A malária é uma das principais doenças parasitárias no mundo, e o aumento da resistência aos antimaláricos atualmente utilizados dificulta o controle dessa parasitose. Assim, é de interesse a descoberta de novas vias metabólicas que sirvam de alvos para o desenvolvimento de drogas para combater essa doença. Nosso laboratório, nos últimos anos, tem investido na caracterização de intermediários e produtos finais da via de isoprenóides. Baseando-se na presença das vias 2-C-metil-D-eritritol 4-fosfato e do chiquimato, decidimos verificar se ocorriam as biossínteses de retinóides e vitamina K no ciclo intraeritrocitário do P. falciparum, através da análise de produtos metabolicamente marcados com [1(n)-3H]-pirofosfato de geranilgranila por análises cromatográficas (HPLC e TLC), além da espectrometria de massas. Não identificamos a presença de retinal, retinol e ácido retinóico no ciclo intraeritrocitário do P. falciparum. Já a biossíntese de vitamina K precisa ser estudada com mais profundidade, pois há indícios de biossíntese, em especial da menaquinona-4. / Malaria is one of the most important parasitic diseases in the world. The spread of resistance to the antimalarials impairs the parasite\'s control. Therefore, is necessary the discovery of new metabolic routes to allow new antimalarials development. Our group has been studying the isoprenoid pathway, characterizing the intermediate and secondary products of this pathway. Based on the presence of 2-C-methyl-D-erythritol 4-phosphate and shikimate pathways, we decided to investigate the occurrence of retinoids and vitamin K biosynthesis in P. falciparum, through the chromatography of [1(n)-3H]Geranylgeranylpyrophosphate labeled products of intraerythrocytic stages of P. falciparum, and mass espectometry analysis. The retinal, retinol and retinoic acid were not identified in P. falciparum. The results indicate the menaquinone-4 biosynthesis, although deeper investigation is necessary.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-16Bitstream added on 2014-06-13T18:31:22Z : No. of bitstreams: 1 carvalho_db_me_sjrp.pdf: 1649413 bytes, checksum: 0ac4714a8b68e2f57418348441007ee7 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O sistema sangüíneo ABO (sABO) é o mais importante sistema na compatibilidade de grupos sangüíneos. Muitas pesquisas têm mostrado associações deste sistema com várias doenças infecciosas, inclusive a malária. Este estudo avaliou a associação entre os genótipos do sistema histo-sangüíneo ABO e a malária não grave causada pelo Plasmodium falciparum. A genotipagem dos grupos sangüíneos do sistema ABO foi feita de acordo com o protocolo de PCR/ RFLP, em amostras de indivíduos maláricos e não maláricos de áreas da Amazônia brasileira. O genótipo homozigoto ABO*O01O01 foi prevalente tanto nos maláricos quanto nos doadores de sangue. O genótipo ABO*AB representou cerca de 3% da população infectada e 5% da não infectada. Não foram verificadas diferenças estatisticamente significantes na comparação das freqüências alélicas e genotípicas do sABO entre pacientes e grupo controle, mesmo quando foram analisados apenas indivíduos com infecções puras de P. falciparum. A freqüência do sABO na Amazônia brasileira pode estar relacionada com a baixa freqüência de malária grave pelo P. falciparum. Portanto, os genótipos encontrados no sistema ABO dos indivíduos maláricos e não maláricos pode promover relevantes informações, para o entendimento da epidemiologia da malária grave por P. falciparum na Amazônia brasileira. / The ABO blood system (sABO) is the most important system on the blood groups compatibility. Several studies have shown its associations with various infectious diseases, including malaria. This study evaluated the association between the ABO histo-blood genotypes and non-severe malaria caused by Plasmodium falciparum. PCR/RFLP protocol had be used for both ABO blood group system genotyping in malaria suffering individuals and blood donors, from malaria areas of the Brazilian Amazon. The homozygous genotype ABO*O01O01 was prevalent in both malaria and the blood donors. The genotype ABO*AB represented about 3% of the infected population and 5% of non-infected. No statistically significant differences were observed in sABO genotypic and allelic frequencies of patients and the control group, even when individuals were analyzed only with pure infection of P. falciparum. The frequency of sABO in the Brazilian Amazon may be related to the low frequency of non-severe malaria P. falciparum. Therefore, the genotypes found in the ABO blood system in malaric and non-malaric individuals can promote relevant information for the understanding of the severe malaria by P. falciparum epidemiology in the Brazilian Amazon. Keywords: Malaria; ABO blood group system; Plasmodium falciparum.

