Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-12T14:21:53Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PolimorfismoExon1Gene.pdf: 550227 bytes, checksum: 435a4d06507b6272adc8948ba5d9c613 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-01T16:34:31Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PolimorfismoExon1Gene.pdf: 550227 bytes, checksum: 435a4d06507b6272adc8948ba5d9c613 (MD5) / Made available in DSpace on 2014-04-01T16:34:31Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PolimorfismoExon1Gene.pdf: 550227 bytes, checksum: 435a4d06507b6272adc8948ba5d9c613 (MD5) Previous issue date: 2006 / Neste estudo analisamos a freqüência de polimorfismos no éxon 1 do gene 2 da lectina Ligadora de manose (MBL) em indivíduos expostos à malária causada por Plasmodium vivax. Foram analisadas amostras de 81 indivíduos primoinfectados e 250 não infectados. Os indivíduos não infectados constituíram dois grupos, um que relatou nunca ter tido malária e o outro que teve de 1 a 4 ou mais episódios da doença. No grupo infectado, foi investigada a associação entre os polimorfismos e a suscetibilidade à infecção, a intensidade dos sinais e sintomas clínicos e a parasitemia. As mutações foram identificadas por reação em cadeia da polimerase e análise de restrição. O grupo infectado apresentou distribuição de freqüências alélicas e genotípicas diferente do grupo não infectado. As freqüências dos alelos MBL*A, MBL*B, MBL*C e MBL*D nos indivíduos infectados foram 64,20%, 19,75%, 0,00% e 15,43%, nos não infectados que nunca tiveram malária as freqüências foram 72,96%, 14,80%, 3,06% e 9,18%, e nos não infectados que relataram episódio prévio de malária as freqüências foram 74,67%, 14,81%, 2,30% e 8,22%, respectivamente. O alelo MBL*B foi associado à sintomatologia intensa e ao aumento na parasitemia, enquanto o alelo MBL*D foi associado às parasitemias mais baixas. No grupo não infectado, a distribuição das freqüências alélicas e genotípicas variou com o número de episódios e o tempo decorrido após o último episódio de malária. As variantes MBL*B e MBL*D contribuíram para essa variação. Esse foi o primeiro estudo para avaliar o impacto desses polimorfismos do gene da lectina ligadora de manose na resposta imune inata em indivíduos expostos naturalmente à malária causada por Plasmodium vivax. / In this study we evaluated the frequency of polymorphism in exon 1of gene 2 of mannose-binding lectin (MBL) in individuals exposed to malaria caused by Plasmodium vivax. We analized 81samples from primo-infected and 250 from non- infected individuals. In the non-infected group were included individuals that reported never had malaria and individuals that have had 1 to 4 or more malaria episodes. In this study we analized the association between polymorphisms and susceptibility to infection, intensity of signals and clinical symptoms and parasitaemia. Polymorphisms in MBL gene were detected by polymerase chain reaction (PCR) and restriction analysis. The infected group presented allele and genotype frequency distribution different from non-infected group. The frequencies of the alleles MBL*A, MBL*B, MBL*C and MBL*D in infected individuals were 64.20%, 19.75%, 0.00% and 15.43%, in non-infected individuals that had never had malaria the frequencies were of 72.96%, 14.80%, 3.06% and 9.18%, and in non-infected individuals that have reported previous episode of malaria the frequencies were 74.67%, 14.81%, 2.30% and 8.22%, respectively. The allele MBL*B was associated with intense symptomology and increase in parasitemia, whereas the allele MBL*D was associated with lower parasitemias. In non-infected group, allele and genotype frequency varied according to the number of episodes and the time after the last malaria episode. The alleles MBL*B and MBL*D contributed for this variation. This was the first study to evaluate the impact of these polymorphisms of MBL gene in innate immune response of individuals naturally exposed to malaria caused by Plasmodium vivax.

