Les phosphoantigènes (P-Ag) de P. falciparum (P.f.) induisent une forte activation et une expansion des lymphocytes T (LT) Vγ9Vδ2 par un mécanisme encore mal décrit. Les LT Vγ9Vδ2 actives inhibent le cycle sanguin de P. f. par des médiateurs cytotoxiques solubles, inhibant ainsi la capacité invasive des mérozoites. Il a été montre in vitro que des LT Vγ9Vδ2 activés par les P-Ag peuvent présenter des antigènes et activer les LT αβ, agissant ainsi comme des cellules présentatrices d’antigènes (APC). Cette fonction n’a cependant pas été démontrée dans un contexte physiopathologique. Le but de ce projet est i) d’étudier les mécanismes d’activation des LT Vγ9Vδ2 par les stades sanguins P. f. et ii) d’evaluer le potentiel APC des LT Vγ9Vδ2 stimules par P. f. Nous montrons que l’activation des LT Vγ9Vδ2 par des globules rouges parasites par P. f. (GRP) intacts ne dépend ni d’un contact cellulaire, ni de l’expression de butyrophiline par le GRP. Les LT Vγ9Vδ2 sont activés par des molécules contenues dans les surnageants de culture de GRP, ayant les caractéristiques de P-Ags et étant libérées lors de la rupture des GRP. In vitro, les LT Vγ9Vδ2 stimules par les GRP expriment des marqueurs de surface associés à un rôle d’APC et cross-présentent un antigène modèle à une lignée T CD8 spécifique. In vivo, nous montrons une expression augmentée des marqueurs APC à la surface de LT Vγ9Vδ2 de patients infectés par P. falciparum. L’ensemble de ces données suggèrent que les P-Ag libérés par les GRP dans le milieu extracellulaire pourraient activer les LT Vγ9Vδ2 à distance, et ouvrent de nouvelles perspectives quant au rôle des LT Vγ9Vδ2 dans la réponse immunitaire adaptative anti-palustre. / P. falciparum derived phosphoantigens (P‐Ag) induce potent activation and expansion of Vγ9Vδ2 T-cells by a poorly described mechanism. Activated Vγ9Vδ2 T cells inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. In vitro, P-Ag activated Vγ9Vδ2 T lymphocytes have been shown to present antigens and induce αβ T lymphocyte responses, i.e. to act as an antigen presenting cell (APC). Whether this activity can be involved in a pathophysiological context is unknown. The aim of this PhD project is to a) investigate the mechanisms of Vγ9Vδ2 T cell activation by blood stage P. falciparum and b) assess the potential of P. falciparum activated Vγ9Vδ2 T cells to display APC functionality. We show that Vγ9Vδ2 T-cell activation by intact iRBCs is independent of iRBC contact and butyrophilin expression. Blood stage culture supernatants can potently activate Vγ9Vδ2 T-cells and bioactivity is found to be attributable to P-Ags released at the time of parasite egress from the RBC. In vitro iRBC stimulated Vγ9Vδ2 T cells up-regulate surface expression of APC associated markers and can cross-present a model antigen to specific CD8 T cell responders. In vivo we demonstrate an increase in surface expression of APC makers on Vγ9Vδ2 T cells from P. falciparum infected patients.Altogether, these data outline a framework whereby P‐Ag release by iRBC into extracellular milieu can promote activation of distant Vγ9Vδ2 T cells, and opens the door to a new aspect of Vγ9Vδ2 T cell contribution to P. falciparum adaptive immune responses.

