. Chez la plupart des eucaryotes, les protéine kinases sont impliquées dans des fonctions cellulaires essentielleset constituent une cible privilégiée pour la conception de nouveaux médicaments. Dans cecadre, nous nous sommes intéressés à la voie de transduction de l’AMP cyclique et en particulier à la sous-unité catalytique de la protéine kinase AMPc dépendante (PfPKAc)dont le rôle essentiel reste mal défini chez P. falciparum. Deux approches complémentaires ont été choisies pour étudier cette kinase :1) au niveau biochimique par le clonage, l’expression, la purification et la caractérisation enzymatique de la PfPKAc. L’objectif était d’obtenir une enzyme active in vitro de façon à pourvoir mesurer les constantes enzymatiques de la PfPKAc et conduire les premiers essais d’inhibitions.2) au niveau cellulaire en analysant les conséquences de l’inhibition par des ARN interférents spécifiques des transcrits de la PfPKAc. Le développement parasitaire mais également le transcriptome global ont été étudiés de manière à préciser les voies métaboliques liées à cette kinase plasmodiale.L’ensemble de ces études précise la compréhension de la voie de transduction de l’AMP cyclique et de la PfPKA qui pourrait conduire au développement de nouvelles voies thérapeutiques. / Nowadays, the increase of risks associated with malaria results from the development of resistance of Plasmodium falciparum strains to antimalarial drugs. This situation and the lack of an effective vaccine require the development of new antimalarial strategies. Untilnow, molecular mechanisms controlling the life cycle of malaria parasites, are still poorly understood. In most eukaryotes, protein kinases are implicated in essential cellular functions and represent attractive targets for the development of new drugs. In this context, we focused on the signaling pathway implicating cAMP and particularly the catalytic subunit of cAMP-dependent protein kinase (PfPKAc), whose function is still unclear in P. falciparum. Two complementary strategies were chosen to study this kinase:1) at the biochemical level by the cloning, expression, purification and enzymatic characterization of the PfPKAc. The objective was to obtain an in vitro active PfPKAc to evaluate the kinetic constants of PfPKAc and to conduct the first inhibition studies.2) at the cellular level by studying the consequences of PfPKAc transcripts inhibition byspecific interfering RNAs. The parasite growth but also the overall transcriptome werestudied to specify the metabolic pathways associated with this plasmodial protein kinase.All of these studies improve the understanding of cAMP transduction pathway and PfPKA,which could allow the development of new therapeutic approaches.

Le paludisme reste l'une des plus redoutables maladies infectieuses avec plus de 200 millions d'infections et près de 430 000 décès chaque année, principalement des enfants de moins de 5 ans vivant en Afrique subsaharienne. L'espèce Plasmodium falciparum est responsable de la grande majorité de la mortalité. Le contrôle de l'endémie palustre reste encore aujourd'hui un problème majeur de santé publique, notamment à cause des résistances aux antipaludiques développées par les parasites. L'apparition de ces résistances s'opère par la pression de sélection médicamenteuse, et leur diffusion progressive se fait principalement via le déplacement des hôtes infectés. Cependant, la dynamique d'émergence, de diffusion et de persistance des parasites résistants résulte d'interactions complexes entre les antipaludiques, l'Homme, le parasite et le vecteur. Le travail présenté ici participe à la démarche de lutte contre le paludisme en proposant tout d'abord un état des lieux de la résistance de Plasmodium aux antipaludiques utilisés au Cameroun, avec des outils moléculaires, phénotypiques et cliniques. Une deuxième partie explore, in vitro, les possibles conséquences d'une utilisation prolongée des dérivés d'artémisinine sur le phénotype de P. falciparum, alors que la résistance à cette molécule est déjà installée. Le modèle in vitro utilisé a permis de mettre en évidence un nouveau profil de pluri-résistance suite à des pressions continues à l'artémisinine. Enfin, une dernière partie de ce travail analyse le rôle du moustique dans l'épidémiologie des résistances et montre que la sporogonie favoriserait la diffusion des allèles minoritaires, résistants ou non, présents chez l'Homme. L'ensemble de ces travaux confirme la multiplicité des facteurs agissants sur la dynamique de résistance et la complexité de leurs interactions rendant toute prévision très spéculative. Même si une