A caracterização fenotípica de Plasmodium falciparum permite conhecer o padrão de sensibilidade do parasito às drogas antimaláricas utilizadas em países endêmicos. No presente estudo avaliamos fenotipicamente isolados clínicos de P. falciparum provenientes do Continente Africano e do Caribe. A sensibilidade à dihidroartemisinina (DHA: 4 - 1.000 nM), artesunato (AS: 0,1 - 100 nM), lumefantrina (LMF: 3,1 - 200 nM) e mefloquina (MFQ: 0,2 - 1.000 nM) foi investigada por meio de quatro técnicas: (a) ensaio de sensibilidade ex-vivo e in vitro, (b) ensaio de dormência, (c) ensaio de citometria de fluxo e (d) ensaio de sobrevivência do trofozoíto jovem (Ring Stage Survival Assay - RSA). Nos experimentos ex-vivo e in vitro, os IC50 estabelecidos foram 0,4 - 66,6 nM para DHA; 3,8 - 48,8 nM para LMF; 0,3 - 25,9 nM para AS e 2 - 439 nM para MFQ. No ensaio de dormência, esquizontes foram observados na amostra de referência NF54 de P. falciparum e na amostra clínica S-01/15 após pressão com 62,5 nM, 250 nM e 1.000 nM de DHA. O período de recuperação variou de 4 a 40 dias. Para LMF, houve maturação para o estágio de esquizonte no isolado de referência no sétimo e décimo segundo dia após a exposição a 66,6 nM e 200 nM da droga, respectivamente. Esquizontes foram visualizados no isolado clínico FS-08/15 de P. falciparum depois da pressão com 100 nM de AS, com recuperação de 0 a 28 dias, portanto sem apresentar dormência. Na citometria de fluxo, trofozoítos jovens viáveis de P. falciparum marcados com Rodamina 123 e DAPI foram observados nas máximas concentrações de DHA (1.000 nM) e LMF (200 nM). Finalmente no RSA, a taxa de crescimento (TC) e porcentagem de supervivência (PS) do isolado de referência foi 2,92 e 4,19%, respectivamente, frente a 700 nM de DHA. O mesmo isolado pressionado com 3.500 nM de LMF apresentou 3,6 de TC e 2,25% de PS. A avaliação microscópica dos ensaios de sensibilidade ex-vivo e in vitro subestima a resposta de P. falciparum à terapia combinada com artemisinina (ACT). Nossos resultados sugerem que a dormência, principal mecanismo de tolerância às artemisininas (ART), não aconteceria em todos os isolados clínicos de P. falciparum. A citometria de fluxo avaliou com acurácia a viabilidade parasitária. No presente estudo, pela primeira vez foi reportada a dormência de P. falciparum à LMF / The phenotypic characterization of Plasmodium falciparum is useful for the knowledge of parasite sensitivity against antimalarial used in endemic countries. In this study we evaluated the sensitivity of clinical isolates of P. falciparum from the African continent and the Caribbean. The sensitivity to dihydroartemisinin (DHA: 4 - 1,000nM), artesunate (AS: 0.1 - 100 nM), lumefantrine (LMF: 3.1 to 200 nM), and mefloquine (MFQ: 0.2 to 1,000 nM) was investigated through four techniques: (a) ex vivo and in vitro microtests, (b) dormancy assay, (c) flow cytometry assay and (d) survival assay using young trophozoites (Ring Stage Survival Assay - RSA). In the ex vivo and in vitro experiments, the IC50 was calculated and was 0.4 - 66.6 nM for DHA; 3.8 - 48.8 nM for LMF; 0.3 - 25.9 nM for AS and 2 - 439 nM for MFQ. According to dormancy assays, schizonts were observed in the P. falciparum reference isolate NF54 and in the clinical isolate S-01/15 after pressure with 62.5 nM, 250 nM and 1,000 nM DHA. The recovery period ranged from 4 to 40 days. For LMF was observed the growth to the schizont stage in NF54, in the days 7 e 12 after exposure to 66.6 nM and 200 nM of the drug, respectively. Schizonts were seen in the P. falciparum clinical isolate FS-08/15 after pressure with 100 nM of AS, right after incubation period, with no dormancy of trophozoites. In flow cytometry assays, viable young trophozoites of P. falciparum labeled with DAPI and Rhodamine 123 were observed at the maximum concentrations of DHA (1,000 nM) and LMF (200 nM). Finally in RSA, the growth rate (GR) and percentage of survival (PS) of the reference isolate was 2.92 and 4.19%, respectively, after pressure with 700 nM of DHA. The same isolate pressed with 3,500 nM of LMF presented GR of 3.6% and PS of 2.25%. In conclusion, microscopic evaluation of ex vivo and in vitro sensitivity tests underestimates the P. falciparum response to artemisinin-based combination therapy (ACT). Our results suggest that the dormancy, main mechanism of tolerance to artemisinin (ART), is not presented in all clinical isolates of P. falciparum. Flow cytometry was able to confirm the parasite viability accurately. In this study, for the first time the dormancy of P. falciparum after pressure with LMF was reported