Os genes var de Plasmodium falciparum codificam a proteína PfEMP1 expressa na superfície de eritrócitos infectados e que medeia os fenômenos de citoaderência e \"rosetting\". Ambos os fenômenos estão diretamente associados à malária grave, e seu domínio mais N-terminal, DBL1alfa, media especificamente \"rosetting\". Análise de seqüências DBL1alfa de isolados brasileiros e de outros países revelou que a similaridade entre elas não pode predizer origem geográfica. Com o objetivo de determinar se existem seqüências DBL1alfa associadas à malária grave, analisamos as seqüências DBL1alfa expressas em parasitas obtidos de pacientes brasileiros com esta manifestação clínica e encontramos que as seqüências predominantemente expressas apresentavam uma ou duas deleções de cisteínas. Significativamente, apesar de freqüentes no genoma de parasitas de pacientes com malária não grave, essas seqüências foram raramente expressas. Esses dados demonstram a primeira associação de seqüências PfEMP1 expressas e malária grave em pacientes da Amazônia Brasileira. / Plasmodium falciparum var genes code for PfEMP1, a protein expressed on the surface of infected erythrocytes, and which mediates cytoadherence and rosetting. Both phenomena are directly associated with severe malaria and the most N-terminal domain, DBL1alfa, specifically mediates rosetting. DBL1alfa sequence analysis from Brazilian and worldwide isolates revealed that sequence similarities cannot predict geographical origin. To determine whether there are DBL1alfa sequences associated with severe malaria, we examined expressed var DBL1alfa sequences in patients with severe malaria from the Brazilian Amazon and found that the predominantly expressed DBL1alfa sequences from these parasites lacked 1-2 cysteine residues. Significantly, these sequences were amply found on the genomic repertoire of parasites from patients with mild malaria and yet they were rarely expressed. These data demonstrate the first association of particular PfEMP1 expressed sequences and severe malaria in patients from the Brazilian Amazon.

O desenvolvimento de uma vacina contra malária causada por P. falciparum é prejudicado pelo alto nível de polimorfismo apresentado pelos antígenos desse parasito. O dimorfismo alélico é um padrão no qual os alelos observados de um gene se encontram divididos em duas famílias. A proteína dimórfica MSP-6 se associa à proteína MSP-1 (também dimórfica) na superfície do merozoíto. Genes de msp-6 de 21 isolados obtidos de pacientes do Brasil, mais 2 isolados da Tanzânia, África, foram seqüenciados para estudo da diversidade nucleotídica e distribuição geográfica dos alelos. As duas famílias possuem distribuição global. Não foi verificada associação entre o dimorfismo de MSP-1 e MSP-6. O gene ortólogo de msp-6 em P. reichenowi, grupo irmão de P. falciparum, foi seqüenciado para estudos evolutivos. Os alelos dimórficos de MSP-6 aparentam ter surgido de uma população ancestral polimórfica, tendo sido mantidos no presente por seleção balanceada. O alto grau de conservação encontrado dentro de cada família alélica torna MSP-6 um potencial alvo de uma vacina contra a malária. / The development of a vaccine against malaria caused by Plasmodium falciparum has been hampered by the high level of antigen polymorphism exhibited by this parasite. Allelic dimorphism is a pattern in which every observed allele of a gene is clearly grouped into one of two families. The dimorphic protein MSP-6 forms a complex with MSP-1 (also dimorphic) on merozoite surface. The msp-6 genes were sequenced in isolates obtained from 21 patients from Brazil, plus 2 isolates from Tanzania, Africa, to study nucleotide diversity and geographic distribution of alleles. Both families are globally distributed. Moreover, no association was observed between the MSP-1 and MSP-6 allelic types. Orthologous gene of msp-6 in P. reichenowi, chimpanzee parasite and sister group of P. falciparum, was sequenced for evolutionary studies. Dimorphic alleles of MSP-6 seem to have originated from an ancestral polymorphic population and are maintained by balancing selection. The high degree of conservation observed within each allelic family makes MSP-6 an promising target for vaccine development.

Estudamos o papel de quinases de Plasmodium na modulação, via derivados de triptofano, no ciclo celular do parasita. Foram realizadas análises com melatonina, N-acetilserotonina, triptamina e serotonina, avaliadas através de microscopia e citometria de fluxo. Parasitas deficientes para as proteínas PfNEK2, PfNEK3 e PfMAP1 apresentaram respostas similares ao parasita selvagem enquanto pfpk7- e pfeik1- não apresentaram resposta aos compostos indólicos. Estes dados sugerem envolvimento das quinases PfPK7 e PfeIK1 na transdução de sinal via compostos derivados de triptofano na modulação do ciclo intraeritrocítico em P. falciparum. Ensaios de medidas de cálcio intracelular mostraram que a ausência de pfpk7- leva a uma atenuação na liberação de cálcio promovida por melatonina, sugerindo que a ativação desta quinase é importante no aumento de cálcio citosólico. Estes resultados ampliaram a compreensão dos mecanismos moleculares que regulam resposta a melatonina. / We evaluated the role of Plasmodium kinases in the modulation, via tryptophan derivatives, of the parasite cell cycle. We performed analysis with melatonin, N-acetylserotonin, tryptamine and serotonin through blood smears and flow cytometry analyzes. Parasites deficient in the proteins PfNEK2, PfNEK3 and PfMAP1 showed similar responses to the wild type while pfpk7- e pfeik1- did not respond to the indolic compounds. This data suggest the involvement of the kinases PfPK7 and PfeIK1 in melatonin signal transduction for cell cycle modulation in P.falciparum. Intracellular calcium measurement assays showed that pfpk7- diminished the ability of melatonin to rise cytosolic calcium concentration, suggesting that this kinase is involved with cytosolic calcium increase. These results expand the understanding of the molecular mechanisms that regulate melatonin-dependent cell cycle modulation.

