Leishmania et Trypanosoma brucei sont des protozoaires parasites responsables de graves parasitoses de distribution mondiale. Aucun vaccin n'est disponible contre ces maladies dont le traitement reste basé sur un nombre limité de médicaments coûteux, souvent toxiques et peu efficaces, problème auquel s'ajoute celui des chimiorésistances. D'où l'urgente nécessité de trouver de nouvelles cibles pour le développement de nouveaux traitements qui soient à la fois efficaces, non ou moins toxiques et à un coût plus accessible. Les Trypanosomatidés, dont les génomes ont été entièrement séquencés, présentent de nombreuses originalités dans leur biologie cellulaire et moléculaire, par exemple un ADN mitochondrial unique et extrêmement complexe appelé kinétoplaste. Leur développement suit également un "double" cycle cellulaire répliquant, d'une part, classiquement le noyau et, d'autre part, l'ensemble "corps basal-ADN mitochondrial" dont la ségrégation correcte conditionne la cytodiérèse. Ils possèdent par ailleurs deux types de protéasomes, un classique (26S) et un de type procaryote, plus spécifique et absent chez l'homme, le complexe HslVU. Nous avons montré que HslVU est localisé exclusivement dans l'unique mitochondrie des parasites, et qu'il est, chez T. brucei, essentiel pour la survie de ces organismes. En effet, son inhibition par ARN interférence entraine un blocage de la cytodiérèse suivi par une mort cellulaire. Le premier objectif de cette thèse a été de tenter de mieux comprendre le rôle de HslVU dans le cycle cellulaire associé au kinétoplaste chez ces parasites possédant déjà un protéasome classique. Mettant un terme à plusieurs publications contradictoires, nous avons confirmé la localisation mitochondriale de ce complexe chez Leishmania et chez T. brucei. Nous montrons pour la première fois qu'il est tout aussi essentiel dans les formes sanguines, celles présentes chez l'hôte mammifère, que dans les formes procycliques. Nous montrons aussi un rôle différencié des différentes sous-unités du complexe dans le déroulement du cycle cellulaire associé au kinétoplaste. Le deuxième objectif de cette thèse a été d'identifier de nouvelles protéines mitochondriales régulatrices du cycle cellulaire associé au kinétoplaste. Pour ce faire, nous avons développé une approche de criblage par ARN interférence "semi-systématique" sur 104 protéines mitochondriales, principalement de fonction inconnue. Si l'inhibition de l'expression de la majorité de ces protéines (62) n'a aucun effet sur la croissance cellulaire, celle des 42 restantes induit une baisse moyenne ou sévère de cette croissance. De façon surprenante, cette inhibition modifie significativement et avec plus ou moins d'ampleur le déroulement du cycle cellulaire, suggérant qu'il est dépendant de multiples fonctions cellulaires. Finalement, ce travail valide le protéasome HslVU comme une cible thérapeutique pertinente tout particulièrement à l'adresse des formes sanguines de T. brucei. La différenciation fonctionnelle de HslU1 et HslU2 et l'activité indépendante de HslV donnent une image plus complexe sur le fonctionnement de ce protéasome. Les données d'ARN interférence pour leur part nous orientent vers une régulation du cycle cellulaire très intégrée à l'ensemble des activités cellulaires. / Leishmania and Trypanosoma brucei are protozoan parasites responsible for worldwide distributed severe diseases. No vaccine is available and the treatment relies upon a limited number of drugs, which are costly, often toxic and not highly efficient, and for which resistances are increasing. Hence the necessity to urgently discover novel drug targets with the aim of developing new drug treatments which would be more efficient, less toxic and if possible cheaper.Trypanosomatids, of which the genome has been entirely sequenced, exhibit numerous peculiarities in their cell and molecular biology, for example a single and complex mitochondrial DNA network termed kinetoplast. Also, their development follows a ‘double' cell cycle ensuring the replication of, on the one hand, the nucleus (classical mitosis) and on the other hand, the “basal body-kinetoplast” whole, of which the correct segregation conditions cytokinesis. They