L’initiation de la traduction est un processus complexe qui fait intervenir une douzaine de facteurs d’initiation. L’élément clef de ce mécanisme est le facteur eIF4E qui grâce à sa liaison avec la coiffe, recrute l’ensemble du complexe d’initiation au niveau de l’ARNm et permet l’assemblage du ribosome au voisinage du codon d’initiation. Chez Arabidopsis thaliana, il existe à coté de la protéine eIF4E, une isoforme : eIF(iso)4E. Ces deux protéines participent à l’initiation de la traduction. L’existence de ces deux protéines évoque un phénomène de redondance fonctionnelle qui est attestée par la létalité du double mutant alors que les simples mutants sont viables. Cependant, l’étude phénotypique de mutants pour les gènes eIF4E et eIF(iso)4E a permis de montrer que cette redondance est partielle et inégale. En effet, les mutants pour le gène eIF4E présentent un retard de croissance, un retard de floraison, une baisse de la fertilité, une sénescence précoce et une activité traductionnelle réduite. Inversement, le phénotype des plantes mutantes pour le gène eIF(iso)4E est comparable à celui du sauvage. Les mutations dans les gènes eIF4E et eIF(iso)4E induisent une hypersensibilité à la lumière Enfin, en présence d’un inhibiteur de TOR la croissance de la racine des plantes de la lignée mutante pour le gène eIF(iso)4E est moins inhibée que celle des plantes de la lignée sauvage. / More than 12 initiation factors are involved in eukaryotic translation initiation. The key step of this mechanism is the binding of eIF4E with the cap of the mRNA. This step allows the recruitment of the initiation complex and the assembly of the ribosome close to the start codon. Arabidopsis thaliana encodes a second eIF4E protein: eIF(iso)4E. Those two proteins perform translation initiation. The existence of those two proteins suggests that they may be functionally redundant. Double mutant lethality testifies for functional redundancy. However, phenotypic studies of mutant lines for gene eIF4E and eIF(iso)4E showed that redundancy is partial and unequal. Indeed, the eIF4E mutant lines exhibit growth delay in rosette and roots, bolting delay, impaired fertility and early senescence in leaves. Translational activity is also largely impaired. On the contrary, a mutant line for the eIF(iso)4E gene has the same phenotype as wild type line. Mutant lines for eIF4E and eIF(iso)4E are more sensitive to light and accumulate anthocyanins even in normal light. On the molecular level, the amounts of mRNA of genes that are involved in high light response and their association to polysomes increase. When plants are grown on media containing a TOR inhibitor, AZD-8055, plants of the eIF(iso)4E mutant line show less root growth inhibition compared to wild type and eIF4E mutant lines. This result suggests that eIF(iso)4E could be targeted by the TOR pathway.

Les glioblastomes (GB) sont des tumeurs cérébrales extrêmement agressives, très hypoxiques qui récidivent dans les champs d'irradiation malgré un traitement bien conduit qui associe radio et chimiothérapie. L'hypoxie tumorale et les facteurs de transcription inductibles par l'hypoxie (HIF-1alpha et HIF-2alpha) sont des facteurs importants de résistance aux thérapeutiques anti-cancéreuses, en particulier dans les glioblastomes. Récemment, un nouveau partenaire de HIF-2alpha a été identifié. Il s'agit de la protéine Int6 également connue sous le nom d'eIF3e, sous-unité " e " du facteur d'initiation de la traduction eIF3. De façon intéressante, des études ont montré que les niveaux d'expression d'eIF3e étaient associés à l'état d'avancement tumoral, dans les cancers du sein et du colon. Ces données de la littérature suggèrent que le facteur Int6/eIF3e pourrait être une cible particulièrement intéressante dans les cellules de glioblastome. Au cours de ma thèse et dans l'optique de vérifier cette hypothèse, j'ai démontré sur un plan fonctionnel que l'inhibition d'Int6/eIF3e dans différentes lignées de GB diminue la prolifération en provoquant un arrêt du cycle cellulaire en phase G1 et en augmentant la mort cellulaire, notamment par apoptose et par une augmentation des processus autophagiques. Sur un plan moléculaire j'ai mis en évidence que cette diminution de la survie des cellules de glioblastome en réponse à l'inhibition d'Int6/eIF3e est en partie causée par une réduction de l'expression des HIFs. De plus, l'étude de la biosynthèse protéique et en particulier de la traduction, via l'analyse des ARNm associés aux polysomes combinée à une analyse microarray, a permis de mettre en évidence une modulation de la traduction d'ARNm spécifiques permettant d'expliquer les phénotypes observés. Dans une perspective thérapeutique, j'ai pu démontrer que l'association de l'inhibition d'Int6/eIF3e avec les radiations ionisantes potentialise les effets de l'irradiation sur ces cellules de GB. Enfin sur un plan clinique j'ai mis en évidence une corrélation entre la perte d'expression d'Int6/eIF3e et une augmentation de la survie des patients atteints de GB. En conclusion, mes travaux de thèse ont permis de valider