Submitted by Chaylane Marques (chaylane.marques@ufpe.br) on 2015-03-12T19:27:55Z No. of bitstreams: 2 Dissertação de Mestrado - Janaína - Versão final biblioteca.pdf: 2425301 bytes, checksum: f7164df63d1331f1d568db3ea2c93c22 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T19:27:55Z (GMT). No. of bitstreams: 2 Dissertação de Mestrado - Janaína - Versão final biblioteca.pdf: 2425301 bytes, checksum: f7164df63d1331f1d568db3ea2c93c22 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-05-14 / FACEPE; CNPQ / Os tripanossomatídeos são protozoários unicelulares que apresentam uma biologia molecular bastante divergente dos demais eucariotos e são responsáveis por doenças negligenciadas que afetam milhares de pessoas. A carência de drogas mais eficazes contra estes patógenos justifica a busca da compreensão dos seus processos celulares. Um desses processos é a síntese proteica que, nos eucariotos, é dividida em quatro etapas, sendo a primeira, a iniciação, a mais complexa. A iniciação da tradução é dependente dos fatores de iniciação eucarióticos (eIFs), dentre os quais destaca-se o complexo eIF4F, cuja subunidade eIF4E é responsável pelo reconhecimento do cap presente na extremidade 5’ do mRNAs. Em Leishmania major e Trypanosoma brucei foram identificados seis homólogos conservados de eIF4E, sendo os EIF4E5 e 6 identificados mais tardiamente. O presente trabalho buscou contribuir com a caracterização funcional destas proteínas, iniciando-se com uma análise comparativa das suas sequências onde foram identificados motivos conservados presentes em outros homólogos de eIF4E. Em T. brucei, experimentos de localização subcelular mostraram que ambas as proteínas são citoplasmáticas, porém com alguma localização nuclear. Utilizando RNAi na fase procíclica do parasita, somente TbEIF4E5 mostrou-se essencial a viabilidade celular enquanto que as células depletadas do TbEIF4E6 apresentaram alterações morfológicas. Através da purificação de fosfoproteínas constatou-se que ambos os fatores são fosforilados e a predição in silico dos possíveis sítios revelou a presença de múltiplos resíduos passíveis de fosforilação. Já em L. major seus respectivos homólogos foram expressos em bactéria e utilizados para a produção de soros policlonais. Esses soros foram capazes de detectar a expressão do LmEIF4E5 em extratos de formas promastigotas de Leishmania enquanto para LmEIF4E6 não foi observada expressão compatível com o peso molecular esperado. Os resultados e as ferramentas moleculares produzidas contribuem tanto para a compreensão da função desempenhada por estas proteínas, como para o conhecimento do processo de tradução nos tripanossomatídeos.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T19:22:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-JanaínaNascimento.pdf: 2428494 bytes, checksum: 1f074c8519d239e65f167723cc34b5cf (MD5) / Made available in DSpace on 2017-04-06T19:22:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-JanaínaNascimento.pdf: 2428494 bytes, checksum: 1f074c8519d239e65f167723cc34b5cf (MD5) Previous issue date: 2012-05-14 / Os tripanossomatídeos são protozoários unicelulares que apresentam uma biologia molecular bastante divergente dos demais eucariotos e são responsáveis por doenças negligenciadas que afetam milhares de pessoas. A carência de drogas mais eficazes contra estes patógenos justifica a busca da compreensão dos seus processos celulares. Um desses processos é a síntese proteica que, nos eucariotos, é dividida em quatro etapas, sendo a primeira, a iniciação, a mais complexa. A iniciação da tradução é dependente dos fatores de iniciação eucarióticos (eIFs), dentre os quais destaca-se o complexo eIF4F, cuja subunidade eIF4E é responsável pelo reconhecimento do cap presente na extremidade 5’ do mRNAs. Em Leishmania major e Trypanosoma brucei foram identificados seis homólogos conservados de eIF4E, sendo os EIF4E5 e 6 identificados mais tardiamente. O presente trabalho buscou contribuir com a caracterização funcional destas proteínas, iniciando-se com uma análise comparativa das suas sequências onde foram identificados motivos conservados presentes em outros homólogos de eIF4E. Em T. brucei, experimentos de localização subcelular mostraram que ambas as proteínas são citoplasmáticas, porém com alguma localização nuclear. Utilizando RNAi na fase procíclica do parasita, somente TbEIF4E5 mostrou-se essencial a viabilidade celular enquanto que as células depletadas do TbEIF4E6 apresentaram alterações morfológicas. Através da purificação de fosfoproteínas