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Dissertação (mestrado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Tecnologias Química e Biológica, 2018. / Submitted by Robson Amaral (robsonamaral@bce.unb.br) on 2018-05-11T15:04:54Z No. of bitstreams: 1 2018_AnaLauraAlfonsoPérez.pdf: 2516747 bytes, checksum: b579caf86dfe900c3615afb57174b809 (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2018-06-07T13:22:17Z (GMT) No. of bitstreams: 1 2018_AnaLauraAlfonsoPérez.pdf: 2516747 bytes, checksum: b579caf86dfe900c3615afb57174b809 (MD5) / Made available in DSpace on 2018-06-07T13:22:17Z (GMT). No. of bitstreams: 1 2018_AnaLauraAlfonsoPérez.pdf: 2516747 bytes, checksum: b579caf86dfe900c3615afb57174b809 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). / O sistema de expressão baseado na utilização da levedura Komagataella phaffii tem sido utilizado com sucesso na produção de uma grande variedade de proteínas heterólogas. Esta levedura reúne características como fácil manipulação molecular, crescimento celular rápido, capacidade de realizar modificações pós-traducionais e secreção eficiente de proteínas, além de atingir altas densidades celulares com grande produção da proteína heteróloga. Uma das proteínas de grande interesse na indústria biofarmacêutica e cosmética é o fator de crescimento epidermal humano (hEGF). O objetivo desse estudo foi desenvolver um sistema para a produção de hEGF na levedura K. phaffii. Para isso foram utilizados os peptídeos sinais dos genes αF, SUC2 e PHO1. Três vetores foram construídos, cada um deles contendo um dos peptídeos sinal. Foram construídos os vetores de expressão sob o controle do promotor do PGK1 e utilizados para transformação de K. phaffii M12-K, uma linhagem mutante para o gene KEX1. Os clones recombinantes foram confirmados por PCR de colônia e foram crescidos em meio mínimo para avaliar a cinética de crescimento. Os clones positivos foram selecionados e utilizados na expressão em frasco empregando meio complexo. A presença do hEGF foi avaliada por SDS-PAGE e confirmada por Western blot na presença de um anticorpo específico. Com isso, um clone de cada sistema foi escolhido para a purificação do hEGF por cromatografia de exclusão molecular e RP-HPLC. A partir desses experimentos foi confirmada a presença do hEGF e o polipeptídeo foi parcialmente purificado. Finalmente, foi escolhido o clone 1αF para otimizar o processo de purificação, adicionando uma etapa em HILIC. O hEGF foi satisfatoriamente purificado. O tamanho correto e a sequência do N-terminal igual ao EGF presente em humanos foram confirmados por espectrometria de massas e sequenciamento automático. Os resultados obtidos mostram que o hEGF foi produzido com sucesso em K. phaffii com a sequência primária correta. / The expression system based on the utilization of the yeast Komagataella phaffii has been used successfully used in the production of a large variety of heterologous proteins. This yeast combines several important features such as easy molecular manipulation, rapid cell growth, ability to perform post-translational modifications and efficient secretion of proteins, in addition to large heterologous protein production at high cell densities. One of the proteins of great interest in the biopharmaceutical and cosmetic industry is the human epidermal growth factor (hEGF). The aim of this study was to develop a system for hEGF production in K. phaffii. For this, the 3 different signal peptides sequences from genes αF, SUC2 and PHO1 were tested. Three vectors were constructed, each containing one of the signal peptides. Expression vectors were constructed under the control of the PGK1 promoter and used for transformation of K. phaffii M12-K, a strain mutant for the KEX1 gene. Recombinant clones were confirmed by colony PCR and grown in minimal medium to evaluate growth kinetics. Positive clones were selected and used in flask expression using complex media. The presence of hEGF was assessed by SDS-PAGE and confirmed by western blot with a specific antibody. One clone from each system was chosen for the production and purification of hEGF by molecular-exchange chromatography and RP-HPLC. From these experiments the presence of hEGF was confirmed and the polypeptide