also possess tow types of proteasomes, one classical one (26S) and one of the prokaryotic type, more specific and absent in human, the HslVU complex. We have shown that HslVU is located exclusively in the single mitochondrion of these parasites and, in T. brucei, that it is essential to parasite's survival. Indeed, its RNAinterference-based knockdown leads to a cytokinesis block followed par cell death. The first aim of this work was to try to better understand the role of HslVU in the ‘kinetoplast-associated' cell cycle in these parasites that already possess a classical proteasome. Putting an end to several contradictory publications, we confirmed the mitochondrial location of this complex in Leishmania and T. brucei. For the first time, we also demonstrate that it is just as essential in bloodstream forms (those present in the mammalian host) than in procyclic forms. We finally show a differentiated role for the different subunits of the complex in the progress of the kinetoplast-associated cell cycle.The second aim of this work was to identify novel mitochondrial proteins which would participate in the regulation of the kinetoplast-associated cell cycle. To do this, we developed a ‘semi-systematic' screening approach using RNA interference for 104 mitochondrial proteins, most of them being of unknown function. If the inhibition of most of these proteins (64) had no effect on cell growth, that of the 42 remaining ones induced a moderate or severe growth defect. Surprisingly, this inhibition yielded significant and more or less visible modifications of the cell cycle progress, suggesting that the latter is dependent upon multiple cell functions.Finally, this study validates the HslVU proteasome as a pertinent drug target, particularly for the bloodstream forms of T. brucei. The functional differentiation of HslU1 and HslU2 and the independent activity of HslV are intriguing and give a complex picture of the functioning of this proteasome. On the other hand, the RNA interference data suggest a cell cycle regulation which would be highly integrated to the whole of the cell activities.

O SL trans-splicing (do inglês, spliced leader trans-splicing) catalisado pelo spliceossomo em Trypanosoma brucei é responsável pelo processamento dos pré-mRNAs policistrônicos em mRNAs maduros. Esta maquinaria é associada a partir de pequenas partículas ribonucleoproteicas nucleares (snRNPs) U1, U2, U4/U6 e U5 constituídas de pequenos RNAs nucleares (snRNAs), um complexo canônico de sete proteínas Sm (SmB, SmD3, SmD1, SmD2, SmE, SmF e SmG) e fatores proteicos específicos. O núcleo de proteínas Sm de T. brucei apresenta variações com funções desconhecidas, como a substituição do heterodímero SmD3/SmB por Sm16,5K/Sm15K na snRNP U2, e de SmD3 por SSm4 na snRNP U4. Na primeira parte deste trabalho, investigou-se a interação destes diferentes complexos Sm recombinantes com os snRNAs U2, U4 e U5 obtidos por transcrição in vitro. Todos os complexos apresentaram alta afinidade pelo snRNA cognato. Observou-se, ainda, que apenas o núcleo Sm que contém Sm16,5K/Sm15K associado ao snRNA U2 interage com alta afinidade com U2A/U2B. Adicionalmente, foi obtida a estrutura cristalográfica de U2A/U2B de T. brucei, que revela uma organização similar àquela já descrita para ortólogas de Homo sapiens. Entretanto, há um desvio de pelo menos 6 Å no ponto médio da alça carregada positivamente no domínio RRM de U2B para a acomodação do snRNA U2. Além disso, observou-se uma longa hélice-α adicional na extremidade C-terminal de U2A. A análise dos três núcleos Sm de T. brucei a partir da combinação de modelagem molecular e espalhamento de raios-X a baixo ângulo revela estruturas de barril-β altamente torcido com interior carregado positivamente para interação com snRNAs. A principal diferença entre as estruturas encontra-se nas extremidades C- e N-terminal dos variantes de proteínas Sm, possivelmente para interação com U2A no braço 1 do spliceossomo, no caso de Sm15K/Sm16,K, e com U5-220K e U5-200K no corpo do spliceossomo, no caso de SSm4. Na segunda parte deste trabalho, a expressão homóloga da proteína U5-200K de T. brucei completa e do produto truncado no seu cassete helicase/ATPase/Sec63 