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Estudos relacionados à malária autóctone em regiões de baixa transmissão no Brasil ganham cada vez mais relevância científica e epidemiológica, pois revelam a manutenção desse cenário em regiões de Mata Atlântica remanescente. No sudeste do Estado de São Paulo, a ocorrência de surtos no município de Juquitiba tem sido foco de pesquisas sobre a prevalência de Plasmodium na população, com registros de casos assintomáticos. Relatos de ocorrência da doença ou da presença de anticorpos antiplasmodiais em gestantes nessa região não haviam sido descritos anteriormente. Embora infecções por P. falciparum em gestantes tenham sido amplamente abordadas na literatura, a interação entre P. vivax e P. malariae com esta coorte imunodeprimida foi pouco explorada até o momento. Nesse estudo nós monitoramos trimestralmente a circulação de Plasmodium em gestantes atendidas em cinco Unidades de Saúde de Juquitiba. Para isso foi empregado o diagnóstico por gota espessa e metodologias moleculares sensíveis para detecção do parasito, além de ensaios imunológicos para avaliação de parâmetros imunes humorais. Desse modo, foram detectadas infecções por P. vivax e P. malariae em gestantes, incluindo casos assintomáticos. A alta prevalência de anticorpos IgG nesta população mostrou importante exposição das gestantes ao Plasmodium. Em regiões com perfil semelhante ao apresentado neste estudo, o diagnóstico de malária poderia ser indicado no seguimento pré-natal / Studies related to autochthonous malaria in low transmission areas in Brazil have acquired scientific and epidemiological relevance, since they suggest continued transmission in remaining Atlantic Forest regions. In the Southeast of São Paulo State, outbreaks in the municipality of Juquitiba has been focus of studies on the prevalence of Plasmodium in the population, with reports of asymptomatic cases. Data on the occurrence of the disease or presence of antiplasmodial antibodies in pregnant women in this region were not described previously. Although P. falciparum infections in pregnant women have been widely addressed in the literature, the interaction of P. vivax and P. malariae with this immunocompromised cohort were poorly explored to date. In this study, we monitored quarterly the circulation of Plasmodium in pregnant women in five health facilities in Juquitiba. For this purpose, we performed diagnosis by thick blood film and sensitive molecular protocols for parasite DNA detection, as well immunological assays in order to evaluate humoral immune parameters. Through these tools, it was possible to detect infections due to P. vivax and P. malariae in pregnant women, including asymptomatic cases. The high prevalence of IgG antibodies showed a significant exposure of this population to Plasmodium. In regions with a similar profile presented in this study, the diagnosis of malaria might be indicated in prenatal care

Os diversos aspectos da cadeia de transmissão da malária autóctone são importantes para o estabelecimento de estratégias de intervenção. Entre abril de 2001 e março de 2004, 65 pacientes e 1.777 habitantes foram avaliados em nove municípios da região montanhosa do Espírito Santo. Foram realizados: gota espessa, esfregaço fino, PCR Multiplex, reação de imunofluorescência indireta (IFI) para detecção de anticorpos contra antígenos de estágios eritrocitários de Plasmodium e ELISA para detecção de anticorpos contra peptídeos sintetizados a partir da porção repetitiva da proteína circunsporozoíta (CSP) das variantes de P. vivax e do P. malariae. Foram capturados anofelíneos no peridomicílio, com pesquisa, por PCR Multiplex, de DNA de Plasmodium. O mesmo foi pesquisado também em alguns símios locais. Os pacientes tinham 35,11 + 16 anos, em média. A maioria era do gênero masculino (51 ou 78,5%), 42 (64,6%) residiam em área rural, 23 (35,4%) eram agricultores e oito (12,3%) estudantes. Não houve viagens relevantes. Sessenta e dois (95,4%) nunca haviam tido malária. Vinte e quatro (36,9%) declararam ter entrado na mata. Predominaram a febre, a cefaléia e os calafrios. A febre era episódica em 63 (96,9%), a cada 48 horas em 48 (73,8%) e a cada 24 horas em 15 pacientes (23,1%). O baço foi impalpável em 26 (42,6%). Foi evidenciado o P. vivax em 47 de 48 pacientes e o P. malariae naquele restante, por características morfológicas e pela PCR Multiplex. Esta foi positiva para P. vivax em 45 dos 48, para P. malariae em um e negativa em dois. A IFI foi positiva, para P. malariae, em seis de sete testados, para IgM, e em todos os sete para IgG. Para o