was partially purified. Finally, clone 1αF was chosen to optimize the purification process by adding one step in HILIC. The hEGF was successfully purified. Correct size and N-terminal sequence equal to EGF present in humans were confirmed by mass spectrometry and Edman sequencing. Together, our results show that hEGF was successfully produced in K. phaffii with the proper primary structure. / El sistema de expresión basado en la utilización de la levadura Komagataella phaffii ha sido utilizado con éxito en la producción de una gran variedad de proteínas heterólogas. Esta levadura reúne características como fácil manipulación molecular, rápido crecimiento celular, capacidad de realizar modificaciones pos-traduccionales y eficiente secreción de proteínas, además de llegar hasta altas densidades celulares con elevada producción heteróloga. Una de las proteínas de gran interés en la industria biofarmacéutica y cosmétic es el factor de crecimiento epidérmico humano (hEGF). El objetivo de este estudio foi desarrollar un sistema para la producción de hEGF en la levadura K. phaffii. Para eso fueron utilizadas los péptidos señales de los genes αF, SUC2 e PHO1. Fueron construidos tres vectores, cada uno de ellos conteniendo una de las sequencias señales. Los vectores fueron construidos sobre el control del promotor del gen PGK1 y fueron utilizados para la transformación de la cepa M12-K de K. phaffii, que es mutante para el gen KEX1. Los clones recombinantes fueron confirmados mediante PCR de colonias y fueron crecidos en medio mínimo para analizar la cinética de crecimiento. Los clones positivos fueron seleccionados y utilizados para expresión en frasco utilizando medio complejo. La presencia de hEGF fue analizada por SDS-PAGE e confirmada por western blot en presencia de un anticuerpo específico. A partir de ahí, una muestra de cada sistema fue escogida para la purificación de hEGF utilizando cromatografía de exclusión molecular y RP-HPLC. A partir de esos experimentos fue confirmada la presencia de hEGF y el polipeptídeo fue parcialmente purificado. Finalmente, fue escogido el clon 1αF para optimizar el proceso de purificación, adicionando una cromatografía en HILIC. El hEGF fue satisfactoriamente purificado. El tamaño correcto y la secuencia del N-terminal igual al EGF presente en humanos fueron confirmados por espectrometría de masas y sequenciamiento automático. Los resultados obtenidos muestran que fue producido hEGF en K. phaffii, con la sequencia primária correcta.

La couche cornée, couche la plus superficielle de l'épiderme, assure une fonction de barrière multifonctionnelle vitale pour l'organisme. Le maintien de cette barrière dépend de la fonctionnalité des cellules sous-jacentes, les kératinocytes granuleux, dernière couche de cellules vivantes de l'épiderme. Les kératinocytes granuleux contiennent dans leur cytoplasme de nombreux organites tubulo-vésiculaires sécrétoires, appelés corps lamellaires (CL). Les CL jouent un rôle majeur dans la formation et le maintien de cette barrière en déversant leur contenu (lipides, lipases, protéases, inhibiteurs de protéases, peptides antimicrobiens,...) à la transition couche cornée - couche granuleuse. Le trafic intracellulaire des CL est donc un processus déterminant dans l'homéostasie de la couche cornée. Cependant, les mécanismes moléculaires impliqués dans la régulation de ce trafic ne sont pas élucidés. Précédemment, au laboratoire, une analyse systématique par spectrométrie de masse des protéines associées aux CL a permis d'identifier plusieurs GTPases de la famille Rab et certains de leurs effecteurs. Ces protéines étant des régulateurs majeurs du trafic vésiculaire dans tous les types cellulaires, elles pourraient jouer un rôle dans le routage des CL et, ainsi, dans la fonctionnalité de la barrière épidermique. L'objectif de ma thèse était d'identifier les GTPases Rab et effecteurs impliqués dans la régulation du trafic intracellulaire des CL. Dans un premier temps, j'ai mis en évidence que la GTPase Rab11a est fortement exprimée dans les kératinocytes granuleux où elle est partiellement associée aux CL. J'ai montré que la déplétion de Rab11a dans un modèle tridimensionnel d'épiderme reconstruit in vitro réalisée grâce à la