Issued also as thesis, Université de Paris. / "Bibliographie": p. [195]-209.

No description available.

No description available.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [129]-135).

No description available.

As espécies do gênero Trypanosoma parasitam vertebrados de todas as classes (peixes, anfíbios, répteis, aves e mamíferos) e possuem ciclos de vida com alternância entre vertebrados e invertebrados. A maioria das espécies se desenvolve em artrópodes hematófagos, que podem pertencer a diversas ordens e famílias. A maioria das espécies não é patogênica, T. cruzi é a única espécie patogênica para o homem nas Américas. Estudos realizados com algumas espécies de tripanossomas apontam uma grande complexidade do ciclo silvestre. Ressalta-se o fato que existam poucos trabalhos realizados no estado de Minas Gerais em animais silvestres. Até o momento, poucos estudos avaliaram os pequenos mamíferos terrestres e morcegos como reservatórios silvestres destes parasitas neste estado, com ausência de estudos com outros grupos de vertebrados. O presente projeto tem por objetivo principal, o conhecimento da diversidade de parasitas do gênero Trypanosoma em animais silvestres da ilha pluvial e Estação ecológica de Pirapitinga, Minas Gerais através do isolamento, caracterização molecular e estudos filogenéticos com marcadores tradicionais. Foram realizadas duas campanhas de captura nos meses de outubro de 2013 e março de 2014 totalizando 183 pequenos mamíferos terrestres, de 12 espécies pertencentes, a três diferentes Ordens (Calomys callosus, Cerradomys subflavus, Rhipidomys sp., Akodon sp., Hylaemys megacephalus, Delomys sp., Oligoryzomys sp., Didelphis albiventris, Micoreus sp., Gracilinanus agilis, Monodelphis domestica e Cabassous unicinctus, a espécie mais abundante foi Calomys sp, capturados com pitfalls e Shermann. Foram capturados 57 indivíduos de morcegos, com o auxilio de redes de neblina, de seis diferentes espécies (Glossophaga soricina, Artibeus sp., Platyrrhinus sp., Noctilio albiventris, Myotis sp., Choeronicus minor), a espécie mais abundante foi Glossophaga soricina. Todos os quirópteros foram negativos para tripanossomatídeos e dentre os pequenos mamíferos somente oito exemplares da espécie Monodelphis domestica foram positivas para o parasita, porém foram estabelecidas nove culturas (um dos animais estava parasitado por duas espécies de tripanossomas). Os isolados de M. domestica foram identificados como T. cruzi e uma nova espécie com morfologia distinta, mas agrupada nas filogenias com SSU rDNA e gGAPDH no Clado Lagartos/ Cobras. Esta nova espécie foi denominada T. gennarii. Os anuros e répteis foram capturados através de busca ativa e foram capturados 14 indivíduos de repteis pertencentes a 6 espécies e 88 indivíduos de anuros pertencentes a 4 espécies. Do total de anuros capturados 7 (7,95%) apresentaram hemocultura positivas e 2 (2,27%) de Leptodactylus latrans foram estabelecidas e criopreservadas com morfologia compatível a parasitas do gênero Trypanosoma. Filogenias baseadas em SSU rDNA segregou os isolados do Cerrado em um novo grupo denominado AN05 e a inclusão destes isolados evidenciaram um outro grupo, AN06 compostos de isolados obtidos de flebotomíneos. / The species of the genus Trypanosoma parasites of all vertebrate classes (Fish, amphibians, reptiles, birds and mammals) and have life cycles alternating between vertebrates and invertebrates. Most species develops in blood-sucking arthropods, which may belong to different orders and families. Most species are not pathogenic, T. cruzi is the only species pathogenic to humans in the Americas. Studies with some species of trypanosomes indicate a great complexity of the sylvatic cycle in biomes. We highlight the fact that there are few studies in the state of Minas Gerais in wild animals. To date, few studies have evaluated small terrestrial mammals and bats as wild reservoirs of these parasites in this state, with no studies with other groups of vertebrates. This project\'s main objective, knowledge of Trypanosoma parasites of the genus diversity in wild animals of the rain Island and Ecological Pirapitinga Station, Minas Gerais through isolation, molecular characterization and phylogenetic studies with traditional markers. Two arrest campaigns were conducted between October 2013 and March 2014 totaling 184 small terrestrial mammals, 12 species belonging to three different Orders (Calomys callosus, Cerradomys subflavus, Rhipidomys sp., Akodon sp., Hylaemys megacephalus, Delomys sp., Oligoryzomys sp., Didelphis albiventris, Micoreus sp., Gracilinanus agilis, Monodelphis domestica e Cabassous unicinctus, the most abundant species was Calomys callosus, captured with pitfalls and Shermann. The bats were captured with mist net and caught 57 individuals from six different species (Glossophaga soricina, Artibeus sp., Platyrrhinus sp., Noctilio albiventris, Myotis sp., Choeronicus minor), the most abundant species was Glossophaga soricina. All were negative for trypanosomatids bats and small mammals among only eight copies of Monodelphis domestica species were positive for the parasite, but nine cultures were established (one animal was infested by two species of trypanosomes). Isolates of M. domestica were identified T. cruzi and a new species with distinct morphology, but grouped in phylogenies with SSU rDNA and gGAPDH in Clade Lizards/ Snakes. This new species was named T.gennarii. Frogs and reptiles were captured through active search and were captured 14 individuals of reptiles belonging to 6 species of frogs and 88 individuals belonging to four species. Of the total of frogs captured 7 (7,95%) had positive blood culture and 2 (2.27%) of Leptodactylus latrans were established and cryopreserved with morphology compatible with the parasites of the genus Trypanosoma. Phylogenies based on SSU rDNA segregated the Cerrado isolated in a new group called AN05 and the inclusion of these isolates showed another group of compounds AN06 isolates from sand flies.