A malária é causada por parasitas Plasmodium falciparum, e embora vários aspectos ainda sejam desconhecidos, é sabido que a regulação do ciclo intraeritrocítico é crítica para a compreensão do ciclo celular e patogênese. A melatonina modula o ciclo de P. falciparum promovendo a sincronização, mas, o mecanismo de transdução de sinal é parcialmente caracterizado, envolvendo variações citosólicas de cálcio, AMPc e ativação da PKA. Modificações pós-traducionais participam na via de sinalização, e diversas proteínas quinase podem estar envolvidas na sinalização por melatonina. eIF2α fosforilado é capaz de ativar a tradução de mRNAs em resposta a situações desfavoráveis. O genoma de P. falciparum codifica três quinases cujo substrato é eIF2α: PfeIK1, PfeIK2 e PfPK4. Investigamos o papel da PfeIK1 na via de transdução de sinal de melatonina usando cepas nocaute para PfeIK1. Além disso, os efeitos de metabólitos da degradação do heme sobre a fosforilação de eIF2α. Sugerimos que o mecanismo de fosforilação e defosforilação de eIF2α possam ser relevantes para a resposta do parasita a hemina ou biliverdina. Nossos dados indicam a PfeIK1, juntamente com a PfK7 e PKA, como quinases-chaves no controle do desenvolvimento durante o ciclo intraeritrocítico. / Malaria is caused by Plasmodium falciparum parasites, and although some aspects are still unknown, its established that the intraerythrocytic cycle regulation is critic for understanding the cell cycle and pathogenesis of the parasite. Melatonin modulates the cycle of P. falciparum promoting synchronization; however, the signal transduction mechanism is partially characterized, and it contains cytosolic variations of calcium, AMPc and PKA activation. Post-translational modifications participate in this signal pathway, and several kinase proteins may be involved in melatonin signaling pathway. Phosphorylated eIF2α is able to activate mRNAs translation in stress conditions. The genome of P. falciparum encodes three kinases whose substrate is eIF2α: PfeIK1, PfeIK2 e PfPK4. We investigate the role of PfeIK1 in melatonin signaling pathway by using knockout strains for PfeIK1. Furthermore, we investigate the effects of heme degradation metabolities in eIF2α phosphorylation. We suggest that the phosphorylation and dephosphorylation mechanisms of eIF2α may be relevant for parasite response to heme and billiverdin. Our data indicates PfeIK1, PfK7 and PKA as key kinases for the development control during intraerythrocytic cycle.