A malária é uma doença tropical de grande relevância para a saúde pública no mundo. O objetivo do presente estudo foi construir a curva endêmica das espécies de Plasmodium falciparum e vivax, no período de 2003 a 2012 no Garimpo do Lourenço, verificando o efeito da periodicidade e da sazonalidade. Trata-se de um estudo ecológico, de série temporal e de caráter descritivo e exploratório, cujos dados foram obtidos do SIVEP-Malária, e o número de casos notificados ao mês foi utilizado como a variável dependente. Na amostra desta série 69% dos infectados eram do sexo masculino. As taxas cumulativas de infecção por gênero foram similares, sendo a infecção por P.vivax responsável por 75,4% das infecções em mulheres e por 72% do total de infecção em homens. As taxas por faixa etária foram de 72% entre 15 a 49 anos e de 39,6% em menores de 15 anos. Quanto à escolaridade, 66,57% não estudaram ou tinham menos de quatro anos de escolaridade. Observou-se um total de 12.357 infecções, de 12.056 casos novos e de 301 lâminas de verificação de cura (LVC). A infecção em garimpeiros foi responsável por cerca de 40% dessas infecções. Na avaliação da curva endêmica, realizada por meio do teste Augmented Dickey-Fuller, observou-se a ausência de estacionariedade das duas espécies (p > 0,05). A periodicidade, avaliada pelo teste G de Fisher, não evidenciou diferença estatisticamente significativa para os períodos da infecção no curso do ano para nenhuma das duas espécies (p > 0,05). O contexto observado nas zonas de garimpo da Amazônia Brasileira poderiam ser apoiadas por ações de controle ambiental e pesquisas, direcionados aos mosquitos transmissores, ao comportamento das espécies e aos aspectos climáticos, além do apoio à rede de serviços de saúde, de forma a interromper a cadeia de transmissão e controlar a endemia em patamares reduzidos nessa região garimpeira específica. / Malaria is a tropical disease of great relevance for the world’s public health. The objective of the present study was to construct the endemic curve of the species falciparum and vivax during the period starting from 2003 until 2012 in gold mining of Lourenço, assessing the effect of periodicity and seasonality. The present is an ecological time-series, descriptive and exploratory study, whose data were obtained from the SIVEP-Malária, using the number of cases per month as the dependent variable. 69% of those infected were male. Cumulative rates of infection by gender were similar, where vivax was responsible of 75.4% of the infections in females, and of 72% in males. Rates by age were 72% in those between 15 to 49 years old, and of 39.9% in those younger than 15 years old. Regarding literacy, 66.57% of the sample had never studied or had less than four years of schooling. It was observed a total of 12357 infections, 12056 new cases and 301 cure verification slides (CVS). Infection of gold miners was responsible of around 40% of the cases. Endemic curve assessment was performed using the Augmented Dickey-Fuller test, showing absence of seasonality of the two species (p > 0.05). Periodicity was assessed using the Fisher’s G test that did not show statistically significant difference for the infection periods along the year among the two species (p > 0.05). The context observed in the mining areas of the Brazilian Amazon could be supported by environmental control measures and research, targeted to mosquitoes, the behavior of species and climatic aspects and the support to the network of health services in order to stop the transmission chain and control endemic at low levels that specific gold mining region.