constatou-se que ambos os fatores são fosforilados e a predição in silico dos possíveis sítios revelou a presença de múltiplos resíduos passíveis de fosforilação. Já em L. major seus respectivos homólogos foram expressos em bactéria e utilizados para a produção de soros policlonais. Esses soros foram capazes de detectar a expressão do LmEIF4E5 em extratos de formas promastigotas de Leishmania enquanto para LmEIF4E6 não foi observada expressão compatível com o peso molecular esperado. Os resultados e as ferramentas moleculares produzidas contribuem tanto para a compreensão da função desempenhada por estas proteínas, como para o conhecimento do processo de tradução nos tripanossomatídeos. / Trypanosomatids are unicellular protozoans characterized by a divergent molecular biology, when compared to other eukaryotes. They are responsible for neglected diseases which affect thousands of people and the lack of more effective drugs against these diseases justifies a better understanding of their basic processes. One of these is protein synthesis (translation), formally divided into four stages, the first of which, initiation, is the most complex. Translation initiation is entirely dependent on the “eukaryotic Initiation Factors” (eIFs), such as the complex eIF4F. eIF4E is an eIF4F subunit which is responsible for the recognition of the cap on the 5’ end of mRNAs. Six conserved eIF4E homologues were found in Leishmania major and Trypanosoma brucei, with EIF4E5 and 6 being the latest ones identified. This work aimed to contribute with the functional characterization of these proteins, starting with a comparative analysis of their sequences where conserved motifs also found in other eIF4E homologues were identified. In T. brucei, subcellular localization experiments indicated that both proteins are predominantly cytoplasmic, but with minor nuclear localization. Using RNAi in the procyclic stage, only TbIF4E5 proved to be essential for cell viability, while cells depleted of TbEIF4E6 showed morphological abnormalities. Through phosphoprotein affinity purification, both proteins were observed to be phosphorylated, and the in silico prediction of possible phosphorylation sites revealed the presence of multiple putative sites. Regarding the L. major homologues, the corresponding proteins were expressed in bacteria and used to produce polyclonal antisera which were able to detect the expression of LmEIF4E5 in Leishmania promastigotes. However the expression observed for LmEIF4E6 is not consistent with its expected molecular weight. The results and molecular tools produced in this work contribute not only to the understanding of the functional role of these proteins, but also to the knowledge of the translation process in trypanosomatids.

Made available in DSpace on 2014-06-12T18:01:41Z (GMT). No. of bitstreams: 2 arquivo3642_1.pdf: 6130351 bytes, checksum: 0d75c1901369305978b575398eefcd51 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os tripanossomatídeos são caracterizados por processos moleculares atípicos como o controle pós-transcricional da expressão gênica, através da degradação de mRNA e controle da tradução. Na maioria dos eucariotos, a iniciação da tradução começa com a ligação do complexo eIF4F (composto pelas subunidades eIF4A, eIF4E e eIF4G) ao cap monometilado presente na região 5 dos mRNAs, permitindo seu reconhecimento pelo ribossomo e outros eIFs participantes. A atividade do eIF4F é reforçada pela proteína de ligação a cauda poli-A (PABP), que atua através de uma interação direta com o eIF4G. Vários homólogos às subunidades do eIF4F e PABP foram previamente identificados nos tripanossomatídeos, como cinco homólogos do eIF4G (EIF4G1-5) e múltiplos homólogos das demais subunidades (EIF4E1-4, EIF4AI e III e PABP1-2) e suas funções vem sendo investigadas em Leishmania major e Trypanosoma brucei. Como parte deste estudo, nós demonstramos que tanto o LmEIF4G3 como o LmEIF4G4 interagem com o LmEIF4AI, embora mutações nos aminoácidos LNK destas proteínas abolam suas interações com o LmEIF4AI. As proteínas LmEIF4G3 e LmEIF4G4 também se ligam respectivamente aos homólogos LmEIF4E4 e LmEIF4E3 e diferentes aminoácidos foram associados a estas interações. A substituição dos resíduos FSL no LmEIF4G3 abole sua interação com o LmEIF4E4 e a mutagênese dos resíduos IL no LmEIF4G4 abole sua ligação ao LmEIF4E3. Adicionalmente, nós demonstramos que as proteínas LmEIF4G3 e LmEIF4G4 ligam-se à LmPABP1 e nós também sugerimos funções distintas para o TbEIF4AI e TbEIF4AIII em ambas as formas procíclica e sanguínea de T. brucei. Em síntese, nossos resultados indicam uma maior complexidade na iniciação da tradução nos tripanossomatídeos e provavelmente a mesma é regulada por pelo menos dois diferentes complexos eIF4F

