Ce projet d’interface Chimie/Biologie repose sur les expertises complémentaires de deux équipes. Il concerne la conception et le développement d’inhibiteurs dirigés contre les Kinétoplastidés (trypanosomes, leishmanies). Il est en effet urgent de développer de nouvelles stratégies thérapeutiques pour répondre à la chimiorésistance et à la toxicité des médicaments actuellement utilisés contre ces parasites. Le métabolisme et le transport des polyamines étant essentiel chez les parasites, ils constituent des cibles thérapeutiques d’intérêt contre les Kinétoplastidés. Le projet intègre la synthèse de nouveaux dérivés polyamines spécifiques des parasites, l’évaluation sur des modèles in vitro de leishmaniose et de trypanosomose africaine, ainsi qu’une évaluation sur trypanothione réductase. La mise au point d’une méthode de quantification du transport de polyamine a également été initiée. Cinquante-quatre composés, répartis en trois séries chimiques, ont été synthétisés et évalués. Un grand nombre d’entre eux présentent des activités antiparasitaires de l’ordre du micromolaire et des évaluations in vivo sont actuellement en cours avec le composé le plus prometteur. / This project is at the interface of chemistry and biology and relies on the expertise of two different teams. This thesis involves the design and development of inhibitors directed against Kinetoplastids. It is urgent to develop new therapeutic strategies to respond to drug resistance and toxicity of currently used drugs against these parasites. Polyamine metabolism and transporter have been demonstrated as essential for parasite growth. Therefore, these systems are potential drug targets for development of antikinetoplastid compounds. We chose to synthesize polyamine derivatives and evaluate their biological activity against Kinetoplatids. Fifty-four compounds, divided into three chemical series, have been synthesized and evaluated. Many have shown a micromolar biological activity in vitro against parasite. In vivo evaluation is foreseen for the most promising derivative.

Le parasite Trypanosoma brucei est retrouvé en Afrique sub-Saharienne et est responsable de la maladie du sommeil chez l’homme et de la Nagana chez les animaux. Il cause de graves problèmes sanitaires et économiques car il affecte le bétail. La vaccination est impossible à cause de la variation antigénique. Les traitements actuels sont difficiles à mettre en place avec des effets secondaires importants. Il est donc urgent de trouver de nouvelles cibles thérapeutiques afin de développer de nouveaux médicaments. T. brucei possède un flagelle unique qui émerge de la cellule par une structure appelée la poche flagellaire (FP). Cette FP est une invagination de la membrane plasmique. Elle est nécessaire à la survie du parasite car c’est le seul site d’endo- et d’exocytose. Au cou de la FP on trouve le collier de la poche flagellaire (FPC) en forme d’anneau. Le FPC est composé de nombreuses protéines dont BILBO1 qui est nécessaire à la biogenèse de la FP et du FPC. De nombreux partenaires de BILBO1 ont été identifiés. Dans cette thèse, je caractérise deux d’entre eux : FPC5, une kinésine putative et FPC9, une synaptotagmine putative. J’ai pu montrer que FPC5 est localisée aux corps basaux mais aussi au FPC. Cette protéine n’est pas essentielle à la survie des parasites bien que des phénotypes de croissance et de ségrégation de la FP apparaissent après induction de l’ARNi. Nous ne sommes pas parvenus à prouver sa fonctionnalité, cependant j’ai pu montrer que son domaine moteur est capable de lier les microtubules. FPC9 est trouvée au niveau de la zone de transition du flagelle. L’ARNi contre cette protéine n’étant pas effectif, nous ne pouvons pas conclure quant à sa fonction dans la cellule. / Trypanosoma brucei is a parasite found in sub-Saharan Africa and is responsible for sleeping sickness in humans and Nagana in animals. It is the source of serious health and economic problems because it kills livestock. Vaccination is not possible because of antigenic variation and current treatments are difficult to implement or have toxic side effects. For these reasons it is urgent to find new therapeutic targets in order to develop effective treatments. T. brucei has a single copy flagellum that emerges from the cytoplasm through a unique structure called the Flagellar Pocket (FP). This pocket is an invagination of the pellicular membrane and because it is the sole site of endo- and exocytosis, it is essential for parasite survival. At the neck of the FP there is a cytoskeletal structure: the Flagellar Pocket Collar (FPC) that forms a “ring” around the flagellum. The FPC consists of numerous proteins, including the first to be identified - BILBO1, which is necessary for FP and FPC biogenesis. A number of potential BILBO1 partners were identified. In this thesis I characterize two of these proteins: FPC5, a putative kinesin and FPC9, a putative synaptotagmin. I show that FPC5 localizes mainly in the basal body area, but also at the FPC. This protein is not essential for parasite survival although reduced FP segregation and growth phenotypes appear after RNAi induction. We are not able to prove its functionality, however I could show its motor domain is able to bind microtubules. FPC9 is found in the transition zone of the flagellum. However RNAi knockdown against this protein was not efficient, so we are currently unable to define a function for this protein.