A malária é uma doença tropical de grande relevância para a saúde pública no mundo. O objetivo do presente estudo foi construir a curva endêmica das espécies de Plasmodium falciparum e vivax, no período de 2003 a 2012 no Garimpo do Lourenço, verificando o efeito da periodicidade e da sazonalidade. Trata-se de um estudo ecológico, de série temporal e de caráter descritivo e exploratório, cujos dados foram obtidos do SIVEP-Malária, e o número de casos notificados ao mês foi utilizado como a variável dependente. Na amostra desta série 69% dos infectados eram do sexo masculino. As taxas cumulativas de infecção por gênero foram similares, sendo a infecção por P.vivax responsável por 75,4% das infecções em mulheres e por 72% do total de infecção em homens. As taxas por faixa etária foram de 72% entre 15 a 49 anos e de 39,6% em menores de 15 anos. Quanto à escolaridade, 66,57% não estudaram ou tinham menos de quatro anos de escolaridade. Observou-se um total de 12.357 infecções, de 12.056 casos novos e de 301 lâminas de verificação de cura (LVC). A infecção em garimpeiros foi responsável por cerca de 40% dessas infecções. Na avaliação da curva endêmica, realizada por meio do teste Augmented Dickey-Fuller, observou-se a ausência de estacionariedade das duas espécies (p > 0,05). A periodicidade, avaliada pelo teste G de Fisher, não evidenciou diferença estatisticamente significativa para os períodos da infecção no curso do ano para nenhuma das duas espécies (p > 0,05). O contexto observado nas zonas de garimpo da Amazônia Brasileira poderiam ser apoiadas por ações de controle ambiental e pesquisas, direcionados aos mosquitos transmissores, ao comportamento das espécies e aos aspectos climáticos, além do apoio à rede de serviços de saúde, de forma a interromper a cadeia de transmissão e controlar a endemia em patamares reduzidos nessa região garimpeira específica. / Malaria is a tropical disease of great relevance for the world’s public health. The objective of the present study was to construct the endemic curve of the species falciparum and vivax during the period starting from 2003 until 2012 in gold mining of Lourenço, assessing the effect of periodicity and seasonality. The present is an ecological time-series, descriptive and exploratory study, whose data were obtained from the SIVEP-Malária, using the number of cases per month as the dependent variable. 69% of those infected were male. Cumulative rates of infection by gender were similar, where vivax was responsible of 75.4% of the infections in females, and of 72% in males. Rates by age were 72% in those between 15 to 49 years old, and of 39.9% in those younger than 15 years old. Regarding literacy, 66.57% of the sample had never studied or had less than four years of schooling. It was observed a total of 12357 infections, 12056 new cases and 301 cure verification slides (CVS). Infection of gold miners was responsible of around 40% of the cases. Endemic curve assessment was performed using the Augmented Dickey-Fuller test, showing absence of seasonality of the two species (p > 0.05). Periodicity was assessed using the Fisher’s G test that did not show statistically significant difference for the infection periods along the year among the two species (p > 0.05). The context observed in the mining areas of the Brazilian Amazon could be supported by environmental control measures and research, targeted to mosquitoes, the behavior of species and climatic aspects and the support to the network of health services in order to stop the transmission chain and control endemic at low levels that specific gold mining region.

Nosso grupo tem caracterizando diversos produtos da via de isoprenóides em Plasmodium falciparum que é promissora para o desenvolvimento de novas drogas. Dentre os compostos isoprenicos identificados estão as vitaminas K1 e E que apresentam uma cadeia isoprenica fitil. Em plantas o fitol é originado da degradação da clorofila. mas, como ainda não existem evidências da presença de clorofila no parasito, sugerimos que este possa ser formado pela degradação dessas vitaminas. Parte do fitol seria ligado a ácidos graxos utilizado como constituinte de membrana, e parte sofreria fosforilações, formando as vitaminas E e K1 como uma segunda via de biossíntese desse composto, assim como ocorre em Arabidopsis thaliana. Analises por HPLC e TLC utilizando o marcador [3H]GGPP, confirmaram a presença de fitol nos estágios intraeritrocíticos. Além disso, mostramos que o [3H]-fitol pode ser convertido em fitil-P e fitil-PP por uma atividade fitol quinase utilizando nucleotídeos como doador fosfato. / Our group has characterized differents products from isoprenoid pathway in Plasmodium falciparum which is promising for the development of new drugs. Among the isoprenics compounds identified there are the vitamins K1 and E which have a isoprenic phytyl side chain . In plants the phytol is obtained from the chlorophyll degradation. Until now theres no evidence that support its presence in the paraste. Our hypothesis that is phytol can be produced by the degradation of these vitamins E and K1. In the parasite phytol would fulfill to functions: used as a constituent of membrane, bounded to fatty acids and; be converted through phosphorylations vitamins E and K1 as a second biosynthetic pathway as well as in Arabidopsis thaliana. Analisys through and HPLC and TLC with radioactive [3H]GGPP confirmed the presence of phytol in intraerythrocytic stages. In addition, we showed that [3H]-phytol is converted into phytyl-P and phytyl-PP through a phytol kinasey using nucleotides as Phosphate donor.