Orientador: Regina Maria Barretto Cicarelli / Banca: Marcia Aparecida da Silva Graminha / Banca: Maurício Bacci Junior / Resumo: A família Trypanosomatidae compreende um grande número de protozoários parasitas, incluindo importantes agentes etiológicos de doenças humanas. Entre os causadores de doenças, destaca-se o Trypanosoma brucei (responsável pela Doença do Sono) e o Trypanosoma cruzi (agente causador da Doença de Chagas). Ambos possuem como mecanismo de processamento dos seus mRNAs o "trans-splicing", que envolve a excisão de íntrons e a união dos éxons de dois transcritos independentes, o splice-leader, e o pré-mRNA aceptor. As reações de cis e trans-splicing in vitro com extrato nuclear de células HeLa já foram padronizadas e utilizada como modelo em diversos experimentos. Entretanto, em tripanosomas, as únicas referências sobre a reação de transsplicing in vitro são de Vianna et. al., (2001) e Skaked et. al., (2010), com extratos de parasitas preparados de modos diferentes. A reação com células permeáveis do Trypanosoma cruzi foi usada como modelo para análise de drogas tripanocidas na reação de trans-splicing (Barbosa et. al., 2007); porém, não é um sistema livre de células conforme mencionado acima. Diante disso, este trabalho propõe reproduzir a reação de "trans-splicing" in vitro com extratos nucleares livre de parasitas utilizando as formas epimastigotas de T. cruzi e/ou as formas prociclicas de T. brucei e uma sequência parcial de alfa tubulina de T. cruzi como pré-mRNA aceptor. A padronização desta reação in vitro possibilitará avanços no entendimento da maquinaria do trans-spliceossomo, tornando-se também um modelo interessante para avaliação de mecanismo de ação de drogas tripanocidas / Abstract: Trypanosomatidae family comprises a large number of protozoan parasites, including important etiological agents of neglected human diseases. Among the diseases' agents, Trypanosoma brucei (responsible for sleeping sickness) and Trypanosoma cruzi (causative agent of Chagas' disease) are the most important parasites. Both have trans-splicing as a mechanism for the processing of its mRNA, which involves the excision of introns and union of exons form two independent transcripts, the spliceleader (SLRNA), and the acceptor pre-mRNA. The reaction of cis-and transsplicing in vitro with nuclear extract of HeLa cells has already been standardized and used as a model in several experiments. However, in trypanosomes, the only references on the reaction of trans-splicing in vitro are from Vianna et. al., (2001) and Skaked et. al., (2010), using parasitefree extracts prepared on different methods. For trypanocidal drugs analyze, trans-splicing reaction using T. cruzi permeable cells was used as a model (Barbosa et. al., 2007), but this is not the same as free-cell nuclear extract as mentioned above. Thus, this work shows trans-splicing in vitro reaction using nuclear extracts either from T. cruzi epimastigote forms and/or T. brucei procyclic forms reacting with the T. cruzi alpHa-tubulin cloned sequence as pre-mRNA acceptor. The standardization of this in vitro reaction will be able to promote advances to understanding the transspliceosomo machinery, as well as occurred in mammalian cis- and/or trans-splicing, becoming also an interesting model for evaluating trypanocidal drugs interference / Mestre