PhD (Biochemistry) / Department of Biochemistry / The main malaria agent, Plasmodium falciparum expresses an Hsp70 (PfHsp70-1) which plays a significant role in parasite survival. PfHsp70-1 is distinct in that it possesses glycine-glycine-methionine-proline (GGMP) tetrapeptide repeats in its C-terminal domain. To date, the GGMP motif of PfHsp70-1 has not been studied. The motif is positioned within the C-terminal lid segment of PfHsp70-1. The motif is also about seven residues upstream the terminal EEVD residues that are responsible for the interaction of PfHsp70-1 with its functional regulators (co-chaperones). P. falciparum Hsp70/Hsp90 organizing protein (PfHop) constitutes one of the functional regulators of PfHsp70-1. PfHop allows PfHsp70-1 and its chaperone partner, PfHsp90 to form a functional partnership. Given the proximity of the GGMP repeats to the C-terminus of PfHsp70-1, it was postulated in this study that the GGMP repeat residues may regulate attachment of PfHop to PfHsp70-1. Hence, this study hypothesized that the GGMP repeat motif is important for the interaction between PfHop and PfHsp70-1 as well as the chaperone activity of PfHsp70-1. Two variants in which the N-terminal and the C-terminal GGMP repeats were conservatively substituted were generated. E. coli Hsp70 (DnaK) lacks a GGMP motif. Thus, the GGMP motif of PfHsp70-1 was introduced into E. coli DnaK in order to generate a third GGMP variant. Recombinant forms of PfHsp70-1, DnaK, and their GGMP variants were heterologously expressed in E. coli XL1 Blue cells. The proteins were purified to homogeneity by using a combination of Ni-NTA affinity chromatography, ion exchange, and size exclusion chromatography. Purified proteins were then biophysically characterized using CD spectroscopy and tryptophan fluorescence. Findings from this study revealed that there were minimal secondary structural differences between PfHsp70-1, DnaK and their GGMP variants. In order to investigate the chaperone function of PfHsp70-1, DnaK and the GGMP variants, a complementation assay in E. coli dnak756 cells whose Hsp70 is functionally compromised was conducted. The PfHsp70-1 GGMP variants were able to suppress the thermosensitivity of the E. coli cells. However, the Investigation of the role of GGMP motif of Plasmodium falciparum Hsp70-1 on the chaperone function of the protein and its interaction with a co-chaperone, PfHop ii DnaK-G variant failed to confer cytoprotection to the E. coli dnak756 cells. To further validate the findings from the complementation assay, the ability of the recombinant proteins to suppress aggregation of heat stressed Malate dehydrogenase (MDH) was elucidated. PfHsp70-1 had better MDH aggregation suppression capabilities than its GGMP variants. Overall, findings from the MDH aggregation suppression assay suggest that the GGMP repeats may contribute towards substrate binding. Substrate binding might be dependent on the specific positioning of a particular repeat in the GGMP motif of PfHsp70-1. Furthermore, the ATPase activity of PfHsp70-G632 and PfHsp70-G648 was significantly reduced compared to PfHsp70-1 (wild type). However, PfHsp70-G632 had the lowest ATPase activity. Interestingly, the ATPase activity of PfHsp70-G632 was enhanced in the presence of synthetic Hsp70 model peptide substrates. Slot blot and ELISA approaches confirmed that the GGMP mutations partially abrogated the interaction of PfHsp70-1 with PfHop. Altogether, the findings suggest that the GGMP motif of PfHsp70-1 has marginal effects on the structure of PfHsp70-1. In conclusion, this study provides the first direct evidence that the GGMP motif is important for the chaperone function of PfHsp70-1 as well as its interaction with PfHop. / NRF

En Afrique tropicale, là où le paludisme est fortement endémique, la plupart des individus sont semi-immuns et les infections asymptomatiques sont très répandues. Ainsi la détection de parasites dans le sang de malades fébriles n'est pas un critère suffisant pour distinguer le paludisme des autres causes de fièvre. A Dielmo, un village du Sénégal d'environ 500 habitants en 2010, un suivi épidémiologique continu très étroit du paludisme a débuté en 1990. Dans ce village où la transmission est pérenne, la mise en place de moyens de lutte et de prévention contre le paludisme de plus en plus efficaces a profondément transformé l'épidémiologie du paludisme. Dans ce travail, nous analysons l'impact de ces interventions sur les prévalences parasitaires, les densités parasitaires et les critères diagnostiques du paludisme et nous mesurons l'évolution du paludisme clinique à Plasmodium falciparum, P. malariae et P. ovale de juin 1990 à décembre 2010. Les données parasitologiques et cliniques ont été analysées par régression logistique à effet aléatoire pour étudier la relation entre les densités parasitaires et le risque de fièvre. Les prévalences parasitaires des trois espèces plasmodiales ont considérablement diminué lors de l'abandon de la chloroquine en traitement de première ligne et de son remplacement par des combinaisons thérapeutiques, puis sont devenues presque nulles après la mise en place de moustiquaires imprégnées d'insecticides à longue durée d'action. Les seuils pyrogéniques calculés nous ont permis de mesurer la densité d'incidence des accès palustres et d'étudier l'impact des mesures de lutte sur la morbidité palustre dans la population. / In tropical Africa, where malaria is highly endemic, most people are semi-immune and asymptomatic infections are widespread. Thus, the detection of malaria parasites in the blood of febrile patients is not a sufficient criterion for distinguishing malaria from other causes of fever. In Dielmo, a Senegalese village of about 500 inhabitants in 2010, a very closely continuous epidemiological monitoring of malaria began in 1990. In this village where the transmission is perennial, the establishment of more effective means of control and prevention against malaria have profoundly changed the epidemiology of malaria. In this work, we analyze the impact of these interventions on the parasite prevalences, the parasite densities and the malaria diagnostic criteria and we measure the evolution of Plasmodium falciparum, P. malariae and P. ovale clinical malaria from June 1990 to December 2010 in Dielmo. Parasitological and clinical data are analyzed in a random effect logistic regression to investigate the relationship between parasite density and fever risk. The prevalence of the three Plasmodium species decreased dramatically with the abandonment of chloroquine as first line treatment and his replacing with the combination therapies and became almost zero after the introduction of long lasting insecticidal nets. Pyrogenic thresholds calculated enabled us to measure the incidence density of malaria and to study the impact of intervention methods on malaria morbidity in the population.