meilleure connaissance des phénomènes sociétaux, épidémiologiques, biologiques et pharmacologiques impliqués dans les résistances reste une priorité, la surveillance phénotypique et génotypique régulière sur le terrain apparait à ce jour, le meilleur outil pour adapter au mieux les stratégies de contrôle du paludisme. / Malaria remains one of the most terrible infectious diseases with more than 200 million infections and 430,000 deaths each year, mostly children under five years old in sub-Saharan Africa. Plasmodium falciparum is responsible for the vast majority of malaria mortality cases. Control of malaria still remains a major public health problem, in particular because of resistances to antimalarials that parasites developed. The apparition of these resistances is due to the drug pressure, and their progressive diffusion is mainly via the travelling of infected hosts. However, the dynamics of emergence, diffusion and persistence of resistant parasites result from complex interactions between the antimalarials, the Human, the parasite and the vector. The work presented here participates in the malaria control process by first proposing an inventory of Plasmodium resistance to antimalarials used in Cameroon, thanks to molecular, phenotypic and clinical tools. A second part explores the possible consequences of prolonged use of artemisinin derivatives on the P. falciparum phenotype, in areas where resistance to this molecule is already established. The in vitro model used showed that continuous artemisinin pressures induced a new pluri-resistance profile. Finally, a last part analyses the role of the mosquito in the epidemiology of resistances and shows that the sporogony favours the diffusion of minority alleles, resistant or not, presented in humans. All this work confirms the multiplicity of forces acting on the dynamics of resistances and the complexity of their interactions making any prediction very speculative. Even if better knowledge of the societal, epidemiological, biological and pharmacological phenomena involved in resistances is a priority, regular phenotypic and genotypic surveillance in the field remains the best tool for adapting malaria control strategies.

L’objectif général de cette thèse était d’étudier le rôle de la protéine soluble HLA-G (sHLA- G) dans la variabilité des réponses à l’infection par P. falciparum chez la femme enceinte et son nouveau-né pendant ses deux premières années de vie. Précisément, nous avons étudié, chez les mères pendant la grossesse et leurs nourrissons de la naissance à 2 ans, les relations entre les niveaux de sHLA-G et l’infection palustre au niveau périphérique et placentaire. Nous avons également étudié les polymorphismes génétiques situés dans la région 3’UTR du gène HLA- G chez les mères et leurs nourrissons par la détermination des fréquences alléliques, génotypiques et haplotypiques afin évaluer l’impact de ces polymorphismes sur la cinétique d’expression de sHLA-G dans un contexte d’infection palustre. Nos résultats ont montré une association entre le niveau élevé de sHLA-G chez les nourrissons et l’augmentation du risque d’infection palustre au cours du trimestre suivant. Ces niveaux élevés de sHLA-G dans le sang de cordon ont été également associés au faible poids de naissance du nouveau-né. De même, nous avons trouvé une forte corrélation entre les niveaux de sHLA-G maternels dans le sang périphérique à l’accouchement et ceux de l’enfant dans le sang du cordon à la naissance. Nous avons aussi montré l’existence de trois profils d’expression de sHLA-G chez les individus inclus dans l'étude. Certains individus expriment la protéine à chaque prélèvement (HLA-G ++) alors que d’autres l’expriment par intermittence (HLA-G +-) ou ne l’expriment pas (HLA-G --). Le risque de développer un accès palustre chez les mères était respectivement trois fois plus élevé (p=0,001, OR=3,47;p=0,008, OR=3,14) chez celles appartenant au groupe HLA-G (++) et HLA-G (+-) que le groupe HLA-G (--). L’analyse génétique de la région 3’UTR du gène nous a permis de mettre en évidence huit sites polymorphes dans cette région et de construire six haplotypes correspondant (UTR 1, 2, 3, 4, 5, 6). Nous avons aussi montré chez les mères une association entre l’allèle T en position +3001 (C/T) et une expression plus fréquente de sHLA-G tandis que l’allèle C en position +3003 (T/C) et l’haplotype UTR-4 ont été associés à une expression moins fréquente de la protéine. Ces associations n’ont pas été mises en évidence chez les enfants. L’ensemble de ces résultats suggère l'implication de sHLA-G dans la sensibilité à l'infection