P. vivax, entre 50, 47 (94%) foram positivos para IgM e 48 (96%) para IgG. Entre 50 pacientes, pelo ELISA, 25 (50%) tinham anticorpos contra variantes do P. vivax ou contra o P. malariae. As freqüências individuais foram: 22 (44%) para a VK 210, 11 (22%) para a VK 247, 10 (20%) para o P. vivax-like e 10 (20%) para o P. malariae. Entre 253 amostras dos habitantes testadas na IFI para o P. malariae, o resultado foi positivo em 15,8% (40/253) para IgM e em 44,6% (113/253) para IgG. Para o P. vivax , em 1.701, foram 6,2% (105/1701) para IgM e 37,7% (641/1.701) para IgG. Foram detectados anticorpos contra a CSP em 615 de 1.702 amostras (36,1%). Foram 433 (25,4%) para a VK210, 258 (15,1%) para P. malariae, 108 (6,3%) para a VK 247 e 182 (10,7%) para P. vivax -like. A PCR Multiplex, em 1.527 amostras, detectou P. vivax em 23, P. malariae em 15, P. falciparum em nove e P. falciparum e P. malariae em um. Entre 785 espécimes de anofelíneos, com 10 espécies, foi encontrado DNA de P. vivax em um conjunto de exemplares de A. evansae. O P. malariae/brasilianum foi identificado pela PCR Multiplex em dois de cinco símios da região, em um também pelo esfregaço fino. Existem dois possíveis cenários para a transmissão. No primeiro, ela seria inter-humana, com vetores Nyssorhynchus secundários. Em um segundo, viria do reservatório símio, por indivíduos adentrando o ambiente florestal. / The several aspects of the transmission cycle of the indigenous malaria are important to base on the intervention strategies. From April 2001 to March 2004, 65 patients and 1,777 inhabitants were evaluated in nine Municipalities of the highlands of Espírito Santo State. Laboratory methods included: thick and thin smears, Multiplex PCR, imunnofluorescent assay to detect antibodies against crude blood-stages antigens of the Plasmodium genus (IFA) and ELISA to detect antibodies against synthetic peptides corresponding to the repetitive region of the Circumsporozoite protein of P. vivax variants and P. malariae. Anopheline mosquitoes were captured nearby the houses, being screened by Multiplex PCR in the search for Plasmodium DNA. The same test was also applied to some local wild monkeys. Patients had 35.11 + 16 years old in average. Most of them were males (51 or 78.5%), 42 (64,6%) lived in the rural environment, 23 (35.4%) were farmers and eight (12.3%) were students. There was no relevant history of travel. Sixty-two (95.4%) of them had never experienced malaria before. Twenty- four (36.9%) of them informed excursions inside the forest. The predominant symptoms were fever, headache and chills. Fever was periodic in 63 patients (96.9%), recurring each 48 hours in 48 of them (73.8%) and each 24 hours in 15 (23.1%). Spleen was not palpable in 26 patients (42.6%). Morphologic aspects and PCR results disclosed P. vivax as the agent involved in 47 of the 48 cases so screened. Multiplex PCR was positive for P. vivax in 45 of 48 tested, for P. malariae in another one and negative for the two remaining. IFA tested positive for IgM against P. malariae in six of seven evaluated samples, and for IgG against the same parasite in all of the seven. For P. vivax , the figures were 47 of 50 (94%) for IgM antibodies and 48 of 50 (96%) for IgG antibodies. From fifty patients whose samples were screened by ELISA, 25 (50%) were positive for P. vivax variants or P. malariae. The results considering each one of the tested peptides were: 22 (44%) for VK 210, 11 (22%) for VK 247, 10 (20%) for P. vivax -like e 10 (20%) for P. malariae. Among 253 population samples screened in search for P. malariae antibodies at IFA, 40 (15.8%) were positive for IgM antibodies and 113 (44,6%) for IgG antibodies. The search for P. vivax antibodies by the same technique in1,701 samples, resulted in 105 (6.2%) positive for IgM antibodies and in 641 positive for IgG antibodies. Anti-CSP antibodies were detected in 615 of 1,702 tested samples (36.1%). Among these 615, the positive results for each one of the tested peptides were: 433 (25,4%) for VK210, 258 (15,1%) for P. malariae, 108 (6,3%) for VK 247 e 182 (10,7%) for P. vivax-like. Multiplex PCR detected P. vivax DNA in 23 out of 1,527 tested samples, as it did for P. malariae in 15 of them, for P. falciparum in nine of them and both for P. malariae and P. falciparum in one of them. Among 785 mosquito specimens, representing 10 Anopheline species, P. vivax DNA was found in a set of some A. evansae specimens. P. malariae/brasilianum was identified by Multiplex PCR in two of five wild monkeys screened, in one of them also by thin smear. There are two possible scenarios to explain this transmission cycle. The first one bears malaria as a disease transmitted exclusively among human beings by secondary Nyssorhynchus vectors present nearby the houses. In a second scenario, the malaria is acquired after the simian reservoir when the human beings make excursions inside the forest.