N-terminal levou à copurificação de um subcomplexo de snRNP U5 composto por U5-220K, U5-116K, U5-40K e U5-Cwc21, sendo que a proteína recombinante completa ainda copurificou as proteínas Sm. Experimentos de imunolocalização mostraram que a proteína U5-200K truncada não é direcionada ao núcleo, como é o caso da proteína completa. As células que expressam a proteína truncada apresentaram um defeito de crescimento significativo, e os processamentos de pré-mRNA por cis- e SL trans-splicing foram ligeiramente afetados, já que a proteína truncada não entra no núcleo, onde deveria exercer sua atividade. Os resultados apresentados indicam a formação de um subcomplexo de snRNP U5 ainda no citoplasma, sendo que as proteínas Sm devem ser um sinal para o seu transporte nuclear mediado por importina-β. Em leveduras, a proteína Aar2 substitui U5-200K no citoplasma, regulando assim a biogênese de snRNP U5, porém esta proteína não foi identificada em T. brucei. Os resultados apresentados neste trabalho contribuem como o primeiro estudo estrutural de proteínas spliceossomais de um parasita do homem e também com novas informações sobre a biogênese das partículas ribonucleoproteicas U2 e U5 de T. brucei. / The spliced-leader (SL) trans-splicing catalyzed by the spliceosome in Trypanosoma brucei is responsible for processing polycistronic pre-mRNAs into mature mRNAs. The spliceosome machinery is assembled by small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 that are composed by small nuclear RNAs (snRNAs), a canonical complex of Sm proteins (SmB, SmD3, SmD1, SmD2, SmE, SmF, SmG) and specific factors. The Sm core peculiarly varies in T. brucei, where SmD3/SmB are replaced by Sm16.5K/Sm15K in U2 snRNP and SmD3 is substituted by SSm4 in U4 snRNP. In the first part of this thesis, we investigated the interaction of the different recombinant Sm cores with U2, U4 and U5 snRNAs obtained by in vitro transcription. All the protein complexes bind the cognate snRNA with high affinity. Only the Sm core that contains Sm16.5K/Sm15K associated with U2 snRNA interacts with the recombinant U2A/U2B subcomplex. Additionally, the crystallographic structure of T. brucei U2A/U2B was obtained, showing an overall organization similar to the one observed in the human counterpart. However, we observed a 6 Å deviation in the medium point of a positively charged turn in the RRM motif of U2B to accommodate U2 snRNA. Besides, a long α -helix was observed in the C-terminal region of U2A. Structural analysis of Sm core variations in T. brucei was proceeded using molecular modelling techniques associated with small angle X-ray scattering. The quaternary structure models show seven Sm proteins as β-barrels with positively charged interior for cognate snRNA interaction. The main difference among these Sm core structures resides in the C- and N-terminal regions of the variant proteins, probably enabling the interaction of Sm15K/Sm16,5K with U2A in the spliceosomes arm 1, and the association of SSm4 with U5-220K and U5-200K in the spliceosomes body. In the second part of this thesis, homologous expression of full-length and N-terminally truncated U5-200K from T. brucei led to the copurification of a U5 snRNP subcomplex containing U5-220K, U5-116K, U5-40K and U5-Cwc21. The full-length U5-200K construct also copurified Sm proteins. Immunolocalization experiments showed that the truncated U5-200K protein is not directed to the nucleus as is the case for the full-length protein. Cells that expressed the truncated protein showed a significant growth defect and the pre-mRNA processing by cis- and SL trans-splicing was negatively affected since the truncated protein did not enter the nucleus where it should be active. The results suggest that a subcomplex of U5 snRNP begins to be assembled in the cytoplasm and the Sm proteins may be the signal for the nuclear transport mediated by β-importin. In yeast, Aar2 replaces U5-200K in the cytoplasm in another regulation step. However, Aar2 has not been identified in T. brucei. The results presented here contribute with the first structural study of spliceosomal proteins of a human parasite and give new insights into the biogenesis of U2 and U5 snRNPs in T. brucei.