The flagellum of Trypanosoma brucei contains a family of antigenically related EF-hand calcium-binding proteins which are called the calflagins. Genomic Southern blots indicated that multiple copies of calflagin genes occur in T brucei. All of the copies were contained in a single 23 kb Xhol-Xhol fragment. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. Two new members of the calflagin family were found from genomic clone sequences. The deduced amino acid sequences of the genomic clones showed the calflagin genes were arranged tandemly along the genomic fragments and were similar to previously described calflagins. The calflagin genes were related by two unrelated 3' flanking sequences. An open reading frame that was unrelated to any calflagin was found at the 5' end of the 2.5 kb genomic fragment. Each encoded protein (~24,000u) contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. In general, variability among the T. brucei calflagins is greater than related proteins in T. lewisii and T. cruzi. This variability results from amino acid substitutions at the amino and carboxy termini, and duplication of internal segments.

No description available.

Tripanossomas de mamíferos constituem um dos grupos mais complexos da família Trypanosomatidae, abrangendo parasitas com ciclos de vida e estruturas populacionais heterogêneos. De acordo com a diversidade, filogenias baseadas em genes SSUrDNA e gGAPDH segregaram estes parasitas em 4 Clados principais: T. brucei, T. cruzi, T. theileri e T. lewisi. Catepsinas L e B (CATL e CATB), as principais atividades proteolíticas dos tripanossomas, participam não apenas na degradação de proteínas como também em eventos biológicos como diferenciação, invasão celular, virulência e evasão do sistema imune. Comparamos os perfis proteolíticos de enzimas CATL em tripanossomas patogênicos e não patogênicos e também isolamos e sequenciamos os domínios catalíticos dos genes CATL e CATB em diversas espécies dos principais clados. Os resultados provaram a utilidade destes marcadores no diagnóstico e genotipagem de T. cruzi, T. rangeli, T. theileri e T. congolense, assim como na construção de filogenias robustas da família Trypanosomatidae, congruentes com os marcadores tradicionais. / Trypanosomes of mammals comprise one of the most complex groups of the family Trypanosomatidae, including parasites with heterogeneous life cycles and population structures. According to such diversity, phylogenetic analyzes based on SSUrDNA and gGAPDH genes segregate these parasites in 4 major clades: T. brucei, T. cruzi, T. lewisi and T. theileri. Cathepsins L and B (CATL and CATB), the main proteolytic activities of trypanosomes, are not only involved in protein degradation but also in biological events such as cell differentiation, cell invasion, virulence, and evasion from the immune system. We comparatively analysed the CATL proteolytic profiles in pathogenic and non-pathogenic trypanosomes, and isolated and sequenced the catalytic domains of CATB and CATL genes in several species of the major clades. Our results demonstrated the usefulness of both markers in the diagnosis and genotyping of T. cruzi, T. rangeli, T. congolense and T. theileri as well as in the construction of robust phylogenies of the family Trypanosomatidae, congruent with traditional markers.