Thesis advisor: Marc Muskavitch / The ubiquitin-proteasome system (UPS), composed of classes of proteins central to the process of cellular protein turnover in eukaryotes, is essential to the life cycle of the malaria parasite, Plasmodium falciparum. Although the UPS has been well characterized in other organisms, the extent of its involvement in different stages of P. falciparum growth and development has not been investigated in depth. MG132, a small-molecule proteasome inhibitor known to target the 20S proteasome core (part of the catalytic center for selective protein degradation), has been used successfully in many research studies that require proteasome inhibition. We present data supportive of the conclusion that MG132 is highly effective as a tool for P. falciparum research. In this thesis, I describe the effects of partial and complete proteasome inhibition on parasite growth and development by the use of variable concentrations of MG132. I also assess the effects of MG132 on 20S P. falciparum proteasome enzymatic activities. I have generated parasite lines that exhibit tolerance, or low-level resistance, to MG132, through intermittent compound exposure. Sequencing of the catalytic β-5 subunit of the MG132-tolerant parasites reveals non-synonymous point mutations in three tolerant parasite lines. The use of MG132 as a tool compound for study of the UPS in P. falciparum facilitates research into detailed roles of the proteasome using reversible partial and complete inhibition. MG132-tolerant lines are also valuable tools for studying the genesis of different levels of drug resistance and cross-resistance in parasite evolution. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Neste trabalho, usamos um modelo murino de recrudescência da malária associada à gravidez, com camundongos BALB/c infectados por Plasmodium berghei ANKAGFP com o objetivo de avaliar a resposta imune associada ao recrudescimento da infecção. Observando as populações de células do baço por citometria de fluxo notou-se um aumento no percentual de células CD4+, CD19+, TCR <font face=\"Symbol\">g<font face=\"Symbol\">d+, GR1+ nos animais infectados, em relação aos controles não infectados. Não foram observadas diferenças no percentual de leucócitos placentários. Além disso, nossos resultados mostraram que no baço, linfócitos TCD4+ e TCD8+ estão mais ativados em fêmeas recrudescentes, enquanto que na placenta, estes animais apresentaram um aumento na ativação de células TCD4+. A relação IgG2a/IgG1 em animais ecrudescentes e não recrudescentes indicou um aumento de 3 vezes na produção de IgG2a que indica um padrão Th1 de resposta imune. A análise de qRT-PCR concomitantemente à citometria de fluxo evidenciou que células dendríticas são os reservatórios do parasita durante o estágio crônico da doença, que antecede o recrudescimento. Juntos, esses dados sugerem que a recrudescência durante a gestação está diretamente ligada ao dano tecidual, provavelmente relacionado a uma exacerbada resposta Th1 na placenta. / In this work, we used a murine model of recrudescence of malaria associated with pregnancy, consisting of BALB / c mice infected with Plasmodium berghei ANKAGFP to evaluate the immune response associated with the recrudescence of the disease. Observing the spleen cell populations by flow cytometry, an increase in the percentage of CD4+, CD19+, TCR <font face=\"Symbol\">g<font face=\"Symbol\">d+, GR1+ cells was observed in the infected animals, compared to the uninfected controls. No differences were observed in the percentage of placental leukocytes. Furthermore, our results showed that in the spleen, CD4+ and CD8+ cells were in a more active state in recrudescent females, while in the placenta, these animals exhibited an increase in CD4+ T cell activation. The ratio IgG2a/IgG1 in recrudescent vs non-recrudescent animals showed a 3-fold increase in the production of IgG2a indicating a Th1 immune response. qRT-PCR analysis, together with flow cytometry, showed that dendritic cells were the reservoirs of the parasite during the chronic stage of the disease, preceding the recrudescence. Together, these data suggest that recrudescence during pregnancy is directly linked to tissue damage, probably related to an exacerbated Th1 response in the placenta.