As espécies do gênero Trypanosoma parasitam vertebrados de todas as classes (peixes, anfíbios, répteis, aves e mamíferos) e possuem ciclos de vida com alternância entre vertebrados e invertebrados. A maioria das espécies se desenvolve em artrópodes hematófagos, que podem pertencer a diversas ordens e famílias. A maioria das espécies não é patogênica, T. cruzi é a única espécie patogênica para o homem nas Américas. Estudos realizados com algumas espécies de tripanossomas apontam uma grande complexidade do ciclo silvestre. Ressalta-se o fato que existam poucos trabalhos realizados no estado de Minas Gerais em animais silvestres. Até o momento, poucos estudos avaliaram os pequenos mamíferos terrestres e morcegos como reservatórios silvestres destes parasitas neste estado, com ausência de estudos com outros grupos de vertebrados. O presente projeto tem por objetivo principal, o conhecimento da diversidade de parasitas do gênero Trypanosoma em animais silvestres da ilha pluvial e Estação ecológica de Pirapitinga, Minas Gerais através do isolamento, caracterização molecular e estudos filogenéticos com marcadores tradicionais. Foram realizadas duas campanhas de captura nos meses de outubro de 2013 e março de 2014 totalizando 183 pequenos mamíferos terrestres, de 12 espécies pertencentes, a três diferentes Ordens (Calomys callosus, Cerradomys subflavus, Rhipidomys sp., Akodon sp., Hylaemys megacephalus, Delomys sp., Oligoryzomys sp., Didelphis albiventris, Micoreus sp., Gracilinanus agilis, Monodelphis domestica e Cabassous unicinctus, a espécie mais abundante foi Calomys sp, capturados com pitfalls e Shermann. Foram capturados 57 indivíduos de morcegos, com o auxilio de redes de neblina, de seis diferentes espécies (Glossophaga soricina, Artibeus sp., Platyrrhinus sp., Noctilio albiventris, Myotis sp., Choeronicus minor), a espécie mais abundante foi Glossophaga soricina. Todos os quirópteros foram negativos para tripanossomatídeos e dentre os pequenos mamíferos somente oito exemplares da espécie Monodelphis domestica foram positivas para o parasita, porém foram estabelecidas nove culturas (um dos animais estava parasitado por duas espécies de tripanossomas). Os isolados de M. domestica foram identificados como T. cruzi e uma nova espécie com morfologia distinta, mas agrupada nas filogenias com SSU rDNA e gGAPDH no Clado Lagartos/ Cobras. Esta nova espécie foi denominada T. gennarii. Os anuros e répteis foram capturados através de busca ativa e foram capturados 14 indivíduos de repteis pertencentes a 6 espécies e 88 indivíduos de anuros pertencentes a 4 espécies. Do total de anuros capturados 7 (7,95%) apresentaram hemocultura positivas e 2 (2,27%) de Leptodactylus latrans foram estabelecidas e criopreservadas com morfologia compatível a parasitas do gênero Trypanosoma. Filogenias baseadas em SSU rDNA segregou os isolados do Cerrado em um novo grupo denominado AN05 e a inclusão destes isolados evidenciaram um outro grupo, AN06 compostos de isolados obtidos de flebotomíneos. / The species of the genus Trypanosoma parasites of all vertebrate classes (Fish, amphibians, reptiles, birds and mammals) and have life cycles alternating between vertebrates and invertebrates. Most species develops in blood-sucking arthropods, which may belong to different orders and families. Most species are not pathogenic, T. cruzi is the only species pathogenic to humans in the Americas. Studies with some species of trypanosomes indicate a great complexity of the sylvatic cycle in biomes. We highlight the fact that there are few studies in the state of Minas Gerais in wild animals. To date, few studies have evaluated small terrestrial mammals and bats as wild reservoirs of these parasites in this state, with no studies with other groups of vertebrates. This project\'s main objective, knowledge of Trypanosoma parasites of the genus diversity in wild animals of the rain Island and Ecological Pirapitinga Station, Minas Gerais through isolation, molecular characterization and phylogenetic studies with traditional markers. Two arrest campaigns were conducted between October 2013 and March 2014 totaling 184 small terrestrial mammals, 12 species belonging to three different Orders (Calomys callosus, Cerradomys subflavus, Rhipidomys sp., Akodon sp., Hylaemys megacephalus, Delomys sp., Oligoryzomys sp., Didelphis albiventris, Micoreus sp., Gracilinanus agilis, Monodelphis domestica e Cabassous unicinctus, the most abundant species was Calomys callosus, captured with pitfalls and Shermann. The bats were captured with mist net and caught 57 individuals from six different species (Glossophaga soricina, Artibeus sp., Platyrrhinus sp., Noctilio albiventris, Myotis sp., Choeronicus minor), the most abundant species was Glossophaga soricina. All were negative for trypanosomatids bats and small mammals among only eight copies of Monodelphis domestica species were positive for the parasite, but nine cultures were established (one animal was infested by two species of trypanosomes). Isolates of M. domestica were identified T. cruzi and a new species with distinct morphology, but grouped in phylogenies with SSU rDNA and gGAPDH in Clade Lizards/ Snakes. This new species was named T.gennarii. Frogs and reptiles were captured through active search and were captured 14 individuals of reptiles belonging to 6 species of frogs and 88 individuals belonging to four species. Of the total of frogs captured 7 (7,95%) had positive blood culture and 2 (2.27%) of Leptodactylus latrans were established and cryopreserved with morphology compatible with the parasites of the genus Trypanosoma. Phylogenies based on SSU rDNA segregated the Cerrado isolated in a new group called AN05 and the inclusion of these isolates showed another group of compounds AN06 isolates from sand flies.