palustre. Cette sensibilité serait, en partie, corrélée à l’inhibition des réponses anticorps spécifiquement dirigées contre P. falciparum. sHLA-G pourrait donc à terme devenir un bio-marqueur de susceptibilité au paludisme chez la femme enceinte et chez le nouveau-né au cours des premières années de vie. / The general objective of this thesis was to study the role of soluble HLA-G protein (sHLA-G) in the variability of individual response to malaria during pregnancy and during the first 2 years of infant life. Actually, we assessed the relationships between sHLA-G and malaria infection in peripheral and placental blood. We also investigated the effect of polymorphisms in the 3’UTR region of HLA-G gene in 400 mothers and their infants on the kinetic of sHLA-G expression three times during pregnancy and at 6, 9, 12, 18, 24 months of life in a context of malaria infection. Our results showed that high levels of sHLA-G increased the risk of malaria at the subsequent trimester in infants and were associated with low birth weight. We also showed a strong correlation between the plasmatic sHLA-G level of the mothers at delivery and those of newborns in cord blood. We found that the risk of developing malaria in mothers was respectively three fold higher in the HLA-G (++) (OR=3.47; p=0.001) and HLA-G(+-)(OR=3.14, p=0.008) groups compared to HLA-G (--) group. Besides, we described eight polymorphic sites in the 3’UTR corresponding to six haplotypes (UTR 1, 2, 3, 4, 5, 6) and showed in mothers, an association between the allele T at position +3001 (C/T) and a higher frequency of sHLA-G expression. However, the allele C at position +3003 (T/C) and UTR-4 were associated to a lower frequency of sHLA-G expression. In infants, no association was observed between alleles or haplotypes and expression of the soluble protein. Overall, these results suggest that sHLA-G is implicated in malaria susceptibility. This could be partly, related to the inhibition of P. falciparum-specific antibody responses. Therefore, sHLA-G might be useful as a bio- marker of malaria susceptibility during pregnancy and during the first years of infancy.

Dans ce travail de thèse, nous avons développé et optimisé une stratégie originale pour la caractérisation des extrémités N-terminales des protéines et des sites de clivages protéolytiques. Elle s’appuie sur la dérivation chimique spécifique des amines N-terminales et nous l’avons adapté à différents types d’échantillons biologiques. L’application de cette stratégie dans des études en biologie nous a permis d’apporter plusieurs éléments de réponse à différentes problématiques. Nous avons ainsi caractérisé les peptides de transit des protéines mitochondriales humaines et ainsi validé/corrigé expérimentalement leurs prédictions dans les banques de données. Nous avons aussi appliqué cette stratégie à l’étude du protéome du parasite P. falciparum. La mise au point de la dérivation N-terminale de protéines immobilisées dans un gel SDS PAGE nous a permis d’étudier le mécanisme d’export des protéines de ce parasite vers sa cellule hôte et de déterminer le rôle des acides aminés impliqués dans cet export. Un réactif de dérivation marqué aux isotopes stables permet d’effectuer des études différentielles des processus protéolytiques que subissent les protéines. Cette stratégie quantitative a été appliquée à l’étude du protéome hépatique du rat soumis au jeûne expérimental. D’autres applications de l’analyse protéomique en biologie sont aussi présentées dans ce manuscrit. / In this manuscript, we describe the development and the optimization of an original strategy for the characterization of protein N-termini and protease cleavage sites. The strategy is based on specific chemical derivation of alpha-amines. We applied this method to the characterization of mitochondrial proteins’ transit peptides which allowed us to experimentally validate/correct their prediction in protein databases. In another study, the strategy was applied to the analysis of the proteome of the malaria parasite Plasmodium falciparum. The optimization of ingel N-terminal derivation and its application to the study of parasite exported proteins allowed us to determine the role of implicated amino acid residues in the signaling and export mechanism of these proteins to the host cell. To enable differential studies of proteolysis, we introduced an isotope labeled derivation reagent. This quantitative method was applied in the context of a study of the rat liver proteome after experimental long-term fasting. Other applications of proteomics in biology are also presented in this manuscript.