technique d'interférence à l'ARN, induit une diminution de la densité et de la sécrétion des CL. L'extinction de Rab11a entraîne également une baisse du taux de céramides et de cholestérol, une désorganisation des lipides intercornéocytaires et une augmentation de la perméabilité de la couche cornée. Dans les épidermes reconstruits déplétés, les composants des CL non-sécrétés sont adressés vers une voie de dégradation lysosomale. Dans un deuxième temps, j'ai observé que la perte d'expression de Rab11a induit une anomalie de distribution du moteur moléculaire Myosine-Vb, effecteur majeur de Rab11a. J'ai donc analysé les conséquences de la déplétion de Myosine-Vb dans les épidermes reconstruits et montré qu'elle induit un phénotype comparable à celui observé lors de la déplétion de Rab11a. L'ensemble de ces résultats suggèrent fortement que le complexe bipartite Rab11a-Myosine-Vb constitue un régulateur majeur de la biogenèse des CL et ainsi, de l'homéostasie de la barrière épidermique. Mon travail de thèse est la première étape du décryptage des voies moléculaires impliquées dans la biogenèse des CL et contribue à une meilleure compréhension du rôle du trafic intracellulaire dans la constitution de la barrière épidermique. Il pourrait permettre de caractériser les mécanismes physiopathologiques associés à un défaut de trafic des CL. / The stratum corneum, the most superficial layer of the epidermis, provides a multifunctional protective barrier which is vital for the organism. The maintenance of this barrier is directly dependent on the underlying granular keratinocytes which are the last living cells in the epidermis. The granular keratinocytes contain in their cytoplasm numerous tubulovesicular secretory organelles called lamellar bodies (LB). LB play a major role in the establishment and the maintenance of the epidermal barrier by releasing their content (lipids, lipases, proteases, protease inhibitors, antimicrobial peptides,...) at the junction between the stratum corneum and the stratum granulosum. Because of LB importance in the maintenance of the stratum corneum homeostasis, the regulation of their trafficking deserves further study.Previously, in my laboratory, a proteomic characterization of LB by mass spectrometry has identified several Rab family GTPases and some of their effectors. In any cell type, from yeast to human, Rab GTPases are considered as major regulator of vesicular trafficking. Thus, I postulated that these proteins could play a role in the regulation of LB routing in the cytoplasm of granular keratinocytes. In this context, the aim of my thesis was to determine which Rab GTPases and effectors are involved in this process. In a first step, I demonstrated that Rab11a is strongly expressed in granular keratinocytes where it is associated with LB. I showed that Rab11a silencing using RNA interference technique in an in vitro tridimensional model of reconstructed human epidermis induces a decrease of LB density and secretion in granular keratinocytes. The Rab11a depletion also leads to a decrease of ceramide and cholesterol level and a disorganization of intercorneocyte lipids, generating a defective epidermal barrier. In depleted reconstructed epidermis, there is a missorting of non-secreted LB components, driven to the lysosomal degradation pathway. In a second step, I observed that Rab11a silencing affects distribution of its effector, the molecular motor Myosin-Vb. So, I analyzed the consequences of Myosin-Vb depletion in the model of reconstructed epidermis and I demonstrated that the phenotype obtained is similar that of a Rab11a depleted epidermis. Taken together, these results strongly suggest that the bipartite complex Rab11a-Myosin-Vb is able to regulate the biogenesis of LB in granular keratinocytes. Thus, this molecular complex is a crucial regulator of the epidermal barrier homeostasis. My thesis work is a first step in the deciphering of the molecular pathway involved in LB biogenesis. It is a breakthrough in the comprehension that membrane dynamic in the granular keratinocytes is a major regulator of epidermal barrier. It may contribute to a better understanding of pathophysiological mechanisms related to dysregulated LB trafficking in skin diseases.