Tripanossomas de mamíferos constituem um dos grupos mais complexos da família Trypanosomatidae, abrangendo parasitas com ciclos de vida e estruturas populacionais heterogêneos. De acordo com a diversidade, filogenias baseadas em genes SSUrDNA e gGAPDH segregaram estes parasitas em 4 Clados principais: T. brucei, T. cruzi, T. theileri e T. lewisi. Catepsinas L e B (CATL e CATB), as principais atividades proteolíticas dos tripanossomas, participam não apenas na degradação de proteínas como também em eventos biológicos como diferenciação, invasão celular, virulência e evasão do sistema imune. Comparamos os perfis proteolíticos de enzimas CATL em tripanossomas patogênicos e não patogênicos e também isolamos e sequenciamos os domínios catalíticos dos genes CATL e CATB em diversas espécies dos principais clados. Os resultados provaram a utilidade destes marcadores no diagnóstico e genotipagem de T. cruzi, T. rangeli, T. theileri e T. congolense, assim como na construção de filogenias robustas da família Trypanosomatidae, congruentes com os marcadores tradicionais. / Trypanosomes of mammals comprise one of the most complex groups of the family Trypanosomatidae, including parasites with heterogeneous life cycles and population structures. According to such diversity, phylogenetic analyzes based on SSUrDNA and gGAPDH genes segregate these parasites in 4 major clades: T. brucei, T. cruzi, T. lewisi and T. theileri. Cathepsins L and B (CATL and CATB), the main proteolytic activities of trypanosomes, are not only involved in protein degradation but also in biological events such as cell differentiation, cell invasion, virulence, and evasion from the immune system. We comparatively analysed the CATL proteolytic profiles in pathogenic and non-pathogenic trypanosomes, and isolated and sequenced the catalytic domains of CATB and CATL genes in several species of the major clades. Our results demonstrated the usefulness of both markers in the diagnosis and genotyping of T. cruzi, T. rangeli, T. congolense and T. theileri as well as in the construction of robust phylogenies of the family Trypanosomatidae, congruent with traditional markers.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2016-07-07T12:13:56Z No. of bitstreams: 1 Tese_DIP_JoãoVictorLeiteDias.pdf: 6357155 bytes, checksum: 49e964fd560fe7feb582301c409eab99 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2016-07-07T12:14:08Z (GMT) No. of bitstreams: 1 Tese_DIP_JoãoVictorLeiteDias.pdf: 6357155 bytes, checksum: 49e964fd560fe7feb582301c409eab99 (MD5) / Made available in DSpace on 2016-07-07T12:14:08Z (GMT). No. of bitstreams: 1 Tese_DIP_JoãoVictorLeiteDias.pdf: 6357155 bytes, checksum: 49e964fd560fe7feb582301c409eab99 (MD5) Previous issue date: 2015 / Made available in DSpace on 2016-07-08T18:48:04Z (GMT). No. of bitstreams: 3 Tese_DIP_Jo?oVictorLeiteDias.pdf.txt: 355863 bytes, checksum: d476e278a3e7e647d0e295e1c759d53d (MD5) Tese_DIP_Jo?oVictorLeiteDias.pdf: 6357155 bytes, checksum: 49e964fd560fe7feb582301c409eab99 (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil / A região do Vale do Jequitinhonha foi uma área de intensa transmissão vetorial da infecção chagásica no século passado, tendo sido uma das primeiras áreas do Brasil a realizar ações de combate a populações de triatomíneos domiciliados. Com os níveis de controle alcançados e a virtual eliminação do Triatoma infestans no país, outras espécies continuam como alvo das atividades de vigilância. Esse estudo teve por objetivo analisar a ocorrência e distribuição de triatomíneos nas áreas urbana e rural do município de Diamantina, Vale do Jequitinhonha, Minas Gerais, além de analisar a infecção por Trypanosoma cruzi em vetores e eventuais reservatórios da infecção. Os triatomíneos foram coletados durante as ações de vigilância entomológica entre setembro de 2011 e agosto de 2014. Os insetos positivos para tripanossomatídeos tiveram o conteúdo intestinal submetido a ensaio para determinação da linhagem de T. cruzi. A distribuição dos triatomíneos na área urbana foi analisada por meio de estatística espacial cotejando com dados de vegetação. A distribuição dos insetos na área rural foi analisada por meio de Modelos Lineares Generalizados empregando dados obtidos de sensores remotos. Avaliaram-se os conhecimentos a respeito de triatomíneos e doença de Chagas por meio de questionário entre moradores de localidades com diferentes níveis históricos de infestação triatomínica domiciliar. Mamíferos silvestres e sinantrópicos foram capturados e examinados quanto à infecção por T. cruzi. Foi realizada análise morfométrica de asas e análise molecular por meio