Bakgrund: Malaria är en av de allvarligaste infektionssjukdomarna i världen. De allvarligaste malariafallen orsakas främst av Plasmodium falciparum som sprids av Anopheles-myggor. Ett vaccin med långvarigt och potent skydd skulle kunna minska dödligheten, men också minska behovet av kontrollåtgärder och problemet med läkemedelsresistens. Subenhetsvaccin är den vaccintyp som kommit längst i kliniska studier men dessa uppvisar begränsad effekt. Helparasitvaccin ger en bredare immunitet vilket kan ge ett mer fullständigt skydd. Syfte: Syftet med denna litteraturstudie var att jämföra effekt och varaktighet i skydd mot P. falciparum hos de två P. falciparum sporozoit (PfSPZ)-helparasitvaccinkandidaterna: RAS (Radiation-attenuated sporozoites) och CPS (Chemoprophylaxis and sporozoites), samt att undersöka betydelsen av vaccindos och administreringssätt. Metod: Arbetet är en litteraturstudie baserat på 14 vetenskapliga studier vilka har erhållits via sökning i PubMed. De aspekter som avhandlas är: vaccineffekt och dess varaktighet, immunsvar och dess korrelation till vaccineffekt, betydelsen av dos och administreringssätt samt vaccinens säkerhetsprofil. Resultat: Litteraturstudien visade att RAS-vaccin och CPS-vaccin kan ge ett potent samt säkert kort- och långvarigt skydd mot homolog kontrollerad human malaria-infektion (CHMI) vid immunisering via myggor eller venös inokulation. Dosen har stor betydelse för vaccineffekten och CPS-vaccin kan uppnå potent skydd vid mycket lägre doser än RAS-vaccin. En del immunmekanismer har visat sig korrelera med skydd men CD8+ T-celler i levern verkar ha störst betydelse för långvarigt sterilt skydd. Det långvariga skyddet mot heterolog kontra homolog CHMI är bristfälligt för både RAS-vaccin och CPS-vaccin. Slutsats: En potent vaccineffekt uppnås med PfSPZ-vaccin mot homolog CHMI vid tillräckligt hög dos, men inte ett långvarigt skydd mot heterolog CHMI vilket begränsar användningen i endemiska områden. / Background: Malaria is still one of the most common infectious diseases in the world and there is an overwhelming threat to the development of resistance to different control methods such as drugs and insecticides. A durable vaccine with sterile protection would reduce and maybe eradicate the disease. The most serious cases of malaria are caused by Plasmodium falciparum that is transmitted through the bites of infected Anopheles mosquitoes. The life cycle of malaria is extremely complex and different vaccine candidates have effects at different stages. Naturally acquired immunity develops gradually after many years of clinical episodes but never becomes sterile. RTS,S is the only vaccine candidate who has been in phase III clinical trials. Unfortunately this vaccine has limited efficacy, like many other subunit vaccines, due to rapidly diminishing antibody titers. Whole parasite vaccines have the ability to generate a greater quantity and breadth of antigenic exposure within both the humoral and cellular immunity. This results in stronger immune response and can provide sterile protection. The development of whole parasite vaccines has mainly focused on the pre-erythrocytic stage and the most tested vaccine candidates that are in early clinical trial are radiation-attenuated sporozoites (RAS), chemoprophylaxis and sporozoites (CPS) and genetically attenuated parasites (GAP). Aim: The purpose of this literature study is to examine and compare the vaccine efficacy and durability towards P. falciparum of the two whole parasite vaccine candidates: RAS and CPS and to examine the importance of dose and different routes of administration. Methods: Fourteen different clinical studies were selected from PubMed to be included in this literature study. Different variables were selected for study: the vaccine efficacy and it´s durability after controlled human malaria infection (CHMI) using P. falciparum parasites homologous or heterologous to the vaccine strain, the correlation between the immunogenicity and protection, the importance of the dose and different kinds of administration and vaccine