>Magister Scientiae - MSc / Human African trypanosomiasis is the world’s third most important parasitic disease affecting human health after malaria and schistosomiaisis. The world health organization estimates approximately 60 million people at risk in sub-Saharan Africa and up to 50,000 deaths per year caused by trypanosomiasis. Current management of human African trypanosomiasis relies on active surveillance and chemotherapy of infected patients. Efforts to develop a vaccine to immunize the human host have been hampered by antigenic variation of the parasites cell coat. The advent of the genome era has opened up opportunities for developing novel strategies for interrupting the transmission cycle of trypanosomes, specifically using any of the three players,the human host, the tsetse fly vector and/or the parasite. The human genome has been deciphered and the genomes of several trypanosome species have been sequenced. Sequencing of additional neglected trypanosome species is in progress. The tsetse fly genome is currently being sequenced as part of the genomic activities of the International Glossina genome initiative (IGGI). In an attempt to support the tsetse fly sequencing effort, expressed sequence tags (ESTs) from various tissues and developmental stages of Glossina morsitans have been generated.In this study, tsetse fly EST data was analyzed using bioinformatics approaches, focusing on transcripts encoding serpin genes implicated in the immune defenses of tsetse flies. Glossina morsitans homologues to Drosophila melanogaster serpin4, serpin5, and serpin27A and Anopheles gambiae serpin10 were identified in the tsetse fly EST contigs. Comparison of the reactive center loop of tsetse fly serpins with human α-1-antitrypsin suggests that these tsetse serpins are inhibitory. Preliminary EST clustering did not succeed in assembling 3564 Tsal encoded ESTs into one contig. In this study, these ESTs were assembled together with three published Tsal cDNAs. A total of 29 Tsal-encoded contigs were generated. An analysis of the sequence variation within the Tsal EST assembled contigs identified five single base mismatches namely A-T, T-A, G-T and T-G.Results from this study form a basis onto which genetic and biochemical experimental studies can be designed, a process that will be successfully carried out once we have a reference genome. Specifically, studies aimed at genetic modification of tsetse flies towards populations that are inhabitable to trypanosomes. Ultimately, this will supplement current vector control strategies towards elimination of human African trypanosomiasis.

Trypanosoma brucei and related parasites are causative agents of severe diseases that affect global health and economy. T. brucei is responsible for sleeping sickness in humans (African trypanosomiasis) and a wasting disease in livestock. More than 100 years after T. brucei was identified as the etiological agent for sleeping sickness, available treatments remain inadequate, complicated by toxicity, lengthy and expensive administration regiments, and drug-resistance. There is clear need for the development of a new antitrypanosomal drugs. Due to the unique evolutionary position of these early diverging eukaryotes, trypanosomes posses a number of biological properties unparalleled in other organisms, including humans, which could prove valuable for new drug targets. One of the most distinctive properties of trypanosomes is their mitochondrial DNA, called kinetoplast DNA (kDNA). kDNA is composed of over five thousand circular DNA molecules (minicircles and maxicircles) catenated into a topologically complex network. Replication of kDNA requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication and at least five DNA polymerases. Three of these (POLIB, POLIC, and POLID) are related to bacterial DNA polymerase I and are required for kDNA maintenance and growth. Each polymerase appears to make a specialized contribution to kDNA replication. The research described in this dissertation is a significant contribution to the field of kDNA replication and the advancement of kDNA replication proteins as putative drug targets for sleeping sickness. Functional characterization of POLIB indicated that it participates in minicircle replication but is likely not the only polymerase contributing to this process. Gene silencing of POLIB partially blocked minicircle replication and led to the production of a previously unidentified free minicircle species, fraction U. Characterization of fraction U confirmed its identity as a population of dimeric minicircles with non-uniform linking numbers. Fraction U was not produced in response to silencing numerous other previously studied kDNA replication proteins but, as we demonstrated here, is also produced in response to POLID silencing. This common phenotype led us to hypothesize that POLIB and POLID both participate in minicircle replication. Simultaneously silencing both polymerases completely blocked minicircle replication, supporting a model of minicircle replication requiring both POLIB and POLID. Finally, we demonstrate that disease-causing trypanosomes require kDNA and the kDNA replication proteins POLIB, POLIC, and POLID. These data provide novel insights into the fascinating mechanism of kDNA replication and support the pursuit of kDNA replication proteins as novel drug targets for combating African trypanosomiasis.

No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

This thesis aimed to identify the trypanocidal mode of action of non-natural chamuvarinin analogs, and to assess the previously uncharacterized secondary alternative oxidase (AOX2) as a possible drug target of the trypanosomatids. The drugs used to treat infections with Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are highly toxic and are increasingly becoming less effective as the parasites develop resistance, therefore new drugs against the diseases are desperately needed. Non-natural analogs of chamuvarinin were tested for trypanocidal activity to determine the structure activity relationships of the compounds against insect-form T. cruzi and Leishmania spp. This identified several potent and selective analogs, which retained good activity against the medically relevant intracellular forms of the parasites. Photoaffinity labeling was utilized to identify the mode of action and protein target(s) of the chamuvarinin analogs. The analogs were shown to deplete ATP levels and to induce mitochondrial dysmorphia and mitochondrial oxidative stress. Photoaffinity labeling confirmed the mitochondrial localization of the protein target(s) of these compounds, however the exact protein target(s) were unable to be identified by protein pull-down and mass spectrometry. The previously uncharacterized secondary alternative oxidases (AOX2) are conserved throughout the human-infective trypanosomatids and are absent from mammalian cells, thus making an attractive drug target if the protein is essential. The AOX2 of T. brucei, T. cruzi and L. major were expressed in Escherichia coli to characterize the enzymatic activity of the proteins. T. brucei AOX2 was successfully purified and shown to be an ubiquinol oxidase, which contains bound iron (III). The role of AOX2 within the trypanosomatids was determined by biochemical phenotyping and genetic manipulation of T. brucei, T. cruzi and L. major, which indicated that AOX2 is an essential mitochondrial oxidase in the three trypanosomatids, with a putative role in energy production, and therefore is an attractive multi-trypanosomatid drug target.