Made available in DSpace on 2016-08-17T18:39:34Z (GMT). No. of bitstreams: 1 3220.pdf: 7184046 bytes, checksum: d31437c1aa1937336c7b8cb91918b19b (MD5) Previous issue date: 2010-08-06 / Universidade Federal de Minas Gerais / Malaria disease, caused mainly by Plasmodium falciparum parasite, afflicts about 500 million people and causes nearly one million deaths every year. For the development of new drugs against this disease, one possible approach is to identify an enzyme that plays a key role in P. falciparum development and presents significantly different properties from the corresponding human one. These differences can be exploited in the design of specific inhibitors of the parasite s protein, thus, three different enzymes were selected as possible targets. As there are evidences suggesting that increasing oxidative stress can effectively inhibit the growth of the malarial parasite the enzyme Glutathione Reductase of P. falciparum (PfGR), responsible for the parasite s antioxidant defense, has become a potential target for the design and development of inhibitors. The second target was the P. falciparum Dihydrofolate Reductase-Thymidylate Synthase (PfDHFR-TS), and in this case blocking its action stops the dTMP production and DNA synthesis in the parasite. The third chosen target was the P. falciparum Lactate Dehydrogenase (PfLDH), whose inhibition interrupts the ATP formation and thus causing the death of the parasite. So that a family of arilloxazines compounds, together with chloroquine and methylene blue, were studied by means of docking simulations in the binding sites of these enzymes and also in the corresponding human enzymes for comparison. The three-dimensional structures of the enzymes and of chloroquine and methylene blue were obtained from the Protein Data Bank (PDB). The structures of the arilloxazines compounds, in turn, were obtained by molecular modeling with HyperChem 6.01 and MOPAC2009 programs, using as starting models similar crystallographic structures deposited in the Cambridge Structural Database. Docking simulations were performed using GOLD 4.0.1. The docking results showed that the enzymes PfGR and PfDHFR-TS are not the preferential targets of chloroquine. For the methylene blue it was possible to elucidate its binding mode in hGR and PfGR. Regarding the arilloxazines it was possible to show that they present their higher affinity for hGR, followed by PfGR, hDHFR, PfDHFR-TS, PfLDH and hLDH. In the case of GRs, the interface site was the preferred binding site. The results suggest that if arilloxazines compounds with higher affinity for PfGR are desirable then a pentafluorophenyl should be attached at the N10 position, as in the 2e compound. When searching for arilloxazines with higher affinity for PfLDH, it seems to be desirable a carboxymethyl group at the N3 position (as in 5b) and a pentafluorophenyl group at N10 (as in 2e). Finally, the results suggest that in general the studied arilloxazines probably will present a higher affinity for hDHFR than PfDHFR-TS. All these results are an important starting point for the design of new arilloxazines ligands so that they can be used as lead compounds in the search for new drugs against malaria. / A malária, causada principalmente pelo Plasmodium falciparum, atinge cerca de 500 milhões de pessoas e causa aproximadamente um milhão de mortes todos os anos. Para o desenvolvimento de novos fármacos contra esta doença, uma das abordagens possível é identificar uma enzima que desempenhe papel vital no desenvolvimento do P. falciparum e apresente propriedades significantemente diferentes das enzimas humanas correspondentes, de modo que tais diferenças possam ser exploradas no design de inibidores específicos à proteína do parasita. Existem evidências sugerindo que aumentar o estresse oxidativo pode inibir eficientemente o crescimento do parasita causador da malária e, portanto, a enzima Glutationa Redutase do P. falciparum (GRPf), responsável por sua defesa antioxidante, tornou-se um alvo em potencial para o desenvolvimento de inibidores. Também, o bloqueio da ação da Diidrofolato Redutase-Timidilato Sintase do P. falciparum (DHFR-TSPf) interrompe a produção de dTMP e a síntese de DNA no parasita. Ainda, espera-se que a inibição da Lactato Desidrogenase do P. falciparum (LDHPf) interrompa a produção de ATP no parasita e, consequentemente, cause sua morte. Portanto, estudou-se o comportamento de compostos da família das ariloxazinas, da cloroquina e do azul de metileno nos sítios de ligação destas enzimas, além das enzimas humanas correspondentes para fins de comparação, por meio de cálculos de docking. As