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T18:30:18Z No. of bitstreams: 1 josemarciofernandesdasilva.pdf: 606722 bytes, checksum: 480e6122c6670cee9053857fed232d2e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:18:46Z (GMT) No. of bitstreams: 1 josemarciofernandesdasilva.pdf: 606722 bytes, checksum: 480e6122c6670cee9053857fed232d2e (MD5) / Made available in DSpace on 2017-08-07T19:18:46Z (GMT). No. of bitstreams: 1 josemarciofernandesdasilva.pdf: 606722 bytes, checksum: 480e6122c6670cee9053857fed232d2e (MD5) Previous issue date: 2013-05-09 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / As dificuldades de adequação, aplicação e manutenção das estratégias de controle nos locais onde a malária é endêmica contribuem, em grande parte, para que a doença seja considerada o maior flagelo da humanidade. Neste contexto, destaca-se o amplo desenvolvimento de resistência dos plasmódios aos mais variados compostos que foram ou que ainda são utilizados na terapêutica antimalárica, o que faz do tratamento efetivo dos casos um grande desafio a cada ano. Diante disso, torna-se inquestionável a necessidade da descoberta de novas moléculas que possam, no futuro próximo, estar disponíveis para inclusão em medicamentos destinados à cura efetiva da infecção. Este estudo investigou a atividade antiplasmodial de 10 moléculas sintéticas derivadas de quinolinas em cultura de P. falciparum, bem como sua atividade citotóxica em células HeLa e mononucleares do sangue periférico. Das 10 moléculas, 5 estão conjugadas a sulfonamidas enquanto as demais possuem em sua estrutura um grupo hidrazina. Independentemente do grupamento farmacofórico integrado ao anel quinolínico, nenhuma molécula foi citotóxica para células HeLa ou linfócito humano em baixas concentrações. No que se refere a atividade antimalárica, 60% das moléculas foram altamente ativas contra P. falciparum (CI50 variando de <0,195µg/mL a 3,12µg/mL) e 10% totalmente inativas (CI50>50µg/mL). Ao analisarmos o índice de seletividade das moléculas, somente dois compostos não foram seletivos para o parasito (RMP103 e RMP107). Dentre as moléculas mais seletivas destacaram-se aquelas conjugadas ao grupo hidrazina, sobretudo RMP105 que foi >1800 vezes mais seletiva para plasmódio quando comparada a droga de referência. Portanto, por apresentarem alta atividade antimalárica e baixa toxicidade em células humanas, com elevado índice de seletividade para os plasmódios, as moléculas testadas nesse estudo podem ser consideradas boas alternativas para se tornarem medicamentos antimaláricos e auxiliarem de maneira eficaz no combate a doença. / The difficulties found in adapting, implementing and maintaining control strategies in malaria endemic regions are largely responsible for the spread of this disease, which is considered one of the greatest scourges of mankind. In this context, there is a widespread development of plasmodia resistance to various compounds that have been or are still used in antimalarial therapy, making effective treatment of the cases a constant challenge. Therefore, there is an unquestionable need for discovery of new molecules that may, in the near future, be available for inclusion in effective medicines for the cure of the infection. This study investigated the antiplasmodial activity of 10 synthetic molecules (derived from quinoline) in cultured P. falciparum, and their cytotoxic activity against HeLa cells and peripheral blood mononuclear cells. Of the 10 molecules tested, 5 are combined to sulfonamides, while the others have in their structure a hydrazine group. Regardless of the grouping pharmacophore integrated into the quinoline ring, all the molecules tested were not cytotoxic to HeLa cells or human lymphocytes, at low concentrations. Concerning the antimalarial activity, 60% of the molecules were highly active against P. falciparum (CI50 ranging from <0.195 µg / ml to 3.12 µg / mL) and 10% totally inactive (CI50 > 50μg/mL). By analyzing the selectivity index of molecules, only two compounds were not selective for the parasite (RMP103 and RMP107). Among the more selective molecules, the highlights were those linked to the hydrazine group, especially RMP105 which was >1800 times more selective for plasmodium compared to the reference drug. Therefore, due to their high antimalarial activity and low toxicity in human cells, with a high selectivity for Plasmodium, the molecules tested in these studies can be considered good alternatives to become antimalarial drugs and assist effectively in combating the disease.