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T15:47:35Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Gustavo Barbosa de Lima PPGG UFPE 2016.pdf: 2227933 bytes, checksum: 03616701d6dbf5994db493d4eb7b243c (MD5) / Made available in DSpace on 2017-07-12T15:47:35Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Gustavo Barbosa de Lima PPGG UFPE 2016.pdf: 2227933 bytes, checksum: 03616701d6dbf5994db493d4eb7b243c (MD5) Previous issue date: 2016-03-09 / A família de protozoários tripanosomatídeos apresenta características moleculares diferenciadas dos demais eucariotos, onde a regulação da expressão gênica é feita principalmente em nível pós-transcricional. Como em outros eucariotos, acredita-se que a iniciação da tradução seja uma etapa crítica de controle pós-transcricional, onde atuam diferentes fatores de iniciação da tradução (eIFs). Nesta etapa os pontos centrais são o reconhecimento do mRNA maduro e o recrutamento do ribossomo para dar início ao processo, atividades realizadas pelo complexo eIF4F, formado por três subunidades: eIF4E, eIF4A e eIF4G. Nos tripanosomatídeos foram descritos seis homólogos de eIF4E, a proteína de ligação ao cap. Dois destes, EIF4E3 e EIF4E4, participam da formação de complexos envolvidos no processo de tradução e outros dois, EIF4E5 e EIF4E6, participam de novos complexos de função desconhecida. Destes quatro homólogos, o EIF4E4 já foi caracterizado, de forma que o presente trabalho visa contribuir para o entendimento da importância dos demais (EIF4E3, EIF4E5 e EIF4E6) na viabilidade e taxa de crescimento celular de Leishmania sp. Construções gênicas foram geradas de forma a permitir a deleção das duas cópias gênicas de cada proteína por meio da transfecção de Leishmania e seleção com antibióticos. As três proteínas, e mutantes do EIF4E3, foram ainda expressas em parasitas transgênicos para a realização de experimentos de complementação. Os resultados mostram que os baixos níveis de expressão de EF4E5 e EIF4E6 indicam que as três proteínas parecem ser importantes para a viabilidade celular com funções não sobrepostas. Sítios específicos no EIF4E3 foram também identificados de forma isolada como essenciais para a função da proteína e críticos para a sobrevivência do organismo. Os resultados obtidos neste trabalho mostram a importância do estudo do papel destes homólogos de eIF4E na síntese protéica, assim com seu papel na biologia celular de tripanossomatídeos. / The Trypanosomatid of protozoans display distinct molecular features not seen in other eukaryotes, where the regulation of gene expression is mainly performed at the post-transcriptional level. As in other eukaryotes, it is believed that the initiation of translation is a critical stage of post-transcriptional control, where different initiation factors (eIFs) are active. At this stage, critical steps are the recognition of the mature mRNA and the ribosome recruitment to start the process, activities of the eIF4F complex, consisting of three subunits: eIF4E, eIF4A and eIF4G. In trypanosomatids, six homologues of eIF4E, the cap binding protein, have been described. Two of these, EIF4E3 and EIF4E4, participate in the formation of complexes involved in the translation process and two others, EIF4E5 and EIF4E6, participate in new complexes of unknown function. Among these four homologues, EIF4E4 has been better characterized, so that the present work aims to contribute to the understanding of the remaining homologues (EIF4E3, EIF4E5 and EIF4E6) and study their importance for cell viability and growth rate of Leishmania species. Gene constructs were generated to allow deletion of the two gene copies of each protein by transfection of Leishmania cells and selection with antibiotics. The three proteins and EIF4E3 mutants were also expressed in transgenic parasites in order to carry out complementation experiments. The results show low levels of expression of EF4E5 and EIF4E6 and indicate that the three proteins appear to be important for cell viability with non-overlapping functions. EIF4E3 specific residues were also identified which are essential for the protein to the function and critical for the survival of the organism. The results of this study highlight the importance of the study on the role of eIF4E homologues in protein synthesis, as well as their role for the trypanosomatids cell biology.