Malaria affects over half of the world's population and causes half a million deaths annually, especially in Sub-Saharan Africa. Four species of the apicomplexan Plasmodium parasite (P. falciparum, P. ovale, P. malariae and P. vivax) are responsible for malaria in Africa. Both parasite and host factors contribute to heterogeneity in the risk of developing malaria, clinical manifestation of the disease as well as the number of treatments required to clear parasites. The epidemiology of the different species, and the role of exposure to mixed-species Plasmodium co-infections in generating heterogeneity remains poorly studied. Being an obligate intracellular parasite the blood-stage life cycle of the Plasmodium parasite takes place in the erythrocytes of the human host. The surfaces of these erythrocytes are the medically important ABO blood group antigens that have been reported to influence the susceptibility or otherwise of an individual developing severe malaria. In this thesis I have considered the contributions of the species of Plasmodium parasites and the ABO blood group of the host in driving heterogeneity in human malaria. The aims of this thesis were to determine: (i) the seroepidemiology of the different Plasmodium species in two mesoendemic African populations (Zimbabwe and Sudan); (ii) to determine if heterogeneity in clinical presentations of malaria (history of fever, body temperature and parasitaemia) and response to drug treatment is related to exposure to single vs. mixed-Plasmodium species infection; (iii) the spatial and temporal dynamics of malaria prevalence and Plasmodium species distribution in a mesoendemic village in eastern Sudan; (iv) gene expression changes in 3D7 P. falciparum parasites as they infect erythrocytes of different ABO blood group donors. For aims (i to iii) I developed an enzyme-linked immunosorbent assay using antigens derived from Plasmodium merozoite surface protein 1, also known as MSP-119, to detect IgG antibodies to all four malaria parasite species in Zimbabwean and Sudanese populations. In the Zimbabwean study, plasma samples from 100 individuals each (aged 5-18 years) from three villages (Burma Valley, Mutoko and Chiredzi) were screened for exposure to Plasmodium parasites. In Daraweesh, Sudan, plasma samples from 333 individuals (aged 1-74 years) who had experienced a first malaria episode between 1990 and 2000 were recruited into the study. For study aim (iv) I cultured a single clone of 3D7 P. falciparum parasite using erythrocytes of individuals of different ABO blood group types, harvested parasite RNA and sequenced it to determine gene expression changes in the different hosts. I showed that human IgG antibodies to MSP-119 antigens of the four Plasmodium species are species-specific and do not cross-react. In both study populations almost all antibody responses involved P. falciparum, and single-species responses were almost exclusively directed against P. falciparum antigens. Mixed-species responses accounted for more than a third of responses, and were associated with chloroquine treatment failure, with significantly high proportion of individuals with mixed-species infections requiring repeated treatment with chloroquine/sulfadoxine-pyrimethamine for parasite clearance. This finding highlights the need for a sensitive method for detecting mixed-species malaria infections to enable the assessment of the true prevalence and magnitude of the disease burden caused by the non-falciparum species in endemic populations. Drug treatment failures associated with mixed species infections have significant impact on malaria morbidity and mortality. Treatment failure or partial parasite clearance has the potential to allow dormant liver stages of P. vivax and P. ovale to become a source of parasite reservoir for onward transmission. Furthermore, untreated low-grade chronic infections caused by P. malariae have been reported to cause systemic diseases many years after the primary infection. Spatial analysis of malaria epidemiology showed that malaria parasite transmission in Daraweesh was focal, and that infections are not randomly distributed in the village. Two space-time clusters of significantly increased malaria risk were identified (1993- 1999, and 1998-1999) with marked variations between households, but little or no variation in the species of Plasmodium over time. Similarly, multiple significant clusters were identified for the parasite species; three for P. falciparum, two for P. vivax and P. malariae, and one for P. ovale. These clusters had overlapping time frames, with some of the species significantly infecting the same households. This suggests that even in a small geographic area malaria transmission shows heterogeneity, and that such data can provide useful information to guide malaria control efforts. Finally, I demonstrated that 3D7 P. falciparum parasite growth was similar in the erythrocytes of different blood group donors, and provide preliminary data to show that the non-coding RNA gene, PF3D7_1370800, is differentially expressed in blood group A donors relative to blood groups B and O donors. Further research is needed to better understand the role of this gene in malaria pathology. All together, these findings will aid malaria researchers and other stakeholders in making informed choices about tools for diagnosing Plasmodium species, and control programmes targeting eradication of malaria caused by all Plasmodium species, as is the case of incorporating these findings into current malaria research in Sudan.