de sequenciamento de gene do citocromo B para esclarecer relações de espécies de triatomíneos morfologicamente semelhantes ao Triatoma maculata. Foram capturados 975 triatomíneos pertencentes a oito espécies. Panstrongylus megistus foi a principal espécie capturada, sobretudo em colônias detectadas durante atividades de atendimento às notificações. Triatoma vitticeps foi a espécie mais notificada no município. A infecção por tripanossomatídeos foi observada em 7,4% dos insetos examinados, com destaque para Panstrongylus geniculatus (38,1%) e T. vitticeps (17,6%). Foram detectadas as linhagens TcI, TcII e TcIII do T. cruzi entre triatomíneos. As espécies distribuíram-se em distintas áreas de ocorrência ao longo do município. Os Modelos Lineares Generalizados não mostraram bom poder preditivo para descrever a ocorrência de P. megistus, T. arthurneivai e T. vitticeps. Na área urbana foi observada ocorrência de 140 exemplares de quatro espécies (P. geniculatus, P. megistus, Triatoma arthurneivai e T. vitticeps). A distribuição de imóveis infestados foi agregada para todas as espécies, exceto P. megistus, estando T. vitticeps e P. geniculatus próximos a áreas mais vegetadas e cobertas por campo rupestre. A população reconheceu os triatomíneos independentemente da localidade de residência, porém os moradores de áreas mais infestadas conheciam melhor os serviços de controle e as condutas a se adotar em relação aos insetos. Não foram detectados mamíferos infectados por T. cruzi. Na morfometria geométrica T. maculata e Triatoma pseudomaculata formaram um clado enquanto na análise molecular T. pseudomaculata com Triatoma wygodzinskyi foram agrupados em um clado, sendo T. maculata e T. arthurneivai espécies mais distantes. Os resultados mostram diferenças nos espaços geográficos ocupados pelos triatomíneos no município, além da interação com diferentes linhagens de T. cruzi, contribuindo para um melhor entendimento da ecoepidemiologia das espécies encontradas em Diamantina e indicando a necessária manutenção da vigilância entomológica. / Jequitinhonha Valley region represented an area with intense vector-borne transmission of Chagas disease infection in the past century, and also was one of the first areas in Brazil to carry out activities against domiciliated triatomines. Because of the controlled transmission and elimination of Triatoma infestans in Brazil, other triatomine species remain as target of surveillance. This study aimed to analyze the occurrence and distribution of triatomines in the urban and rural areas of the municipality of Diamantina, Jequitinhonha Valley, Minas Gerais, and to evaluate infection by Trypanosoma cruzi among vectors and possible reservoirs. Triatomines were collected during entomological surveillance between September 2011 and August 2014. Intestinal content of the insects with trypanosomes was evaluated for diagnosis of T. cruzi lineage. Distribution of triatomines in an urban area was analyzed by means of spatial statistics associated to vegetation cover data. Distribution of insects in the rural areas was analyzed by means of Generalized Linear Models with remote sensed data. Knowledge related to triatomines and Chagas disease was evaluated by using a questionnaire among residents in localities with different historical levels of triatomine infestation. Sylvatic and synanthropic mammals were examined about T. cruzi infection. Wing morphometry and sequencing of cytochrome B gene were used to clarify relationships of species morphologically similar to Triatoma maculata. A total of 975 triatomines of eight species were captured. Panstrongylus megistus was the most captured species, especially in colonies detected during searches by health agents in response to previous notification. Triatoma vitticeps was the most notified triatomine. Infection by trypanosomes was observed in 7.4% of examined insects, and Panstrongylus geniculatus (38,1%) and T. vitticeps (17,6%) were most infected species. T. cruzi lineages TcI, TcII and TcIII were detected among triatomines. Triatomine species occurred in distinct areas along the municipality. Generalized Linear Models did not present good predictive power in describing P. megistus, Triatoma arthurneivai and T. vitticeps distribution. In urban area 140 triatomines of four species were captured (P. geniculatus, P. megistus, T. arthurneivai and T. vitticeps). Spatial distribution of infested buildings was clustered for all species, except for P. megistus. T. vitticeps and P. geniculatus were found in more vegetated areas covered by rocky fields. People from all localities were able to recognize triatomines, but