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T16:05:41Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação ADALUCIA DA SILVA PPGG 2016.pdf: 2174365 bytes, checksum: e3305d6246515bbc247cd29783f8b265 (MD5) / Made available in DSpace on 2017-07-12T16:05:41Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação ADALUCIA DA SILVA PPGG 2016.pdf: 2174365 bytes, checksum: e3305d6246515bbc247cd29783f8b265 (MD5) Previous issue date: 2016-03-07 / FACEPE / Os tripanossomatídeos são parasitas flagelados causadores de diversas doenças humanas e que apresentam algumas características moleculares bem distintas. Nestes organismos, o controle da sua expressão gênica é quase exclusivamente pós-transcricional e dados obtidos até o momento sugerem que a etapa de iniciação da tradução tem um papel importante neste controle. Durante a iniciação da tradução de eucariotos, o complexo trimérico eIF4F (formado pelas subunidades eIF4A, eIF4G e eIF4E) tem uma participação importante no reconhecimento do mRNA, facilitando o recrutamento ribossomal para iniciar a síntese proteica. Múltiplos homólogos das subunidades do complexo eIF4F foram identificados em tripanossomatídeos, mas não foi possível elucidar a função específica de cada um deles. Desta forma, este trabalho teve como objetivo o desenvolvimento de um ensaio in vivo para a avaliação do efeito da superexpressão desses homólogos de Trypanosoma brucei na tradução de um mRNA repórter, o qual codifica a proteína GFP (green fluorescent protein). Três linhagens repórter foram obtidas (4212, 4213 e 4235) e sete homólogos das subunidades do complexo eIF4F e um de PABP foram testados nestas linhagens. A análise da superexpressão foi feita através de ensaios de Western blot, seus perfis de crescimento foram avaliados por curvas de crescimento e a expressão de GFP foi avaliada através de citometria de fluxo. Os resultados apresentados mostram que as linhagens repórter 4213 e 4235 são viáveis para serem utilizadas na caracterização das proteínas envolvidas no controle da expressão gênica de tripanossomatídeos. Foi observado que as proteínas EIF4E1 e EIF4E2 provocam diminuição do crescimento, enquanto a superexpressão de EIF4E3, EIF4E4, EIF4E4W279A, EIF4G3, EIF4G4 e PABP1 não alteram o crescimento celular dos parasitas. Apesar da detecção de variações na expressão de GFP durante a superexpressão de alguns dos homólogos testados, não foi possível, contudo, confirmar que estas proteínas aumentam ou diminuem a tradução. Ensaios complementares ainda precisam ser feitos para confirmar a real função destes. / The trypanosomatids are flagellated parasites responsible for several human diseases and which display unique molecular characteristics. Control of gene expression in these organisms is almost exclusively post-transcriptional and the data generated so far suggest that the initiation stage of translation plays an important role in this control. During translation initiation in eukaryotes, the trimeric complex eIF4F (formed by the eIF4A eIF4G and eIF4E subunits) has a relevant role in mRNA recognition and facilitates ribosomal recruitment to start the protein synthesis. Multiples homologues for the eIF4F subunits have been identified in trypanosomes, but it has not been possible to elucidate their specific functions. Thus, this study aimed to develop an in vivo assay to evaluate the effect of the overexpression of these Trypanosoma brucei homologues during the translation of a reporter mRNA encoding for the green fluorescent protein (GFP). Three reporter strains were generated (4212, 4213 and 4235) in which seven homologues of eIF4F subunits and one of their PABP partner were overexpressed. Confirmation of overexpression was carried out by Western blot assays, growth profiles were evaluated by cell counting and GFP expression was assessed by flow cytometry. The results generated show that the reporter lines 4213 and 4235 are feasible for use in the characterization of proteins involved in the control of trypanosomatids gene expression. Overexpression of EIF4E1 and EIF4E2 was seen to induce a reduction in cell growth, while EIF4E3, EIF4E4, EIF4E4W279A, EIF4G3, EIF4G4 and PABP1 did not interfere with growh. Despite the detection of variations in GFP expression during overexpression of some of the homologues tested it was not possible to confirm if these proteins increase or decrease translation. Complementary assays still need to be done to confirm the true function of these factors.

