SUMMARY: Cancers represent one of the main causes of death worldwide. Together with surgery and radiotherapy, chemotherapy constitutes a main therapeutic tool in cancer treatment. However, combat remains challenging because of the intrinsic and/ or acquired resistance mechanisms displayed by cancers to these agents. In order to maintain their continuous growth, multiplication and dissemination, cancer cells display a number of biological hallmarks. Growing evidence of the remarkable association of the protein synthesis process with the onset and progression of cancer has led to extensive revision and research on the role of translation in this disease as well as its potential in therapy. Initiation of translation is especially dysregulated in cancer. Thus, strategies targeting different translation steps, ranging from upstream inhibitors - like mTOR inhibitors - to direct inhibition of specific translation initiation factors, represent potential and selective recent alternatives to conventional chemotherapies. In this work, we have investigated the antiproliferative effects of the previously synthetized harmine derivative CM16, with a particular emphasis on its effects on the protein synthesis of cancer cells. We confirmed CM16 cytostatic effects and showed its selectivity towards cancerous cells. The correlation of the growth inhibition profile of CM16 in the NCI 60-cell-line with those of other protein synthesis inhibitors led us to investigate such potential inhibition in vitro. CM16 induced inhibition of protein synthesis and it seems to specifically affect the initiation phase of translation, as it affected the organization of ribosome and polysomes. Phosphorylation on the initiation factor 2α (eIF2α) could be partly responsible for the inhibitory effect observed, as evidenced in this work. Also, the transcriptomic comparison of cell models displaying different levels of sensitivity to CM16 suggested that EIF1AX, EIF3E and EIF3H could drive, at least partly, their sensitivity to this compound. Proteomic study of glioma cells treated or not with CM16 was then conducted. Although the proteins of the genes mentioned above were not identified by this technic, we evidenced tiny but significant changes in Hs683 glioma cell proteomic profile through LC-MS shotgun approach. Thanks to 2-DE gel comparison, proteins differentially expressed in these conditions were identified, such as HspB1, Ebp1, BTF3, galectin, cofilin, dUTPase, PGAM1 and CK-18. These might be involved in the antiproliferative and protein synthesis inhibitory activities of CM16, particularly when considering their roles in cancer cell biology, bringing additional insights to the elucidation of the mechanism of action of this harmine derivative in cancer cells. / RÉSUMÉ: Les cancers figurent parmi les principales causes de mortalité dans le monde. Avec la chirurgie et la radiothérapie, la chimiothérapie reste une des principales manières de lutter contre le cancer. Néanmoins, en raison des mécanismes de résistance intrinsèques et / ou acquis à ces agents, le traitement du cancer reste difficile. Pour assurer leur prolifération, leur dissémination et le développement de la maladie, les cellules cancéreuses présentent certaines caractéristiques biologiques. La mise en évidence de liens remarquables entre la synthèse protéique et l'apparition et la progression du cancer a conduit à une révision et à une recherche plus approfondie de la dérégulation de la traduction au sein des cellules cancéreuses ainsi que de son potentiel en tant que cible thérapeutique. La phase d’initiation de la traduction est particulièrement dérégulée dans le cancer. Ainsi, les stratégies ciblant différentes étapes de la traduction, depuis l’inhibition des voies de signalisation en amont du processus - comme les inhibiteurs de mTOR - à l'inhibition directe des facteurs spécifiques d'initiation de la traduction, représentent de potentielles alternatives sélectives aux chimiothérapies actuelles. Dans le cadre de ce travail, nous avons étudié les effets antiprolifératifs du composé CM16, un dérivé de l'harmine préalablement synthétisé, et, en particulier, ses effets sur la synthèse des protéines des cellules cancéreuses. Nous avons confirmé les effets cytostatiques du composé CM16 et avons montré sa sélectivité vis-à-vis des cellules cancéreuses. Le profil de réponse des 60 lignées cellulaires cancéreuses du panel du NCI s’est avéré corréler avec ceux d'autres inhibiteurs connus de la synthèse protéique, ce qui nous a conduits à investiguer in vitro cette potentielle inhibition. Le CM16 inhibe la synthèse protéique et semble affecter spécifiquement la phase d'initiation de la traduction étant donné que nous avons observé une désorganisation des ribosomes et polysomes. L’induction de la phosphorylation du facteur d'initiation 2α (eIF2α) pourrait en partie être responsable de l'effet inhibiteur de la synthèse protéique. La comparaison transcriptomique des modèles du NCI présentant des degrés divers de sensibilité au CM16 suggère que EIF1AX, EIF3E et EIF3H puissent, au moins en partie, être impliquées dans la sensibilité des cellules cancéreuses au composé CM16. Nous avons ensuite