estruturas tridimensionais das enzimas foram obtidas no Protein Data Bank (PDB). As estruturas dos inibidores da família das ariloxazinas, por sua vez, foram obtidas por meio de modelagem molecular, utilizando os programas HyperChem 6.01 e MOPAC2009, a partir de estruturas cristalográficas semelhantes obtidas no Cambridge Structural Database; já as estruturas da cloroquina e do azul de metileno foram obtidas também no PDB. Os cálculos de docking destes compostos nos sítios de ligação das enzimas estudadas foram realizados utilizando o programa GOLD 4.0.1. Com base nos resultados de docking, sugere-se que as enzimas GRPf e DHFR-TSPf não são alvos preferenciais da cloroquina. Também, pôde-se elucidar o possível modo de ligação do azul de metileno nas enzimas GRh e GRPf. No geral, foi possível sugerir ainda que as ariloxazinas devam apresentar maior afinidade pela GRh, seguida por GRPf, DHFRh, DHFR-TSPf, LDHPf e LDHh, nesta ordem. Nas GRs, o sítio da interface foi o sítio preferencial de ligação. Para se buscar inibidores da família das ariloxazinas com maior afinidade pela GRPf, sugere-se considerar um pentafluorfenil como substituinte na posição N10, como no composto 2e. Ainda, na busca por ariloxazinas com maior afinidade pela LDHPf, sugere-se considerar um carboximetil na posição N3 (como o de 5b) e um pentafluorfenil na posição N10 (como em 2e). Por fim, foi obtido que as ariloxazinas estudadas possivelmente apresentarão, em geral, uma maior afinidade pela DHFRh do que pela DHFR-TSPf. Estes dados podem ser tomados como ponto de partida para o design de novos compostos da família das ariloxazinas, a fim de que possam atuar como compostos líderes na busca por novos fármacos contra a malária.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-14T08:51:05Z No. of bitstreams: 2 Dissertação - Maríilia Nunes do Nascimento - 2015.pdf: 2988580 bytes, checksum: 941d336cea1a51aeb45d97702067875a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-15T08:54:06Z (GMT) No. of bitstreams: 2 Dissertação - Maríilia Nunes do Nascimento - 2015.pdf: 2988580 bytes, checksum: 941d336cea1a51aeb45d97702067875a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-15T08:54:06Z (GMT). No. of bitstreams: 2 Dissertação - Maríilia Nunes do Nascimento - 2015.pdf: 2988580 bytes, checksum: 941d336cea1a51aeb45d97702067875a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-03 / Malaria is a serious endemic disease caused by parasites of the genus Plasmodium, which affects much of the population, especially in tropical and subtropical areas. Currently, drug therapy makes use of artemisinin or its derivatives associated with a second anti-malarial drug. The shortage of new treatments as well as the spread of parasite resistance to drugs currently available, makes urgent the search and discovery of new targets and new antimalarial drugs. The enzyme deoxyuridine triphosphatase (dUTPase) of Plasmodium falciparum plays an important role in maintaining balance between 2'-deoxyuridine 5'-triphosphate (dUTP) and 2'- deoxitimina 5'-triphosphate (dTTP) in order to avoid the erroneous incorporation uracil on the DNA tape. Thus, the enzyme dUTPase is a potential target for the development of new drugs, and has been validated for the organisms Escherichia coli, Saccharomyces cerevisiae and Mycobacterium smegmatis. This study aimed to carry out quantitative studies of the relationship between structure and activity (QSAR) to a series of β-branched nucleoside inhibitors PfdUTPase, in order to generate robust and predictive models to predict compounds activity untested and that may help to elucidate the important structural requirements for the affinity of this class of compounds. For this, there was the hologram QSAR analysis (HQSAR), comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA). For studies of CoMFA and CoMSIA were tested two methods of calculation of partial charges, the empirical method Gasteiger-Huckel and the semi-empirical method AM1-BCC. Were also tested three structural alignment strategies based on the binder: maximum common substructure, based on the overlap of molecular volumes, and on the basis of morphological similarities; and a strategy based on the 3D coordinates of the enzymeinhibitor complex (molecular docking). The QSAR models generated showed good robustness and external predictability, showing good power correlation and prediction of affinity. The HQSAR contribution maps and contour maps of the CoMFA and CoMSIA indicated the importance of certain groups for affinity, such as the importance of the presence of at least two of trityl rings that contribute both sterically as hydrophobically