Orientadores: Fabio Trindade Maranhão Costa, Stefanie Costa Pinto Lopes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T13:56:29Z (GMT). No. of bitstreams: 1 NaranjoPrado_IsabelCristina_M.pdf: 2482256 bytes, checksum: 0074002c6c9825a945839bdec23357f3 (MD5) Previous issue date: 2014 / Resumo: A malária é responsável por cerca de 300 milhões de casos de infecção e 1 milhão de mortes por ano. No Brasil, em 2012, foram registrados cerca de 1500 casos sendo a malária vivax responsável por 85% destes. Ainda, é frequentemente reportada falha terapêutica aos antimaláricos convencionais (como cloroquina e mefloquina) em infecções por P. falciparum, principalmente, mas também por P. vivax. Sendo assim, terapias combinadas com artemisinina e seus derivados (ACT) são atualmente recomendadas. No entanto, resistência aos derivados da artemisinina já é evidente e a busca por novos compostos com atividade antimalárica é urgente. A violaceína, pigmento violeta extraído de bactérias Gram negativas, demostrou apresentar elevada atividade antimalárica in vitro e in vivo em trabalho anterior de nosso grupo. Neste sentido, este projeto tem como objetivo aprofundar a investigação a respeito da atividade antimalárica da violaceína avaliando seu papel em isolados amazônicos de P. falciparum adaptados recentemente em cultura, e em P. vivax após imediata coleta de sangue de pacientes infectados. Além disso, investigamos o potencial da violaceína como terapia combinada junto ao artesunato no tratamento de parasitas murinos resistentes a este e a outros antimaláricos como a cloroquina. Inicialmente foram testadas dois diferentes tipos de violaceína in vitro contra P. falciparum 3D7: uma comercial extraída de Janthinobacterium lividum (vJl- IC50: 227 nM) e outra extraída de Chromobacterium violaceum (vCv- IC50: 390 nM). Apesar de não termos encontrado uma diferença na atividade antimalárica entre as duas violaceínas, a extraída de C. violaceum teve uma baixa toxicidade em eritrócitos (<400 nM) em células de hepatoma humano (<800 nM). Devido a esta baixa toxicidade, somente vCv foi avaliada quanto sua atividade antimalárica. Demonstramos que vCv apresentou IC50 similar ao encontrado para P. falciparum 3D7 (IC50 média= 419,8 nM) em 7 isolados de campo de P. falciparum. Ainda, em ensaios in vivo, utilizando cepas murinas de P. chabaudi, vCv conseguiu diminuir significativamente (P<0,05) a parasitemia no dia pico da infeção em cepas resistentes à cloroquina (30CQ) e a artesunato e mefloquina (ATNMF1). Adicionalmente, nos testes realizados em P. vivax a vCv parece evitar o amadurecimento parasitário nos quatro isolados testados. Coletivamente, podemos concluir que a vCv apresentou um efeito antimalárico em cepas de P. falciparum e pode ser especialmente útil quando usada em combinação com artesunato no tratamento de camundongos infectados com cepas resistentes. Finalmente, ensaios adicionais de amadurecimento com isolados de P. vivax necessitam ser conduzidos para a comprovação do efeito da vCv nesta espécie / Abstract: Malaria is responsible for about 300 million infections and one million deaths per year. In Brazil in 2012, about 1500 cases were reported and malaria vivax accounts for 85% of these. Still, it is frequently reported treatment failure with conventional antimalarials (as chloroquine and mefloquine) mainly in infections with P. falciparum but also by P. vivax parasite. Because of that combination therapies with artemisinin and its derivatives (ACT) are now currently recommended. In a previous work, our group demonstrated that violacein was able to inhibit the in vitro growth of laboratory strains of P. falciparum and also to strongly control the parasitemia of mice infected with P. chabaudi. This project aims to investigate further the antimalarial activity of violacein evaluating its activity in Amazonian isolates of P. falciparum recently adapted in culture, and in P. vivax isolates immediately after collecting blood from infected patients. Furthermore, we investigate the potential of violacein as combination therapy with artesunate in the treatment of murine strains which are resistant in different levels to artesunate, mefloquine and chloroquine. The antimalarial activity of violacein were initially investigated in vitro against P. falciparum 3D7 using two different types of violacein, one comercial, extracted from Janthinobacterium lividum (vJl-IC50: 227 nM), and another extracted from Chromobacterium violaceum (vCv-IC50: 390 nM) by our collaborators. In spite of no difference in the antimalarial activity between the two violaceins, the one extracted from C. violaceum had the lowest toxicity in erythrocytes (<400 nM) and in human hepatoma cells (<800 nM). Because of this, the antimalarial activity