Le mélanome métastatique est un des cancers les plus agressifs avec une croissance constante du nombre de nouveaux cas par an dans le monde.Deux mutations sont principalement à l’origine de ce cancer : BRAF(V600) et NRAS(Q61). Les principaux traitements sont des thérapies ciblant BRAF lui-même ou MEK, prescrites en monothérapie ou en combinaison. La moitié des patients répondent à ce traitement mais malheureusement rechutent dans les 6 mois à 1 an après le début du traitement. Les mécanismes de résistance décrits passent par une réactivation de la voie des MAPK, de la voie PI3K/Akt/mTOR ou par une dérégulation de l’apoptose.Les deux premières voies de signalisation permettent la régulation d’un complexe : le complexe d’initiation de la traduction eIF4F. Ce complexe est composé de 3 protéines : eIF4E, une protéine qui s’associe à la coiffe 7-methyl-guanosine, eIF4A une hélicase et eIF4G une protéine échafaudage dont un des rôles est de maintenir la cohésion de ce complexe.Le complexe eIF4F étant en aval de deux voies dérégulées dans le mélanome, le but de ma thèse a été de mettre en évidence une potentielle implication de ce complexe dans la résistance aux thérapies ciblant BRAF et MEK.Pour cela, dans un premier temps, nous avons traiter des lignées cellulaires de mélanome BRAF(V600) sensibles et résistantes aux anti-BRAF et anti-MEK. Nous avons ensuite quantifié les interactions eIF4E-eIF4G, synonymes d’activation du complexe eIF4F, et les interactions eIF4E-4EBP1, synonymes d’inhibition de la traduction par une technique de Proximity Ligation Assay (PLA). Ces expériences ont permis de conclure que les inhibiteurs de BRAF et de MEK induisaient une dissociation du complexe dans les lignées sensibles, alors qu’il reste maintenu dans les lignées résistantes. Ces résultats ont été confirmés sur des tumeurs de patients répondeurs et non répondeurs à ces mêmes traitements. Par la suite, nous avons reproduits ces expériences avec un panel de lignées cellulaires mutées en NRAS(Q61) et avons observé les mêmes résultats.Nous avons donc pu mettre en évidence l’implication du complexe eIF4F dans la résistance aux thérapies ciblant la voie des MAPK dans les mélanomes mutés en BRAF et en NRAS, mais également découvert une nouvelle cible thérapeutique potentielle.La seconde partie de cette thèse a consisté au test de plusieurs inhibiteurs connus et spécifiques d’eIF4A (silvestrol, flavaglines, hippuristanol, patéamine A) ou de l’interaction eIF4E-eIF4G (4EGI1). Tous ces inhibiteurs ont sensibilisé les lignées résistantes aux thérapies ciblées mais aucun ne pouvait être potentiellement utilisable en clinique.L’utilisation d’un vecteur bicistronique dans le cadre d’un criblage de drogue a permis d’identifier 4 flavaglines qui inhibent significativement le complexe d’initiation de la traduction. L’une d’entre elles (FL3) a été testé in vivo dans un modèle de xénogreffes issues de lignées cellulaires de mélanomes résistantes. Les résultats ont montré un effet synergique de cette drogue en combinaison avec le vemurafenib (anti-BRAF) ainsi qu’une inhibition de la croissance tumorale. En conclusion, nous avons identifié une nouvelle cible thérapeutique et un nouveau biomarqueur de résistance aux thérapies ciblées dans deux contextes mutationnels de mélanome différents : BRAF et NRAS. / In BRAF(V600) or NRAS(Q61)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS–RAF–MEK–ERK mitogen-activated protein kinase (MAPK) signal transduction pathway or on activation of the alternative PI(3)K–AKT–mTOR pathway (which is ERK independent). These two pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex. By using an in situ method to detect the eIF4E-eIF4G and eIF4E-4EBP1 interactions, we recently showed that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF and/or anti-MEK in BRAF(V600)-mutant cancer cell lines. We next focused on NRAS-mutant cancer cell lines and found that this complex is also involved in the resistance to anti-MEK compounds. Strikingly, inhibiting the eIF4F complex in BRAF or NRAS-mutated cell lines is able to overcome resistance and to synergize with drugs targeting BRAF or MEK kinases. As a result, eIF4F appears to be a promising therapeutic target in a BRAF or NRAS-mutation context.