O Complexo de pré-replicação em T.brucei é composto por Orc1/Cdc6 e as helicases MCMs. Em um trabalho anterior mostramos que TbOrc1/Cdc6 pode ligar e hidrolisar ATP in vitro. Neste sentido, o objetivo deste trabalho é avaliar a importância da hidrólise e ligação de ATP para a formação e estabilidade do complexo pré-replicação de T.brucei. Para tanto, foram geradas proteínas recombinantes Orc1/Cdc6 de T. brucei mutadas nas regiões Walker A (TbOrc1/Cdc6K79T) ou sensor 2 (TbOrc1/Cdc6R251,252E) incapazes de ligar ou hidrolisar ATP, respectivamente. Finalmente, as células expressando TbOrc1/Cdc6K79T ou TbOrc1/Cdc6R251,252E foram avaliadas quanto a (i ) estabilidade da interação Orc1/Cdc6 -DNA, (ii) capacidade de estabilizar MCM no DNA, (iii) capacidade de replicar seu DNA. A mutação na região sensor 2 de T.brucei (TbOrc1/Cdc6R251,252E) reduziu drasticamente a atividade de ATPase em comparação com a proteína selvagem . TbOrc1/Cdc6 mutado no sitio de ligação ao ATP perdeu a capacidade de interagir com o ATP (TbOrc1/Cdc6K79T). A super expressão desses genes inibiu de forma significativa a proliferação celular, causou ineficiência no carregamento de MCM para o DNA e ocasionou falhas na progressão do ciclo celular, atrasando a fase S. / The pre-replication complex in T.brucei is composed of at Orc1/Cdc6 and MCMs helicases. In a previous paper we showed that TbOrc1/Cdc6 can bind and hydrolyze ATP in vitro. Based on that, the objective of this study is to evaluate the importance of ATP binding and hydrolysis to the formation and stability of the pre - replication complex in T.brucei. For this purpose, T. brucei Orc1/Cdc6 recombinant proteins were generated mutated at regions on Walker A (TbOrc1/Cdc6K79T) and sensor 2 (TbOrc1/Cdc6R251 , 252E) in order to unable the ATP binding and hydrolyzation respectively . Finally , cells expressing TbOrc1/Cdc6K79T or TbOrc1/Cdc6R251 , 252E were evaluated for (i) stability of Orc1/Cdc6 - DNA interaction , (ii) ability to stabilize MCM in DNA , (iii) ability to replicate its DNA . The mutation in the sensor 2 region of T.brucei (TbOrc1/Cdc6R251 , 252E) drastically reduced the ATPase activity compared to the wild-type protein. TbOrc1/Cdc6 mutated in the ATP binding site has lost the ability to interact with ATP (TbOrc1/Cdc6K79T). The overexpression of these genes significantly inhibited cell proliferation causing inefficient loading of MCM DNA and led to failure in cell cycle progression by delaying the phase S.

Trypanosoma cruzi e Trypanosoma brucei são os agentes etiológicos da doença de Chagas e da doença do sono, respectivamente. Ambos são tripanossomatídeos, apresentam um ciclo de vida que alterna entre os hospedeiros mamíferos e os hospedeiros invertebrados e apresentam o metabolismo baseado no consumo de aminoácidos e/ou glicose, dependendo da disponibilidade de nutrientes no ambiente. Neste trabalho foi demonstrado a importância da glutamina (Gln) em diferentes aspectos da biologia do T. cruzi e a relevância da Gln e da enzima glutamina sintetase (GS) para formas sanguícolas de T. brucei. A Gln é transportada pelo T. cruzi e pelo T. brucei a partir do meio externo. Em T. cruzi foi demonstrado que esse transporte é realizado por um único sistema, saturável, específico, dependente de ATP e do gradiente de H+ na menbrana do parasita. Também foi demonstrado que a Gln é importante para replicação das formas amastigotas e epimastigotas, além de promover o processo de metaciclogênese. Tratamento com análogos estruturais da Gln dimuiu a proliferação do estágio epimastigota e também a diferenciação para tripomastigota metacíclico. Além do mais células infectadas e tratadas com os análogos apresentaram redução do número de tripomastigotas que eclodiram das células, demonstrando que a Gln também é importante para os estágios intracelulares. Em formas sanguícolas de T. brucei, a enzima GS é ativa, mas é incapaz de suprir a necessidade de Gln do parasita, fazendo com que seja completamente dependente do transporte a partir do meio externo. A Gln é importante para a proliferação formas sanguícolas e correta progressão do ciclo celular. Em meio sem Gln os parasitas são incapazes de manter a proliferação normalmente, sendo que este processo é dependente da concentração de Gln no meio externo. Também foi demonstrado que a Gln participa do processo de modificação pós-traducional de glutamilação da tubulina. Conclui-se portanto que a Gln é um aminoácido fundamental