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T14:44:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Luana Michels.pdf: 1171595 bytes, checksum: b9997bd5e324e4be251a83985c506a7c (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T14:44:18Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Luana Michels.pdf: 1171595 bytes, checksum: b9997bd5e324e4be251a83985c506a7c (MD5) / Made available in DSpace on 2016-09-22T14:44:18Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Luana Michels.pdf: 1171595 bytes, checksum: b9997bd5e324e4be251a83985c506a7c (MD5) Previous issue date: 2016-03-11 / O aumento da resistência do Plasmodium falciparum dificulta o tratamento da malária, o que leva a utilização de doses mais elevadas dos fármacos e subsequente toxicidade. As nanopartículas com superfície modificada têm sido estudadas com a finalidade de alterar a performance in vivo dos fármacos. O objetivo do presente trabalho foi desenvolver, caracterizar e avaliar a eficácia in vitro, in vivo e a farmacocinética das nanopartículas contendo quinina (QN) com diferentes características de superfície: nanocápsulas revestidas com polissorbato 80; nanocapsulas revestidas com PEG e nanocapsulas preparadas com Eudragit®. As suspensões foram preparados pelo método de nanoprecipitação e caracterizados de acordo com o diâmetro, índice de polidispersão, pH, potencial zeta, teor, taxa de encapsulação e microscopia de força atômica. As nanopartículas que apresentaram os melhores resultados na caracterização e eficácia in vitro, foram escolhidas para a avaliação da farmacocinética e eficácia in vivo, utilizando ratos Wistar e camundongos infectados com o P. berghei. As nanocápsulas apresentaram os melhores resultados na caracterização físico-química, com diâmetro adequado, população monodispersa, potencial zeta mais distante de zero, maior taxa de encapsulação e penetração intra-eritrocitária. Houve um aumento significativo no t1/2 de eliminação de todas as nanocápsulas avaliadas em relação à QN livre. Na eficácia in vivo, as nanocápsulas catiônicas aumentaram a sobrevida em relação à salina e à QN livre, demonstrando que o fármaco incorporado na suspensão com características catiônicas pode alterar a eficácia da QN apresentando-se como uma alternativa potencial para o tratamento da malária. / The increase of Plasmodium falciparum resistance difficult the treatment of malaria, and the use of higher doses of the drug induce toxicity. The coating of nanoparticles have been studied with the purpose of improve the in vivo performance of drugs. The aim of this study was to develop, characterize and evaluate the efficacy in vitro, in vivo and pharmacokinetics of quinine (QN) loaded-nanoparticles with different surface characteristics: polysorbate 80 coated-nanocapsules; PEG coated-nanocapsules and nanocapsules prepared with Eudragit® RS 100. The suspensions were prepared by nanoprecipitation method and characterized according to the diameter, polydispersity, pH, zeta potential, content encapsulation rate and atomic force microscopy. The nanoparticles showed the best results on the characterization and in vitro efficacy were chosen for evaluating the in vivo efficacy and pharmacokinetics, using Wistar rats and mice infected with P. berghei. The nanocapsules showed the best results in the physical-chemical characterization, with appropriate diameter, monodisperse population, zeta potential distant from zero, the higher rate of encapsulation and intra-erythrocyte penetration. There was a significant increase in t1/2 of all nanocapsules evaluated in comparison to free QN. On the efficacy in vivo, cationic nanocapsules increased the survival rate compared to saline and to the free QN, demonstrating that the drug incorporated in the suspension with cationic characteristics can alter the efficacy of QN presenting as a potential alternative for the treatment of malaria.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-05-07T11:52:24Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ConcentracoesPlasmaticasPrimaquina.pdf: 1467763 bytes, checksum: 2b37691a17a357b8c5248bfb0114d8c9 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2014-08-28T16:54:24Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ConcentracoesPlasmaticasPrimaquina.pdf: 1467763 bytes, checksum: 2b37691a17a357b8c5248bfb0114d8c9 (MD5) / Made available in DSpace on 2014-08-28T16:54:24Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ConcentracoesPlasmaticasPrimaquina.pdf: 1467763 bytes, checksum: 2b37691a17a357b8c5248bfb0114d8c9 (MD5) Previous issue date: 2010 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A malária vivax é uma doença que a cerca de 40% da população mundial, utiliza-se no tratamento desta, cloroquina (150 mg) e primaquina (15 mg). Esta é uma 8- aminoquinolina com ação esquizonticida tecidual. Dentre seus efeitos adversos se destaca a capacidade de oxidar a hemoglobina, de maneira dose dependente, que é agravada nos indivíduos com deficiência da glicose-6-fosfato desidrogenase. Ao se considerar a ausência de estudos referentes aos teores de metemoglobina e sua correlação com as concentrações plasmáticas de