Severe malaria caused by Plasmodium falciparum (P. falciparum) remains a leading cause of death in many low and middle income countries. The intraerythrocytic reproduction cycle of the parasite is responsible for all the symptoms and mortality of malaria. The merozoite, first invade a red blood cell (RBC) in the circulation, then grows, develops and multiplies within it by clonal division. Merozoite invasion is a complex process involving dynamic interactions between ligands in the merozoite coat and receptors on the red blood cell membrane. Therefore, filming the complete malaria invasion processes may shed the light on its mechanism. The rationale of this work is that learning how the various ligand-receptor interactions affect invasion phenotypes will lead us to a better understanding of the key biological and biophysical aspects of parasite growth in the blood. The work described has firstly involved the development of an optimised imaging platform for recording egress-invasion sequences. I used live cell microscopy to understand this stage of malarial infection better, by monitoring egress-invasion sequences in live cultures under controlled conditions and addressing the morphology and kinetics of erythrocyte invasion by P. falciparum. In addition, the erythrocyte invasion phenotypes of the various P. falciparum strains were systematically investigated for the first time by live cell microscopy. Furthermore, to better understand genetic recombination affecting erythrocyte invasion phenotypes, progeny from the 7G8 x GB4 cross was compared to their parents. In order to investigate specific receptor-ligand interactions and their distinct functional characterisations at each distinct stage, the enzymes that cleave receptors on the erythrocytes and antibodies targeting ligands on the merozoites were studied and their effects observed using the live-imaging platform. In the results, the functions of ligands on the merozoites demonstrated for the first time distinct and sequential functions of proteins during erythrocyte invasion, which could potentially guide the design of more effective malaria vaccines. In addition, I have designed microfluidic devices for studying blood stage malaria. Polydimethylsiloxane (PDMS) microfluidic devices are optically transparent, non-toxic and have biocompatible features. Building on previous work, I made specific microfluidic devices for achieving a high throughput of egress-invasion observations. Infected red blood cells were delivered into a microfluidic device channel containing cage-like "nests". The nests were designed to selectively trap these stiff, egress-ready cells, in order to obtain streams of merozoites on maturation. Uninfected RBCs were delivered from another input into a long serpentine channel co-flowing with the egressed merozoites. The results indicated that, during P. falciparum erythrocyte invasion under flow conditions, the morphological effect on erythrocytes and the kinetic properties show significant differences to those in static conditions. In addition, with optimised flow rates, it is possible to reach higher throughput of egress-invasion observations than static conditions. Both the static and flow experiments carried out in this study highlight important mechanisms and processes of malaria invasion, and represent new ways of studying blood stage malaria. Precise and high throughout recording of single-event host-pathogen interaction events will allow us to address a new area of fundamental biological questions in future work.