OLIVEIRA, Lauana Pereira de. Células epidérmicas mucilaginosas facilitam a manutenção hídrica foliar em espécies do semiárido? 2015. 48 f. Dissertação (Mestrado em Ecologia e Recursos Naturais)-Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by ELAINE PEREIRA (ellainec.pereira@gmail.com) on 2017-11-07T12:55:08Z No. of bitstreams: 1 2015_dis_lpoliveira.pdf: 307896 bytes, checksum: cf11aba686b239f2a332da5235af7dcd (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-11-24T19:55:09Z (GMT) No. of bitstreams: 1 2015_dis_lpoliveira.pdf: 307896 bytes, checksum: cf11aba686b239f2a332da5235af7dcd (MD5) / Made available in DSpace on 2017-11-24T19:55:09Z (GMT). No. of bitstreams: 1 2015_dis_lpoliveira.pdf: 307896 bytes, checksum: cf11aba686b239f2a332da5235af7dcd (MD5) Previous issue date: 2015 / In environments with severe water deficit, plants have mechanisms that allow the absorption of water and maintenance of water status. Mucilage secretion by plants has been reported as a way to reduce transpiration and water loss, to maintain moisture in tissues and water potential and as a carbohydrate reserve. However, experimental evidence supporting the role of mucilages in these environments is scarce. To survey if the occurrence of mucilaginous epidermal cells increases leaf water potential, we evaluated the water uptake, water potential and gas exchange of leaves of leguminous species with and without mucilage in the epidermis. Our results indicated that mucilage present in epidermal cells is not always directly involved with water absorption. Tests with the Lucifer Yellow apoplastic tracer (LY) showed that there was foliar water absorption, but there was no retention of LY in mucilaginous cells. Though, the species with mucilage absorbed water through the epidermis and the leaf water potential was less negative than in L. ferrea, indicating that these polysaccharides in the epidermis help in the water economy of the plant. / Em ambientes com forte escassez hídrica, as plantas apresentam mecanismos que permitem a absorção de água e a manutenção do status hídrico. A secreção de mucilagem pelas plantas tem sido reportada como uma forma de reduzir a transpiração e a perda de água, de manter a umidade nos tecidos e o potencial hídrico e como reserva de carboidrato. Entretanto, evidências experimentais que comprovem o papel das mucilagens nesses ambientes são escassas. Para testar se a ocorrência de células epidérmicas mucilaginosas aumenta o potencial hídrico foliar, avaliamos a absorção hídrica, o potencial hídrico e as trocas gasosas das folhas de espécies de leguminosas com e sem mucilagem na epiderme. Os nossos resultados indicaram que a mucilagem presente nas células epidérmicas nem sempre está envolvida diretamente com absorção de água. Os testes com o traçador apoplástico Lucifer Yellow (LY), mostraram que houve absorção hídrica foliar, mas não houve retenção do LY nas células mucilaginosas. No entanto, as espécies com mucilagem, absorveram água pela epiderme e o potencial hídrico foliar foi menos negativo do que em L. ferrea, o que indica que esses polissacarídeos na epiderme auxiliam na economia hídrica da planta.

Made available in DSpace on 2014-06-11T19:29:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-03-02Bitstream added on 2014-06-13T20:19:04Z : No. of bitstreams: 1 nascimento_sm_me_araiq.pdf: 2513485 bytes, checksum: 3687c4c4c8aaef5a5668ed797b952c8a (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O peeling químico consiste na aplicação de um ou mais agentes esfoliantes para obter, primeiramente, destruição e posterior regeneração de parte da epiderme e derme. O peeling de fenol é o método mais profundo de peeling químico, onde o agente penetra na epiderme, causando sua necrose e induzindo reação inflamatória controlada não só na epiderme como também na derme, estimulando a síntese de colágeno, determinante no processo de rejuvenescimento facial. O presente trabalho envolveu diversas etapas. A primeira foi a determinação da composição da formulação comercialmente utilizada, análise realizada com o auxílio das técnicas de Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-MS), Cromatografia Líquida de Alta Eficiência (HPLC) e Ressonância Magnética Nuclear de Carbono 13 (C13-RMN), chegando à composição de fenol a 50% e ácido salicílico a 13% em etanol. Foram realizados ensaios biológicos preliminares com camundongos (Mus musculus) para aprendizado do manuseio e observação dos efeitos macroscópicos do peeling. Foram realizados ensaios com coelhos (Oryctolagus cunniculus), onde foram selecionados alguns marcadores