para sobrevivência do T. cruzi e do T. brucei. / Trypanosoma cruzi and Trypanosoma brucei are the etiologic agent of Chagas disease and sleeping sickness, respectively. Both parasites are trypanosomatids that have a complex life cycle, which alternates between a mammalian host and insect vector. T. cruzi and T. brucei are able to use carbohydrates and amino acids as energy source, depending on availability of nutrients in the different environments that parasites go through in the life cycle. In this work we demonstrate that glutamine (Gln) is an important metabolite that participates in many biological processes in T. cruzi, and the relevance of the enzyme glutamine synthetase and Gln in bloodstream forms of T. brucei. T. cruzi and T. brucei are able to uptake Gln from the medium. T. cruzi incorporate Gln through a single and saturable transport system. Gln uptake system is dependent on ATP intracellular levels and H+ gradient and is a highly specific system. Also was demonstrated that Gln is important to replicative stages amastigote and epimastigote, and promotes the metacyclogenesis process. The treatment with Gln analogs impared the epimastigote replication and the differentiation from epimastigote to trypomastigote metacyclic. Moreover, analogs treatment in the infected cells decrease the number of trypomastigotes released from the cells, suggesting that Gln is important to intracellular development of T. cruzi. This work also demonstrates that the enzyme glutamine synthetase is active in bloodstream forms from T. brucei, but is not enough to produce the amount of Gln required by the parasite. T. brucei, bloodstream forms are completely dependent of Gln uptake from the medium. The proper proliferation rate and correct cell cycle progress are dependent of Gln concentration in the medium. Moreover Gln participates in the tubulin glutamylation process in bloodstream forms; this is a post translational modification that is important to microtubules dynamics and cytokinesis process. We concluded that Gln is a fundamental amino acid to maintenance of T. cruzi and T. brucei.

Les parasites de la classe des Kinetoplastidae, comprenant notamment les trypanosomes et les leishmanies, sont responsables pour plusieurs maladies d’importance socio-économique et de santé publique. La maladie du sommeil, la maladie de Chagas et la leishmaniose, classées comme maladies tropicales négligées (NTD) par l’Organisation mondiale de la santé (OMS) et la Surra, reportée par l’Organisation pour l’alimentation et l’agriculture, des Nations Unies (FAO). La Trypanosomiase Animale Africain sub-saharienne entraîne la mort de 3 millions bovins par an accompagné d'une perte annuelle de l'économie de 4,5 milliards de dollars américains. La leishmaniose cutanée, une maladie zoonose, présente 1,5 millions de nouveaux cas chaque année.Trypanosoma brucei (T. brucei) est un ancien eucaryote, utilisé comme organisme modèle dans le laboratoire pour l’étude des cils et des flagelles. Le remodelage du cytosquelette des trypanosomes est essentiel pour la morphologie cellulaire, le positionnement et la division des organites. L’étude des protéines essentielles du cytosquelette permet de mieux comprendre les processus cellulaires. Ces protéines pourraient également constituer des cibles potentielles pour des traitements thérapeutiques. Les trypanosomes échappent au système immunitaire de l’hôte en modifiant périodiquement les antigènes de présent à leur surface. En effet ces antigènes de surface sont endocytés, ainsi que les anticorps de l’hôte qui y sont attachés, au niveau d’une structure appelée la poche flagellaire (FP). TbBILBO1 est une protéine structurelle du collier de la poche flagellaire (FPC), essentielle à la biogenèse du FPC et à la survie du parasite. En raison du rôle majeur de la protéine TbBILBO1 dans le parasite, des partenaires de TbBILBO1 ont été recherchés.Dans ce travail, j’ai pu caractériser une nouvelle protéine essentielle du cytoskelette, la protéine FPC6, partenaire de TbBILBO1, qui se situe au niveau du complexe FPC/Complexe du Hook de T. brucei. L’ARN interférence de FPC6 conduit à une mort rapide des formes sanguines des trypanosomes, accompagnée d’un blocage de l’endocytose. Ensuite, j’ai produit un nanobody (Nb48), dirigé contre TbBILBO1, dans le système d’expression bactérien. Je l’ai également exprimé dans les lignées de trypanosomes. Le Nb48 reconnait TbBILBO1 sur les trypanosomes