réalisé une étude du profil protéomique des cellules de gliomes traitées ou non par le CM16. Bien que les cibles identifiées ci-dessus n’ont pu être identifiées par cette technique, de légères mais significatives différences dans le protéome des cellules de gliomes traitées avec le CM16 ont été mises en évidence par LC-MS shotgun. Grâce à étude comparative de gels en deux dimensions, des protéines différentiellement exprimées dans ces conditions ont été identifiées, telles que HspB1, Ebp1, BTF3, galectine 1, cofiline, dUTPase, PGAM1 et CK-18. Celles-ci pourraient être impliquées dans les effets antiprolifératifs et inhibiteurs sur la synthèse protéique induits par le CM16, notamment suite à leurs rôles dans la biologie tumorale, contribuant ainsi à l'élucidation du mécanisme d'action de ce dérivé harmine dans les cellules cancéreuses. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

A doença de Chagas é um problema de saúde pública, com dados preocupantes referentes ao número de pessoas contaminadas e daquelas que ainda permanecem expostas ao risco de infecção. A dificuldade do combate a Trypanosoma cruzi, agente etiológico da doença, está intimamente relacionada às interações existentes entre o parasito e o hospedeiro, sendo que até o momento, nenhum medicamento ou substância tem demonstrado real eficácia ao combate ao parasito. Em estudos recentes realizados por nosso grupo de pesquisa, os ácidos ursólico e oleanólico demonstraram um bom potencial tripanocida, além de um efeito imunomodulatório, promovendo a inibição da produção de IFN-? quando de suas utilizações por via intraperitoneal em elevadas concentrações. Considerando essas evidências encontradas em relação a essas substâncias e os efeitos promovidos no processo de avaliação biológica, propusemos como objetivo avaliar o potencial terapêutico dos triterpenos ácido ursólico e ácido oleanólico na fase crônica da infecção chagásica e suas associações ao benzonidazol, fármaco referência indicado ao tratamento da parasitose, verificando a possibilidade de geração de benefícios sobre a patogênese da infecção crônica experimental. A quantificação de linfócitos T CD4+ e T CD8+, e das citocinas IL-2, IL-10, IL-12, IFN-? e TNF-??pela técnica de citometria de fluxo, utilizando o kit BD Cytometric Bead Array®, indicaram que essas substâncias não apresentam efeitos imunomodulatórios significativos sobre a resposta Th1 e Th2 nessa fase da doença, na comparação entre grupos infectados e tratados e aquele que recebeu apenas solvente. O parasitismo tecidual determinado por Real Time PCR e as observações realizadas em cortes histológicos, mostraram, respectivamente, baixo número de cópias de DNA de T. cruzi, e ausência de ninhos amastigotas, porém, com marcante presença de infiltrado inflamatório em todos os grupos. Assim, os dados obtidos levam à conclusão de que apesar da atividade apresentada pelos triterpenos ácidos em estudo na fase aguda da doença de Chagas, o mesmo não ocorre na fase crônica, como seria desejável para favorecer uma melhora do quadro patológico, mesmo não havendo a cura da doença, considerando as conseqüências da infecção de longo prazo. / Chagas disease is a public health problem, with disturbing data about the number of infected people and those who remain at risk of infection. The difficulty of eliminating Trypanosoma cruzi, etiologic agent of the disease is closely related to the interactions between the parasite and the host, and until now, no medicine or substance has demonstrated real effectiveness against the parasite. In recent studies by our research group, the ursolic and oleanolic acids revealed considerable trypanocidal activity, and an immunomodulatory effect, promoting the inhibition of IFN-? with intraperitoneal administration in high concentrations. Considering these evidences and the effects observed during the biological evaluation, our objective was to evaluate the biological potential of the triterpenoids ursolic acid and oleanolic acid in the chronic phase of chagasic infection and their associations to benznidazole, the reference drug indicated for the treatment of this disease, verifying the possibility of benefits on the pathogenesis of experimental chronic infection. Quantification of T CD4+ and T CD8+ cells and of the cytokines IL-2, IL-10, IL-12, IFN-? and TNF-? by flow cytometry using the kit BD cytometric Bead Array®, indicated that these substances do not exhibit significant immunomodulatory effects on Th1 and Th2 responses in this stage of the disease, comparing infected and treated groups and that who received only solvent. The tissue parasitism determined by Real Time PCR and in histological observations showed, respectively, low copy number of T. cruzi DNA, and absence of amastigote nests, however, with marked inflammatory infiltration in all groups. Thus, our data lead to the conclusion that despite the activity presented by the triterpene acids studied in the acute phase of Chagas disease, the same does not occur in the chronic phase, as would be desirable to the reduction of the pathological conditions, even if there is no cure of the disease, considering the consequences of long term infection.