to interact with the hydrophobic site of the parasite enzyme, non-existent in the human enzyme. The drug design based on information obtained from 2D and 3D QSAR, generated 121 molecules grouped into 18 clusters. Two hits with approximate power to one of the most active compounds of the series stood out by presenting appropriate physicochemical properties. / A malária é uma doença endêmica grave, causada por parasitos do gênero Plasmodium, que afeta grande parte da população, em especial nas áreas tropicais e subtropicais. Atualmente, o tratamento farmacológico faz uso de artemisinina ou de seus derivados associado a um segundo fármaco antimalárico. A escassez de novos tratamentos assim como a disseminação da resistência do parasito aos fármacos atualmente disponíveis, torna urgente a busca e descoberta de novos alvos e novos fármacos antimaláricos. A enzima deoxiuridina trifosfatase (dUTPase) de Plasmodium falciparum desempenha um papel importante na manutenção do equilíbrio entre 2’-desoxiuridina 5’-trifosfato (dUTP) e 2’-deoxitimina 5’-trifosfato (dTTP), a fim de evitar a incorporação errônea de uracila na fita do DNA. Dessa forma, a enzima dUTPase é um alvo potencial para o desenvolvimento de novos fármacos, e já foi validada para os organismos Escherichia coli, Saccharomyces cerevisiae e Mycobacterium smegmatis. Este trabalho teve como objetivo a realização de estudos quantitativos de relação entre estrutura e atividade (QSAR) para uma série de nucleosídeos β-ramificados inibidores da PfdUTPase, com a finalidade de se gerar modelos robustos e preditivos para predizer a atividade de compostos não testados e que possam auxiliar na elucidação dos requisitos estruturais importantes para a afinidade desta classe de compostos. Para isso, realizou-se a análise de holograma QSAR (HQSAR), análise comparativa de campos moleculares (CoMFA) e a análise comparativa dos índices de similaridade molecular (CoMSIA). Para os estudos de CoMFA e CoMSIA, foram testados dois métodos de cálculo de cargas parciais, o método empírico Gasteiger-Huckel e o método semi-empírico AM1-BCC. Foram também testadas três estratégias de alinhamento estrutural baseadas no ligante: máxima subestrutura comum, baseada na sobreposição de volumes moleculares, e em função da similaridade morfológica; e uma estratégia baseada nas coordenadas 3D do complexo enzima-inibidor (docking molecular). Os modelos de QSAR gerados apresentaram boa robustez e preditividade externa, demostrando bom poder de correlação e predição da afinidade. Os mapas de contribuição de HQSAR e os mapas de contorno do CoMFA e CoMSIA indicaram a importância de determinados grupos para a afinidade, como por exemplo, a importância da presença de ao menos dois anéis tritila que contribuem tanto estericamente como hidrofobicamente para interação com o sítio hidrofóbico da enzima do parasito, inexistente na enzima de humanos. O planejamento de fármacos baseado nas informações obtidas do QSAR 2D e 3D, gerou 121 moléculas agrupadas em 18 clusters. Dois hits com potência aproximada a um dos compostos mais ativos da série se destacaram por apresentar propriedades físico-químicas apropriadas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dentre as espécies florestais nativas da Amazônia utilizadas por populações tradicionais na terapêutica de diversos problemas de saúde estão a Geissospermum argenteum Woodson, uma Apocynaceae, e a Minquartia guianensis Aubl., da família Olacaceae. Entre os problemas de saúde está a malária, que no Brasil e no mundo ainda é um grave problema de saúde pública. Outro agravo à saúde de importância são infecções microbianas, pois os micro-organismos têm desenvolvido resistência aos agentes antimicrobianos. Neste trabalho, foram coletadas amostras de cascas, folhas e galhos de G. argenteum e M. guianensis, em área de floresta, na Vila Apiaú, município de Mucajaí RR. As amostras foram submetidas à extração metanólica a quente e extração aquosa, Amostra de extrato metanólico de cascas de G. argenteum foi submetida a particionamento líquido-líquido, resultando nas frações hexânica, clorofórmica, acetato de etila e metanol/água. A fração clorofórmica foi selecionada para cromatografia em coluna com sistema gradiente de solventes, obtendo-se 38 frações, as quais foram analisadas através de CCD e destas a fração Cr10 foi selecionada para cromatografia preparativa, a partir da qual foi obtida a fração F6, que analisada em CCD mostrou-se positiva para alcalóides. Os extratos metanólicos e aquosos de ambas as espécies, e frações primárias provenientes de extratos G. argenteum foram testados em ensaios in vitro para atividade