Les lymphomes diffus à grandes cellules B (DLBCL) sont la forme de lymphomes non–Hodgkiniens agressifs la plus fréquente chez l’adulte, et sont très hétérogènes à la fois sur le plan biologique et clinique. Bien que plus de la moitié des patients peuvent être guéris avec le traitement standard R-CHOP (combinant la polychimiothérapie CHOP aux anti-CD20 comme le rituximab) 30 à 40 % des patients échappent ou sont réfractaires au traitement, déterminant des morbidités et mortalités importantes liées au nombre limité d’alternatives thérapeutiques. L’objectif des travaux de cette thèse était centré sur les mécanismes de résistance thérapeutique dans ces lymphomes, et plus particulièrement les résistances au rituximab. Dans une première partie de la thèse, nous avons étudié le rôle de facteurs endogènes neuropeptidiques, les neurotrophines (NTs), et l’implication de leur signalisation dans la survie des cellules tumorales et leur sensibilité à l’apoptose induite par le rituximab. Nous avons montré dans un premier temps que les cellules B tumorales des patients présentaient des taux parfois élevés de neurotrophines (NGF, BDNF) et de leurs récepteurs de haute (Trk) et de basse affinité p75NTR. Puis les résultats obtenus in vitro sur des lignées cellulaires humaines de DLBCL et in vivo (xénogreffes tumorales) ont mis en évidence l’existence d’un axe de survie BDNF/TrkB/p75NTR pouvant interférer avec l’efficacité de l’immunothérapie. Cet axe contribue à la survie des cellules tumorales et pourrait aussi participer à la résistance thérapeutique aux anti-CD20 en modulant l’expression du CD20 à la surface des exosomes. En effet ces microvésicules, sécrétées en grande quantité par les cellules B tumorales, expriment le CD20 à leur surface et seraient à ce titre impliquées dans l’échappement thérapeutique en réalisant des « récepteurs –leurres » vis-à-vis des anticorps thérapeutiques. Dans une 2e partie de cette thèse, nous avons évalué dans les DLBCL le rôle potentiel de nouvelles cibles émergentes en oncologie, les prohibitines (PHBs) et le facteur d’initiation de la traduction eIF4A. Pour cela, nous avons utilisé un ligand de ces acteurs cellulaires de la famille des flavaglines, le FL3. Nous avons montré que le FL3 présente un effet apoptotique très important in vitro sur les lignées cellulaires de DLBCL et in vivo sur les tumeurs induites chez la souris. Nos travaux ont permis d’en préciser les mécanismes moléculaires, mettant en évidence le rôle des PHBs en lien notamment avec l’activation d’Erk1/2, et la formation et l’activité du complexe eIF4F dans la survie des cellules de DLBCL. Les données préliminaires obtenues à partir des biopsies de patients montrent, de plus, que la forte expression de PHB1 pourrait avoir une valeur pronostique dans ces lymphomes. L’ensemble de nos travaux ont permis de mettre en évidence de nouvelles voies de survie et d’échappement thérapeutique dans les DLBCL, qui pourraient permettre d’identifier aussi de nouveaux marqueurs biologiques à valeur diagnostique et/ou pronostique pour le choix de thérapies ciblées. / Diffuse large B cell Lymphomas (DLBCL) are the most aggressive and heterogeneous biological and clinical form of non-Hodgkin lymphomas in adults. Although more than 50% of patients can be cured with standard therapy R-CHOP (combining the CHOP chemotherapy to the anti-CD20 as rituximab) 30 to 40% of patients exhibit primary refractory disease or relapse after initial response to therapy, determining morbidities and significant mortality related to the limited number of treatment options. The aim of this thesis was focussed on therapeutic resistances of these lymphomas, notably those of rituximab. In the first part of this thesis, we have evaluated the role of endogenous factor signaling, the neurotrophins (NTs), in DLBCL cell survival and sensitivity to the cytotoxicity of rituximab. We showed first that a high expression of neurotrophines (NGF, BDNF) and their high (Trk) and low (p75NTR) affinity receptors was often found in tumor B cells of DLBCL patients. Results obtained in vitro, on human cell lines of DLBCL, but also in vivo (xenografts) showed evidence of a survival BDNF/TrkB/p75NTR axis that