Orientador: Jayme Augusto de Souza Neto / Resumo: A malária é uma doença ocasionada por protozoários do gênero Plasmodium e transmitida ao homem por meio da picada de mosquitos do gênero Anopheles. No Brasil a maior parte dos casos da doença concentra-se na região Amazônica, onde grande parte das infecções é causada por Plasmodium vivax, e o A. darlingi é o principal vetor. Estudos recentes indicam que a microbiota e que o sistema imune dos mosquitos do gênero Anopheles, em particular, componentes do sistema complemento, possuem uma importante função na determinação da competência vetorial, podendo modular o desenvolvimento do parasita no mosquito. Apesar da importância epidemiológica de A. darlingi, pouco se sabe a cerca da composição de sua microbiota intestinal, e sobre as interações moleculares deste vetor com P. vivax. Neste cenário, temos como principais objetivos: 1) Avaliar o papel de LRIM1 (Leucine-rich repeat protein 1), um dos genes do complemento, na interface A. darlingi-P.vivax, e 2) Averiguar determinados aspectos da interação entre microbiota- A.darlingi- P.vivax. Quanto às análises de LRIM1, as topologias das árvores filogenéticas mostram maior relação filogenética entre A. darlingi e A. albimanus, apresentando-os como táxons irmãos. Para a realização dos ensaios funcionais, sintetizamos RNA dupla-fita com base na sequência codificadora de LRIM1 de A. darlingi e microinjetamos no tórax de fêmeas desta espécie, as quais foram posteriormente submetidas a uma infecção por P. vivax. Quanto aos ensaios funcionais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Malaria is a disease caused by protozoa of the genus Plasmodium and transmitted to humans through the bite of mosquitoes of the genus Anopheles. In Brazil, most cases of the disease are concentrated in the Amazon region, where most of the infections are caused by Plasmodium vivax, and A. darlingi is the main vector. Recent studies indicate that the microbiota and the immune system of Anopheles mosquitoes, in particular components of the complement system, play an important role in the determination of vector competence and can modulate the development of the parasite in the mosquito. Despite the epidemiological importance of A. darlingi, little is known about the composition of its intestinal microbiota, and about the molecular interactions of this vector with P. vivax. In this scenario, we have as main objectives: 1) To evaluate the role of LRIM1 (Leucine-rich repeat protein 1), one of the complement genes, in the interface A. darlingi-P.vivax, And 2) To ascertain some aspects of the interaction between microbiota- A.darlingi- P. vivax. As for the LRIM1 analyzes, the topologies of the phylogenetic trees show a higher phylogenetic relationship between A. darlingi and A. albimanus, presenting them as sister taxa. To perform the functional assays, we synthesized double-stranded RNA based on the coding sequence of A. darlingi LRIM1 and microinjections in the thorax of females of this species, which were subsequently subjected to a P. vivax infection. Regarding the functional tes... (Complete abstract click electronic access below) / Mestre

Estudos relacionados à malária autóctone em regiões de baixa transmissão no Brasil ganham cada vez mais relevância científica e epidemiológica, pois revelam a manutenção desse cenário em regiões de Mata Atlântica remanescente. No sudeste do Estado de São Paulo, a ocorrência de surtos no município de Juquitiba tem sido foco de pesquisas sobre a prevalência de Plasmodium na população, com registros de casos assintomáticos. Relatos de ocorrência da doença ou da presença de anticorpos antiplasmodiais em gestantes nessa região não haviam sido descritos anteriormente. Embora infecções por P. falciparum em gestantes tenham sido amplamente abordadas na literatura, a interação entre P. vivax e P. malariae com esta coorte imunodeprimida foi pouco explorada até o momento. Nesse estudo nós monitoramos trimestralmente a circulação de Plasmodium em gestantes atendidas em cinco Unidades de Saúde de Juquitiba. Para isso foi empregado o diagnóstico por gota espessa e metodologias moleculares sensíveis para detecção do parasito, além de ensaios imunológicos