the residents in areas with more infestation knew better the existence of triatomine control service and reported desirable attitudes about the detection of insects. T. cruzi-infected mammals were not detected. At geometric morphometrics T. maculata and Triatoma pseudomaculata were grouped into a clade, whereas in molecular analysis T. pseudomaculata and Triatoma wygodzinskyi were grouped into a clade and T. maculata and T. arthurneivai remained more distant to them. Results presented differences in geographic space occupied by triatomines in the municipality, with interaction with distinct lineages of T. cruzi, contributing to improved understanding of ecoepidemiology of the triatomines species found in Diamantina, and indicating the necessity of a sustained entomological surveillance.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2018-01-17T14:43:51Z No. of bitstreams: 1 PATRÍCIA PEREIRA FARIAS.PDF: 3725473 bytes, checksum: 27a790c0c992ee9cf80615ba7199bc05 (MD5) / Made available in DSpace on 2018-01-17T14:43:51Z (GMT). No. of bitstreams: 1 PATRÍCIA PEREIRA FARIAS.PDF: 3725473 bytes, checksum: 27a790c0c992ee9cf80615ba7199bc05 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As doenças parasitárias são um grave problema de saúde pública em diversos países e estão distribuídas, principalmente, em áreas endêmicas em países da África, Ásia, América Central e do Sul. Entre estas doenças estão a doença de Chagas e a doença do Sono, reconhecidas como negligenciadas. Há uma necessidade de tratamentos mais eficientes para essas doenças devido a toxicidade, baixa eficácia e segurança dos fármacos existentes, além da dificuldade de administração e evolução de resistência. O grupo de pesquisa da Dra. Núbia Boechat (Farmanguinhos/FIOCRUZ), vem realizando estudos com análogos nitroimidazólicos sintetizados considerando o megazol como protótipo, molécula ativa contra Trypanosoma cruzi, porém com efeitos mutagênicos e genotóxicos. Estes derivados apresentaram atividade contra o T. cruzi, com menor efeito genotóxico quando comparados com o megazol. Através deste trabalho, a relação estrutura-atividade dos derivados nitroimidazólicos (40a, 40b e 41a-41h) foi realizada e através dos descritores eletrônicos HOMO e LUMO, observou-se que grupos volumosos e com caráter retirador de elétrons do anel nitroimidazólico apresentam relação direta com a atividade. A avaliação do perfil toxicológico in silico confirmou que o composto 41a, mais ativo da série, não apresentou citotoxicidade em células sanguíneas humanas in vitro. O modelo da enzima nitrorredutase de T. cruzi, construído por modelagem comparativa, pode ser utilizado nos estudos de docagem molecular, os quais sugeriram que o tamanho da molécula, a possibilidade de interação com os resíduos His503 e Tyr545 e interações hidrofóbicas do tipo π-π com o cofator FMN podem contribuir para a atividade de derivados nitroimidazólicos no sítio ativo da enzima nitrorredutase. Através dos estudos de docagem molecular, sete novos derivados otimizados foram propostos (PR01 a PR07), dentre os quais o PR03, considerado como melhor ligante planejado, apresentou interações no sítio ativo similares às observadas para o protótipo 41a. Desta forma, os resultados obtidos neste trabalho podem ser úteis a novas pesquisas e podem contribuir para o desenvolvimento de novos protótipos contra o T. cruzi / Parasitic diseases are a major public health problem in many countries, and they are distributed primarily in endemic areas in Africa, Asia, Central and South America. Among them, there are Chagas disease and African trypanosomiasis, known as neglected. There is a need for better treatments for these diseases due to toxicity, low efficacy and safety of the existing drugs, besides the difficulty of administration and evolution of resistance. The research group of Dr. Núbia Boechat (Farmanguinhos/FIOCRUZ), has been conducting studies with nitroimidazole analogs synthesized through the prototype megazol (active molecule against trypanosoma, but with mutagenic and genotoxic effects). In this work, the structure activity relationship of the nitroimidazole derivatives (40a, 40b and 41a-41h) was performed and it was observed through the electronic descriptors HOMO and LUMO that groups with electron withdrawing character display relation with activity. In silico toxicological studies confirmed that the most active compound 41a did not show cytotoxicity in human blood cells in vitro. T. cruzi type I nitroreductase constructed by comparative modeling, can be used in molecular docking studies, which suggested that the size of the molecule, the possibility of interaction with the residues His503 and Tyr545, and hydrophobic interactions of the π- Π with the FMN cofactor may contribute to the activity of nitroimidazole derivatives in the active site of the nitroreductase. From molecular docking studies, seven new optimized derivatives were proposed (PR01 to PR07), among them PR03 was considered as the best planned molecule, it displayed similar active site interactions to those observed for prototype 41a. Thus, the results obtained in this work may be useful to new research and may contribute to the development of new prototypes against T. cruzi