Little is known about the generation of Leishmania morphology and the function of morphology in trypanosomatids, despite every species having characteristic cell shapes and undergoing changes in morphology between life cycle stages. To address this I analysed morphogenesis of the cell body and flagellum through the cell cycle of the Leishmania insect (promastigote) life cycle stage using a novel method for determining cell cycle stage from cell size and DNA content. This showed cell body morphology is generated by growth and then remodelling of cell shape around mitosis and cytokinesis. Mathematical modelling of flagellum growth indicated flagellum length continues to increase over multiple cell cycles and does not reach a defined length. I also observed little link between the cell cycle and flagellum length regulation during differentiation to the mammalian macrophage-inhabiting (amastigote) life cycle stage. Analysis of motility showed the diverse flagellar lengths of promastigote Leishmania cells bestow different swimming abilities, and the capacity of Leishmania promastigotes for highly directional swimming differs sharply from trypomastigote Trypanosoma brucei. This difference did not arise from altered flagellar beating therefore appeared to be linked to morphology. Together these indicate the mechanisms of cell body morphogenesis, flagellum length regulation, life cycle stage differentiation and the swimming abilities of the cells the morphogenetic processes generate differ significantly between Leishmania and T. brucei. These insights motivated the programming of automated micrograph analysis tools based on a new DNA staining method to support similar future morphometric analyses. This is the first comprehensive comparison of morphogenesis and function of morphology in a promastigote and a trypomastigote and, by considering these new insights in the context of existing molecular biology and the morphological diversity across many trypanosomatid species, give insight into basic Leishmania biology, the shared molecular mechanisms underlying morphogenesis and the potential functions of the diverse morphologies which are seen in different trypanosomatid species and life cycle stages.

Sleeping sickness, caused by Trypanosoma brucei, is a deadly disease that affects some of the poorest countries in sub-Saharan Africa. Although the disease prevalence is declining, strengthening of the current control efforts, including introduction of more adequate chemotherapeutic options, is needed to prevent the re-emergence of yet another epidemic. Nitroaromatic compounds, such as nifurtimox (in combination with eflornithine) and fexinidazole (in clinical trials), have been recently introduced for the treatment of the second stage of sleeping sickness. These compounds are believed to act as pro-drugs that require intracellular enzymatic activation for antimicrobial activity. Here, the role of the bacterial-like nitroreductase TbNTR as a nitrodrug activating enzyme is examined through overexpression and knock-out studies in T. brucei. Multiple attempts to purify soluble recombinant TbNTR from E. coli were unsuccessful, because the recombinant protein was found to be membrane associated. In keeping with the role of TbNTR in nitrodrug activation, loss of an NTR gene copy in T. brucei was found to be one, but not the only, mechanism that may lead to nitrodrug resistance. Furthermore, in the bloodstream form of T. brucei, resistance was relatively easy to select for nifurtimox, with no concurrent loss of virulence and at clinically relevant levels. More worryingly, nifurtimox resistance led to a decreased sensitivity of these parasites to other nitroaromatic compounds, including a high level of cross-resistance to fexinidazole. Conversely, generation of fexinidazole resistance resulted in cross-resistance to nifurtimox. Should these findings translate to the field, emerging nitrodrug resistance could reverse all recent advances in the treatment of sleeping sickness, made since the introduction of eflornithine 20 years ago. Therefore, all efforts should be made to ensure nitroaromatic drugs are used only in drug combination therapies against sleeping sickness, in order to protect them from emerging resistance.

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.