can interfere with the efficacy of immunotherapy. This axis promotes survival of tumor cells and may also participate in rituximab resistance in regulating CD20 expression at the surface of exosomes. Indeed, these microvesicles, secreted in large amounts by the tumoral B cells, express the CD20 and would be involved in the therapeutic escape acting as decoy receptors upon rituximab exposure. In the second part of the thesis, we evaluated in DLBCL the potential role of new oncogenic targets, PHBs proteins and the initiation factor of translation eIF4A. To this end, we used one of their ligands, a synthetic flavagline named FL3. We showed that FL3 determines a strong apoptosis in vitro on DLBCL cell lines and in vivo on tumors induced in mice (xenografts). Our works have clarified the molecular mechanisms, demonstrating involvement of PHBs, in correlation with ERK1/2 activation, and eIF4F complex formation and activity. Preliminary data obtained in patient biopsies showed a high expression of PHB1 in tumor B cells that may be decisive for cell survival and patient outcome lymphomas.Overall, present results show evidence of new survival and rituximab escape mechanisms in DLBCL, that should allow to identify new diagnostic and prognostic biomarkers for alternative therapeutic options.

No description available.

L’ARN hélicase à boite DEAD de la levure S.cerevisiae Ded1 est une protéine essentielle dont la fonction a été conservée au cours de l’évolution. Ses homologues fonctionnels sont impliqués dans le développement et le cycle cellulaire. Ded1 a longtemps été associée à l’étape de scanning de la région 5’UTR des ARNm au niveau de l’initiation de la traduction. Nous avons utilisé différentes approches comme les co-immunoprécipitations, des analyses de spectrométrie de masse, des tests de complémentation génétique, de séparation des complexes sur gradients de saccharose, des expériences de localisation in situ et d’enzymologie pour montrer que Ded1 interagissait physiquement avec des complexes cytoplasmique et nucléaire de liaison à la coiffe des ARNm. Nous avons également montré que Ded1 peut passer du noyau vers le cytoplasme par différentes voies d’export nucléaire. De façon intéressante, ses partenaires protéines sont capables de stimuler son activité ATPase. De plus, nous avons montré qu’il existait un lien génétique entre Ded1 et ses partenaires. Nous avons également montré que Ded1 colocalise partiellement avec ses partenaires dans des gradients de saccharose, suggérant que Ded1 pourrait être associée à certains mRNPs. Nos résultats encore préliminaires indiquent que Ded1 pourrait s’associer à d’autre ARNs coiffés. Ainsi, Ded1 pourrait remodeler les complexes associés à différentes étapes de la vie des ARN coiffés. / The budding yeast DEAD-box RNA helicase Ded1 is an essential yeast protein that is closely related to a subfamily of DEAD-box proteins that are involved in developmental and cell-cycle regulation. Ded1 is generally considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5' 7-methylguanosine cap to the AUG start codon. We have used IgG pulldown experiments, mass spectroscopy analyses, genetic experiments, saccharose gradients, in situ localizations, and enzymatic assays to show that Ded1 is a cap-associated factor that actively shuttles between the cytoplasm and the nucleus. We show that Ded1 physically interacts with various cap-associated factors and that its enzymatic activity is stimulated by these factors. By using various mutated proteins, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these factors on saccharose gradients, but the peak of Ded1 sediments slightly heavier than for the other factors, which suggests that Ded1 is predominately associated with a subset of the mRNPs. Finally, purification of the protein complexes associated with Ded1 and subsequent analysis by nanoLC-MS/MS indicates that Ded1 is associated with both nuclear and cytoplasmic mRNPs. Preliminary experiements showed that Ded1 can associate with other capped RNA. We conclude that Ded1 may function as a remodeling factor that is needed to form the different complexes associated with the different processing steps of the capped RNA.