Chagas disease is endemic in a region from Mexico to Argentina. In Brazil, chronic cases predominate with approximately two million infected individuals. Central nervous system involvement, triggering stroke in chagasic patients with myocardiopathy, has occurred in high incidence, usually due to cardiac emboli originating from apical aneurysm, mural thrombi and atrial fibrillation. However, the occurrence of stroke can also occur in individuals with Chagas disease (CD) even without a history of cardiopathy. This study aims to evaluate cerebral hemodynamics in individuals with serological diagnosis for CD through Transcranial Doppler(TCD) in the various chronic forms of the disease. A cross-sectional study was carried out in twelve settlements in the rural area of municipality of Umbaúba, State of Sergipe, where 617 individuals answered a questionnaire on socio-epidemiological conditions and collected blood sample for the performance of serology for Trypanosoma cruzi. There was a prevalence of 12.1% (75/617) of seropositivity for CD, 64% had a primary school level and 33.3% were illiterate, 78.7% worked in agriculture and 92% had a family income of up to one minimal salary. Of these seropositive individuals, 71 underwent clinical examination and electrocardiogram, echocardiogram, chest X-ray, and contrast-enhanced radiography of the esophagus and colon to determine the clinical form of CD. For the TCD, 96 individuals participated, 59 seropositive for CD and 37 seronegative. These individuals had a mean age of 54 ± 11 years and female sex in 62.5%. Among the chronic clinical forms of CD, 22 individuals were in the indeterminate, 17 cardiac, 16 cardiodigestive and 4 digestive forms. The values of mean velocity of the middle cerebral artery (MVMCA) and pulsatility index did not change in the individuals examined. Correlating the mean velocity of the middle cerebral artery with the age and clinical forms of Chagas disease and control, it was observed that in the cardiac and indeterminate forms and control there was a reduction of the MVMCA with the increase of the age in a similar way. However, with the cardiodigestive and digestive forms, the opposite occurred, increasing MVMCA with increasing age. No signs of microemboli were evident. The present study did not show alteration of the cerebral hemodynamics among the clinical chronic forms and control, probably due to individuals with CD being in a controlled phase of the disease, not presenting during the research symptoms or cardiac signals. / A doença de Chagas é endêmica numa região que abrange desde o México até a Argentina. No Brasil, atualmente predominam os casos crônicos com aproximadamente dois milhões de indivíduos infectados. O acometimento do sistema nervosa central, desencadeando acidente vascular cerebral (AVC) nos indivíduos chagásicos com miocardiopatia tem ocorrido em alta incidência, geralmente em decorrência de êmbolos cardíacos originados de aneurisma apical, trombos murais e fibrilação atrial. Entretanto, a ocorrência de AVC também pode advir de indivíduos com doença de Chagas (DC) mesmo sem história de cardiopatia. Este estudo tem por objetivo avaliar a hemodinâmica cerebral nos indivíduos com diagnóstico sorológico para DC por meio do Doppler Transcraniano (DTC) nas diversas formas crônicas da doença. Foi realizado um estudo transversal em doze povoados da zona rural do município de Umbaúba, Estado de Sergipe, onde 617 indivíduos responderam a um questionário sobre as condições sócio epidemiológicas e coletado amostra de sangue para realização de sorologia para Tripanosoma cruzi. Houve uma prevalência de 12,1% (75/617) de soropositividade para DC, 64% apresentavam nível de escolaridade primária e 33,3% eram analfabetos, 78,7% trabalhavam na lavoura e 92% tinham renda familiar de até um salário mínimo. Desses indivíduos soropositivos, 71 foram submetidos ao exame clínico e realização de eletrocardiograma, ecocardiograma, radiografia de tórax e radiografia contrastada de esôfago e cólon para determinar