Int6/eIF3e comme une nouvelle cible essentielle pour la survie des cellules de glioblastome en régulant la traduction d'ARNm spécifiques. / Glioblastomas (GB) are very aggressive and malignant brain tumors, with frequent relapses despite an appropriate treatment combining surgery, chemotherapy and radiotherapy. In GB, hypoxia is a characteristic feature and activation of the Hypoxia Inducible Factors (HIF-1a and HIF-2a) has been associated with resistance to anti-cancer therapeutics. Int6, also named eIF3e, is the "e" subunit of the translation initiation factor eIF3, and was identified as novel regulator of HIF-2a. Interestingly, studies led in colon and breast cancers showed that high expression of eIF3e was correlated with a bad prognosis. These data suggest that Int6/eIF3e could become an interesting target in cancer cells and particularly in glioblastoma. During my graduate work and to assess this hypothesis, I first demonstrated at a functional level that Int6/eIF3e inhibition leads to a decreased GB cell proliferation induced by a cell cycle arrest in G1 phase and an increased cell death, especially apoptotic and autophagic processes in various GB cell lines. At the molecular level, I showed that this decreased GB cell survival in response to Int6/eIF3e inhibition was partly due to a reduction in HIFs expression. Moreover, the study of protein synthesis mechanisms, and particularly translation, via polysome-bound mRNA and microarray analyses revealed a modulation in the translation of specific subsets of mRNA, which could potentially explain our phenotypes. From a therapeutic prospect, I demonstrated that combination of Int6/eIF3e inhibition with ionizing radiations potentiates radiation effects on GB cells. Finally, immunohistochemistry studies showed that low expression of Int6/eIF3e is correlated with a longer survival for patients with GB, suggesting a potential relevance for Int6/eIF3e as a prognostic marker. In conclusion, my doctoral work allowed us to identify Int6/eIf3e as a new target essential for glioblastoma cell survival through the regulation of translation of specific mRNA subsets.

Submitted by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:24:49Z No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Approved for entry into archive by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:25:15Z (GMT) No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Made available in DSpace on 2014-11-26T16:25:15Z (GMT). No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Células-tronco (CT) são células autorrenováveis e não especializadas, com o potencial de diferenciação multidirecional. Células-tronco de tecido adiposo (CT-TA) são um tipo de células-tronco adultas multipotentes, de fácil isolamento e cultura. Nos últimos anos, CT-TA têm mostrado grande potencial para engenharia de tecidos e terapias baseadas em células. Apesar do interesse em aplicações clínicas deste tipo de célula, os mecanismos moleculares fundamentais a sua autorrenovação e diferenciação ainda não foram completamente elucidados. miRNAs são pequenos RNAs não-codificadores, com 21-25 nucleotídeos de comprimento, que tem se mostrado como importantes reguladores da expressão gênica em nível póstranscricional. miRNAs podem atuar por meio de clivagem direta de mRNAs alvo ou através da repressão da tradução, dependendo da complementaridade entre o mRNA e a sequência do miRNA. Perfis de miRNAs de CT adultas sugerem que estes pequenos reguladores podem contribuir para as propriedades intrínsecas das CT. Para entender melhor os mecanismos de ação dos miRNAs em CT-TA, miRNAs associados ao polissomos de CT-TA foram isolados durante a diferenciação celular. Procurando miRNAs reguladores das etapas iniciais de diferenciação ou envolvidos na autorrenovação de CT, as culturas de células foram induzidas a diferenciação adipogênica durante 72 h. O lisado celular foi submetido à ultracentrifugação em gradiente de sacarose para separar monosomos, polissomos e fração livre de ribossomos. O RNA total associado aos ribossomos foi extraído, os fragmentos de RNA (<200 nt) foram enriquecidos e a seleção de tamanho de fragmentos de RNA apropriados ocorreu durante a preparação das amostras para o sequenciamento em larga escala. As amostras foram sequenciadas utilizando a plataforma SOLiD ™, e as frações polissomais de culturas Não Induzida e 72h de indução foram comparadas e dezesseis miRNAs foram identificados. miRNAs encontrados em um trabalho prévio do grupo foram adicionados a esses dados, e sete miRNAs (hsamiR-29b-1-5p, hsa- miR-29c-5, hsa-miR-30c-5p, hsa-miR-143-5p, hsa-miR-210-3p, hsa-miR-210- 5p e hsa-miR-6775- 5p) foram testados por RT-qPCR para confirmar a expressão diferencial, sendo que um deles (hsa-miR-210-5p) mostrou diferença estatisticamente significativa. / Stem cells (SC) are self-renewing and non-specialized cells with the potential of multi-directional differentiation. Adipose Stem Cell (ADSC) is a type of multipotent adult stem cell, easy to isolate and culture. In the past few years, hADSCs have shown great potential for tissue engineering and cell-based therapies. Despite the interest in clinical applications of this kind of cell, the molecular mechanisms underlying their self-renewal and differentiation have yet to be fully elucidated. miRNAs are small noncoding RNAs, 21-25 nucleotides in length, that have been shown to be important regulators of posttranscriptional gene expression. miRNAs can act through direct cleavage of target mRNAs or through translational repression, depending of complementary pairing between the mRNA and miRNA sequence.miRNA profile of adult SCs suggests that these small regulators can contribute to the intrinsic properties of SCs. To better understand the mechanisms of action of miRNAs in hADSCs, we isolate miRNAs associated to polysomes of hADSC during cellular differentiation. Looking for miRNAs regulators of early steps of differentiation or involved in ADSC self-renewing, cell cultures were induced to adipogenic differentiation for 72 h. The cell lysate was submitted to ultracentrifugation on a sucrose gradient to separate monosomes, polysomes and the fraction free of ribosomes. The total RNA associated to ribosomes was extracted and the RNA fragments (<200 nt) were enriched and the size selection of appropriate RNA fragments occurred during the preparation of samples for deep sequencing. The samples were sequenced using SOLiD™ platform, and polysomal fraction of cell cultures non induced and 72h of induction were compared and sixteen miRNAs were identified. miRNAs found in a previous work of our group were added to these data and seven miRNAs (hsa-miR-29b-1-5p, hsa-miR-29c-5, hsa-miR-30c-5p, hsa-miR- 143-5p, hsa-miR-210-3p, hsa-miR-210-5p e hsa-miR-6775-5p) were tested by RTqPCR to confirm differential expression, and one of them (hsa-miR-210-5p) showed statistical significant difference.

Infection of barley (Hordeum vulgare) leaves with the rust fungus, Puccinia hordei, causes changes in the host protein synthesis. This thesis analyses these changes in the barley cultivar Triumph following inoculation of 7-day-old leaves with either a virulent or an avirulent race of P. hordei. The initial approach was to isolate membrane-bound polysomes from infected leaves, translate them in vitro and analyse the translation products. These products include the integral membrane proteins which were expected to be involved in the response of the host to the pathogen. A method based on differential centrifugation in the presence of a ribonuclease-inhibiting buffer was developed for separating membrane-bound polysomes from the rest of the cytoplasmic polysomes. Membrane-bound polysomes were found to comprise one fifth of the total polysomes in the leaves. Analysis of the translation products of membrane-bound polysomes by SDS-PAGE showed them to be of higher average molecular weight than those from free polysomes. Comparison of polypeptides produced by membrane-bound polysomes from healthy and inoculated plants showed some differences however the low yield of membrane-bound polysomes made it difficult to obtain conclusive results. Thus it was decided to isolate total polysomes by including 1% Triton X-100 in the extraction buffer. Polysomes were extracted from 12 to 72 h after inoculation. Infection caused a decline in yield of polysomes during this period when compared with healthy leaves of the same age. Polysomes isolated 16 h after inoculation with the virulent race were 20% less efficient at translation than polysomes from control leaves. In contrast polysome isolated from leaves inoculated with the avirulent race were 20% more efficient. Analysis of the labelled translation products by SDS-PAGE and fluorography showed relative increases in the synthesis of some proteins by 16 h after inoculation with either race when compared to products from healthy leaves. Protein synthesis in the infected plants was further analysed by in vivo labelling and one- and two-dimensional PAGE. The fluorographs revealed increased synthesis of a group of proteins from 58 to 116 kDa starting 12 h after inoculation with either race of P. hordei; confirming the results from the polysome translations. Two polypeptides with molecular weights of about 66 kDa were found to increase following infection only with the virulent race. By three days after inoculation with either fungal race the most obvious change in protein synthesis was a marked decrease in the synthesis of the two most prominent polypeptides with molecular weights of 15 and 51 kDa which were considered to be the subunits of ribulose bisphosphate carboxylase. The elicitor hypothesis, in attempting to explain cultivar-specific resistance in plants, postulates that resistance is controlled by the interaction of specific fungal elicitors and plant receptors and that this interaction which only occurs between resistant hosts and avirulent pathogens triggers specific gene expression leading to resistance. This hypothesis does not fit the situation in the barley-P. hordei interaction as protein synthesis showed similar changes following infection with either a virulent or an avirulent race.