Malgré les thérapies anti-cancéreuses ciblées, beaucoup de patients récidivent à cause de la résistance aux traitements qui constitue un problème clinique majeur. Cette résistance est soutenue par la reprogrammation métabolique et traductionnelle. La synthèse des protéines oncogéniques fait appel au complexe d’initiation de la traduction eucaryote 4F (eIF4F), compris des facteurs : eIF4A, eIF4E et eIF4G. Il est ainsi possible de cibler la synthèse protéique spécifique aux cellules cancéreuses par des inhibiteurs de l’initiation de la traduction. Nous proposons que le ciblage de la machinerie traductionnelle, via l’inhibition du eIF4A (eIF4Ai), affecte particulièrement les cellules cancéreuses. Mais est-ce qu’il est en mesure d’atténuer la résistance aux inhibiteurs de kinase (IKs) et d’en empêcher l’adaptation et la reprogrammation métabolique? L’efficacité des eIF4Ais est vérifiée par l’évaluation de la croissance et de la mort cellulaire (Annexine V) dans des cellules de mélanome, sensibles et résistantes aux IKs. Celle-ci est accompagnée de la réduction de la synthèse protéique évaluée par profilage polysomique, de la baisse des cibles de l'eIF4Ai (BCL-2, CDK4...) quantifiées par western-blots, d’un important stress bioénergétique mesuré par Seahorse et du contrôle des principales voies métaboliques analysées par GCMS. L’analyse du profilage polysomique, de l’ARNseq et du métabolome permettront de mettre en évidence les réseaux qui soutiennent l’efficacité des eIF4Ais et les mécanismes moléculaires sous-jacents à l’efficacité de cette thérapie. Ces derniers seront par la suite validés par des approches génétiques évaluant les principaux gènes et voies métaboliques qui y sont impliqués. Cette étude comblera de grosses lacunes dans les connaissances relatives aux mécanismes moléculaires qui soutiennent la résistance aux IKs afin d’améliorer leur efficacité en clinique. / Despite advances in research and development of targeted cancer therapies, many patients relapse due to treatment resistance. This is the case of melanoma resistant to BRAF inhibitors (BRAFi). 40-50% of melanoma cancer cells express the constitutively active oncoprotein BRAFV600E, leading to metabolic and translational reprogramming that is proposed to support resistance to cancer therapies. Studies have shown the importance of the eukaryotic translation initiation complex 4F (eIF4F), including factors: eIF4A, eIF4E and eIF4G, in the oncogenic proteins’ synthesis (e.g. growth factors, metabolic factors, etc.). The cancer cells translatome is distinct from the normal cells translatome. It is thus possible to target protein synthesis specific to cancer cells using molecules that inhibit translation initiation, the limiting phase in this process, affecting the cancer cells specifically. We propose that targeting the translational machinery via eIF4A inhibitors (eIF4Ai) would attenuate resistance to kinase inhibitors (KIs) and we seek to dissect the underlying molecular mechanisms. The efficacy of eIF4Ais in BRAFi sensitive and/or resistant melanoma is evaluated showing that eIF4Ais inhibit cell growth and induce melanoma cells death, using growth curves and FACS (Annexin_V). This is accompanied by a protein synthesis reduction, evaluated by polysome profiling showing a decrease in eIF4Ais treated cells and western-blots showing a decrease in translational targets of eIF4Ai (BCL-2, CDK4, etc.). A significant bioenergetic stress is measured by Seahorse and the control of the main metabolic pathways including glycolysis, TCA cycle and amino acids metabolism is analyzed by GCMS. Then the analysis of the translatome by polysome profiling and RNAseq and of the metabolome by GCMS/LCMS and Seahorse shed light on the translational networks that dictate metabolic reprogramming supporting eIF4Ai efficacy. Finally, the molecular mechanisms underlying the efficacy of eIF4Ai will be identified using genetic approaches to validate the main genes and metabolites and/or corresponding metabolic pathways involved in the response to eIF4Ai of the kinase inhibitor resistant melanoma. This study will fill large gaps in knowledge about the molecular mechanisms that support resistance to KIs in order to improve their clinical efficacy.