fixés par immunofluorescence et dans les extraits totaux de protéines dénaturées. L’analyse par résonance plasmonique de surface (SPR) a confirmé une haute affinité du Nb48 pour TbBILBO1. L’expression de Nb48 dans le parasite T. brucei en tant qu’intrabody demontrant que ce nanobody pouvait être exprimé de manière fonctionnelle, capable de reconnaitre spécifiquement sa cible protéique, TbBILBO1, intra-cellulaire et de bloquer sa fonction conduit à un effet trypanocide rapide. Ces études ouvrant ainsi la voie pour de nouvelles utilisations potentielles thérapeutiques dans le traitement des trypanosomiases. / Kinetoplastid parasites, including trypanosomes and leishmania, are responsible for several diseases of socio-economic and public health importance worldwide. These include the Neglected Tropical Diseases: Sleeping Sickness, Chagas disease and Leishmaniasis, as classified by the World Health Organisation (WHO) and the global wasting disease of animals, Surra, as reported by the Food and Agricultural Organisation of the United Nations (FAO). Animal African Trypanosomiais (AAT) causes the death of 3 million cattle per year in sub-Saharan Africa, with an annual loss of 4.5 billion US dollars to the African economy. Cutaneaous leishmaniasis is a zoonotic disease, with 1.5 million new cases reported globally each year.Trypanosoma brucei is an ancient, early diverging eukaryote, used as a model organism in the laboratory for studying eukaryotic cilia and flagella. Remodelling of the trypanosome cytoskeleton is essential for cell morphology, organelle positioning and division. Study of essential proteins of the cytoskeleton provides insight into intracellular processes and could provide potential targets for therapeutic interventions. Trypanosomes evade the host immune system by periodically changing their external surface coat, which is endocytosed, along with any attached host antibodies, via a structure called the flagellar pocket. TbBILBO1 is a structural protein of the Flagellar Pocket Collar (FPC) that is essential for FPC biogenesis and parasite survival. Due to the importance of TbBILBO1 for the parasite, protein partners were investigated.In my thesis, I describe, firstly, the characterisation of a novel and essential cytoskeletal protein, FPC6, of the FPC/Hook complex of T. brucei; FPC6 is a partner of TbBILBO1. RNAi Knock-down of FPC6 protein leads to rapid cell death in the blood-stream form of the parasite accompanied with a block in endocytosis. Secondly, I describe the purification and intracellular expression of a nanobody (Nb48), raised against TbBILBO1. The purified Nb is able to identify TbBILBO1 in fixed trypanosomes and denatured protein. Surface Plasmon Resonance analysis confirmed a high affinity of Nb48 to TbBILBO1. Expression of Nb48 as an intrabody in T. brucei, reveals that it binds precisely to its target, TbBILBO1 and leads to rapid cell death. Further exploration of the potential uses of this trypanocidal nanobody is warranted.

No description available.

Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. Results: The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. Conclusion: Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the bloodbrain- barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemolymphatic as well as neurological stages of sleeping sickness.

Trypanosoma brucei, un parasite flagellé unicellulaire, est responsable de la trypanosomiase humaine africaine aussi connue comme la maladie du sommeil.Les microtubules (MTs) sous-pelliculaires, le quartet de MTs (MTQ), le flagelle (F) et le collier de la poche flagellaire (CPF) sont les principaux composants du cytosquelette dutrypanosome. À ce jour, une seule protéine du CPF, BILBO1, a été identifiée et caractérisée.Dans cette étude, nous montrons in vivo que BILBO1 forme des polymères capables deconstruire un échafaudage qui permet l’ancrage de protéines partenaires. Ainsi, un crible en double hybride chez la levure a identifié plusieurs protéines partenaires de BILBO1,notamment une nouvelle protéine appelée FPC4. Nous démontrons que FPC4 est une protéine spécifique des kinétoplastides, localisée au CPF mais aussi au hook-complex, une structure proche du CPF. L’interaction FPC4 – BILBO1 est démontrée in vitro et in vivo, etles domaines d'interaction identifiés. En outre, nous démontrons in vivo et in vitro que FPC4est une protéine associée aux microtubules. Nos données suggèrent