primaquina nos pacientes com malária vivax, justifica-se a realização deste estudo empregando-se como ferramentas a monitorização das concentrações sanguíneas de primaquina e sua correlação com os teores de metemoglobina. Neste sentido, foi realizado seguimento clínico-laboratorial de 20 pacientes com malária vivax antes (D0) e após três (D3), sete (D7) e quatorze (D14) dias iniciado o tratamento, bem como a validação do método para determinação de primaquina por cromatografia líquida de alta eficiência (CLAE). A metemoglobinemia foi avaliada pela técnica de Hegesh et al. (1970) e a glicose-6-fosfato desidrogenase pelo teste colorimétrico de Brewer et al. (1962). A metodologia validada demonstrou parâmetros aplicáveis à determinação de primaquina, cujos teores médios em D3, D7 e D14 foram de 227±106 ng/mL, 191±97 ng/mL e 160±128ng/mL. Não foram obervadas diferenças significativas nas concentrações do fármaco quanto ao sexo dos pacientes participantes e nos diversos dias do estudo. Os teores médios de metemoglobina em D0, D3, D7 e D14 foram de 1,15±0,9%, 4,1±2%, 5,7±2% e 3±1,4%, respectivamente. Foi observado aumento no teor de metemoglobina após administração do fármaco, sem diferença quanto ao sexo. Não foi observada correlação significativa entre os teores de metemoglobina e as concentrações plasmáticas de primaquina em ambos os sexos. Os coeficientes de correlação de Pearson para os sexos masculino e feminino foram 0.8296 e 0.8137, respectivamente. Foi observada deficiência da expressão da enzima glicose-6- fosfato desidrogenase em seis pacientes do sexo masculino sem diferenças entre os teores de metemoglobina e das concentrações plasmáticas de Primaquina, quando comparados com pacientes com expressão normal da enzima. / The vivax malaria is a disease that effects around 40% of the world, to treat it, chloroquine (150 mg) and primaquine (15 mg). This is an 8-aminoquinoline with tissue schizonticide action. Among the adverse effects enhance the capacity to hemoglobin oxidation, dose-dependent, which is exacerbated in individuals with glucose-6-phosphate dehydrogenase deficiency. When considering the lack of studies concerning the methemoglobin levels and its correlation with primaquine concentrations plasma in patients with vivax malaria, is justified this study using as tools to monitor the plasma primaquine concentrations and its correlation with methemoglobin levels. In this sense, it was followed up clinically and laboratory findings of 20 patients with vivax malaria before (D0) and after three (D3), seven (D7) and fourteen (D14) days starting the treatment, as well as validation of the method for primaquine determination by high performance liquid chromatography (HPLC). Methemoglobinemia was evaluated using the method of Hegesh et al. (1970) and glucose-6-phosphate dehydrogenase by colorimetric method of Brewer et al. (1962 ). The methodology validated was demonstrated efficient for primaquine determination, whose average levels at D3, D7 and D14 were 227 ± 106 ng / mL, 191 ± 97 ng / mL and 160 ± 128 ng/mL. In the analysis according to gender was not observed differences significant in the drug levels in several days of study. The average methemoglobin levels in D0, D3, D7 and D14 were 1.15 ± 0.9%, 4.1 ± 2%, 5.7 ± 2% and 3 ± 1.4%, respectively. There was an increase in the methemoglobin level after drug administration, without difference by gender. There was not significant correlation between the methemoglobin levels and primaquine concentrations plasma in both sexes. The coefficients of Pearson correlation for males and females were 0.8296 and 0.8137, respectively. We observed impaired expression of the enzyme glucose-6-phosphate dehydrogenase in six male patients without differences between the methemoglobin levels and primaquine concentrations plasma, compared with patients with expression normal of the enzyme.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2015-06-08T17:59:33Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoRespostaInflamatoria.pdf: 1919267 bytes, checksum: c50878eb5031e9aca92984be34efef40 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2015-06-18T13:09:55Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoRespostaInflamatoria.pdf: 1919267 bytes, checksum: c50878eb5031e9aca92984be34efef40 (MD5) / Made available in DSpace on 2015-06-18T13:09:55Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoRespostaInflamatoria.pdf: 1919267 bytes, checksum: c50878eb5031e9aca92984be34efef40 (MD5) Previous issue date: 2015-02 / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A malária cerebral (MC) é uma das complicações mais graves resultante da infecção por P. falciparum e a principal causa de morte em crianças. O quadro de MC apresenta uma patogênese complexa, associado a complicações neurológicas provenientes de uma resposta imunológica exacerbada, bem como eventos hemorrágicos. Estudos descrevem uma retinopatia associada ao quadro, juntamente com um intenso processo de astrogliose nas proximidades de vasos que nutrem o tecido retiniano. O presente trabalho buscou caracterizar o processo inflamatório e as possíveis alterações neuroquímicas e eletrofisiológicas no tecido retiniano de camundongos albino suíço, quando inoculados com a cepa ANKA de Plasmodium berghei (PbA). Camundongos albino suíço foram infectados com cepa PbA. Para