No description available.

Le cancer représente l'un des problèmes majeurs en santé publique. Jusqu'à présent, en parallèle de l'intervention chirurgical, plusieurs traitements ont été mis au point et largement utilisés en thérapie clinique telles que les chimiothérapies. Cependant, leur efficacité est parfois limitée et couplée à des effets secondaires très néfastes, laissant les patients dans une impasse thérapeutique. Par conséquent, de nouvelles approches thérapeutiques doivent être développées sur de nouvelles cibles avérées en oncologie afin d'apporter des soins personnalisés aux patients. La première partie de mon travail de thèse a été dédiée à la compréhension des mécanismes moléculaires de la nucléotidase cytosolique de type II (cN-II), une enzyme du métabolisme des purines dont l'implication dans des phénomènes de résistance à des traitements anticancéreux a pu être démontrée. Aussi, une étude sur la cinétique enzymatique à l'état pré-stationnaire et stationnaire a été entreprise sur la forme sauvage et une forme mutée de l'enzyme lui conférant une activité accrue fortement impliquée dans les cas de résistance. Par cette approche, il a été possible de décortiquer le mécanisme cinétique, de définir l'étape cinétiquement limitant afin d'identifier les intermédiaires prépondérants de la réaction pouvant être ciblés pour le développement de nouveaux inhibiteurs. Cette étude cinétique est présentée dans ce premier volet de la thèse. En second lieu, mon travail s'est focalisée sur un second membre de cette famille d'enzyme qui est l'ecto-5'-nucléotidase (CD73). Cette enzyme exprimée sous forme dimérique à la surface extracellulaire régule la concentration en adénosine extracellulaire (par hydrolyse de l'adénosine monophosphate), ce dernier étant un puissant immunosuppresseur de la réponse immune anticancéreuse. L'objectif de mon travail de thèse a été de développer de nouveaux inhibiteurs de type allostérique en utilisant une approche basée sur la structure tridimensionnelle et la dynamique moléculaire. Une des étapes clés a été tout d'abord de mettre au point un système expression hétérologue afin d'obtenir l'enzyme recombinante en quantité suffisante pour les études enzymatiques ultérieures. Différents systèmes d'expression ont été testés et seul le système en cellules d'insecte infectées par le baculovirus a permis d'obtenir l'enzyme active en grande quantité. En parallèle, une étude in silico a permis de reproduire la dynamique fonctionnelle de l'enzyme requise pour sa fonction. A partir de ses données, un criblage virtuel d'une chimiothèque de 324 000 molécules a été réalisé sur le site de dimérisation et a permis d'identifier 33 composés chefs de files. Parmi, ces composés, dix molécules se sont avérés être de puissants inhibiteurs de CD73 (Ki < 1 µM) avec un mécanisme d'inhibition de type allostérique ou non-compétitif. La cytotoxicité des composés a été évaluée sur des lignées cellulaires transformées ou tumorales montrant un effet uniquement à des concentrations très élevées (supérieures à 100 µM). L'étude des relations structure-fonction devrait permettre à présent de proposer de voies d'optimisation afin d'améliorer l'efficacité des composés les plus actifs afin d'aboutir à de nouveaux candidats médicaments. / Cancer burden still remains a major worldwide health problem. To date, several types of conventional anticancer treatments are widely used in clinical. However, the alternative effects of these treatments often leave patients impaired. Therefore, it is required to understand the unique medical needs of individual patients and to conduct effective, high–quality research focusing on the not yet identified oncotargets.The first part of my thesis is dedicated to decipher molecular basis of cN-II reaction. This study characterizes the steady state and transient state kinetics of cN-II wild type and hyperactive mutant which involved in cancer treatment resistance. Furthermore, the characterization of the rate-limiting step and reaction intermediates gave insights into the binding mechanisms and the development of small molecules inhibitors of cN-II.In the second part of this work, we aimed to investigate allosteric inhibitors of CD73 using structure-based drug design approach. In this study the suitable protein expression system was established for the production of sufficient quantities of fully active CD73. This work followed by in silico studies, including molecular dynamics, virtual screening, and hits identification and in vitro hits validations and kinetics characterizations. The cytotoxicity of the most powerful inhibitors exhibited on different cell types was determined. SAR studies gave insights into the binding mode of best compounds and function.