de toxicidade para a determinação de toxicidade aguda cardíaca, hepática e renal. Para todos os grupos experimentais os testes forneceram resultados negativos, mostrando a ausência de toxicidade. Ensaios de mimetização in vitro da reação ocorrente na pele, com o acompanhamento por Cromatografia em Camada Delgada (CCD), não forneceram resultados satisfatórios. Para a caracterização da pele e o entendimento do mecanismo de ação do peeling, foram realizados ensaios com camundongos (Mus musculus), onde foram utilizadas as técnicas de Histologia, Espectrometria Raman por Transformada de Fourier (FT-Raman) e Tomografia por Coerência Óptica (OCT). / Chemical peeling consists of the application on the skin of one or more exfoliating agents to obtain first destruction and then regeneration of part of the epidermis and dermis. The phenol peel is the deepest peeling method, where the agent of the peeling penetrates in the epidermis, causing a controlled wound and determining the cutaneus renewal after the synthesis of new collagen fibers. The present work involved various stages. The first was the determination of the composition of the trade sample, using Gas Chromatography coupled to Mass Spectrometry (GC-MS), High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Ressonance of the Nucleus C13 (13C-NMR), concluding that it is composed by 50% phenol, 13% salicylic acid in ethanol. Preliminary biological assays were performed with mice (Mus musculus) with the aim of learning the techniques of handling and the observation of the macro effects of the peeling. Rabbits (Oryctolagus cunniculus) were used to other assays, where some toxicity markers were selected and analysed to determine acute heart, liver and kidney toxicity. For all the experimental groups the results were negative. For the skin characterization and the comprehension of the action mechanism of the peeling, assays were performed with mice (Mus musculus), where it was taken into account the analysis of Histology, Fourier Transform Infrared Spectrometry (FT-Raman) and Optical Coherence Tomography (OCT)

Resumo: O peeling químico consiste na aplicação de um ou mais agentes esfoliantes para obter, primeiramente, destruição e posterior regeneração de parte da epiderme e derme. O peeling de fenol é o método mais profundo de peeling químico, onde o agente penetra na epiderme, causando sua necrose e induzindo reação inflamatória controlada não só na epiderme como também na derme, estimulando a síntese de colágeno, determinante no processo de rejuvenescimento facial. O presente trabalho envolveu diversas etapas. A primeira foi a determinação da composição da formulação comercialmente utilizada, análise realizada com o auxílio das técnicas de Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-MS), Cromatografia Líquida de Alta Eficiência (HPLC) e Ressonância Magnética Nuclear de Carbono 13 (C13-RMN), chegando à composição de fenol a 50% e ácido salicílico a 13% em etanol. Foram realizados ensaios biológicos preliminares com camundongos (Mus musculus) para aprendizado do manuseio e observação dos efeitos macroscópicos do peeling. Foram realizados ensaios com coelhos (Oryctolagus cunniculus), onde foram selecionados alguns marcadores de toxicidade para a determinação de toxicidade aguda cardíaca, hepática e renal. Para todos os grupos experimentais os testes forneceram resultados negativos, mostrando a ausência de toxicidade. Ensaios de mimetização in vitro da reação ocorrente na pele, com o acompanhamento por Cromatografia em Camada Delgada (CCD), não forneceram resultados satisfatórios. Para a caracterização da pele e o entendimento do mecanismo de ação do peeling, foram realizados ensaios com camundongos (Mus musculus), onde foram utilizadas as técnicas de Histologia, Espectrometria Raman por Transformada de Fourier (FT-Raman) e Tomografia por Coerência Óptica (OCT). / Abstract: Chemical peeling consists of the application on the skin of one or more exfoliating agents to obtain first destruction and then regeneration of part of the epidermis and dermis. The phenol peel is the deepest peeling method, where the agent of the peeling penetrates in the epidermis, causing a controlled wound and determining the cutaneus renewal after the synthesis of new collagen fibers. The present work involved various stages. The first was the determination of the composition of the trade sample, using Gas Chromatography coupled to Mass Spectrometry (GC-MS), High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Ressonance of the Nucleus C13 (13C-NMR), concluding that it is composed by 50% phenol, 13% salicylic acid in ethanol. Preliminary biological assays were performed with mice (Mus musculus) with the aim of learning the techniques of handling and the observation of the macro effects of the peeling. Rabbits (Oryctolagus cunniculus) were used to other assays, where some toxicity markers were selected and analysed to determine acute heart, liver and kidney toxicity. For all the experimental groups the results were negative. For the skin characterization and the comprehension of the action mechanism of the peeling, assays were performed with mice (Mus musculus), where it was taken into account the analysis of Histology, Fourier Transform Infrared Spectrometry (FT-Raman) and Optical Coherence Tomography (OCT) / Orientador: Younès Messaddeq / Coorientador: Hérida Regina Nunes Salgado / Banca: Eduardo Maffud Cilli / Banca: Marcos Antônio Corrêa / Mestre

Orientador: Maria Beatriz Puzzi, Maria Helena Stangler Kraemer / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T03:54:34Z (GMT). No. of bitstreams: 1 Souto_LuisRicardoMartinhao_M.pdf: 2402921 bytes, checksum: a79b6ae181ce1b24d01ec608815d8bf7 (MD5) Previous issue date: 2005 / Resumo: A obtenção de uma pele humana que apresente derme e epiderme, reconstruída a partir de células isoladas de pacientes, possibilita a realização de enxertos autólogos de pele reconstruída em laboratório (in vitro) em pacientes com áreas doadoras escassas além de permitir ensaios com substâncias químicas e drogas in vitro e não mais in vivo. A partir da cultura de fibroblastos humanos, é possível obter um número suficiente de células que podem ser injetadas em uma matriz de colágeno bovino tipo I que, mantida imersa em meio de cultura, específico para fibroblastos, permite a formação de uma derme humana reconstruída in vitro. Sobre essa derme, através de cultura de queratinócitos e melanócitos humanos, forma-se uma epiderme diferenciada levando à formação de uma pele humana reconstruída in vitro, constituída de derme e epiderme associadas. Essa pele humana formada é, histologicamente, semelhante à pele humana in vivo. Na derme, identifica-se o tecido colágeno, com suas células, e a matriz extracelular organizados paralelamente à epiderme. Esta se desenvolve em várias camadas. Não há distinção entre derme e epiderme no experimento controle, onde não foi utilizado o colágeno bovino tipo I / Abstract: The technique to obtain human skin presenting dermis and epidermis reconstructed from cells isolated from patients allows the performance of autologous grafts of skin reconstructed in laboratory (in vitro) on patients with scarce donor sites, in addition to permitting trials with chemical substances and drugs no more in vivo, but in vitro. It is possible to obtain a sufficient number of cells from human fibroblast culture that can be injected in bovine collagen type I matrix and kept submerged in a specific culture medium for fibroblasts. This will permit the formation of human dermis reconstructed in vitro. On this dermis, through culture of human keratinocytes and melanocytes, a differentiated epidermis is formed, leading to the creation of human skin reconstructed in vitro, composed of associated dermis and epidermis. This human skin is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with its cells and extracellular matrix organized in parallel to the epidermis, which is developed in several layers / Mestrado / Patologia Clinica / Mestre em Ciências Médicas

Mon travail de thèse a été principalement de comprendre comment les voies de signalisations contrôlent la différenciation et la morphogenèse de l'embryon de vertébré. Les communications entre cellules sont à la base du développement des métazoaires et de leurs évolutions et sont souvent impliquées dans les pathologies humaines. J'ai profité de la puissance des approches fonctionnelles chez le xenope pour essayer de comprendre comment les signaux BMP, FGF, Nodal et Notch sont intégrés dans le temps et l'espace afin de coordonnées différentes décisions cellulaires. Premièrement, nous avons montré que la voie Nodal est active avant la transition mid-blastuléene et permet l'induction du mesedoderme à travers l'auto régulation de l'expression de ces ligands Xnr5 et Xnr6 (Skirkanish et al. soumis à Development). Deuxièmement, j'ai montré que différent ligand de la voie Nodal contrôlent séquentiellement l'induction du mesendoderm et les mouvements de gastrulation (Luxardi et al., Development, 2010). Troisièmement, j'ai montré qu'un cinquième ligand de la voie Nodal, Xnr4, contrôle la régionalisation médio latérale de la plaque neurale ouverte et la neurogenèse. Quatrièmement, nous avons montré qu'une famille de microARN, nir449, contrôle la différenciation des cellules multi-ciliées à travers son action sur un ligand de la voie Notch, Delta-1 (Marcet et al. Nature Cell Biology, en révision). Enfin, j'ai découvert une nouvelle fonction des signaux BMP dans le control de la spécification des épithéliums muco cilié. / My PhD work generally addressed how signaling pathways control differentiation and morphogenesis in the vertebrate embryo. intercellular communication is the basis of metazoan development and evolution and is often involved in human pathologies. I take advantage of the power of functional approaches in the Xenopus embryo, to try and understand how BMP, FGF, Nodal and Notch signals are intragrated in time ans space to coordinate vatious cellular decisions. First, we showed that Nodal signaling is activated before the mid blastula transition and allow mesendoderm induction through the auro regulation of the expression of its ligands Xnr5 and Xnr6 (Skirkanish et al., submitted to development). Second, I have demonstrated that in a gastrulation movements (Luxardi et al., Development, 2010). Third, I have demonstrated that a fifth Nodal ligand, Xnr4, control medio-lateral patterning of the open neural plate and neurogenesis. Froth, we showed that a microRNA family, mir449, controls differenciation of multiciliated cells through the regulation of the Notch ligand Delta-1 (Marcet et al. Nature Cell Biology, in revision). Last, I have discovered a novel function of the BMP pathway in the control of cell type specification within the epidermal mucocialiary epithelium

La protéine E4F1 a été identifiée comme une cible cellulaire de l'onco-protéine virale E1A au cours de l'infection par l'adénovirus de type V. Bien que très peu étudiée jusqu'à présent, les travaux du laboratoire d'accueil indiquent cependant qu'E4F1 est une protéine multifonctionnelle ayant à la fois un rôle de facteur de transcription mais fonctionnant également comme une E3 ligase vis-à-vis d'autres régulateurs transcriptionnels tels que les membres de la famille p53. E4F1 se situe au carrefour de plusieurs voies de signalisation fréquemment altérées au cours de la progression tumorale, notamment les voies impliquant les suppresseurs de tumeurs Rb et p53. In vivo, E4F1 est impliqué dans l'homéostasie des cellules souches à travers différents mécanismes encore non élucidés. L'objectif de mon projet de thèse est d'identifier et de caractériser les voies de signalisation impliquant E4F1 dans l'homéostasie des cellules souches. Pour répondre à cette question, j'utilise diverses techniques : Histologie, IHC,FACS, QPCR et des tests clonogénique sur des cellules issues des épidermes de souris E4F1 WT et KO. / E4F1 protein has been first identified in the 80's as a cellular target of the adenoviral oncoprotein E1A. E4F1 is an atypical multifunctional protein with two different activities. In fact, it can acts as a transcriptional factor thanks to its DNA binding domain and exhibits either positive or negative transcriptional activities depending on the promoter context. In addition to its transcriptional activities, E4F1 is also an atypical E3-ligase for other transcription factors, including the p53 tumor suppressor. A growing body of evidence suggests that the multifunctional protein E4F1 is involved in signaling pathways that play essential roles during normal development and tumorigenesis. Moreover, we have shown that skin defect is one of the most striking phenotype of E4F1 conditional knockout mice. E4F1 inactivation in the entire skin or in the basal compartment of the epidermis (K5 specific knockout mice) induces skin homeostasis defects, as evidenced by transient hyperplasia in the interfollicular epithelium and alteration of keratinocyte differentiation, followed by loss of cellularity in the epidermis and severe skin ulcerations. E4F1 depletion alters clonogenic activity of epidermal stem cells (ESCs) ex vivo and ends in exhaustion of the ESC pool in vivo, indicating that the lesions observed in the E4F1 mutant skin result, at least in part, from cell-autonomous alterations in ESC maintenance. The clonogenic potential of E4F1 KO ESCs is partially rescued by Bmi1 overexpression, or by Ink4a/Arf overexpression or p53 depletion. In additions, in vivo, skin phenotype of E4F1 KO mice is also delayed in animals with Ink4a/Arf and E4F1 compound gene deficiencies. Taking together, our data show the essential role of E4F1 protein into Bmi1-Arf-p53 pathway to regulate ESC-dependent skin homeostasis.

Como qualquer inseto holometábolo, a abelha Apis mellifera sofre metamorfose completa, apresentando grandes mudanças na forma e fisiologia quando passa do estágio larval para o estágio de pupa (muda metamórfica). Após esta muda, com o prosseguimento do desenvolvimento, o tegumento pupal (cutícula e a epiderme subjacente), extensivamente remodelado, é substituído pelo tegumento adulto, definitivo, que passa por intensa melanização e esclerotização. Eletroforese bidimensional e espectrometria de massas foram utilizadas neste trabalho para caracterizar as mudanças do padrão proteico no tegumento em desenvolvimento de operárias e zangões. No total foram identificadas 51 proteínas diferentes no tegumento torácico extraído de larvas, pupas e adultos (adultos-faratos). Quatorze proteínas foram identificadas como genuinamente cuticulares: Apidermina-3,1-like, Apidermina-2, Cuticular proteins analogous to peritrophins-3C e 3D, AmelCPR3, 12, 16 e 27, Glicoproteína SgAbd-2-like, e cinco outras proteínas homólogas à proteínas cuticulares de outras espécies de insetos contendo um domínio de ligação à quitina. As proteínas diferiram principalmente quantitativamente entre as fases de desenvolvimento e sexo, e poucas diferenças qualitativas foram observadas. Por exemplo, Apidermina-2 é típica de tegumentos fortemente esclerotizados e pigmentados. As diferenças quantitativas foram destacadas pela comparação da abundância de algumas proteínas e seus respectivos RNA mensageiros (utilizando RT-PCR em tempo real) entre as fases de desenvolvimento e entre os sexos. Várias proteínas cuticulares mostraram mais de uma forma molecular, aparentemente derivadas de modificações pós-traducionais. Além de conferir suporte experimental para a validação de genes de A. mellifera preditos, ou não-anotados, nossos dados forneceram novas informações sobre as proteínas que atuam no tegumento em desenvolvimento. / As a holometabolous insect, the honey bee undergoes complete metamorphosis, displaying a marked change in shape and physiology when passing from the larval to the pupal stage (metamorphic molt). As development progresses, the extensively remodeled pupal integument (cuticle and subjacent epidermis) is replaced by the adult integument, which undergoes intense sclerotization and melanization. Two-dimensional electrophoresis and mass spectrometry were here used to characterize the changing protein patterns in the developing integument of workers and drones. Overall, we identified 51 different proteins in the thoracic integument extracted from larvae, pupae and adults (pharate adults). Fourteen proteins were identified as genuine cuticular proteins: Apidermin-3,1-like protein, Apidermin-2, Cuticular Proteins Analogous to Peritrophins-3C and 3D, AmelCPR3, 12, 16 and 27, Glycoprotein SgAbd-2-like, and 5 other proteins homologous to cuticular proteins from other insect species, and containing the chitin-binding domain. Integument proteins mainly differed quantitatively among the developmental stages and sexes, although few qualitative differences have also been detected. For example, Apidermin-2 is typical of the heavily pigmented and sclerotized integument. The quantitative differences were highlighted by comparing the levels of some of these proteins and their respective mRNAs (using RT-qPCR) among the developmental phases and between sexes. It is noteworthy that several cuticle proteins showed more than one molecular form, apparently derived from post-translational modifications. In addition to give experimental support for validation of predicted, or unannotated, honey bee genes, our data provided new information on proteins acting in the metamorphosing integument.