fortement que FPC4est impliquée dans le processus de séparation des CPFs au cours du cycle cellulaire. Nos résultats mettent en évidence un lien étroit entre le MtQ et le CPF et l'implication probable duhook-complex. Enfin, nous mettons en évidence une structure analogue au hook-complex chez les Leishmanies. L’interaction BILBO1 – FPC4 représente une nouvelle cible thérapeutique et sera caractérisée plus avant. / Trypanosoma brucei, a unicellular flagellated parasite, is responsible for the human African trypanosomiasis also known as sleeping sickness. Sub-pellicular microtubules (MT), the MT quartet (MtQ), the flagellum (F) and the Flagellar Pocket Collar (FPC) are the main components of the T. brucei cytoskeleton. To date, only a single FPC protein, BILBO1, has been identified and characterized. In this study we demonstrate in vivo that BILBO1 forms polymers able to build a scaffold structure that anchors partner proteins. As such, a yeast-2-hybrid screen identified several BILBO1 interacting protein partners. We demonstrate that FPC4 is a kinetoplastid-specific protein, which is localized at the FPC and at the hook complex. Its specific interaction with BILBO1 has been demonstrated in vitro and in vivo, and the interacting domains identified. Furthermore, we demonstrate that FPC4 is a microtubule binding protein. Our data strongly suggest that FPC4 is involved in the separation of the old and the newly formed FPC during the cell cycle. Altogether, our results demonstrate a tight connection and interplay between the MtQ and the FPC and the likely involvement of an adjacent third structure, the hook complex. Finally, we highlight a structure similar to the hook-complex in Leishmania. The BILBO1 – FPC4 interaction represents a new therapeutic target and will be characterized further.

This thesis primarily focuses on the FoF1-ATP synthase/ATPase complex in the parasitic protist, Trypanosoma brucei. Instead of its normal aerobic function to synthesize ATP, it is required to hydrolyze ATP to maintain the m in the infective bloodstream stage of T. brucei and the related parasite, T. b. evansi. To better understand the composition, structure and function of this druggable target, my work focused on deciphering the function of three of the unique Euglenozoa specific subunits that comprise this complex molecular machine. Furthermore, the ADP/ATP carrier, which provides substrates for the FoF1-ATP synthase/ATPase, was functionally characterized and evaluated if it is physically associated with the complexes of the oxidative phosphorylation pathway.

O SL trans-splicing (do inglês, spliced leader trans-splicing) catalisado pelo spliceossomo em Trypanosoma brucei é responsável pelo processamento dos pré-mRNAs policistrônicos em mRNAs maduros. Esta maquinaria é associada a partir de pequenas partículas ribonucleoproteicas nucleares (snRNPs) U1, U2, U4/U6 e U5 constituídas de pequenos RNAs nucleares (snRNAs), um complexo canônico de sete proteínas Sm (SmB, SmD3, SmD1, SmD2, SmE, SmF e SmG) e fatores proteicos específicos. O núcleo de proteínas Sm de T. brucei apresenta variações com funções desconhecidas, como a substituição do heterodímero SmD3/SmB por Sm16,5K/Sm15K na snRNP U2, e de SmD3 por SSm4 na snRNP U4. Na primeira parte deste trabalho, investigou-se a interação destes diferentes complexos Sm recombinantes com os snRNAs U2, U4 e U5 obtidos por transcrição in vitro. Todos os complexos apresentaram alta afinidade pelo snRNA cognato. Observou-se, ainda, que apenas o núcleo Sm que contém Sm16,5K/Sm15K associado ao snRNA U2 interage com alta afinidade com U2A/U2B. Adicionalmente, foi obtida a estrutura cristalográfica de U2A/U2B de T. brucei, que revela uma organização similar àquela já descrita para ortólogas de Homo sapiens. Entretanto, há um desvio de pelo menos 6 Å no ponto médio da alça carregada positivamente no domínio RRM de U2B para a acomodação do snRNA U2. Além disso, observou-se uma longa hélice-α adicional na extremidade C-terminal de U2A. A análise dos três núcleos Sm de T. brucei a partir da combinação de modelagem molecular e espalhamento de raios-X a baixo ângulo revela estruturas de barril-β altamente torcido com interior carregado positivamente para interação com snRNAs. A principal diferença entre as estruturas encontra-se nas extremidades C- e N-terminal dos variantes de proteínas Sm, possivelmente para interação com U2A no braço 1 do spliceossomo, no caso de