caracterização do quadro de malária cerebral experimental (MCE) foram avaliados diversos parâmetros, como surgimento dos sinais clínicos, curva de sobrevivência, parasitemia (%), ganho de massa corpórea, permeabilidade vascular e quantificação de citocinas (TNF-α, IL-6 e IL-10) no tecido cortical. Para avaliarmos alterações na funcionalidade do tecido retiniano, utilizamos eletrorretinograma de campo total. Para a avaliação dos sistemas de neurotransmissão foi realizado ensaio de liberação e captação de glutamato e GABA que, posteriormente foi quantificado por Cromatografia Líquida de Alta Eficiência. Para análise da resposta inflamatória foi realizada a quantificação de citocinas (TNF-α, IL-6 e IL-10) no tecido retiniano. Após a caracterização do quadro de MCE nós observamos a diminuição da amplitude de onda-b de cones e bastonetes, bem como aumento do tempo implícito de bastonetes, respostas mistas em diferentes intensidades e potencial oscilatório. Observamos aumento na liberação e captação de glutamato e, ainda, a ativação de uma via antiinflamatória no tecido retiniano. Este trabalho nos permitiu validar o modelo murino de MCE e caracterizar, pela primeira vez, alterações na funcionalidade do tecido retiniano, acompanhada de alterações no sistema glutamatérgico, bem como ativação de uma via antiinflamatória no tecido retiniano. / Cerebral malaria (CM) is one of the most serious complications resulting from infection by P. falciparum and the leading cause of death in children. The CM frame has a complex pathogenesis associated with neurological complications arising in an enhanced immune response as well as hemorrhagic events. Studies describing retinopathy associated with the frame, together with an intense process of astrogliosis in the vicinity of retinal vessels that nourish the tissue. This paper sought to characterize the inflammatory process and the possible neurochemical and electrophysiological changes in the retinal tissue of Swiss albino mice, when inoculated with Plasmodium berghei ANKA strain (PbA). Swiss albino mice were infected with PbA strain. To characterize the above experimental cerebral malaria (ECM) was evaluated several parameters, such as onset of clinical signs, survival curves parasitemia (%) and body mass gain, vascular permeability and quantification of cytokines (TNF-α, IL-6 and IL-10) in the cortical tissue. To evaluate changes in retinal tissue functionality, use full-field electroretinography. For the evaluation of neurotransmitter systems release assay was performed and uptake of glutamate and GABA which was then quantified by High Performance Liquid Chromatography. The inflammatory response analysis was performed to quantify the cytokines (TNF-α, IL-6 and IL-10) in retinal tissue. After characterizing the MCE framework we observe a reduction in the amplitude of b-wave of rods and cones, as well as increase the implicit time of rods, mixed responses at different intensities and oscillatory potential. We observed an increase in the release and glutamate uptake and also the activation of an anti-inflammatory pathway in retinal tissue. This study allowed us to validate the murine model of MCE and characterize for the first time, changes in the retinal function accompanied by changes in the glutamatergic system as well as activation of the inflammatory pathway in retinal tissue.

Orientador: Ricardo Luiz Dantas Machado / Banca: Gerhard Wunderlich / Banca: Joseli de Oliveira Ferreira / Banca: Carlos Eugênio Cavasini / Banca: Dorotéia Rossi Silva Souza / Resumo: A malária é uma das principais causas de morbidade e mortalidade nas áreas tropicais e subtropicais do mundo. O desenvolvimento de uma resposta imune eficaz é capaz de reduzir a mortalidade e os sintomas clínicos da doença. No entanto, este é um processo complexo, e um dos objetivos dos imunologistas é entender as razões pelas quais os indivíduos diferem em suas respostas imunes contra o parasito. O objetivo do presente estudo foi avaliar a influência de polimorfismos em genes coestimulatórios do sistema imune na resposta imune humoral contra proteínas de estágio sanguíneo do Plasmodium vivax, principal espécie causadora de malária no Brasil. Para tanto, nós genotipamos, pelo método de PCR-RFLP, nove SNPs em sete genes (CD28, CTLA4, ICOS, CD86, CD40, CD40L e BLYS). A amostra foi constituída por 227 indivíduos infectados com P. vivax no município de Goianésia do Pará, no Estado do Pará. As respostas de anticorpos IgG específicos contra as proteínas N- (ICB2-5) e C-terminal (MSP-119) da MSP-1, da DBP e da AMA-1 do P. vivax foram determinadas por ELISA. IgM e as subclasses de IgG contra a ICB2-5 também foram avaliadas. Para estudar os polimorfismos dos genes coestimulatórios, nós primeiramente investigamos o impacto da estratificação da população na distribuição dos polimorfismos com o auxilio de marcadores informativos de ancestralidade e demonstramos que a frequência dos SNPs ICOS +1564T>C, CD40L -726T>C e CD86 +1057G>A varia de acordo com a ancestralidade. Polimorfismos em genes coestimulatórios foram associados com a resposta de anticorpos contra proteínas do estágio sanguíneo do P. vivax, mais especificamente contra a DBP, e as porções N- e C-terminal da MSP-1. Além disso, haplótipos formados pelos genes CD28, CTLA4 e ICOS foram associados com a resposta de anticorpos IgG4 contra a região N-terminal da MSP-1. Este é o primeiro estudo de associação genética envolvendo polimorfismos em genes ... / Abstract: Malaria is one of the main causes of morbidity and mortality in the tropics and subtropics areas of the world. Although the immunity is only partial, it is important in reducing the amount of illness and death caused by malaria. However, the immunity against malaria is complex, and one of the main goals of vaccine developers is to understand why people differ in their immune response to the parasite. The present research aims to investigate the genetic mechanisms related to humoral immune response against P. vivax blood stages antigens, predominant malaria species in Brazil. Nine single nucleotide polymorphisms (SNPs) in 7 genes (CD28, CTLA4, ICOS, CD86, CD40, CD40L e BLYS) were determined by PCR-RFLP. A total of unrelated 227 individuals infected with P. vivax from the Goianésia do Pará, Pará state, participated in this study. Level and prevalence of IgG antibodies against N-terminal (ICB2-5) and C-terminal (MSP-119) regions of MSP-1, DBP and AMA-1 of P. vivax were measured by ELISA. First, we evaluate the influence of genomic ancestry on distributions of co-stimulatory genes polymorphisms in an admixed Brazilian population using ancestry informative markers. ICOS, CD40L and CD86 polymorphisms were associated with genomic ancestry. There were significant association between CD28 -372G>A, ICOS +1564T>C, and CD40L -726T>C SNPs with antibodies anti-DBP prevalence. Moreover, CD40 -1C>T and CD86 +1057G>A SNPs were associated with antibody levels anti-PvMSP-119. The CD28 -372G>A and CD40 -1C>T SNPs were associated with IgM prevalence against ICB2-5. Haplotypes formed by polymorphisms in CD28, CTLA4, and ICOS genes were associated with IgG4 antibodies against ICB2-5. This is the first study to associate polymorphisms in costimulatory genes with humoral immune response against P. vivax. These data may add important information for understanding the immunological aspects involved in vivax malaria / Doutor

Determina la eficacia de las pruebas DELI y MSF para calcular la IC50 de drogas antimaláricas mefloquina, quinina y cloroquina obtenidas de aislamientos de P. falciparum provenientes de pobladores de la comunidad de Padre Cocha en Iquitos-Perú. Se realizó un estudio cuantitativo descriptivo, prospectivo de corte transversal. La muestra fueron 16 muestras de sangre con diagnóstico de malaria confirmado por gota gruesa. Se realizaron los dos ensayos de sensibilidad in vitro (DELI y MSF) a cada muestra. Se determinaron tres factores de eficacia para el presente estudio; porcentaje de éxito, coeficiente de determinación de curva (R2) y coeficiente de variación (CV). Se hizo un análisis descriptivo y estadístico de los factores de eficacia mediante las pruebas de Wilcoxon y McNemar- Bowker para muestras pareadas con p < 0.05. Las medias aritméticas de los valores de IC50 con el ensayo DELI fueron para cloroquina 231.26 nM, quinina 101.17 nM y mefloquina 16.03 nM. Las medias de los valores de IC50 con el ensayo MSF fueron para cloroquina 227.52 nM, quinina 142.46 nM y mefloquina 35.07 nM. El porcentaje de éxito del cálculo de la IC50 para las tres drogas fueron el 50% (8/16) y 87.5% (14/16) en los ensayos MSF y DELI respectivamente, estas diferencias fueron estadísticamente significativas (p < 0.05). Sin embargo, en el análisis entre los porcentajes de éxito entre drogas, no presentaron diferencias para CQ y QN y si presentaron diferencias para MQ (p < 0.05). No hay diferencias significativas entre los valores de R2 entre las pruebas MSF y DELI. El porcentaje de éxito de CV positivos aumentó de 37.5% con el ensayo MSF a 81.25% con el ensayo DELI, estas diferencias fueron significativas (p < 0.05). Se concluye que el ensayo DELI es más eficaz que el ensayo MSF para calcular la IC50 de las drogas CQ, QN Y MQ. / Tesis

Plasmodium falciparum, one of the parasites that cause clinical malaria, is a continuous public health concern, especially in Asia and Africa. Unfortunately, the parasite has developed resistance to many drugs created to treat and prevent the disease. Artemisinin and its derivatives are the new gold standard for treatment of malaria, yet treatment failures in clinical studies are starting to be reported. Clearly, artemisinin resistance needs to be characterized and dealt with accordingly. In support of the Gates Foundation Artemisinin Consortium, we conducted a blinded study to elucidate the phenotypic response of artemisinin derivatives of parasites derived from patient blood samples from Cambodia and Thailand. Blood samples containing Plasmodium falciparum were cultured and then assayed using SYBR green as an indicator to obtain drug IC50s. The data suggested that many isolates are not demonstrating resistance to artemisinin. However, a select few are showing some resistant characteristics in the form of elevated IC 50s, especially to some of the drugs already identified in previous studies as drugs having resistant characteristics. Compared to studies conducted within the past ten years, no significant changes in parasite susceptibility to the artemisinin drugs have been observed. Additional analysis of clinical outcomes, therapeutic drug levels, and molecular markers needs to be completed before it can be assumed that artemisinin resistance has emerged.

Les plastes sont des organites semi-autonomes qui dérivent d'un événement unique d'endosymbiose entre une cyanobactérie et une cellule eucaryote ancestrale. Le plaste le mieux caractérisé est le chloroplaste des cellules de plantes et d'algues vertes. À la différence des systèmes membranaires eucaryotes qui sont en général riches en phospholipides, les membranes des plastes végétaux se composent à plus de 70% de galactoglycérolipides (monogalactosyldiacylglycérol, MGDG ; digalactosyldiacylglycérol, DGDG). Leur synthèse est assurée par des galactosyltransférases, les MGDG synthases (EC 2.4.1.46) et les DGDG synthases (EC 2.4.1.241) localisées dans les membranes de l'enveloppe qui limitent l'organite. Chez les plantes, les galactolipides sont essentiels pour la biogenèse des plastes mais aussi à la synthèse de membranes extraplastidiales sous certaines conditions physiologiques telles qu'en carence de phosphate.La plupart des parasites Apicomplexes (protozoaires parasites à mode de vie intracellulaire) possèdent une structure plastidiale, non photosynthétique, contenant un ADN circulaire. Cet organite, baptisé apicoplaste, a pour origine phylogénétique une endosymbiose secondaire entre deux eucaryotes, avec ingestion d'une algue rouge unicellulaire par un protozoaire ancestral, suivie d'une disparition de la plupart des structures subcellulaires de l'algue. Des protéines codées par des gènes nucléaires sont importées dans l'apicoplaste et impliquées dans des voies métaboliques typiques des plantes telles que la biosynthèse d'acides gras par le système FASII.Des lipides aux propriétés chromatographiques proches de celles du MGDG et du DGDG ont pu être détectés dans des extraits de Plasmodium falciparum et Toxoplasma gondii, suggérant l'existence d'une voie de biosynthèse des galactolipides comparable à celle existant dans le chloroplaste. Au moyen d'une série d'anticorps dirigés contre le DGDG, un premier objectif de ce travail de thèse a été de caractériser la localisation d'un épitope de structure proche du digalactolipide chloroplastique (DGLE pour digalactolipid-like epitope) chez P. falciparum et déterminer l'évolution de la distribution subcellulaire de cet épitope au cours du cycle cellulaire du parasite. Ces études suggèrent qu'un lipide de structure proche du DGDG est associé à des systèmes membranaires en périphérie de la cellule, en particulier le complexe membranaire interne. Des parasites transgéniques exprimant une MGDG synthase exogène de plante ont été générés. L'accumulation remarquable à la fois de MGDG et de DGDG chez ces transformants démontre d'une part que l'enzyme de plante est fonctionnelle, catalysant la synthèse de MGDG, et d'autre part qu'une glycosyltransférase de P. falciparum est capable ensuite de catalyser de grandes quantités de DGDG. Le rôle possible de cette glycosyltransférase dans la synthèse du DGDG reste à établir. Un second objectif de cette thèse était d'évaluer le potentiel dans P. falciparum de composés inhibant la synthèse des galactolipides de plante dans une visée thérapeutique. Un criblage à haut débit robotisé d'une banque de 24.000 molécules nous a permis d'identifier des inhibiteurs de la MGDG synthase 1 d'Arabidopsis thaliana, parmi lesquels deux composés présentent un effet inhibiteur mesuré par une CI50 de l'ordre 10 µM sur l'activité MGDG synthase. Nous avons pu caractériser l'effet de ces molécules comme compétiteurs de la fixation du diacylglycérol. Une activité inhibitrice de la prolifération de P. falciparum a pu être mesurée in vitro bien qu'aucune enzyme homologue à la MGDG synthase n'ait pu être identifiée in silico sur les banques de données relatives aux Apicomplexes. Une diversification de la structure du châssis moléculaire a été conduite afin d'améliorer 1) l'effet herbicide et 2) l'inhibition de la croissance des parasites et de développer ainsi de nouvelles molécules antipaludiques, qui puissent être qualifiées de médicaments herbicides. L'étude de 250 analogues a permis de progresser de façon substantielle dans le sens d'une meilleure sélectivité avec des composés actifs à 200 nM.Il n'est pas exclu que les molécules sélectionnées pour leur capacité à inhiber une activité MGDG synthase exercent, chez P. falciparum, un effet sur une autre cible. Nous avons conduit une expérience visant à isoler la(les) protéine(s) cible(s) des molécules bioactives par chromatographie d'affinité, puis identifié ces protéines après digestion trypsique par spectrométrie de masse. Une perspective de ce travail consiste à caractériser certaines de ces cibles candidates.Le développement de candidats-médicaments est un processus long selon le schéma classique, depuis la validation d'une cible jusqu'aux essais cliniques. La nouvelle classe de compétiteurs du diacylglycérol caractérisée dans ce travail de thèse présente des propriétés intéressantes dans une visée thérapeutique, qu'il sera important d'optimiser dans l'avenir.

Le paludisme, ou malaria, est une maladie infectieuse qui touche plus de 350 millions d'êtres humains et qui tue chaque année 2,5 millions de personnes à travers le monde. Les parasites responsables de la malaria sont des apicomplexes du genre Plasmodium, essentiellement P. falciparum. Le génome de P. falciparum, est séquencé depuis octobre 2002, et présente un des taux les plus faibles de gènes annotés, avec ~60 % de gènes sans fonction attribuée. Il est difficile, voire impossible, d'identifier dans le génome de P. falciparumi, certains gènes, responsables de fonctions mesurées biochimiquement chez le parasite, par similarité avec des séquences homologues caractérisées dans d'autres organismes. Cette difficulté rencontrée lors des recherches automatiques d'homologie est une limite à tout projet exploratoire du génome malarial fondé sur la phylogénie moléculaire. En particulier, l'inventaire des séquences héritées de l'algue ancestrale, qui a réalisé l'endosymbiose secondaire qui caractérise le phylum des Apicomplexa (sous génome d'origine algale dans lequel il est possible de rechercher des cibles pour des médicaments herbicides), peut être rendu incomplet. Les caractéristiques atypiques du génome et du protéome de Plasmodium, résumées sous le terme de biais compositionnel (en particulier un pourcentage en adénosine+thymidine supérieur à 80%), ont été soupçonnées d'être un cas limite pour les outils d'analyse de séquence existants. L'objet de cette thèse a donc été d'examiner l'influence possible de ce type de biais sur les méthodologies de comparaisons de séquences et de façon plus approfondie sur leurs statistiques.<br />Nous avons proposé des développements théoriques nouveaux, associés à la statistique de la Z-value introduite par Lipman et Pearson pour évaluer la significativité d'un score d'alignement de deux séquences protéiques: (1) le théorème TULIP permettant de déduire un majorant de la probabilité d'un score d'alignement de séquences (i.e. la P-value) par la valeur 1/Z-value2 et (2) la déduction des propriétés remarquables de la distribution des Z-values à partir de quelques hypothèses sur l'évolution des protéines dans le contexte de la théorie de la fiabilité des systèmes. Ces développements théoriques ont permis certaines avancées sur le plan pratique de l'identification de séquences homologues initialement non détectées par le théorème de Karlin-Altschul et d'étayer la relation entre les scores d'alignements et l'information mutuelle, au sens de la théorie de l'information.<br />En construisant un espace de configuration des protéines homologues, permettant une expression du théorème TULIP et ayant une cohérence avec la théorie synthétique de l'évolution, nous avons déduit une méthode de reconstruction de phylogénies de séquences protéiques à l'aide des Z-values. Les phylogénies moléculaires reconstruites par cette méthode sont concordantes avec celles obtenues à partir d'alignements multiples et permettent par ailleurs de résoudre certaines incohérences rapportées avec les méthodes de reconstruction phylogéniques classiques.<br />En prenant en compte le modèle statistique que nous avons élaboré, nous avons entrepris une première analyse de l'évolution du biais en acides aminés chez Plasmodium corrélativement à l'évolution du biais en acides nucléiques dans le génome malarial et en fonction de la divergence évolutive, établie en prenant le génome non biaisé d'Arabidopsis thaliana comme référence. Nous avons observé que le biais des séquences malariales était corrélé au pourcentage de divergence avec leurs homologues végétaux. Nos analyses suggèrent de plus que le biais est vraisemblablement la conséquence d'une évolution au niveau nucléique. Nous avons examiné la possibilité de construire une famille de matrices tenant compte de cette dissymétrie dans le cas de la comparaison de Plasmodium et d'Arabidopsis. Ces matrices appelées DirAtPf, possèdent (1) une sensibilité théorique et (2) une spécificité supérieure aux familles de matrices existantes.<br />Les perspectives des travaux présentés dans ce mémoire incluent une progression de l'annotation automatique de Plasmodium falciparum et la mise en place d'une procédure statistiquement robuste et phylogénétiquement consistante pour caractériser le sous-génome algal du parasite malarial.