Phosphatases of the haloacid dehalogenase superfamily are one of the cell's tools for dephosphorylation of many diverse endogenous and exogenous compounds. This work is aimed at enzymes Tt82 and cytosolic purine 5'-nucleotidase II (cN-II), two members of this large enzyme superfamily. The Tt82 originates in the hyperthermophilic archaeon Thermococcus thioreducens. Up to date, there is only a small amount of knowledge about properties and biological function of this enzyme. Based on its sequence and structure, it was predicted that the Tt82 should possess a phosphatase catalytic activity. Consequently, potential substrates of the Tt82 were proposed by the molecular docking. In this work, the phosphatase activity of the Tt82 was confirmed together with several of its substrates: AMP, D-glucose 1-phosphate, D-glucose 6-phosphate and p-nitrophenyl phosphate (pNPP). Activity towards AMP and pNPP was then characterized by steady-state kinetics at 37 řC and 60 řC. In consistence with its thermophilic origin, the Tt82 showed markedly higher activity towards both substrates at 60 řC. Nonetheless, the effectivity of the Tt82 catalytic activity towards these substrates was actually very low. This leads to assumption, that the identified substrates are probably not biologically relevant. On the other hand, it is quite...

No description available.

Mon projet de thèse s'inscrit dans un vaste projet de caractérisation de protéines nouvellement identifiées dont l'expression est sélective à certaines régions du cerveau. Cette expression sélective pouvant être liée aux phénomènes de dégénérescence neuronale qui caractérisent les maladies neurodégénératives, ces protéines constituent donc des biomarqueurs potentiels. Une étude structurale et physico-chimique a été effectuée sur une dizaine de protéines, dont la protéine CRYM murine (mCRYM) qui fait parti de la famille des Cytosolic- 3,3',5-triiodo-L-thyronine(T3)-Binding Protein car elle régule la concentration en hormone thyroïdienne T3 libre dans la cellule. mCRYM appartient également à la famille des µ-crystallines et à la superfamille des µ-crystallines/Ornithines Cyclodésaminases. Les protéines présentant des homologies pour ces trois familles sont la plupart différentes par leur fonction (enzymatique ou structurale), leur localisation tissulaire et leurs caractéristiques physico-chimiques. Cette diversité est due au recrutement de gènes de la superfamille des crystallines pour diverses fonctions métaboliques tout en conservant le taxon spécifique des crystallines. Je suis parvenue à résoudre sa structure cristallographique complexée au NADP(H) et à l'hormone thyroïdienne T3 à une résolution de 1,75 Å. La protéine mCRYM est un exemple intéressant d'évolution par son appartenance à différentes familles de protéines et, à ce jour, aucune activité enzymatique n'a été identifiée. Sa caractérisation structurale et thermodynamique a donc permis de mettre en évidence les différences et les similitudes avec ses homologues enzymatiques et d'émettre des hypothèses quant à son évolution moléculaire. Ces résultats soulèvent de nouvelles questions concernant son rôle physiologique : mCRYM est-elle une enzyme ou une protéine structurale ? Comment intervient le couple redox NADPH/NADP+ pour réguler l'action génomique et/ou non génomique de l'hormone T3 ? L'hormone T3 est-il le seul ligand physiologique de CRYM dans le cerveau ?

Dissertation is focused on the application of optical measurement methods using techniques of optical microscopy and fluorescence microscopy in measurements of electromechanical characteristics of isolated cardiac cells and clusters of differentiated cardiomyocytes. The first proposed method uses a practical combination of fluorescence microscopy equipped with fluorescent fast and high-resolution camera and atomic force microscopy for simultaneous measurement of calcium transients and contraction of cardiomyocyte clusters. The signals obtained undergoes filtration, processing and analysis. Result function parameters obtained by analyzing signals after application of caffeine are evaluated by comparison with functional parameters obtained during the control measurement. The second proposed method is applied to the cardiomyocyte clusters for the purpose of cardiomyocyte contraction signals measurement. The signals obtained by optical methods are analyzed and compared with the reference signal obtained using atomic force microscopy. Optical measurement method of cell contractins based on detection of cell ends using adjusting of microscopy images by re-sharpening and fluorescence method for cardiomyocyte contractions measurements were designed to increase realiability in simultaneous measurement of cell contractions simultaneously with calcium transients in isolated cardiomyocytes experiments.

No description available.

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal. / The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal. / The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.