Sm15K/Sm16,K, e com U5-220K e U5-200K no corpo do spliceossomo, no caso de SSm4. Na segunda parte deste trabalho, a expressão homóloga da proteína U5-200K de T. brucei completa e do produto truncado no seu cassete helicase/ATPase/Sec63 N-terminal levou à copurificação de um subcomplexo de snRNP U5 composto por U5-220K, U5-116K, U5-40K e U5-Cwc21, sendo que a proteína recombinante completa ainda copurificou as proteínas Sm. Experimentos de imunolocalização mostraram que a proteína U5-200K truncada não é direcionada ao núcleo, como é o caso da proteína completa. As células que expressam a proteína truncada apresentaram um defeito de crescimento significativo, e os processamentos de pré-mRNA por cis- e SL trans-splicing foram ligeiramente afetados, já que a proteína truncada não entra no núcleo, onde deveria exercer sua atividade. Os resultados apresentados indicam a formação de um subcomplexo de snRNP U5 ainda no citoplasma, sendo que as proteínas Sm devem ser um sinal para o seu transporte nuclear mediado por importina-β. Em leveduras, a proteína Aar2 substitui U5-200K no citoplasma, regulando assim a biogênese de snRNP U5, porém esta proteína não foi identificada em T. brucei. Os resultados apresentados neste trabalho contribuem como o primeiro estudo estrutural de proteínas spliceossomais de um parasita do homem e também com novas informações sobre a biogênese das partículas ribonucleoproteicas U2 e U5 de T. brucei. / The spliced-leader (SL) trans-splicing catalyzed by the spliceosome in Trypanosoma brucei is responsible for processing polycistronic pre-mRNAs into mature mRNAs. The spliceosome machinery is assembled by small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 that are composed by small nuclear RNAs (snRNAs), a canonical complex of Sm proteins (SmB, SmD3, SmD1, SmD2, SmE, SmF, SmG) and specific factors. The Sm core peculiarly varies in T. brucei, where SmD3/SmB are replaced by Sm16.5K/Sm15K in U2 snRNP and SmD3 is substituted by SSm4 in U4 snRNP. In the first part of this thesis, we investigated the interaction of the different recombinant Sm cores with U2, U4 and U5 snRNAs obtained by in vitro transcription. All the protein complexes bind the cognate snRNA with high affinity. Only the Sm core that contains Sm16.5K/Sm15K associated with U2 snRNA interacts with the recombinant U2A/U2B subcomplex. Additionally, the crystallographic structure of T. brucei U2A/U2B was obtained, showing an overall organization similar to the one observed in the human counterpart. However, we observed a 6 Å deviation in the medium point of a positively charged turn in the RRM motif of U2B to accommodate U2 snRNA. Besides, a long α -helix was observed in the C-terminal region of U2A. Structural analysis of Sm core variations in T. brucei was proceeded using molecular modelling techniques associated with small angle X-ray scattering. The quaternary structure models show seven Sm proteins as β-barrels with positively charged interior for cognate snRNA interaction. The main difference among these Sm core structures resides in the C- and N-terminal regions of the variant proteins, probably enabling the interaction of Sm15K/Sm16,5K with U2A in the spliceosomes arm 1, and the association of SSm4 with U5-220K and U5-200K in the spliceosomes body. In the second part of this thesis, homologous expression of full-length and N-terminally truncated U5-200K from T. brucei led to the copurification of a U5 snRNP subcomplex containing U5-220K, U5-116K, U5-40K and U5-Cwc21. The full-length U5-200K construct also copurified Sm proteins. Immunolocalization experiments showed that the truncated U5-200K protein is not directed to the nucleus as is the case for the full-length protein. Cells that expressed the truncated protein showed a significant growth defect and the pre-mRNA processing by cis- and SL trans-splicing was negatively affected since the truncated protein did not enter the nucleus where it should be active. The results suggest that a subcomplex of U5 snRNP begins to be assembled in the cytoplasm and the Sm proteins may be the signal for the nuclear transport mediated by β-importin. In yeast, Aar2 replaces U5-200K in the cytoplasm in another regulation step. However, Aar2 has not been identified in T. brucei. The results presented here contribute with the first structural study of spliceosomal proteins of a human parasite and give new insights into the biogenesis of U2 and U5 snRNPs in T. brucei.