antimalárica frente à cepa cloroquino-resistente, K1, de Plasmodium falciparum, nas concentrações de 50 e 5 μg/mL. Posteriormente, amostras ativas foram avaliadas em 7 diluições para estabelecer a relação dose-resposta e valores de concentração inibitória mediana (CI50). Amostras provenientes das duas espécies também foram testadas contra as cepas bacterianas, Staphylococcus aureus, Streptococcus mutans e Escherichia coli, e contra a levedura Candida albicans. A atividade antimicrobiana foi avaliada através de difusão em ágar e a concentração inibitória mínima (CIM) por microdiluição em placas. Para as frações acetato de etila, clorofórmica, hexânica e hidroalcoólica obtida de cascas de G. argenteum e a fração Cr10 foi realizada a bioautografia para S. aureus. Na atividade antimalárica o extrato metanólico de casca foi ativo, com CI50 de 4,6 μg/mL e a fração clorofórmica obtida de casca de G. argenteum também foi ativa com CI50 de 2,0 μg/mL. Os extratos de M. guianensis foram considerados inativos para a atividade antimalárica. Na atividade antimicrobiana, através de difusão em ágar, extratos de G. argenteum foram parcialmente ativos contra S. aureus, S. mutans e inativos contra E. coli e C. albicans. A CIM para S. aureus foi de 0,63 mg/mL para a fração metanol/água, para S. mutans, CIM de 0,63 mg/mL e C. albicans com CIM de 0,63 mg/mL. Para cepa de E. coli os extratos foram inativos. Extratos de M. guianensis mostraram-se ativos frente à S. aureus e C. albicans através de difusão em ágar e apresentaram a CIM superior a 1mg/ml para S. aureus, S. mutans e C. albicans. Os extratos de M. guianensis foram inativos contra E. coli. Na autobiografia para S. aureus as frações testadas apresentaram atividade. / Among the natives species from the Amazon Forest used by traditional populations in the treatment of various health problems, including, malaria, wich in Brazil and in the world remains a serious public health problem, are Geissospermum argenteum Woodson, an Apocynaceae, and Minquartia guianensis Aubl, Olacaceae family. Another important health problem is the microbial infections, because the microorganisms have developed resistence to antimicrobial agents. In this work, samples were collected from bark, leaves and twigs of G.argenteum and M. guianensis in a forestall area in Apiaú village, city of Mucajaí RR. The samples were extracted by hot methanol and water extraction, resulting in methanol and aqueous extracts. Sample of methanol extract of bark of G.argenteum was submitted to liquid-liquid partitioning, resulting in fractions hexane, chloroform, ethyl acetate and methanol/water. The chloroform fraction was selected for fractionation using chromatography colunn and solvent gradient system, resulting in 38 fractions, which were analyzed by TLC and the fraction Cr10 was selected for preparative chromatography, from which the fraction F6 was obtained, analyzed in CCD was positive for alkaloids. The methanol and aqueous extracts of both species and fractions of extracts from primary G.argenteum were tested in vitro assays for antimalarial activity against the chlroquine-resistant K1 Plasmodium falciparum at concentrations of 50 and 5 mg/mL. Subsequently, active samples were evaluated in 7 diluitions to establish the dose-response and median inhibitory concentration values (IC50). Samples from two species were also tested against bacterial strains, Staphylococcus aureus, Streptococcus mutans and Escherichia coli, and against the yeast Candida albicans. Antimicrobial activity was evaluated by agar diffusion and minimum inhibitory concentration (MIC) by microdilution plates. For fractions of ethyl acetate, chloroform, hexane and water-alcohol obtained from barks of G.argenteum, fraction Cr10 and bioautography was performed fo S. aureus. Antimalarial activity in the methanol extract of bark was active, with IC50 of 4,6 mg/mL and chloroform fraction obtained from bark of G.argenteum was also active with IC50 of 2,0 mg/mL. The extracts of M. guianensis were considered inactive for antimalarial activity. In antimicrobial activity by agar diffusion, extracts of G.argenteum were partially active against S. aureus, S. mutans and inactive against E. coli and C. albicans. The MIC for S. aureus was 0,63 mg/mL per fraction methanol/water, S. mutans, MIC of 0,63 mg/mL and C. albicans with an MIC of 0,63 mg/mL. For the strain of E.coli there was no activity. Extracts of M. guianensis were active against the S. aureus and C. albicans by agar diffusion and presented more than 1 mg/mL MIC for S aureus, S.mutans and C. albicans. The extracts of M. guianensis were inactive against E. coli.