only of vCv was evaluated against 7 field isolates of P. falciparum showing a similar IC50 to that found for P. falciparum 3D7 (IC50= 419.8 nM). The antimalarial activity was also evaluated in murine strains of P. chabaudi showing a significant (P < 0.05) parasitemia decrease in the peak day in the two clones of P. chabaudi tested, one resistant to chloroquine (30CQ) and another resistant to artesunate and mefloquine (ATNMF1). Additionally, in P. vivax vCv was capable to reduce the parasite maturation in the four isolates tested. Therefore we can conclude that the vCv has an antimalarial effect on field isolates of P. falciparum and can be especially useful when used in combination with artesunate in the treatment of mice infected with resistant strains. Moreover, more assays should be conducted using blood infected with P. vivax to corroborate its effect in inhibiting the maturation of trophozoites / Mestrado / Imunologia / Mestra em Genética e Biologia Molecular

Le long de la frontière entre la Thaïlande et le Myanmar (TMB), le paludisme se caractérise par une forte hétérogénéité de la transmission, une forte prévalence en porteurs sub-microscopiques et par l’émergence de la résistance à l’artémisinine chez Plasmodium falciparum. L'identification précoce des « foyers » infectieux et leurs éliminations sont nécessaires pour contenir la résistance à l'artémisinine. L'objectif de cette thèse était de démontrer l’intérêt d’utiliser des biomarqueurs sérologiques de l'exposition humaine aux piqûres d'anophèles (gSG6-P1) et au Plasmodium (CSP & MSP1-19) pour quantifier le contact homme-vecteur et identifier les foyers résiduels de transmission. Des papiers filtres contenant du sang ont été prélevés sur une cohorte de 2600 personnes suivie tous les 3 mois jusqu'à 18 mois et analysés par dosage immuno-enzymatique (ELISA). Nos résultats ont montré que les niveaux de réponse IgG à l'antigène gSG6-P1 variaient selon le village, la saison et l'âge et étaient positivement corrélés à l'abondance des espèces anophèles et des vecteurs primaires de paludisme. Une association significative et positive a été observée entre la réponse de l'anticorps au gSG6-P1 et le taux d'inoculation entomologique (EIR), démontrant ainsi que l'hétérogénéité de la transmission du paludisme était directement associée à un comportement de piqûre hétérogène. Des études complémentaires ont montré que le biomarqueur salivaire était pertinent pour détecter des variations micro géographiques dans la transmission à P. falciparum. Cela s’est traduit par des chevauchements significatifs entre les foyers infectieux à P. falciparum et ceux à forts répondeurs en anticorps anti-salive d’anopheles (gSG6-P1). Dans l'ensemble, ces résultats indiquent que le biomarqueur salivaire d'Anopheles est prometteur pour les études épidémiologiques et pourrait guider la mise en œuvre d’interventions de lutte antivectorielle « ciblées » afin d'éliminer les foyers résiduels de paludisme. / Malaria along the Thailand-Myanmar border (TMB) displays geographical heterogeneity and is characterized by high prevalence of submicroscopic carriage and the emergence artemisinin resistance in P. falciparum. Timely identification and elimination of remaining P. falciparum transmission “hotspots” is essential to contain artemisinine resistance. The aim of this study was to address the relevance of using serological biomarkers of human exposure to anopheles bites (gSG6-P1) and Plasmodium antigens to identify remaining sources of transmission and to measure spatial and temporal changes in human vector contact along the TMB. Blood spots were collected in filter papers among a cohort of 2600 people followed every 3 months up to 18 months, and used for analysis by enzyme-linked immunosorbent assay (ELISA). Our findings showed that the levels of IgG responses to gSG6-P1 antigen varied according to village, season, and age and were positively associated with the abundance of total Anopheles species and primary malaria vectors. A significant and positive association was noted between the Antibody response to gSG6-P1 and the entomological inoculation rate (EIR) hence demonstrating that heterogeneity in malaria transmission was directly associated with heterogeneous biting behavior. Further investigations showed that salivary biomarker was relevant to detect small scale variations in P. falciparum malaria. This was supported by scan statistics showing that P. falciparum clusters partially overlap the gSG6-P1 clusters. Altogether, these findings indicates Anopheles salivary biomarker as great potential for epidemiological studies and could be useful to guide the implementation of hotspot–targeted vector control interventions with the aim to achieve malaria elimination.