A doença de Chagas é um problema de saúde pública, com dados preocupantes referentes ao número de pessoas contaminadas e daquelas que ainda permanecem expostas ao risco de infecção. A dificuldade do combate a Trypanosoma cruzi, agente etiológico da doença, está intimamente relacionada às interações existentes entre o parasito e o hospedeiro, sendo que até o momento, nenhum medicamento ou substância tem demonstrado real eficácia ao combate ao parasito. Em estudos recentes realizados por nosso grupo de pesquisa, os ácidos ursólico e oleanólico demonstraram um bom potencial tripanocida, além de um efeito imunomodulatório, promovendo a inibição da produção de IFN-? quando de suas utilizações por via intraperitoneal em elevadas concentrações. Considerando essas evidências encontradas em relação a essas substâncias e os efeitos promovidos no processo de avaliação biológica, propusemos como objetivo avaliar o potencial terapêutico dos triterpenos ácido ursólico e ácido oleanólico na fase crônica da infecção chagásica e suas associações ao benzonidazol, fármaco referência indicado ao tratamento da parasitose, verificando a possibilidade de geração de benefícios sobre a patogênese da infecção crônica experimental. A quantificação de linfócitos T CD4+ e T CD8+, e das citocinas IL-2, IL-10, IL-12, IFN-? e TNF-??pela técnica de citometria de fluxo, utilizando o kit BD Cytometric Bead Array®, indicaram que essas substâncias não apresentam efeitos imunomodulatórios significativos sobre a resposta Th1 e Th2 nessa fase da doença, na comparação entre grupos infectados e tratados e aquele que recebeu apenas solvente. O parasitismo tecidual determinado por Real Time PCR e as observações realizadas em cortes histológicos, mostraram, respectivamente, baixo número de cópias de DNA de T. cruzi, e ausência de ninhos amastigotas, porém, com marcante presença de infiltrado inflamatório em todos os grupos. Assim, os dados obtidos levam à conclusão de que apesar da atividade apresentada pelos triterpenos ácidos em estudo na fase aguda da doença de Chagas, o mesmo não ocorre na fase crônica, como seria desejável para favorecer uma melhora do quadro patológico, mesmo não havendo a cura da doença, considerando as conseqüências da infecção de longo prazo. / Chagas disease is a public health problem, with disturbing data about the number of infected people and those who remain at risk of infection. The difficulty of eliminating Trypanosoma cruzi, etiologic agent of the disease is closely related to the interactions between the parasite and the host, and until now, no medicine or substance has demonstrated real effectiveness against the parasite. In recent studies by our research group, the ursolic and oleanolic acids revealed considerable trypanocidal activity, and an immunomodulatory effect, promoting the inhibition of IFN-? with intraperitoneal administration in high concentrations. Considering these evidences and the effects observed during the biological evaluation, our objective was to evaluate the biological potential of the triterpenoids ursolic acid and oleanolic acid in the chronic phase of chagasic infection and their associations to benznidazole, the reference drug indicated for the treatment of this disease, verifying the possibility of benefits on the pathogenesis of experimental chronic infection. Quantification of T CD4+ and T CD8+ cells and of the cytokines IL-2, IL-10, IL-12, IFN-? and TNF-? by flow cytometry using the kit BD cytometric Bead Array®, indicated that these substances do not exhibit significant immunomodulatory effects on Th1 and Th2 responses in this stage of the disease, comparing infected and treated groups and that who received only solvent. The tissue parasitism determined by Real Time PCR and in histological observations showed, respectively, low copy number of T. cruzi DNA, and absence of amastigote nests, however, with marked inflammatory infiltration in all groups. Thus, our data lead to the conclusion that despite the activity presented by the triterpene acids studied in the acute phase of Chagas disease, the same does not occur in the chronic phase, as would be desirable to the reduction of the pathological conditions, even if there is no cure of the disease, considering the consequences of long term infection.