Les interventions de lutte contre le paludisme, comme la distribution massive de moustiquaires imprégnées et les polythérapies à base d’artémisinine, ont permis depuis les années 2000 de réduire le nombre de décès d’environ 60% au plan mondial, avec de grandes disparités d’un pays à l’autre. Si certains pays sont entrés en phase d’élimination, d’autres, et particulièrement en Afrique sub-Saharienne, doivent encore contrôler la maladie. La mise au point d’un vaccin contre le paludisme reste l’une des voies prioritaires pour la lutte contre la maladie. Parmi les différents candidats vaccins, les vaccins visant à bloquer la transmission chez le moustique vecteur connaissent un intérêt croissant. Dans ce travail de thèse, j’ai évalué l’efficacité d’anticorps anti-AnAPN1 à bloquer la transmission d’isolats naturels de Plasmodium falciparum chez An. coluzzii. L’aminopeptidase 1 est une protéine qui tapisse l’épithélium intestinal du moustique et qui servirait de récepteur aux ookinètes. Nous avons réalisé des essais d’infections expérimentales sur la plateforme d’entomologie de l’OCEAC à Yaoundé. Les anticorps anti-AnAPN1, 4H5B7, ont montré une activité bloquante dose-dépendante et l’inhibition de la transmission varie selon le donneur de gamétocytes. Nous avons alors modélisé les différents paramètres qui pouvaient moduler l’efficacité de l’anticorps et nous avons montré que la multiplicité de l’infection du donneur avait un effet significatif. Nous avons ensuite étudié la dynamique de transmission des génotypes de P. falciparum sous pression d’anticorps à des doses non bloquantes afin d’identifier les génotypes qui ne sont pas sensibles à l’anticorps. Le traitement réduit la diversité génétique de la population de parasites mais nous n’avons pas observé de sélection de génotypes particuliers de P. falciparum suite à l’exposition des moustiques aux anticorps anti-AnAPN1. Les analyses de génétique des populations ont révélé une corrélation positive entre le FIS et la multiplicité d’infection, les gamètes de P. falciparum tendent à s’apparier entre génotypes apparentés dans les populations de forte complexité génétique. Les résultats de ce travail soulignent l’importance de la diversité génétique de la population de gamétocytes pour la transmission chez le moustique vecteur. / Malaria control interventions over the last 15 years have lead to reduce malaria mortality by about 60% worldwide. This large reduction in malaria burden results mostly from effective mass distribution of insecticide-treated mosquito nets and artemisinin-based combination therapies. Some countries have already entered into elimination stage but others, particularly in sub-Saharan Africa, have yet to control the disease. The development of a vaccine against malaria remains a major priority for the fight against the disease. Among the different candidate vaccines, transmission-blocking vaccines have gained interest in the recent years. These vaccines would interrupt transmission from humans to mosquitoes. The anopheline alanyl aminopeptidase N (AnAPN1) is a mosquito-based malaria transmission-blocking vaccine candidate. In this PhD work, we evaluated the efficacy of anti-AnAPN1 antibodies against field isolates of Plasmodium falciparum in Anopheles coluzzii. Anti-AnAPN1 antibodies delivered to mosquitoes through artificial feedings showed a dose-dependent blocking activity and the transmission inhibition varied according to gametocytes donors. We modeled the different parameters that could modulate the antibody efficiency and we showed that the multiplicity of infection in the blood donor has a significant effect. We further explored the transmission dynamics of the co-infecting P. falciparum genotypes at non-blocking doses of antibodies. The mAb treatment reduced the genetic diversity of the parasite population but we did not detect any trace of selection on parasite genotypes. Genetic analyses revealed a positive correlation between FIS and multiplicity of infection, thus P. falciparum gametes mate with close relatives in infections with high genetic complexity. The results of this work highlight the importance of the genetic diversity of gametocyte population for transmission in the mosquito vector