A malária apresenta um alto índice de mortalidade com mais de 500 mil mortes registradas em 2013. Para agravar a situação de saúde pública, foi descrito o surgimento de resistência às drogas usadas na terapêutica da doença. Torna-se necessário a identificação e o estudo de novos alvos antimaláricos. A via MEP se mostra como um potencial alvo para o desenvolvimento de drogas contra P. falciparum uma vez que está ausente em humanos. Nossos objetivos foram avaliar a função da vitamina E biossintetizada pelo parasita, caracterizar a biossíntese de vitamina K1 e o metabolismo de fitol. Esse estudo determinou que a vitamina E biossintetizada pelo parasita atua no sistema redox do parasita. Por outro lado, mostramos que a biossíntese de vitamina K1 é ativa no parasita e detectamos sua forma reduzida. Por fim, observamos que existe uma via de reaproveitamento de fitol em P. falciparum assim como em plantas. O estudo abre oportunidades para um desenvolvimento racional de novos antimaláricos e aprofunda o conhecimento na biologia do parasita. / Malaria has the highest mortality rate with more than 500 000 deaths in 2013. The public health situation gets worse because it has been described the emergence of resistance to common drugs used in the treatment of disease. It is necessary to identify and study of new antimalarial targets. The MEP pathway is a potential target for drug development against Plasmodium falciparum once it is absent in humans. Our objectives were to evaluate the function of vitamin E biosynthesized by the parasite and characterize the biosynthesis of vitamin K1 and the phytol metabolism. This study determined that vitamin E biosynthesized by the parasite operates in the redox system of the parasite. We show the biosynthesis of vitamin K1 is active on parasite and we detected its reduced form. Finally, we demonstrate that there is a phytol salvage pathway in P. falciparum as well as plants. The study opens opportunities for the rational development of new antimalarials and deepens knowledge on parasite biology.

A malária é um problema de saúde global, que hoje acomete aproximadamente 207 milhões de pessoas, e que levou ao óbito cerca de 607.000 indíviduos apenas no ano de 2013. No Brasil, 99% dos casos concentram-se na Amazônia Legal onde infecções por Plasmodium vivax são as principais causas da doença e podem ser fatais. Infecções por Plasmodium spp. podem levar à um quadro respiratório grave, com complicações pulmonares denominadas lesão pulmonar aguda (LPA) e síndrome do desconforto respiratório agudo (SDRA). A LPA/SDRA é caracterizada pela lesão dos alvéolos e do parênquima pulmonar, com perda da função da barreira epitelial do alvéolo e do capilar pulmonar de células endoteliais e, consequentemente, presença de edema pulmonar de origem não-cardiogênica. A diminuição da capacidade de trocas gasosas, aumento da atividade leucocitária e de mediadores inflamatórios nos pulmões resultam em insuficiência respiratória. A dificuldade em se estudar a doença em humanos associado ao desconhecimento dos fatores envolvidos na síndrome faz com que essa disfunção pulmonar torne-se mal compreendida e leve cada vez mais pessoas a óbito. O objetivo central do presente trabalho foi reconhecer e caracterizar o perfil leucocitário pulmonar, os fatores inflamatórios e a morte celular que contribuem para o desenvolvimento da LPA/SDRA associada a malária. Neste trabalho foi utilizado como modelo experimental a associação entre camundongos da linhagem DBA/2 e o parasita murino