Ce travail a eu pour objectif de caractériser les parasites P. falciparum, infectant les femmes enceintes et responsables du paludisme associé à la grossesse. Dans la première partie de ce travail, le phénotype d’adhérence et le profil d'expression des gènes var chez les parasites infectant les femmes enceintes ont été étudiés. Ces analyses ont été réalisées sur des isolats parasitaires collectés dans deux études menées au Bénin entre 2008 et 2013. La première étude, sur des isolats prélevés dans le cadre d'une étude de cohorte de femmes enceintes dans les zones rurales au Bénin, a montré que les parasites qui infectent les femmes dans le premier trimestre de la grossesse expriment déjà un phénotype placentaire. Dans une deuxième étude, portant sur les isolats prélevés en prospective chez des femmes enceintes se présentant en consultations prénatales dans les centres de santé à Cotonou, une analyse plus détaillée des propriétés d'adhérence des hématies parasitées par P. falciparum, a confirmé nos premiers résultats. Cette étude a souligné une plus grande complexité dans les propriétés d’adhérence des isolats obtenus pendant le premier trimestre de grossesse qui adhèrent sur plusieurs récepteurs. Tout au long de la grossesse cette diversité phénotypique s’affine vers des phénotypes typiquement placentaires. Ce travail a également démontré, qu'au-delà de la parasitémie, l'adhérence à la CSA est un facteur important au regard de l’issue défavorable de la grossesse ; les parasites qui infectent les primigestes adhèrent en moyenne plus à la CSA que ceux provenant des multigestes. Conformément aux travaux antérieurs, les études menées au Bénin ont montré que le gène var2csa est le membre de la famille des gènes var qui est préférentiellement transcrit par les parasites infectant la femme enceinte. L’expression de la protéine correspondante à la surface des érythrocytes infectés a été démontrée grâce à des anticorps spécifiques. Cette expression a été étroitement associée à la capacité des isolats à adhérer, in vitro, à la CSA. La deuxième partie de ce travail a examiné le potentiel vaccinal de VAR2CSA par l’exploration des régions peptidiques impliquées dans l'acquisition d'anticorps « anti-adhérence ». En utilisant une approche d'immunisation génétique chez la souris, nous avons été en mesure d'identifier la région minimale de VAR2CSA située dans son extrémité N-terminale comme étant le site qui concentre les épitopes anti-adhérence. Les IgG induites contre la région NTS-DBL2X de VAR2CSA ont été capables d’inhiber l'adhérence de plus de 60% ??des isolats naturels de P. falciparum à la CSA. Cette étude a par ailleurs mis en évidence des propriétés fonctionnelles des IgG qui seraient souche-spécifique ainsi qu’a aidé à formuler une hypothèse concernant la combinaison antigénique des variants de VAR2CSA nécessaires pour garantir une activité optimale sur des isolats de terrain. La dernière partie de ce travail a porté sur l'analyse du polymorphisme des séquences de VAR2CSA dans sa partie N-terminale chez les isolats de terrain. Ces analyses ont démontré l'existence d'une région dimorphique dans le domaine structurellement critique ID1, qui a révélé une association très intéressante avec la survenue d'infections à très forte densité de parasite. Le travail développé dans cette thèse a permis de mettre à jour les connaissances sur les parasites qui infectent les femmes pendant la grossesse et de formuler des hypothèses sur les pistes d'optimisation moléculaire, nécessaires au développement d'un vaccin efficace à base de VAR2CSA. / This thesis aimed to characterize the P. falciparum parasites infecting pregnant women and causing pregnancy-associated malaria (PAM). In the first part of this work, cytoadherence phenotype and var genes expression profile of pregnant women parasites have been investigated on parasite isolates collected in two studies conducted in Benin between 2008 and 2013. The first study on isolates collected as part of a cohort study