safety. Results: According to the findings in the literature study, direct venous inoculation of RAS-vaccine and CPS-vaccine have the ability to give short and longlasting protection against CHMI using P. falciparum parasites homologous to the vaccine strain. The dose is of great importance to the vaccine efficacy and CPS-vaccine has the ability to give potent protection with much lower doses than RAS-vaccine. Some immune mechanisms in the blood correlate with protection but it seems to be the number of CD8+ T-cells in the liver that are of greatest importance for longlasting and steril protection. Whole parasite vaccines are safe but transient parasitemia is common when using CPS-vaccine. Unfortunately, vaccines with longlasting protection against CHMI using P. falciparum parasites heterologous to the vaccine strain has limited efficacy. Conclusion: RAS-vaccine and CPS-vaccine have the ability to give a potent vaccine efficacy against CHMI using P. falciparum parasites homologous to the vaccine strain when used in sufficiently high doses. Longterm protection against CHMI using P. falciparum parasites heterologous to the vaccine strain is limited and this in turn affects the use in endemic areas. In the future, the vaccine effect can be improved by higher doses, more infectious vaccine strains or vaccine cocktails. An alternative to RAS-vaccine and CPS-vaccine could be direct venous inoculation of late arresting GAP.

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.

RNAs não codificantes (ncRNAs) têm ganho crescente prestígio nos últimos anos devido a recentes e contínuas descobertas revelando sua diversidade e importância. Porém, a identificação dessas moléculas ainda é um problema em aberto. Em particular, Plasmodium falciparum é um desafio para a pesquisa de ncRNAs, onde poucos foram identificados até o momento. P. falciparum é o parasita que causa uma malária humana letal. A descoberta de novos ncRNAs neste organismo pode auxiliar no desenvolvimento de novos tratamentos. Este trabalho faz um estudo sobre técnicas computacionais para a predição de ncRNAs e, utilizando como objeto de estudo P. falciparum, propõe uma metodologia de predição que seja aplicável inclusive a genomas com viés composicional. A ênfase deste estudo foi a predição de ncRNAs família-específicos, utilizando o componente RNA da telomerase como objeto de estudo. Este é um importante RNA que, devido à sua alta taxa de mutação, é de difícil identificação. Este RNA ainda não foi identificado em P. falciparum. No entanto, evidências biológicas indicam que este RNA é presente, funcional e deve ser essencial ao parasita, caracterizando-se como um alvo de drogas. Além disso, foi realizado um trabalho preliminar sobre a predição de ncRNAs em geral em P. falciparum utilizando uma abordagem comparativa. / Noncoding RNAs (ncRNAs) have been receiving increasing prestige in the last years due to recent and continuous discoveries revealing their diversity and importance. However, the identification of these molecules is still an open problem. In particular, Plasmodium falciparum is a challenge for the ncRNA research, in which few ncRNAs have been identified. P. falciparum is the parasite that causes a lethal human malaria. The discovery of new ncRNAs in this organism may help in the development of new treatments. This work does a research of computational techniques for the ncRNA prediction and, by using P. falciparum as target, proposes a prediction methodology which is also applicable to compositionally biased genomes. The emphasis of this study was the prediction of family-specific ncRNAs, by using the telomerase RNA component as target. This is an important RNA that has a high mutation rate, being difficult to predict. This RNA has not been identified in P. falciparum, yet. However, biological evidences indicate this RNA is present, functional and might be essential for the parasite, being a drug target. In addition, this work presents preliminary results about the prediction of general ncRNAs in P. falciparum by using a comparative approach.