para avaliação de parâmetros imunes humorais. Desse modo, foram detectadas infecções por P. vivax e P. malariae em gestantes, incluindo casos assintomáticos. A alta prevalência de anticorpos IgG nesta população mostrou importante exposição das gestantes ao Plasmodium. Em regiões com perfil semelhante ao apresentado neste estudo, o diagnóstico de malária poderia ser indicado no seguimento pré-natal / Studies related to autochthonous malaria in low transmission areas in Brazil have acquired scientific and epidemiological relevance, since they suggest continued transmission in remaining Atlantic Forest regions. In the Southeast of São Paulo State, outbreaks in the municipality of Juquitiba has been focus of studies on the prevalence of Plasmodium in the population, with reports of asymptomatic cases. Data on the occurrence of the disease or presence of antiplasmodial antibodies in pregnant women in this region were not described previously. Although P. falciparum infections in pregnant women have been widely addressed in the literature, the interaction of P. vivax and P. malariae with this immunocompromised cohort were poorly explored to date. In this study, we monitored quarterly the circulation of Plasmodium in pregnant women in five health facilities in Juquitiba. For this purpose, we performed diagnosis by thick blood film and sensitive molecular protocols for parasite DNA detection, as well immunological assays in order to evaluate humoral immune parameters. Through these tools, it was possible to detect infections due to P. vivax and P. malariae in pregnant women, including asymptomatic cases. The high prevalence of IgG antibodies showed a significant exposure of this population to Plasmodium. In regions with a similar profile presented in this study, the diagnosis of malaria might be indicated in prenatal care

Os diversos aspectos da cadeia de transmissão da malária autóctone são importantes para o estabelecimento de estratégias de intervenção. Entre abril de 2001 e março de 2004, 65 pacientes e 1.777 habitantes foram avaliados em nove municípios da região montanhosa do Espírito Santo. Foram realizados: gota espessa, esfregaço fino, PCR Multiplex, reação de imunofluorescência indireta (IFI) para detecção de anticorpos contra antígenos de estágios eritrocitários de Plasmodium e ELISA para detecção de anticorpos contra peptídeos sintetizados a partir da porção repetitiva da proteína circunsporozoíta (CSP) das variantes de P. vivax e do P. malariae. Foram capturados anofelíneos no peridomicílio, com pesquisa, por PCR Multiplex, de DNA de Plasmodium. O mesmo foi pesquisado também em alguns símios locais. Os pacientes tinham 35,11 + 16 anos, em média. A maioria era do gênero masculino (51 ou 78,5%), 42 (64,6%) residiam em área rural, 23 (35,4%) eram agricultores e oito (12,3%) estudantes. Não houve viagens relevantes. Sessenta e dois (95,4%) nunca haviam tido malária. Vinte e quatro (36,9%) declararam ter entrado na mata. Predominaram a febre, a cefaléia e os calafrios. A febre era episódica em 63 (96,9%), a cada 48 horas em 48 (73,8%) e a cada 24 horas em 15 pacientes (23,1%). O baço foi impalpável em 26 (42,6%). Foi evidenciado o P. vivax em 47 de 48 pacientes e o P. malariae naquele restante, por características morfológicas e pela PCR Multiplex. Esta foi positiva para P. vivax em 45 dos 48, para P. malariae em um e negativa em dois. A IFI foi positiva, para P. malariae, em seis de sete testados, para IgM, e em todos os sete para IgG. Para o P. vivax, entre 50, 47 (94%) foram positivos para IgM e 48 (96%) para IgG. Entre 50 pacientes, pelo ELISA, 25 (50%) tinham anticorpos contra variantes do P. vivax ou contra o P. malariae. As freqüências individuais foram: 22 (44%) para a VK 210, 11 (22%) para a VK 247, 10 (20%) para o P. vivax-like e 10 (20%) para o P. malariae. Entre 253 amostras dos habitantes testadas na IFI para o P. malariae, o resultado foi positivo em 15,8% (40/253) para IgM e em 44,6% (113/253) para IgG. Para o P. vivax , em 1.701, foram 6,2% (105/1701) para IgM e 37,7% (641/1.701) para IgG. Foram detectados anticorpos contra a CSP em 615 de 1.702 amostras (36,1%). Foram 433 (25,4%) para a VK210, 258 (15,1%) para P. malariae, 