a forma clínica da DC. Para a realização do DTC, participaram 96 indivíduos, 59 soropositivos para DC e 37 soronegativos. Esses indivíduos tiveram uma média de idade de 54± 11 anos e sexo feminino em 62.5%. Dentre as formas clínicas crônicas da DC, 22 indivíduos estavam na forma indeterminada, 17 cardíaca, 16 cardiodigestiva e 4 digestiva. Os valores da velocidade média da artéria cerebral média (VMACM) e do índice de pulsatilidade não apresentaram alteração nos indivíduos examinados. Correlacionando a velocidade média da artéria cerebral média com a idade e as formas clínicas da doença de Chagas e o controle, foi observado que nas formas cardíaca, indeterminada e controle houve redução da VMACM com o aumento da idade de maneira semelhante. Entretanto com as formas cardiodigestiva e digestiva ocorreu o contrário, elevação da VMACM com o aumento da idade. Não foram evidenciados sinais de microêmbolos. O presente estudo não demonstrou alteração da hemodinâmica cerebral entre as formas crônicas clínicas e o controle, provavelmente em decorrência dos indivíduos com DC estarem numa fase controlada da doença, não apresentando durante a pesquisa sintomas ou sinais cardíacos. / Aracaju

African trypanosomes are a major plague in sub-Saharan Africa. They cause sleeping sickness in humans and nagana in cattle. These parasites are transmitted between their mammalian hosts by tsetse flies. They are adapting to their different environments through differentiation processes. These processes involve, amongst other things, the expression of different surface coats. These coats are made of procyclin protein at the insect midgut procyclic stage and of variant surface glycoprotein (VSG) at the mammalian bloodstream stage. At a given time, one VSG is expressed from a single VSG gene out of a repertoire of more than 1500 VSG genes present in the trypanosomes genome. The expressed VSG gene is always located at one of fifteen telomeric polycistronic transcription units called expression sites (ES). The VSG coat is changed regularly in a process called antigenic variation allowing trypanosomes to escape the immune response. The exact mechanism controlling the selection of the active ES is not yet known and controversies have been raised concerning the ES transcription control. Although several molecular factors involved in the ES monoallelic-expression have been identified, none of them seems to be a critical regulator.<p><p>Thus during my thesis we decided to explore two aspects of ES expression: (A) deciphering the level at which this expression is controlled and (B) fishing for new protein factors controlling this expression.<p>A) It is not even clear at which level the ES transcription control takes place. In particular, there has been debate on whether it is taking place at the transcription initiation or elongation level. Previous experiments generated contradictory conclusions and gave rise to two different models. The first model suggested that transcription initiation takes place in all ESs simultaneously. The second model suggested that transcription is initiated in only two ESs, one being fully active and a second being pre-active. These two models were equally able to account for the finding of transcripts from different ES within a trypanosome population provided the pre-active ES differs between individual cells. In order to decide if a single or multiple ES promoters can initiate transcription in a given cell, single cell RT-PCR targeting the beginning of the ES was required. Thus single cell RT-PCR was performed and an analysis of the obtained transcripts showed that transcription initiation is taking place on many ES while only one VSG is transcribed. This permitted the unambiguous conclusion that the monoallelic expression of VSG is exerted by controls operating downstream from transcription initiation, suggesting transcription elongation or RNA processing as critical control steps. <p>B) We have characterized a new nuclear protein, Tb alba3, involved in the repression of silent VSGs. Its invalidation lead to chromatin opening in the silent expression sites and to a raise in their expression. As this protein is cytoplasmic and binding procyclin mRNAs at the procyclic stage, it could be a new versatile factor, shuttling between the cytoplasm and the nucleus and involved both in the inverse regulation of major surface antigens at different differentiation stages and the control of antigenic variation.<p><p>These results enhance our understanding of ES transcription control and of ES monoallelic expression. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished