Les phospholipases A2 sécrétées (sPLA2) font partie d’une grande famille d’enzymes impliquées dans la synthèse d’écosanoïdes, de chimiokines et dans l’expression de molécules d’adhérence. Ce groupe comprend dix isoformes différentes (sPLA2-IB, -IIA, -IIC, -IID, -IIE, -IIF, -III, -V, -X et XII) dont la majorité sont surexprimées en présence de molécules pro-inflammatoires telles que l’interleukine-1β (IL-1 β) et le lipopolysaccharide bactérien (LPS). La sPLA2-IIA fut longtemps considérée comme la principale sPLA2 associée à l’inflammation. Toutefois, un nombre grandissant d’études suggère l’implication d’autres isoformes dans la réponse inflammatoire. Étant donné la similarité structurelle des différentes isoformes de sPLA2, la majorité des inhibiteurs présentement disponibles sont non spécifiques et bloquent simultanément plus d’une sPLA2. De ce fait, encore peu de choses sont connues quant au rôle précis de chacune des sPLA2 dans la réponse inflammatoire. Ayant accès à des souris génétiquement modifiées n’exprimant pas la sPLA2-V (sPLA2-V-/-), nous avons donc investigué le rôle spécifique de la sPLA2-V dans le recrutement leucocytaire induit par le LPS, ainsi que sa capacité à moduler l’expression de certaines molécules d’adhérence. Pour ce faire, nous avons utilisé le modèle inflammatoire de la poche d’air sous-cutanée. L’administration de LPS dans la poche d’air de souris contrôles (WT) entraîne un recrutement leucocytaire important. Cet appel de cellules inflammatoires est cependant significativement diminué chez les souris sPLA2-V-/-. De plus, l’expression des molécules d’adhérence VCAM-1 et ICAM-1 est également diminuée chez les souris sPLA2-V-/- comparativement aux souris WT. Nos résultats démontrent donc le rôle important de la sPLA2-V dans le recrutement leucocytaire et l’expression de molécules d’adhérence induits par le LPS, confirmant ainsi l’implication de cette enzyme dans le processus inflammatoire. / Secretory phospholipases A2 (sPLA2s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA2 isoforms are up-regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA2 (sPLA2-V). Furthermore, it has recently been shown that sPLA2-V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA2-V on LPS-mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA2-V null mice (sPLA2-V-/-) and control wild-type (WT) littermates. We observed that LPS (1 μg/mL)-mediated leukocyte migration in sPLA2-V-/- was attenuated by 52 and 86% after 6 and 12 hours of treatment, respectively, as compared to WT mice. In WT mice, treatment with the cell-permeable sPLA2 inhibitor (12-epi-scalaradial; SLD) reduced LPS-mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA2-V-/- mice. Protein analyses from the air pouch skin were carried out upon LPS-challenge, and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were both significantly reduced in sPLA2-V-/- mice as compared to control WT mice. Together, our data demonstrate the role of sPLA2-V in LPS-induced ICAM-1 and VCAM-1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA2-V in the development of inflammatory innate immune responses.

nÃo hà / A ativaÃÃo das cÃlulas do sistema imunolÃgico à modulada atravÃs dos sinais acionados por uma diversidade de receptores de superfÃcie celular, incluindo os receptores ativadores, receptores coestimuladores e receptores inibidores. Receptores relacionados à molÃcula sinalizadora na ativaÃÃo do linfÃcito (SLAM) tÃm influÃncia na ativaÃÃo imunolÃgica celular. Neste trabalho, investigou-se a funÃÃo de SLAM na resposta imunolÃgica à Leishmania braziliensis, e se a resposta de indivÃduos alto (AP) ou baixo (BP) produtores de IFN-γ seria modulada pela via de sinalizaÃÃo SLAM. CÃlulas monocucleadas do sangue perifÃrico (CMSP) de 43 indivÃduos foram bloqueadas com α-SLAM, rIFN-γ, rIL-12 e fitohemaglutinina, apÃs estimulaÃÃo com L. brazilensis. Verificou-se que L. braziliensis promoveu uma significante reduÃÃo da expressÃo de SLAM nas cÃlulas T, com 120h de cultivo, possivelmente indicando ativaÃÃo desta via na resposta imunolÃgica inicial. A expressÃo de SLAM se comportou de modo diferenciado nos indivÃduos AP e BP. Nos indivÃduos BP, L. braziliensis nÃo alterou a expressÃo de SLAM nas cÃlulas T, na fase inicial da resposta imunolÃgica. O bloqueio da via de SLAM com α-SLAM reduziu significativamente a expressÃo desta proteÃna nos primeiros momentos da resposta imunolÃgica das CMSP estimuladas com L. braziliensis. O bloqueio com α-SLAM, avaliado com 120 horas, nÃo alterou a expressÃo de CD3+SLAM+, em ambos os grupos. As citocinas proinflamatÃrias, rIFN-γ e rIL-12, presentes no microambiente com L. braziliensis, reduziram a expressÃo de SLAM apenas em indivÃduos AP com 6h de sensibilizaÃÃo e nÃo modificaram esta resposta com 120h de cultivo, na presenÃa do antÃgeno. O bloqueio com α-SLAM, na concentraÃÃo de 10μg/ml, nÃo interferiu na produÃÃo das citocinas IFN-γ e IL-13, em ambos os grupos, entretanto aumentou de forma significativa a produÃÃo de IL-10 em indivÃduos AP. O bloqueio da via de SLAM associado à L. braziliensis e rIFN-γ nÃo modificou a produÃÃo de IFN-γ, IL-13 e IL-10. O bloqueio da via de SLAM associado à L. braziliensis e rIL-12 induziu aumento de IFN-γ, nos indivÃduos BP, e aumento de IL-13, nos indivÃduos AP. Os resultados deste trabalho sugerem que, na resposta in vitro de CMSP, sensibilizadas com L. braziliensis, a via de sinalizaÃÃo SLAM atua na modulaÃÃo da resposta Th1 em indivÃduos AP e induz uma condiÃÃo de imunossupressÃo temporÃria nos indivÃduos BP, nÃo descrita anteriormente na literatura. / Immune cells activation is modulated by balancing the signals triggered by a variety of cell surface receptors, including receptor activators, co-stimulating receptors and inhibitory receptors. Receptor-related signaling molecule in lymphocyte activation (SLAM) influences the immune cell activation. In this study we investigated the role of SLAM in immune response of cutaneous leishmaniasis caused by L. braziliensis, as well as if the response of individuals high (HP) or low (LP) IFN-γ producers is modulated by SLAM signaling pathway. Peripheral blood monocuclear cells (PBMC) isolated from 43 health individuals were cultured in vitro with anti-SLAM, rIFN-γ, rIL-12 and phytohemagglutinin in the presence or in the absence of L. braziliensis. It was found that L. braziliensis promoted a significantly reduced SLAM expression in T cells, after 120 h of cultured, possibly indicating activation of this pathway in the initial immune response. SLAM expression behaved differently in HP and LP groups. In LP group, L. braziliensis did not modify SLAM expression in T cells in early immune response. The effect of anti-SLAM on SLAM pathway reduced the expression of this protein in the early stages of the immune response of PBMC stimulated with L. braziliensis. After 120 h the effect of anti-SLAM did not alter CD3+SLAM+ expression in both groups. The proinflammatory cytokines, rIFN-γ and rIL-12, present in the microenvironment with L. braziliensis, reduced SLAM expression only in HP group after 6 h of culture and did not change this response after 120 h. Anti-SLAM at a concentration of 10 μg/ml presented no effect on production of cytokines IFN-γ and IL-13 in both groups, but significantly increased IL-10 production in the HP group. Furthermore anti-SLAM associated with L. braziliensis and rIFN-γ simultaneously did not modify IFN-γ, IL-13 and IL-10 productions. Anti-SLAM associated with L. braziliensis and rIL-12 simultaneously induced an increase of IFN-γ in LP group, and increased IL-13 in HP group. These results suggest that in vitro immune response of PBMC exposed to L. braziliensis, the SLAM signaling pathway acts in modulating Th1 response in HP group and induces a condition of temporary immunosuppression in LP group, not previously described in literature.

A vascularização tem um papel central na progressão tumoral e representa um alvo terapêutico de grande interesse. A inibição da angiogênese tem potencial de retardar a progressão tumoral e inibir metástase. Em decorrência disto, terapias anti-angiogênicas têm demonstrado ser promissora no controle do crescimento tumoral. Segundo a literatura, interferon-? (IFN?, ativador do sistema imune inato e adaptativo) e p19Arf (supressor de tumor e parceiro funcional de p53), quando estudados individualmente, alteram a vasculatura tumoral. Nosso grupo construiu e utilizou vetores adenovirais recombinantes portadores dos cDNAs de INFbeta e p19Arf e observou que a transferência desta combinação de genes induziu morte celular e diminuiu progressão tumoral, resultados foram observados em modelos murinos de melanoma B16 de terapia genica in situ, vacina profilática e vacina terapêutica. Neste trabalho, exploramos a ideia que a combinação dos vetores adenovirais portadores de INFbeta e p19Arf proporcionam efeitos anti-angiogênicos através de seu impacto em células endoteliais. Para averiguarmos essa hipótese, células endoteliais murinas (tEnd) foram transduzidas com os vetores adenovirais, revelando que o vetor Ad-p19 confere inibição da proliferação, formação de tubos, migração e induz aumento na expressão de genes relacionados a via de p53 e morte celular. O vetor Ad-IFNbeta sozinho ou adicionado em combinação com Ad-p19, não teve impacto significante nestes ensaios. Alternativamente, a influencia indireta, ou parácrina, nas células tEnd cultivadas juntamente com as células B16 transduzidas com os vetores adenovirais também foi investigada. Quando as células B16 foram transduzidas com Ad-IFNbeta ou a co-transdução Ad-IFNbeta+Ad-p19 em co-cultura com a linhagem tEnd, houve inibição da proliferação. Não observamos efeito inibitório na tEnd da co-cultura quando as células da B16 foram transduzidas somente com Ad-p19. Seguindo o ensaio de co-cultura, produzimos meio condicionado da B16 transduzida com os vetores e aplicamos esses meios nas células tEnd. Observamos que Ad-IFN, sozinho ou em combinação com Ad-19, diminuiu a viabilidade, proliferação e levou a morte das células tEnd. Neste trabalho, constamos que inibição de células endoteliais pode ser realizada por transdução direta com Ad-19 ou quando estas células são expostas ao ambiente modulado por células tumorais transduzidas com o vetor Ad-IFNbeta. Mesmo que a transferência gênica de ambos IFNbeta e p19Arf não demonstrou ser uma abordagem superior à aplicação dos genes isolados, observamos que nossa abordagem pode ter um impacto importante na inibição da angiogênese pelas células endoteliais / The vasculature plays a central role in tumor progression and represents a therapeutic target of great interest. Inhibition of angiogenesis has the potential to slow down tumor progression and inhibit metastasis. As a result, anti-angiogenic therapies have been shown to be promising for the control of tumor growth. According to the literature, interferon ? (IFN?, activator of the innate and adaptive immune systems) and p19Arf (tumor suppressor and functional partner of p53) when studied individually alter tumor vasculature. Our group has constructed and used recombinant adenovirus vectors carrying the cDNAs of INFbeta and p19Arf and noted that the transfer of this combination of genes induced cell death and decreased tumor progression, as observed in the B16 murine model of in situ melanoma gene therapy as well as prophylactic and therapeutic vaccine approaches. In this study, we explore the idea that the combination of adenoviral vectors bearing INFbeta and p19Arf produce anti-angiogenic effects due to their impact on endothelial cells. To test this hypothesis, murine endothelial cells (tEnd) were transduced with adenoviral vectors, revealing that Ad-p19 vector confers inhibition of proliferation, tube formation, migration and induces increased expression of genes related to the p53 cell death pathway. The Ad-IFNbeta vector alone had no significant impact on these tests. Alternatively, influences on paracrine effects are evaluated on endothelial cells co-cultured with B16 cells that were previously transduced with adenoviral vectors. When the B16 cells were transduced with Ad-IFNbeta or co-transduced with Ad-IFNbeta + Ad-p19, co-culture resulted in the inhibition of proliferation of the endothelial cells. When B16 cells were transduced with Ad-p19 only, co-culture did alter endothelial cell behavior. Following the co-culture assay, we produce conditioned medium from B16 cells that were transduced with the vectors and applied the media on tEnd cells. We noted that conditioned medium derived from B16 transduced with Ad-IFN alone or in combination with Ad-19 decreased the viability and proliferation and induced cell death of tEnd. In this work, we show that inhibition of endothelial cells can be performed directly by transduction with Ad-19 or when such cells are exposed to the environment modulated by tumor cells transduced with Ad-IFNbeta. Even though the gene transfer of both IFNbeta and p19 was not found to be superior to the application of single genes, we observed that our approach may have an important impact on the inhibition of angiogenesis through endothelial cells

Submitted by Fabiana Gonçalves Pinto (benf@ndc.uff.br) on 2017-02-23T17:18:30Z No. of bitstreams: 1 Magali Rezende de Carvalho.pdf: 3727493 bytes, checksum: cd7ca0e5fa89bc698cb1c0a9fe619926 (MD5) / Made available in DSpace on 2017-02-23T17:18:30Z (GMT). No. of bitstreams: 1 Magali Rezende de Carvalho.pdf: 3727493 bytes, checksum: cd7ca0e5fa89bc698cb1c0a9fe619926 (MD5) Previous issue date: 2016 / Mestrado Acadêmico em Ciências do Cuidado em Saúde / Contextualização: Os fatores de crescimento atuam no reparo tecidual emitindo sinais modulatórios estimulando ou inibindo os processos celulares. Objetivo: Analisar a eficácia dos fatores de crescimento no processo cicatricial de úlceras venosas através da busca de evidência na literatura científica. Método: Revisão sistemática segundo as recomendações da Colaboração Cochrane. Critérios de inclusão: Ensaios clínicos randomizados sobre o uso dos fatores de crescimento no tratamento de úlceras venosas; abordando o número total de úlceras cicatrizadas, redução da área e/ou tempo de cicatrização. Exclusão: estudos em andamento, protocolos de pesquisa; artigos que associam fatores de crescimento ao enxerto de pele e estudos que incluíram úlceras de múltiplas etiologias sem análise por subgrupo. Bases de dados consultadas: Ovid MEDLINE(R); Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations; Ovid MEDLINE(R) Epub Ahead of Print; EMBASE; CINAHL Plus with Full Text, Cochrane CENTRAL, LILACS e Web of Science. Também foram consultados a Biblioteca Digital Brasileira de Teses e Dissertações e Google Acadêmico, além da busca manual através da lista de referências dos estudos incluídos. Não houve restrição temporal ou de idioma. A análise estatística foi realizada através do programa Review Manager 5.3 (Colaboração Cochrane). Para variáveis dicotômicas, foram calculados o risco relativo considerando um intervalo de confiança de 95%. A metanálise foi realizada utilizado o modelo de efeito fixo de Mantel-Haenszel quando I2 < 50% e modelo randômico para I2 > 50%. Resultados: A aplicação de fatores de crescimento para tratamento de úlceras venosas não acelerou o reparo tecidual comparado como tratamento padrão/placebo (RR 1,01 [IC 95%: 0,88-1,16]). Resultados semelhantes foram encontrados ao analisar os subgrupos PRP (RR 1,01 [IC 95%: 0,80-1,28]); KGF (RR 0,93 [IC 95%:0,78-1,11]); PDGF (RR 1,17 [IC 95%: 0,78-1,74]); EGF (RR 2,87 [IC 95%: 0,65-12,73; p=0,17]); TGF (RR 1,14 [IC 95%: 0,41-3,15]). Conclusão: Os resultados dessa revisão foram baseados em estudos classificados como moderado a alto risco de viés, portanto, precisam ser interpretados com cautela. Portanto, não há evidências para afirmar que os fatores de crescimento influenciam positivamente na cicatrização de úlceras venosas. Estudos mais robustos, com maior poder e melhor qualidade metodológica são necessários para de determinar melhores recomendações sobre o uso dos fatores de crescimento no tratamento de úlceras venosas. (Registro PROSPERO: CRD42016038390). / Background: Growth factors act in tissue repair by sending modulated signals stimulating or inhibiting cellular processes. Aim: To analyze the efficacy of growth factors in the healing process of venous ulcers by seeking evidence in the scientific literature. Method: Systematic review according to Cochrane Collaboration. Inclusion criteria: Randomized controlled trials using growth factors in the treatment of venous leg ulcers; approaching the total number of healed ulcers, wound area reduction and healing time. Exclusion: ongoing studies, research protocols; articles linking growth factors to the skin graft and studies that included ulcers of multiple etiologies without subgroup analysis. Databases consulted: Ovid MEDLINE (R); Ovid MEDLINE (R) In-Process & Other Non-Indexed Citations; Ovid MEDLINE (R) Epub Ahead of Print; EMBASE; CINAHL Plus with Full Text, Cochrane CENTRAL, LILACS and Web of Science. Also, the Brazilian Digital Library of Theses and Dissertations and Google Scholar were consulted, as well as hand search through the list of references of included studies. There was no time or language restrictions. Statistical analysis was performed using Review Manager 5.3 software (Cochrane Collaboration). For dichotomous variables, the relative risk was calculated considering a 95% confidence interval. The meta-analysis was performed using the fixed-effect model of Mantel-Haenszel when I2 <50% and a random model for I2> 50%. Results: The application of growth factors for the treatment of venous ulcers did not accelerate the healing process compared to standard treatment/placebo (RR 1.01 [95% CI: 0.88 to 1.16]). Similar results were found when analyzing the following subgroups: PRP (RR 1.01 [95% CI: 0.80 to 1.28]); KGF (RR 0.93 [95% CI: 0.78 to 1.11]); PDGF (RR 1.17 [95% CI: 0.78 to 1.74]); EGF (RR 2.87 [95% CI: 0.65 to 12.73, p = 0.17]); TGF (RR 1.14 [95% CI: 0.41 to 3.15]). Conclusion: The results of this review were based on studies classified as moderate to high risk of bias, therefore need to be interpreted with caution. So there is no evidence that the growth factors positively influence the healing of venous leg ulcers. More robust studies, more power and better methodological quality are needed to determine the best recommendations on the use of growth factors in the treatment of venous leg ulcers. (PROSPERO Registration: CRD42016038390).

V. vermiformis est une amibe libre répandue dans l'environnement et les milieux artificiels comme les réseaux d'eau chaude sanitaire (RECS). Il est maintenant bien établi qu'elle joue un rôle de réservoir pour des bactéries pathogènes, comme L. pneumophila. Le contrôle de V. vermiformis dans les RECS représente donc un enjeu sanitaire important. Les amibes libres peuvent passer d'une forme métaboliquement active (trophozoïte) à une forme de résistance, le kyste, lorsque les conditions sont défavorables ce qui leur confère une résistance aux traitements. Malgré la haute prévalence de V. vermiformis dans les RECS, les processus de différenciation et la résistance de ses kystes aux traitements n'ont été que peu étudiés. Nous avons donc investigué les changements morphologiques et ultrastructuraux qui s'opèrent lors de l'enkystement et désenkystement de V. vermiformis. Il en ressort que l'enkystement est un phénomène rapide (9 h) qui conduit à la formation de kystes entourés d'une paroi double couche. Lors du désenkystement, les trophozoïtes n'émergent pas à travers un ostiole comme c'est le cas chez Acanthamoeba. Puis, nous avons étudié l'effet des conditions environnementales et de la concentration cellulaire sur l'enkystement. Nous avons observé que plus la concentration cellulaire est élevée plus l'enkystement est rapide, ce qui suggère l'existence de mécanismes de communication intercellulaire. Enfin, nous avons étudié la résistance des kystes aux traitements utilisés dans les RECS et aux protéases. Ces traitements étaient efficaces, in vitro, pour inactiver les kystes de V. vermiformis. Ces travaux ont permis d'apporter des connaissances de bases sur les processus de d / Vermamoeba vermiformis is a free-living amoeba (FLA) widespread in the environment and artificial environments such as hot water networks. It is now well established that it acts as a reservoir for many pathogenic bacteria, such as Legionella pneumophila. The control of V. vermiformis in artificial environments represents an important health issue. FLA can turn from a metabolic active form (trophozoite) to a resistance form, called cyst, when conditions are unfavorable. Cysts are more resistant to treatments. Despite the high prevalence of V. vermiformis in hot water networks, the processes of differentiation and the resistance of cysts to disinfection treatments have been poorly studied. Therefore we investigated morphological and ultrastructural changes occurring during encystment and excystment of V. vermiformis. It appears that encystment is a fast process (9 h) which leads to the formation of cysts surrounded by a double-layered wall. During excystment, trophozoites do not emerge through an ostiole as is the case with Acanthamoeba. Then, we studied the effect of environmental conditions and cell concentration on encystment. We observed that the higher cell concentration was, the faster the encystment was, which suggests the existence of intercellular communication. Finally, we studied the resistance of cysts to conventional disinfection treatments used in hot water networks and to innovative treatment with proteases. These treatments were effective, in vitro, to inactivate V. vermiformis cysts. This work provides new finding regarding differentiation processes and cysts resistance of V. vermiformis, a free-living amoeba poorly studied.

Depuis plusieurs décennies, la communication intercellulaire par jonctions gap (CIJG) est connue pour être impliquée dans la cancérogenèse. Cette implication semble complexe par le fait que les connexines pourraient augmenter la capacité d’invasion des cellules cancéreuses tout en diminuant leur prolifération. Ceci était particulièrement observé pour la connexine 43 (Cx43) dans le cas de cellules de gliomes. Or, les propriétés d’invasion des gliomes de haut grade, les glioblastomes multiformes (GBM), les rendent difficiles à supprimer par résection chirurgicale et favorisent leur récidive.<br/> Afin de préciser le rôle de la Cx43 dans le contrôle des capacités invasives de cellules de GBM, nous avons utilisé une lignée de cellules de glioblastome humaine U251 exprimant par shRNA des niveaux, en ARNm et protéiques, de Cx43 réduits. Ces clones shRNA des cellules U251 montrent une corrélation entre le niveau d’expression de la Cx43 et le processus d’invasion. Au cours de ce travail, nous avons montré, pour la première fois, que la Cx43 est localisée dans les structures protéolytiques permettant l’invasion, les invadopodes. Nous avons démontré aussi que, par sa localisation, la Cx43 favorise la formation des invadopodes en agissant comme une protéine d’échafaudage qui permet l’interaction de Src de la Cortactine. De plus, l’activité hémicanal de la Cx43, probablement inhibée par le Bisphénol A, possède des effets négatifs sur la cinétique de développement des invadopodes. Une étude du protéome et du sécrétome des cellules U251 et des clones shRNA a permis l’identification des protéines impliquées dans l’invasion et la formation et fonction des invadopodes.<br/> En conclusion, la Cx43 participe au processus invasif des cellules de GBM en favorisant la formation et la fonction des invadopodes. Cette nouvelle fonction de la Cx43 semble être la conséquence de ses propriétés de protéines d’échafaudage et hémicanal, et non de son rôle principalement décrit dans la CIJG. / Since several decades, the gap junction intercellular communication (GJIC) is known to be involved in carcinogenesis. This involvement seems complicated by the fact that connexins could increase cancer cells invasion ability while decreasing their proliferation. This was especially observed for connexin 43 (Cx43) in the case of glioma cells. But high-grade gliomas, glioblastoma multiform (GBM) has invasion properties that make it difficult to remove surgically and promote their recurrence.<br/> To clarify the Cx43 role in the control of GBM cells invasive capacities, we used the GBM U251 cell line expressing Cx43 levels, mRNA and protein, reduced by shRNA strategy. Through this approach, we confirmed that Cx43 expression level is associated with the invasive capacity of GBM cells. Furthermore we have shown, for the first time, that Cx43 is localized in invasive proteolytic structures, the invadopodia. We also show that, by its location, Cx43 promotes invadopodia formation by acting as a scaffolding protein that allows Src and Cortactin interaction. Moreover, Cx43 hemichannel activity, probably inhibited by Bisphenol A, has negative effects on invadopodia kinetics development. A proteome and secretome study of U251 cells and shRNA clones allowed the identification of proteins involved in invasion and invadopodia formation and function.<br/>In conclusion, Cx43 participates in the invasive process of GBM cells by promoting invadopodia formation and function. This new function of Cx43 seems to be the result of its scaffold proteins and hemichannel properties, but not its role described mainly in CIJG.

Beside tumor cells themselves, tumors consist of many non-malignantly transformed cellular elements and an extracellular matrix. This so-called tumor microenvironment, or stroma, significantly influences the biological properties of the tumor through intercellular interactions. In this thesis I have focused on the study of tumor-associated fibroblasts in squamous cell carcinomas of the head and neck, malignant melanoma and glioblastoma. The data show the presence of cells with mesenchymal characteristics, present even in the glioblastoma stroma, which could potentially have a positive effect on proliferative activity and invasiveness of glioblastoma cells. In malignant melanoma, the presence of keratinocytes should also be considered, as they are the major cells of the epidermis influencing tumor melanocytes. The conditioned medium from UVB irradiated keratinocytes and non-irradiated fibroblasts stimulates the invasion of malignant melanoma cells. Targeting the tumor stroma may be a new direction in oncological therapy, so we have focused on the influence of synthetic polyamine on the formation of myofibroblasts, which are an active part of the population of tumor-associated fibroblasts. The tested polyamine prevents the formation of myofibroblasts but has no effect on those already formed nor on...

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La cellule est l’unité à la base de la vie. Elle est généralement délimitée par sa membrane et contient un noyau et du cytoplasme en plus d’autres composantes. Les cellules se divisent afin de maintenir et de perpétuer la vie par duplication de leur matériel génétique et par leur séparation en deux cellules physiquement distinctes durant la cytocinèse. Cependant, la division cellulaire est parfois modifiée et aboutit à la formation d’un tissu contenant plusieurs noyaux bordés d’une membrane unique appelé syncytium. Les syncytia sont fréquemment retrouvés chez les organismes vivants, bien que leurs fonctions et mode de formation restent peu compris. L’organisation en syncytium est conservée chez tous les animaux étudiés à ce jour au niveau de la lignée germinale dans laquelle les cellules partagent un cytoplasme commun par l’intermédiaire d’un pont intercellulaire stable. Dans la majorité des lignées germinales étudiées, les cellules sont directement connectées l’une à l’autre par un pont intercellulaire stable qui émerge de cytocinèses incomplètes. Cependant, certaines lignées germinales sont organisées autour d’une cavité commune à laquelle chaque cellule germinale est connectée. Dans ces lignées germinales, les mécanismes qui mènent à l’expansion du syncytium sont peu compris. Ma thèse décrit l’utilisation de la lignée germinale primordiale de C. elegans à son premier stade larvaire pour mieux comprendre l’organisation, l’expansion et la fonction des lignées germinales syncytiales. En utilisant la microscopie électronique et confocale, j’ai découvert que l’organisation du syncytium est fixée au premier stade larvaire. En effet, les deux cellules germinales primordiales (CGP) sont chacune individuellement connectée à une cavité cytoplasmique centrale par le biais de ponts intercellulaires stables. Nous avons nommé cette cavité le proto-rachis car l’organisation des CGP est identique à l’organisation de la gonade adulte. Chez l’adulte, les ponts intercellulaires qui connectent les cellules germinales au rachis sont stabilisés par des régulateurs d’actomyosine. Nous avons vérifié si cela était également le cas dans la gonade au premier stade larvaire. Tous les régulateurs présents dans la gonade adulte, sont aussi présent dans les ponts intercellulaires des CGP, mais la lignée germinale primordiale est réfractaire à la perturbation de la fonction de ces régulateurs. Ce résultat suggère que les régulateurs d’actomyosine sont organisés de manière très stable au premier stade larvaire. Afin de mieux comprendre comment le syncytium se développe dans la lignée germinale de C. elegans, j’ai ensuite suivi la première division des CGP par microscopie à temps réel. J’ai mis en évidence que l’anneau de cytocinèse se stabilise, puis se déplace vers le proto-rachis jusqu’à qu’il s’y intègre. Ces résultats indiquent que le syncytium se développe par cytocynèse incomplète. De plus, mes résultats montrent que la connexion au proto-rachis est maintenue durant la division des CGP. C’est pourquoi nous proposons un modèle pour l’expansion du syncytium dans lequel l’anneau de cytocinèse stabilise pour connecter une des cellules filles au proto-rachis, tandis que l’autre cellule fille est connecté par l’anneau stable qu’elle aura hérité de la cellule mère. Enfin, pour s’assurer que les mécanismes d’expansion du syncytium observés durant la division des CGP sont conservés au cours du développement de la gonade, j’ai conceptualisé et créé un dispositif de micro-fluidique qui en théorie permettrait de suivre plusieurs séries de division des CGP. En somme, mon travail de doctorat a fourni une caractérisation détaillée de la structure du syncytium dans la lignée germinale de C. elegans au premier stade larvaire, ainsi qu’un modèle pour l’expansion du syncytium. Mes découvertes indiquent que malgré des différences dans l’organisation des syncytia, la cytocinèse incomplète est un mécanisme conservé dans toutes les lignées germinales animales. Des travaux futurs seront nécessaires pour découvrir quelles voies de signalisation moléculaires sont sous-jacentes aux mécanismes de formation des syncytia, et ainsi de mieux comprendre quelle est la fonction de ces structures fascinantes. / The cell constitutes the basic unit of life. It is generally delimited by its membrane and contains a nucleus and cytoplasm amongst other components. To maintain and perpetuate life, cells divide by duplicating their genetic material, and by physically separating into two distinct cells during the process called cytokinesis. However, cell division is sometimes modified and leads to the formation of a tissue in which several nuclei are delimited by a single membrane, called a syncytium. Syncytial tissues are common amongst living organisms, but why and how they form remains unclear. The syncytial architecture is conserved in all studied animal germlines where germ cells share a common cytoplasm through stable intercellular bridges. In most animal germlines, the germ cells are directly connected with one another, and the stable intercellular bridges that connect the cells are known to arise from regulated incomplete cytokinesis. However, some germlines are organized around a central common cavity to which each germ cell is connected. In such germlines, the mechanisms of syncytium expansions remain unknown. My thesis describes the use of the C. elegans germline primordium at the first larval stage to better understand the organization, the expansion, and the function of germline syncytia. Using electron and confocal microscopy I found that the organization of the syncytium is established at the first larval stage. The two germ cells called the primordial germ cells (PGCs) each connect to a central cytoplasmic cavity through stable intercellular bridges. Because this organization is identical to the adult germline where each germ cell is connected to the central rachis, we termed the cavity between the PGCs proto-rachis. In the adult gonad, the intercellular bridges that connect the germ cells to the rachis are stabilized by actomyosin regulators, so I verified if this was also the case in the first larval stage gonad. All the regulators that localize to adult intercellular bridges were also present between the PGCs, but the primordial germ line is refractory to perturbation of these regulators. This suggests that the actomyosin regulators are organized in a very stable manner in the first larval stage germline. I next tracked the first division of the PGCs with live imaging to better understand how the syncytium expands in the C. elegans germline. I found that the cytokinetic ring stabilizes, then displaces towards the proto-rachis until it integrates into the syncytial structures. This finding suggests the syncytium expands by incomplete cytokinesis. In addition, my results indicate that the connection to the proto-rachis was maintained during PGCs division. We therefore propose a model in which the cytokinetic ring stabilizes and connects one of the daughter cells to the proto- rachis while the other cell is connected through the inherited stable ring from the mother cell. Finally, I designed and a created a microfluidic device that in theory would allow us to live image several rounds of PGCs division. This would confirm if the mechanisms of syncytium expansion that we observed during the first division of the PGCs are conserved in further development. My work has provided a detailed characterization of the syncytial structure in the C. elegans germline primordium as well as a model for syncytium expansion. My findings indicate that despite differences in the organization of the syncytium, incomplete cytokinesis is conserved as the mechanism for syncytium expansion in all animal germlines. Further research will be necessary to bring to light the molecular pathways underlying syncytium formation to have a better understanding of the function of these fascinating structures.

Thesis with manuscript / La communication intercellulaire joue un rôle important dans tous les organismes, car elle permet l'échange de molécules bioactives entre les cellules. La plupart des cellules peuvent sécréter des vésicules extracellulaires (VE) délimitées par une membrane, qui peuvent servir de médiateur pour le transfert sélectif de matériel génétique, en particulier de molécules d'ARN, vers des cellules réceptrices et modifier leur phénotype. Pour faciliter le ciblage de l'ARN vers les VE, les protéines de liaison de l'ARN (RBP) interagissent avec les ARN, formant des complexes ribonucléoprotéiques (RNP) et guidant leur localisation vers les sites de production des VE. Alors que la facilitation du transport de l'ARN par les RBP est bien étudiée dans les cellules, leur mécanisme dans les VE reste mal compris. Pour mieux comprendre le chargement sélectif des ARN dans les VE, nous avons effectué un profilage RBP complet du sécrétome libéré par les cellules épithéliales mammaires humaines (HMLE), en comparaison avec le matériel des cellules entières. Nous avons d'abord utilisé la réticulation UV pour lier de manière covalente les RBP et leurs ARN associés, puis nous avons isolé le sécrétome par ultracentrifugation et purifié les RBP par extraction d'ARN lié à des protéines (XRNAX) et par spectrométrie de masse (MS). Grâce à une analyse comparative des RBP dans les environnements cellulaire et extracellulaire, nous avons identifié des collections de protéines associées à l'ARN qui étaient communes aux deux compartiments (n=189), ou qui présentaient une association plus spécifique avec l'ARN dans les échantillons cellulaires (n= 866) ou EV (n=502). Nous avons constaté que les RBP du compartiment extracellulaire (ExRBP) avaient des signatures fonctionnelles distinctes (par exemple, le métabolisme cellulaire, la structure et la modification des cellules, le repliement des protéines) par rapport aux facteurs enrichis en cellules (par exemple, le processus métabolique de l'ARN non codant). Notamment, des RBP EV bien connus, tels que ALIX, ANXA1, hnRNPR, hnRNPQ (SYNCRIP), YBX1, ainsi que des marqueurs EV de tétraspanine (CD9, CD81, TSG101), étaient enrichis dans l'ExRBP, ce qui indique le succès de XRNAX dans la purification des RBP EV. En comparant notre collection d'ExRBP aux signatures MS des VE plus pures dérivées des cellules HMLE, nous avons également pu délimiter les ExRBP qui vi sont susceptibles d'être enrichies en VE (PureEVRBP) par rapport à celles trouvées dans le sécrétome non VE (SecRBP). Il est frappant de constater que le PureEVRBP étaient enrichies en protéines de jonction cellulaire, tandis que le secRBP étaient enrichies en facteurs spliceosomaux, ce qui implique un chargement sélectif des RBP dans les VE ou leur sécrétion dans l'espace extracellulaire. Cette recherche fournit des informations précieuses sur la composition des RBP dans les EV, servant d'ensembles de données protéomiques clés pour élucider davantage les mécanismes modulant le recrutement sélectif des ARN dans les EV et améliorant notre connaissance des rôles des RBP dans la biologie des VE. / Intercellular communication plays an important role in all organisms, as it enables the exchange of bioactive molecules between cells. Most cells can secrete membrane-delimited extracellular vesicles (EVs), which can mediate the selective transfer of genetic material, in particular RNA molecules, to recipient cells and modify their phenotypes. To facilitate the targeting of RNA to EVs, RNA binding proteins (RBPs) are thought to interact with RNAs, forming ribonucleoprotein complexes (RNPs) and guiding their localization to sites of EV production. While the facilitation of RNA transportation by RBPs is well-studied in cells, their mechanism in EVs remain poorly understood. To gain insights into the selective loading of RNAs into EVs, we conducted comprehensive RBP profiling on the secretome released by Human Mammary Epithelial (HMLE) cells, in comparison to whole cell material. We first employed UV cross-linking to covalently link RBPs and their associated RNAs, followed by secretome isolation through ultracentrifugation and purification of RBPs using Protein-Xlinked RNA Extraction (XRNAX) and mass spectrometry (MS). Through a comparative analysis of RBPs in cellular and extracellular environment, we identified collections of RNA-associated proteins that were common to both compartments (n=189), or which exhibited more specific RNA association in cellular (n= 866) or EV (n=502) specimens. We found that extracellular compartment RBPs (ExRBPs) had distinctive functional signatures (e.g. cellular metabolism, cell structure and modification, protein folding) compared to cellular-enriched factors (e.g. non-coding RNA metabolic process). Notably, well-known EV RBPs, such as ALIX, ANXA1, hnRNPR, hnRNPQ (SYNCRIP), YBX1, as well as tetraspanin EV markers (CD9, CD81, TSG101), were enriched in ExRBP, indicating the success of XRNAX in EV RBP purification. By comparing our ExRBP collection to the MS signatures of more pure HMLE cell derived EVs, we were also able to delineate ExRBPs that are likely to be EV-enriched enriched versus those found in the non-EV secretome. Strikingly, pure EV-RBPs were enriched for cell-junction proteins, while general secretome RBPs were enriched for spliceosomal factors, implying selective loading of RBPs into EVs or their secretion into the extracellular space. This research provides valuable insights into the composition of RBPs in EVs, serving as key proteomic datasets to further elucidate iv the mechanisms modulating the selective recruitment of RNAs into EVs and enhancing our knowledge of the roles of RBPs in EV biology.