108 (6,3%) para a VK 247 e 182 (10,7%) para P. vivax -like. A PCR Multiplex, em 1.527 amostras, detectou P. vivax em 23, P. malariae em 15, P. falciparum em nove e P. falciparum e P. malariae em um. Entre 785 espécimes de anofelíneos, com 10 espécies, foi encontrado DNA de P. vivax em um conjunto de exemplares de A. evansae. O P. malariae/brasilianum foi identificado pela PCR Multiplex em dois de cinco símios da região, em um também pelo esfregaço fino. Existem dois possíveis cenários para a transmissão. No primeiro, ela seria inter-humana, com vetores Nyssorhynchus secundários. Em um segundo, viria do reservatório símio, por indivíduos adentrando o ambiente florestal. / The several aspects of the transmission cycle of the indigenous malaria are important to base on the intervention strategies. From April 2001 to March 2004, 65 patients and 1,777 inhabitants were evaluated in nine Municipalities of the highlands of Espírito Santo State. Laboratory methods included: thick and thin smears, Multiplex PCR, imunnofluorescent assay to detect antibodies against crude blood-stages antigens of the Plasmodium genus (IFA) and ELISA to detect antibodies against synthetic peptides corresponding to the repetitive region of the Circumsporozoite protein of P. vivax variants and P. malariae. Anopheline mosquitoes were captured nearby the houses, being screened by Multiplex PCR in the search for Plasmodium DNA. The same test was also applied to some local wild monkeys. Patients had 35.11 + 16 years old in average. Most of them were males (51 or 78.5%), 42 (64,6%) lived in the rural environment, 23 (35.4%) were farmers and eight (12.3%) were students. There was no relevant history of travel. Sixty-two (95.4%) of them had never experienced malaria before. Twenty- four (36.9%) of them informed excursions inside the forest. The predominant symptoms were fever, headache and chills. Fever was periodic in 63 patients (96.9%), recurring each 48 hours in 48 of them (73.8%) and each 24 hours in 15 (23.1%). Spleen was not palpable in 26 patients (42.6%). Morphologic aspects and PCR results disclosed P. vivax as the agent involved in 47 of the 48 cases so screened. Multiplex PCR was positive for P. vivax in 45 of 48 tested, for P. malariae in another one and negative for the two remaining. IFA tested positive for IgM against P. malariae in six of seven evaluated samples, and for IgG against the same parasite in all of the seven. For P. vivax , the figures were 47 of 50 (94%) for IgM antibodies and 48 of 50 (96%) for IgG antibodies. From fifty patients whose samples were screened by ELISA, 25 (50%) were positive for P. vivax variants or P. malariae. The results considering each one of the tested peptides were: 22 (44%) for VK 210, 11 (22%) for VK 247, 10 (20%) for P. vivax -like e 10 (20%) for P. malariae. Among 253 population samples screened in search for P. malariae antibodies at IFA, 40 (15.8%) were positive for IgM antibodies and 113 (44,6%) for IgG antibodies. The search for P. vivax antibodies by the same technique in1,701 samples, resulted in 105 (6.2%) positive for IgM antibodies and in 641 positive for IgG antibodies. Anti-CSP antibodies were detected in 615 of 1,702 tested samples (36.1%). Among these 615, the positive results for each one of the tested peptides were: 433 (25,4%) for VK210, 258 (15,1%) for P. malariae, 108 (6,3%) for VK 247 e 182 (10,7%) for P. vivax-like. Multiplex PCR detected P. vivax DNA in 23 out of 1,527 tested samples, as it did for P. malariae in 15 of them, for P. falciparum in nine of them and both for P. malariae and P. falciparum in one of them. Among 785 mosquito specimens, representing 10 Anopheline species, P. vivax DNA was found in a set of some A. evansae specimens. P. malariae/brasilianum was identified by Multiplex PCR in two of five wild monkeys screened, in one of them also by thin smear. There are two possible scenarios to explain this transmission cycle. The first one bears malaria as a disease transmitted exclusively among human beings by secondary Nyssorhynchus vectors present nearby the houses. In a second scenario, the malaria is acquired after the simian reservoir when the human beings make excursions inside the forest.