of pregnant women in rural areas in Benin, showed that parasites which infect women in the first trimester of pregnancy already express placental phenotype. In a second study on isolates collected prospectively from pregnant women attending antenatal clinics in health centers in Cotonou, the analysis of the adhesion phenotype using multiple host receptors described so far confirmed the first results and highlighted a greater complexity of the binding properties in isolates collected during the first trimester and those obtained from multigravidae. This study also demonstrated that beyond parasitaemia, adhesion to CSA is a major factor for poor pregnancy outcomes. Consistent with previous reports, studies in Benin showed that var2csa was the most transcribed var gene by PAM-isolates and its surface expression on infected erythrocyte (IE) was demonstrated with specific antibodies. This was closely linked to the ability of isolates to adhere to CSA in vitro. The second part of the work investigated the vaccine potency of VAR2CSA by exploring the region of var2csa involved in the acquisition of anti-adhesion antibodies. Using a DNA immunization approach performed in mice, we were able to identify the var2csa minimal region located in its N-terminus as the site that concentrates the anti-adhesion epitopes. The NTS-DBL2X region of VAR2CSA has been found to induce cross-reactive antibodies that inhibit adhesion of more than 60% of field P. falciparum isolates to CSPG. This study highlighted some strain-specific properties in functionality of the antibodies induced and helped formulate a hypothesis of antigenic FCR3 and 3D7 variants combination for optimal activity on field isolates. The last part of this work focused on the analysis of sequence polymorphism in the N-terminal part of VAR2CSA expressed by field isolates. The analysis demonstrated the existence of a dimorphic region within the structurally critical ID1 domain that revealed a very interesting association with the occurrence of infections with very high parasite density. The work developed in this thesis updates knowledge on parasites infecting women during pregnancy and formulates hypotheses on the molecular optimization tracks necessary for the development of an effective VAR2CSA-based vaccine.

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. / Plasmodium species use programmed cell death for the survival of their offspring as some prokaryotic parasites. This study was designed to - assess the gametocytes carrier and their genotypes before/ after treatment and studying their infectivity; - express the catalytic domain (PfMCA1-cd-Sc) of Plasmodium falciparum metacaspase (PfMCA1) in yeast; - Test the “in vitro” anti-plasmodial activity of pyrano and ferro- quinolines derived new synthetic molecules on 3D7 and Dd2 Chloroquine laboratory clones. The 28-day “in vivo” WHO test, molecular markers of resistance and direct feeding; yeast culture, protein expression of P. falciparum metacaspase 1, Western blot, proliferation and survival test, and cell death markers were used to achieve the first two objectives while parasite culture and in vitro tests by the method of fluorimetry in SYBR Green I was used to evaluate the anti-plasmodial activity of new molecules. Results show that post-treatment gametocytes were carriers of point mutations and the most infective in the Chloroquine group. The heterologous expression of PfMCA1 catalytic domain in Saccharomyces cerevisiae resulted in apoptotic clonal death and growth retardation activity-dependent Protease-VAD, showing the involvement of PfMCA1 in the process of cell death. The aromatic substitutes with pyrimidine or benzyldimethylamine ferrocene residues showed satisfactory activity against the methoxyethylidene on 3D7 and Dd2. The data suggest that the structural optimization of these compounds based on pyrimidine and ferrocene is more interesting from the standpoint anti-plasmodial activity for candidate molecules in the near future.