La plus grande sensibilité des souris TLR9-/- a révélé le rôle de ce récepteur dans l'infection par Leishmania major. Les cellules dendritiques (DCs) sont activées de manière TLR9-dépendante par l'ADN du L. major et d'autres Trypanosomatidae et non par l'ADN de vertébré. La nature de l'ADN capable d'activer le TLR9 reste controversée quant à la séquence/charpente de l'ADN et l'implication de cofacteurs se liant avec le TLR9 ou l'ADN. Nous avons démontré l'importance de la séquence d'ADN. Contrairement aux génomes de parasites, l'ADN de vertébré présente une contre-sélection des motifs activateurs du TLR9 au profit des motifs inhibiteurs. De plus, l'activation du TLR9 par l'ADN du parasite est augmentée en présence de la protéine HMGB1, qui se fixe mieux sur l'ADN de parasite que de vertébré. La maturation du TLR9 requiert un clivage protéolytique par des protéases endosomales, dont les cathepsines (Cat) B, S, L et l'asparagine endopeptidase (AEP) qui interviennent différemment dans les macrophages et les DCs. Après infection par L. major, nous avons montré que les souris AEP-/-, CatS-/- et CatL-/- ont une pathologie identique aux souris WT, ce qui peut être dû à la redondance de leur fonction. Etonnamment, les souris CatB-/- sont plus résistantes. Leurs lésions et la charge parasitaire dans les ganglions se résolvent plus rapidement, reflétant une réponse immune plus précoce et un contrôle plus rapide de la réaction inflammatoire.En conclusion, ces résultats contribuent à une meilleure compréhension des mécanismes permettant au TLR9 de discriminer entre l'ADN de pathogène et de vertébré et soulèvent le rôle non protecteur de la cathepsine B dans l'infection par L. major. / As TLR9-deficient mice are more sensitive to Leishmania major infection, we have shown previously that TLR9 receptor mediates this parasite infection. Dendritic cells (DCs) are activated by L. major and other Trypanosomatidae DNA and not by vertebrate DNA. There is an ongoing controversy concerning the properties of DNA required for TLR9 activation, regarding the DNA sequence or backbone or the implication of a cofactor interacting with TLR9 or DNA. We have established the importance of DNA sequences. In contrast to parasite genome, vertebrate genome have counter-selected stimulatory sequences and over-represented inhibitory motifs for TLR9. In addition, host proteins contribute to TLR9-dependent DC activation. HMGB1 enhances TLR9 activation only in the presence of L. major DNA and, surprisingly, HMGB1 binds more abundantly L. major than vertebrate DNA. TLR9 activation requires a proteolytic cleavage by endosomal proteases, as cathepsins (Cat) B, S and L and asparagine endopeptidase (AEP) that have a differential activity in macrophages and DCs. After L. major infection, we have showed that AEP-/-, CatS-/- and CatL-/- mice have a similar pathology than WT mice, likely due to their functionnally redundant activites. In contrast, CatB-/- mice are more resistant to the infection. Their lesion sizes and the parasite burdens in lymph nodes are significantly decreased, reflecting an earlier immune response and a more rapid control of the inflammatory response. In conclusion, our results bring further insights into how TLR9 discriminates between Trypanosomatidae and vertebrate DNA and reveal a non protective role of cathepsin B in L. major infection.

In 2010 the Florence group completed the total synthesis of the natural product chamuvarinin which, in collaboration with the Smith group, was found to be a potent inhibitor of the pathogenic parasite T. brucei. Several simplified analogues were found to maintain this inhibitory activity alongside activity against the related species T. cruzi and L. major. The mechanism of action and structural features of these compounds responsible for the observed biological activity remained elusive despite a large synthetic effort by the Florence group. With the aim of identifying protein targets in trypanosomatids to understand the mechanism of action, several photo-affinity labeling analogs have been successfully synthesised and utilised to identify a primary protein target. This protein target was fully validated and molecule docking to this protein was evaluated in-silico. This computational data was used to evaluate the mode of action and aided in the design of simplified compounds which were found to maintain the previously observed anti-parasitic activity but with decreased toxicity to mammalian cells alongside decreased synthetic complexity. The total synthesis of the natural product ascr#18 is also described alongside the synthesis of photo-affinity labeling analogs. This natural product is implicated in the modulation of pathogen resistance in plants and has potential application in crop production.

Tripanossomas são parasitas obrigatórios de uma grande variedade de hospedeiros vertebrados e invertebrados sendo descritas até hoje centenas de espécies, genótipos, linhagens e DTUs. Análises moleculares foram chaves para perceber a complexidade deste gênero e necessários na predição de histórias evolutivas entre tripanossomas patogênicos e não patogênicos do velho e novo mundo. Além dos marcadores tradicionais (SSU rDNA e gGAPDH), o gene Spliced Leader (SL) tem sido útil na identificação e genotipagem de tripanossomatídeos, mas poucas espécies possuem genes SL bem estudados. Este trabalho caracterizou as estruturas primárias e secundárias do gene SL de várias espécies representantes dos principais clados de tripanossomas, evidenciando seu polimorfismo inter e intra-específico e mostrando sua utilidade como DNA barcoding. Os resultados obtidos permitiram a descrição de novas espécies (T. livingstonei e T. wauwau), a identificação de subgrupos nas DTUs de T. cruzi e suportaram os relacionamentos filogenéticos obtidos com os marcadores tradicionais. / Trypanosomes are obligate parasites found in a great variety of vertebrate and invertebrate hosts, with hundreds of species, genotypes, lineages and discrete typing units (DTUs) being described. Molecular analyses have been essential to understanding the complexity of trypanosomes and to predict the evolutionary history of the non-pathogenic and pathogenic species from the Old and New World. Besides traditional phylogenetic markers (SSU rDNA and gGAPDH), Spliced Leader (SL) gene has proven useful for identifying and genotyping trypanosomatids, but well defined SL sequences are available for only a few species. In this study, SL primary and secondary structures were determined for species representatives of the main trypanosome clades, showing inter and intra-specific variability that rendered them useful for DNA barcoding. SL results allowed the description of new trypanosome species (T. livingstonei and T. wauwau), the identification of subgroups in the T. cruzi DTUs, in addition to support the phylogenetic relationships obtained with traditional markers.