Made available in DSpace on 2015-04-22T22:01:55Z (GMT). No. of bitstreams: 1 Andreia Montoia.pdf: 3824551 bytes, checksum: 75e621941cc5f0f5383f36e0472f3ec2 (MD5) Previous issue date: 2013-03-08 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The growing number of cases of resistance to the common antimalarials chloroquine and the ACTs (artemisinin-based combined therapy) favors the search for new substances with antiplasmodial activity. In 2007, the Amazonian Active Principles Laboratory (LAPAAM) at the National Institute for Amazon Research (INPA) discovered the in vitro antimalarial activity of the indole alkaloid ellipticine (10) against Plasmodium falciparum. A bibliographic search revealed that many indole alkaloids of relatively simple structure exhibited in vitro and in vivo antimalarial activity. In the present study, substances that are structurally related to 10 were obtained by isolation or semi-synthesis and their in vitro antimalarial activity was investigated. 10 and 2-methyl-1, 2, 3 ,4-tetrahydroellipticine (12) were isolated from the alkaline extracts of the bark of carapanaúba (Aspidosperma vargasii, Apocynaceae) by column chromatography. UPLC-MS analysis revealed the presence of 10 and 12 in an infusion of A. vargasii bark. Nitration (HNO3/AcOH) and bromination (Br2/CHCl3) reactions using 10 as starting material formed electrophilic aromatic substitution products 7-nitroellipticine (20, new substance) and a 3:1 mixture of 7,9-dibromoellipticine (17, a new substance) and 9-bromoellipticine (18), respectively. All substances inhibited the K1 strain of P. falciparum in vitro as evidenced by IC50 values 0,19 (10), 1,10 (12), 0,43 (20) and 0,30 (17+18) μg/mL. Compounds 10, 12, 17, 18 and 20 were not cytotoxic to human fibroblasts (IC50 > 50 μg/mL). 10 administered by mouth was highly active in the 4 day suppressive test against Plasmodium berghei in mice and inhibited parasitemia by 92% on the 5th day at a dose of 10 mg/kg/day. In the future, 17, 18 and 20 will be prepared in larger quantity and evaluated for in vivo antimalarial activity. / O aumento de casos de resistência aos antimaláricos comuns cloroquina e TCA (Tratamentos Combinados baseados na Artemisinina) favorece a busca por novas substâncias com atividade antiplasmodial. Em 2007, o Laboratório de Princípios Ativos da Amazônia (LAPAAM) do Instituto Nacional de Pesquisa da Amazônia (INPA) descobriu a atividade antimalárica in vitro contra Plasmodium falciparum do alcaloide indólico elipticina (10). Um levantamento bibliográfico revelou que diversos alcaloides indólicos de estruturas relativamente simples possuem atividade antimalárica in vitro e in vivo. No presente estudo, substâncias relacionadas estruturalmente a 10 foram obtidas por isolamento ou semi-sintése e sua atividade antimalárica in vitro foi investigada. 10 e 2-metil-1, 2, 3, 4-tetraidroelipticina (12) foram isoladas a partir dos extratos alcalinos das cascas de carapanaúba (Aspidosperma vargasii, Apocynaceae) por cromatografia em coluna. Análise por UPLC-ESI-MS revelou a presença de 10 e 12 em uma infusão das cascas de A. vargasii. As reações de nitração (HNO3/HOAc) e bromação (Br2/CHCl3) utilizando 10 como material de partida levou à formação dos produtos de substituição eletrofílica aromática 7-nitroelipticina (20, substância inédita) e uma mistura 3:1 de 7,9- dibromoelipticina (17, substância inédita) e 9-bromoelipticina (18), respectivamente. Todas as substâncias inibiram a cepa K1 de P. falciparum in vitro apresentando valores de IC50 de 0,19 (10); 1,10 (12); 0,43 (20) e 0,30 (17+18) μg/mL. As substâncias 10, 12, 17, 18 e 20 não exibiram toxicidade para fibroblastos humanos (IC50 > 50 μg/mL). 10 via oral exibiu elevada atividade in vivo no teste de supressão de 4 dias contra P. berghei em camundongos e inibiu em 92 % a parasitemia no quinto dia na dose de 10 mg/kg/dia. Futuramente, 17, 18 e 20 deverão ser preparados em maior quantidade e avaliados para atividade antimalárica in vivo.