Plasmodium berghei ANKA. Neste modelo 30-75% dos camundongos desenvolvem sintomas pulmonares agudos (LPA/SDRA) e os demais morrem tardiamente com hiperparasitemia (HP). Desenvolvemos a partir dos parâmetros respiratórios e da parasitemia um modelo preditivo para classificação dos animais em LPA/SDRA ou HP antes do momento da morte com alta sensibilidade e especificidade. Nossos resultados mostram que os macrófagos alveolares e os neutrófilos estão aumentados de maneira significativa nos animais classificados como LPA/SDRA e que a depleção dessas populações promove a sobrevida dos animais e o não desenvolvimento da síndrome. Verificamos também que as células TCD4+ e TCD8+ produzem grandes quantidades de IFN-? nos animais LPA/SDRA, contudo o sistema imunológico desses animais produz grandes quantidades de IL-10 como forma de regular a resposta inflamatória. Em nosso estudo mensuramos a apoptose no tecido pulmonar e verificamos que os animais com LPA/SDRA possuem um número maior de células morrendo em relação aos animais HP. Vimos também que a apoptose de neutrófilos e células dendríticas ocorrem de maneira significativa no lavado broncoalveolar dos animais LPA/SDRA. O estudo da expressão de genes pró e anti-apoptóticos mostrou que há o aumento da expressão de Casp-3, Casp-9, Bad, Bid, Bak, FADD, CAD e Ripk-1 nos animais LPA/SDRA, enquanto que Bcl-XL e Bcl2 estão mais expressos nos animais HP. / Malaria is a global health problem that now affects approximately 207 million people, and led to the deaths of about 607,000 individuals only in 2013. In Brazil, 99% of the cases are concentrated in the Amazon where infections by Plasmodium vivax are the major cause of morbidity and which can also be fatal. Infections with Plasmodium spp. can lead to a serious respiratory condition including pulmonary complications named as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). ALI /ARDS is characterized by damage to the alveoli and the lung parenchyma, loss of epithelial barrier function of the alveoli and pulmonary capillary endothelial cells and, consequently, pulmonary edema noncardiogenic origin. Decreased capacity for gas exchange, increased leukocyte activity and inflammatory mediators in the lungs resulting in respiratory failure. The difficulty in studying human disease associated with lack of knowledge of factors involved in the syndrome and makes the pulmonary dysfunction become misunderstood and take more people to death. The central objective of this study was to determine and characterize the lung leukocytes profile, inflammatory factors and cell death that contribute to the development of ALI / ARDS associated with malaria. In this work was used as an experimental model the association between DBA/2 mice strain and the Plasmodium berghei ANKA murine parasite. In this model, 30-75% of mice develop acute pulmonary symptoms (ALI/ARDS) and the others died too late with hyperparasitaemia (HP). Developed from the respiratory parameters and parasitaemia a predictive model for classification of animals in ALI/ARDS or HP before the moment of death with high sensitivity and specificity. Our results show that alveolar macrophages and neutrophils were increased significantly in animals classified as ALI/ARDS and the depletion of these populations promotes the survival of the animals and not development of the syndrome. We also observed that CD4+ and CD8+ T cells produce large amounts of IFN-? in animals ALI/ARDS, however the immune system of these animals produce large amounts of IL-10 in order to regulate the inflammatory response. In our study we measured apoptosis in lung tissue and found that animals with ALI/ARDS have a larger number of cells dying compared to HP animals. We also saw that apoptosis of neutrophils and dendritic cells occur significantly in BAL of animals ALI/ARDS. The study of the expression of pro and anti-apoptotic genes showed that there is increased expression of Casp-3, Casp-9, Bad, Bid, Bak, FADD, and Ripk CAD-1 in animals ALI/ARDS, as that Bcl XL and Bcl2 are more expressed in HP animals.

Le paludisme est une maladie tropicale causée par un parasite du genre Plasmodium. Chez l’Homme, un niveau élevé de phospholipase A2 sécrétée de groupe IIA humaine (hGIIA) est mesuré dans le plasma des patients impaludés. Cette enzyme est connue pour son rôle antibactérien et pro-inflammatoire. Cependant, son rôle dans le paludisme n’a jamais été exploré. Pour comprendre le rôle in vivo de la hGIIA dans cette pathologie, nous avons entrepris la caractérisation hématologique, histopathologique et immunohistochimique de l’infection de souris C57BL/6, transgéniques (Tg+) pour l’enzyme humaine, par l’espèce murine Plasmodium chabaudi chabaudi 864VD. Ce modèle reproduit un paludisme non létal. Nos résultats ont permis d’établir que les souris Tg+ ont un meilleur contrôle de l’infection au moment du pic de crise parasitaire (J14 post-inoculation), avec une diminution de 27% de la parasitémie, comparé aux souris « littermates » non transgéniques (Tg-). L’injection de hGIIA recombinante aux jours 12, 13 et 14 p.i. (0,125 mg/kg deux fois par jour) à des souris C57BL/6 wild-type (WT) infectées par P. c. chabaudi 864VD provoque une diminution d’environ 19% de la parasitémie à J14 p.i., démontrant un rôle direct de la hGIIA dans la diminution de la population parasitaire. Les données hématologiques montrent que l’infection chez la souris Tg+ provoque une anémie plus durable que chez la souris Tg- et une élévation nettement plus importante du nombre de leucocytes, en particulier des polynucléaires neutrophiles. Chez la souris Tg+ parasitée, on observe aussi l’activation d’un nombre important de lymphocytes et une activation spécifique des monocytes avant le pic de crise. Chez la souris Tg- infectée, les données histologiques mettent en avant une meilleure récupération des lésions histopathologiques du foie et une hyperplasie des lymphocytes B dans la rate, tandis que les souris Tg+ infectées présentent des lésions hépatiques tardives et une hématopoïèse extramédullaire splénique. Les résultats des analyses par RT-qPCR suggèrent que l’ARNm de la hGIIA augmente au pic parasitaire dans le foie des souris Tg+ infectées, mais diminue dans la rate et les cellules sanguines. L’injection de hGIIA recombinante au début de la phase patente est sans effet sur la parasitémie, ce qui laisse supposer que des événements plus tardifs dans l’infection sont nécessaires à l’activité antiparasitaire de l’enzyme. L’étude du rôle des lipoprotéines oxydées comme substrat potentiel de l’activité antiparasitaire de l’enzyme, basée sur des résultats in vitro, est abordée. En conclusion, nos études ont permis de dresser un tableau large de l’infection à Plasmodium chez la souris exprimant la hGIIA, et ouvrent de nouvelles perspectives dans l’analyse du rôle de l’enzyme dans la physiopathologie du paludisme. / Malaria is a tropical disease caused by a parasite of the Plasmodium genus. High levels of circulating human group IIA secreted phospholipase A2 (hGIIA) have been reported in malaria patients. The enzyme is well known for its bactericidal and pro-inflammatory actions. However, so far its role in malaria is unknown. In order to address the in vivo role of hGIIA in malaria, we performed a hematological, histopathological and immunohistochemical characterization of C57BL/6 hGIIA transgenic mice (Tg+ mice) infected with P. chabaudi chabaudi (864VD strain), a murine Plasmodium species and strain which causes non-lethal chronic malaria. Infected Tg+ mice present a 27% reduction of parasitaemia at the peak of infection (D14 post-inoculation, p.i.) compared to infected non-transgenic littermates (Tg- mice). Intraperitoneal injection of recombinant hGIIA at D12, D13 and D14 p.i. (0.125 mg/kg twice a day) into P. chabaudi 864VD-infected WT C57BL/6 mice leads to a 19% reduction of the parasitaemia at D14 p.i., demonstrating the direct and acute role of hGIIA in lowering parasite population and presumably ruling out a potential effect linked to chronic overexpression of hGIIA in Tg+ mice. Hematological data show a durable anemia in Tg+ mice compared to Tg- mice during the infection and an important increase of leucocytes, especially of polynuclear neutrophils. The parasitized Tg+ mouse also presents a higher activation of lymphocytes and a specific activation of monocyte cells at the pic of crisis. In the infected Tg- mouse, histological data show a better histopathological recovery in the liver and B cells hyperplasia in the spleen, whereas the infected Tg+ mouse presents late hepatic injuries and splenic extra-medullar hematopoiesis. RT-qPCR analyses suggest that hGIIA mRNA increases at the pic of infection in the liver of infected Tg+ mice, but decreases in spleen and blood. Intraperitoneal injection of recombinant hGIIA at the patent phase is without effect on parasitaemia, which suggests that later infection events are needed for the enzyme antiparasitic activity. Involvement of oxidized-lipoproteins as potential hGIIA substrates, based on in vitro studies, is discussed. In conclusion, our studies allowed us to elaborate a larger picture of the infection of Plasmodium in the mice expressing hGIIA and open new perspectives in the analysis of the role of the enzyme in malaria pathophysiology.

Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections. / Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

La forme la plus sévère de paludisme est causée par le parasite unicellulaire P. falciparum. Lors de la phase intra-érythrocytaire de son développement, P. falciparum met en place des fonctions métaboliques nécessaires à son développement dans l'érythrocyte, à sa multiplication et enfin à sa dissémination vers d'autres érythrocytes. Comprendre et élucider les structures et les dynamiques du réseau métabolique chez le parasite, permettent de découvrir de nouvelles voies métaboliques et des étapes clefs qui peuvent jouer un rôle important dans le développement du parasite. Elles permettent également de déterminer le mécanisme d'action des agents antipaludiques et de mieux comprendre les résistances associées aux traitements existants. Dans cet objectif, une approche métabolomique ciblée, utilisant la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) a été utilisée. Cette approche consiste en une quantification absolue de métabolites impliqués dans les voies de biosynthèse des phospholipides membranaires du parasite mais également d'autres métabolites qui reflètent son statut métabolique. Nous avons dans un premier temps, déterminé les distributions et les quantités absolues des métabolites présents dans un érythrocyte infecté par P. falciparum en comparaison avec un érythrocyte sain. Nous avons également mis en évidence les perturbations causées par cette infection sur le métabolisme de l'érythrocyte humain ainsi que les différents échanges qu'entretien le parasite avec sa cellule hôte mais également avec le milieu extracellulaire. Le métabolisme phospholipidique de Plasmodium est complexe car il possède plusieurs voies de synthèses opérant à partir de précurseurs initiaux distincts et conduisant à la synthèse d'un même produit final. Dans l'objectif d'étudier la contribution relative des différentes voies métaboliques dans la biosynthèse des phospholipides majoritaires chez P. falciparum (PC et PE), nous avons développé une approche qui consiste à incuber les érythrocytes infectés en présence de précurseurs marqués. / The most severe form of malaria is caused by the single-celled parasite P. falciparum. During the intra-erythrocytic stage of its development, P. falciparum implements several metabolic functions necessary for its development in the erythrocyte, its multiplication and finally to its spread to other erythrocytes. Understand and elucidate the structures and the dynamics of the parasite's metabolic network is useful to discover new metabolic pathways and key steps that may play an important role in the development of the parasite. They also help determine the mechanism of action of antimalarial agents and better understand the resistances associated with available treatments. For this purpose, a targeted metabolomics approach, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. This approach consists of an absolute quantitation of metabolites involved in the biosynthesis of membrane phospholipids of the parasite but also other metabolites that reflect its metabolic status. We initially determined the distributions and the absolute amounts of metabolites in infected erythrocytes in comparison with healthy erythrocytes. We also highlighted the disruption caused by this infection on the metabolism of the human erythrocyte and the various interactions between the parasite and its host cell as well as the extracellular medium. The phospholipids metabolism of Plasmodium is complex because it has several synthetic pathways operating from separate initial precursor and leading to the synthesis of a single end product. With the aim to study the relative contribution of these different metabolics pathways in the biosynthesis of the most important phospholipids in P. falciparum (PC and PE), we have developed an approach that involves incubation of infected erythrocytes in the presence of labeled precursors.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-06-05T21:05:38Z No. of bitstreams: 1 Bruno de Bezerril Andrade - 2010.pdf: 74164438 bytes, checksum: a1cccf3d1f924ff7710fc4a73a190f0a (MD5) / Made available in DSpace on 2012-06-05T21:05:38Z (GMT). No. of bitstreams: 1 Bruno de Bezerril Andrade - 2010.pdf: 74164438 bytes, checksum: a1cccf3d1f924ff7710fc4a73a190f0a (MD5) / Universidade Federal da Bahia. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A malária é considerada uma das mais importantes doenças infecciosas do mxmdo. Esta doença é causada por diversas espécies do protozoário Plasmodium sp., principalmente o Plasmodium falciparum e o Plasmodium vivax, transmitido por mosquitos do gênero Anopheles. Apesar dos esforços governamentais e privados para o desenvolvimento de estratégias para o controle da doença, o panorama atual da malária está piorando, muito em razão do aparecimento de cepas de parasitas resistentes aos medicamentos. Os casos fatais são relatados principalmente na Áfiica e são causados pelo Plasmodium falciparum. Apesar de ser menos letal, a malária causada pelo Plasmodium vivax é mais amplamente distribuída e pode apresentar também alta morbidade e mortalidade. Na maioria das áreas endêmicas, estudos têm identificado vários fatores relacionados à imunidade clínica ou susceptibilidade aos parasitas. Assim, pelo menos quanto à malária causada pelo Plasmodium falciparum, idade, polimorfismos genéticos e exposição repetida ao parasita são considerados importantes determinantes da evolução da doença. Infelizmente, pouco tem sido feito na identificação de fatores preditores consistentes que poderiam ser usados para avaliação clínica. Este quadro é ainda pior para malária causada pelo Plasmodium vivax, provavelmente porque muitos pesquisadores consideram que é uma doença benigna. Além disso, como a maioria do conhecimento atual sobre a patogênese da malária não ajudou a reduzir a ocorrência da infecção e suas complicações, novas abordagens são necessárias para superar este cenário desfavorável. Esta Tese reúne um conjunto de seis manuscritos que visam identificar potenciais determinantes da gravidade da malária em uma área endêmica da Amazônia Ocidental Brasileira. Em primeiro lugar, um método preciso e eficaz para o diagnóstico da malária foi rastreado através da comparação de vários testes, incluindo um software baseado em redes neurais artificiais. O ensaio molecular mostrou-se o mais eficiente para o diagnóstico da malária sintomáticos e assintomáticos. Além disso, a utilização racional de um teste rápido para diagnóstico da malária pode ser promissora em áreas onde há dificuldade na formação continuada dos técnicos diagnósticos. A rede neural artificial indicou que o balanço de citocinas é um forte determinante do quadro clínico. Em outro estudo, uso de sorologia para mensuração de anticorpos IgG contra o sonicado de glândula salivar do vetor Anopheles darlingi mostrou-se útil para a avaliação da exposição ao Plasmodium vivax e também para estimar a imunidade clínica á malária. Em um terceiro estudo com foco na identificação de outros fatores relacionados à imunidade clínica, a exposição natural ao vírus da hepatite B mostrou-se associada à redução da gravidade clínica da malária causada tanto pelo Plasmodium vivax quanto pelo Plasmodium falciparum. No que diz respeito exclusivamente à malária vivax, os casos graves apresentaram uma intensa e desregulada resposta inflamatoria sistêmica. Nestes pacientes, a enzima antioxidante superóxido dismutase-1 surgiu como um excelente marcador da gravidade e mostrou-se envolvida na patogênese da doença grave, na qual há uma liberação de grandes quantidades de heme livre. Em conjunto, os manuscritos desta tese adicionam importantes informações no entendimento dos mecanismos determinantes da gravidade da malária, extremamente / Malaria is considered one of the most important infectious diseases that ever threaten the world. This disease is caused mainly by the infection with Plasmodium falciparum or Plasmodium vivax transmitted by Anopheles mosquitoes. Despite governmental and private efforts for the development of key strategies for the disease control, the actual panorama of the Plasmodium infection is getting worse due to the emergence of drug resistMt parasite strains. The lethal cases are reported mostly in Africa and are caused by Plasmodium falciparum. Albeit being less lethal, Plasmodium vivax infections are more widely distributed can cause high morbidity and eventually death. In most endemic areas, studies have indentified a number of factors related to clinical immunity or susceptibility to the parasites. Thus, at least regarding the falciparum malaria, age, genetic polymorphisms and repeated exposure to Plasmodium are considered most important determinants of the disease outcome. Unfortunately, little has been made in the screening of reliable predicting factors that could be ultimately used for clinical evaluations. This landscape is even worse for vivax malaria, probably because many researches consider it as a benign disease. Moreover, as most of the current knowledge about the malaria pathogenesis did not truly help to relieve the disease burden, new insights are necessary to overcome this unfavorable scenario. This thesis brings together a set of six manuscripts that aim to identify potential determinants of the disease severity linked to the immunopathogenesis in an endemic area from the western Brazilian Amazon. First, a precise and effective method for malaria diagnosis was screening by comparing multiple tests, including a software based of artificial neural networks. The molecular assay showed to be the most efficient for the diagnosis of symptomatic and asymptomatic malaria. In addition, the rational use of a rapid test for the diagnosis of malaria may be promising in areas where there is difficulty in continued training of technical human resources. The artificial neural network indicated that the cytokine balance is a strong determinant of the clinical presentation. In another study, the use of serology for measuring IgG antibodies against the sonicate salivary gland of Anopheles darlingi vector is a promising marker of exposure to Plasmodium vivax and can also estimate the clinical immunity. Intriguingly, the natural exposure to the hepatitis B virus appeared as an important factor associated with reduced clinical severity for both vivax and falciparum malaria. Concerning solely the vivax malaria, severe cases have an intense and unregulated inflammatory response. In these patients, the antioxidant enzyme superoxide dismutase-1 has emerged as an excellent marker of severity and was involved in the pathogenesis of the severe disease in which there is a release of large amounts of free heme. Together, the manuscripts of this thesis add important information in understanding the mechanisms that determine the severity of malaria.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / O parasitismo é uma importante força seletiva em populações, assim como a competição e a predação. Os parasitos sanguíneos podem afetar a coloração da plumagem, a seleção sexual e o sucesso reprodutivo em aves. As aves da região Antártica têm sido mencionadas na literatura como livres de hemoparasitos. A Baía do Almirantado, na Ilha Rei George, Península Antártica, é a maior Baía da região, abrigando diferentes espécies de aves durante o período reprodutivo. Dentre elas, estão duas espécies de skuas, as mais frequentes da Antártica, skua-sub-antártica (Catharacta lonnbergi) e skua-polar-do-sul (C. maccormicki) e três espécies de pinguins, pinguim-antártico (Pygoscelis antarctica), pinguim-papua (P. papua) e pinguim-de-adélia (P. adeliae). Skuas e pinguins são aves que se dispersam durante o inverno austral, podendo ser potenciais reservatórios e transmissores de parasitos, embora resultados negativos de hemoparasitos tenham sido encontrados para diversas outras aves marinhas e também para a região Antártica. O objetivo do presente trabalho foi investigar a presença de hemoparasitos em pinguins e skuas antárticos na Baía do Almirantado. Amostras de lâminas de esfregaço sanguíneo e de sangue para análises moleculares de pesquisa de Plasmodium/Haemoproteus foram coletadas em dois períodos reprodutivos, de dezembro de 2010 a março de 2011 e de dezembro de 2011 a fevereiro de 2012. Um total de 185 amostras de aves foram coletadas, incluindo 120 pinguins e 65 skuas. Skuas foram tiveram resultados negativos para hemoparasitos. As três espécies de pinguins foram positivas para Plasmodium/Haemoproteus , via técnica molecular, incluindo dois P. papua,dois P. antarctica etrês P. adeliae. Apenas um indivíduo confirmado positivo pela técnica molecular, pertencente a P. papua, foi positivo utilizando a técnica de esfregaço sanguíneo, com diagnóstico de Plasmodium sp. Não houve diferença significativa entre indivíduos machos e fêmeas das espécies parasitadas, assim como entre adultos e filhotes. As aves parasitadas (n=7), foram categorizadas abaixo do peso (n=5) e acima do peso (n=2). O presente estudo é o primeiro a relatar hemoparasitos na região Antártica e também é o primeiro registro de presença de hemoprotozoários para as três espécies de pinguins analisadas. A ausência de hemoparasitos em aves antárticas tem sido justificada pela ausência de potenciais vetores na região. Portanto, é possível que os pinguins parasitados tenham adquirido a infecção durante a dispersão por ocasião do inverno austral. No entanto, skuas antárticas também são aves migratórias, que podem atingir regiões com potenciais vetores reconhecidos, mas nunca foram diagnosticadas com hemoparasitos, o que foi reforçado pelos resultados negativos do presente estudo. Nesse caso, acredita-se que skuas, podem ter um sistema imune competente ou que a ausência de hemoparasitos nessas aves seja justificada por confinamentos filogenéticos entre parasito-hospedeiro. Entretanto, pouco se sabe sobre a existência de vetores na Antártica, rotas migratórias das aves da região e especificidade parasito-hospedeiro. Os resultados inéditos encontrados no presente estudo devem, portanto, servir como ponto de partida para o entendimento das interações parasito-hospedeiro, de forma a contribuir para a preservação do ambiente antártico. / Parasitism is an important selective pressure in populations, as well as competition and predation. Blood parasites can affect the color of plumage, sexual selection and reproductive success in birds. Antarctic birds have been mentioned in literature absent of blood parasites. Admiralty Bay is located at King George Island, Antarctic Peninsula, and is the largest bay on the region, harboring different avian species during the reproductive period. Among them, are the two most common skuas of Antarctica, the brown-skua (Catharacta lonnbergi) and the south-polar-skua (Catharacta maccormicki), and tree penguins species, the Chinstrap (Pygoscelis antarctica), the Gentoo (P. papua) and the Adelie (P. adeliae). Skuas and penguins are seabirds that migrating during the southern winter, and may be potential reservoirs and transmitters of parasites. However, negative results of blood parasiteshave been found in several seabirds and also to the Antarctic region. The objective of this study was to investigate the presence of blood parasites in Antarctic penguins and skuas at Admiralty Bay. Blood smears and blood samples for molecular analyses to research Plasmodium/Haemoproteuswere collected in two reproductive periods, from December 2010 to March 2011 and from December 2011 to March 2012. A total of 185 bird samples were collected, including 120 penguins and 65 skuas. Skuas were negative for parasites. The tree species of penguins were positives to Plasmodium/Haemoproteusby molecular analysis, including two P. papua, two P. antarctica and tree P. adeliae. Only one positive penguin by molecular technique, a P. papua, was positive in blood smears, diagnosed with Plasmodium sp. There was no significant difference between male and female individuals of the parasitizedspecies, as well as between adults and chicks. Parasitized birds (n = 7) were categorized as underweight (n=5) and overweight (n=2).The present study is the first to report blood parasites in the Antarctic region and is also the first record of the presence of blood protozoa for the three penguin species analyzed. The absence of blood parasites in Antarctic birds has been justified by the absence of potential vectors in the region. Therefore, it is possible that the parasitized penguins acquired infectionwhen they disperse during southern winter. However, antarctic skuas are migratory birds, and they can reach regions with recognized potential vectors, but have never been diagnosed with blood parasites, what was reinforced by data of the present study. In this case, it is believed that skuas may have a competent immune system, or that the absence of these parasites in these birds is justified by phylogenetic constraints between the host-parasite. Nevertheless, little is known about the existence of vectors in the Antarctica, migratory routes of birds in the region and parasite-host specificity. The inedited results found in this study should therefore serve as a starting point to understand the host-parasite interactions, and to contribute to the preservation of the Antarctic environment.

Um importante fator de patogenicidade de P. falciparum, causador da malária, é a presença de antígenos altamente polimórficos, codificados por famílias multigênicas como var e rif. O grande polimorfismo destes genes em isolados de diferentes regiões contrasta com o fato de existirem, em diferentes isolados de campo brasileiros, alelos conservados. Respostas humorais contra estas proteínas são consideradas importantes na aquisição de proteção contra os sintomas da doença em regiões endêmicas. Portanto, medimos a resposta humoral contra antígenos recombinantes PfEMP1 e RIFIN e detectamos níveis baixos de resposta tanto em indivíduos sintomáticos quanto em indivíduos assintomáticos infectados pelo parasita. Estas respostas foram baixas quando comparadas às respostas contra MSP119 da superfície do merozoíta e parecem ser de curta duração. Com base nestes resultados, as respostas contra domínios DBLa, em situações hipoendêmicas, parecem ocorrer em função da presença do parasita circulante, e não são relacionadas à proteção contra os sintomas clínicos da doença. / An important factor in the pathogenicity of P. falciparum, the causing agent of malaria is the expression of highly polymorphic antigens encoded by the multigene families var and rif. The extreme polymorphism of these genes in strains from different geographical regions is in contrast with the observation of a number of conserved alleles found in Amazonian isolates. The humoral response against these proteins is considered an important factor in the immunity against symptomatic infection in áreas with high transmission. We measured the antibody response against recombinant PfEMP1 and RIFIN antigens and detected low responsiveness of symptomatic and asymptomatic malaria infected individuals. These responses were not only weak when compared to the anti-merozoite surface protein 1 response, but also ceased rapidly after the removal of circulating parasites. On the basis of these results, the response against DBLa seems to be dependent on the presence of parasites and not important for the observed protection against symptomatic infection in hypoendemic settings.