La présence synchrone de métastases hépatiques (MH) et carcinose péritonéale (CP)d'origine colorectale (CRC) est associée à une survie globale médiocre et est traditionnellement considérée comme une contre-indication à l’approche chirurgicale curative. Cependant, suite aux résultats encourageants après traitement chirurgicale, quelques séries ont rapporté une survie prolongée atteignant 3 ans chez des patients sélectionnés, ce qui suggère qu’un traitement chirurgicale curative est possible. À ce jour, en cas de chirurgie majeure hépatique et péritonéale associée, aucune stratégie thérapeutique n'a été établie, Nous avons postulé que la régénération hépatique après une résection hépatique pourrait favoriser la croissance de la CP. Nous avons construit un modèle animal immunnocompetent de CP limitée. L'objectif de l’étude était d'analyser les effets de l’hépatectomie majeure et de la régénération hépatique dans notre modèle murin de PC et le processus d'angiogenèse associé. En outre, nous avons analysée une cohorte prospective multicentrique de patients ayant eu une résection hépatique et une chirurgie cytoréductive avec HIPEC synchrone. L'objectif de cette étude était d'évaluer les outcomes et identifier les facteurs pronostiques afin d'optimiser la sélection des candidats au traitement chirurgical et de déterminer la stratégie chirurgicale optimale. / The synchronous presence of liver metastases (LM) and peritoneal carcinomatosis (PC)from colorectal cancer (CRC) is associated with poor outcome and is traditionally considered acontraindication to any surgical approach. However, few series reported a prolonged survival aftersurgical management, reaching 3 years in selected patients thus suggesting that a curative surgicalmanagement may be possible. To date, no standard management pathway has been established,especially if a major liver and peritoneal surgery has to be performed. We postulated that liverregeneration after liver resection could promote PC growth. We constructed an immunocompetentanimal model of limited PC. The objective of our study was to analyze the effects of major LR andliver regeneration after hepatectomy on peritoneal carcinomatosis growth and the associatedangiogenesis process. Furthermore, we have analyzed a prospective international cohort of patientsundergoing synchronous liver resection and cytoreductive surgery with HIPEC. The aim of this studywas to describe the outcomes, to identify variables potentially related to poor outcome, in order toestablish future guideline for the management of these patients, to optimize the selection of candidatesfor surgical treatment and determine the best surgical strategy.

L'arthrose (OA) est une maladie dégénérative avec un impact remarquable sur la qualité de vie. Pourtant, aucune intervention pharmacologique entièrement appropriée, aucune thérapie biologique ou procédure n'entraînent la dégradation progressive de l'articulation OA. Ici, nous explorons les cellules souches mésenchymateuses (MSC) - précurseurs multi-potentiels de cellules qui peuvent être isolées à partir de différents niveaux de dégradation du cartilage. Nous émettons l'hypothèse que les cellules progénitrices mésenchymateuses (CPM) pourraient servir comme une thérapie potentielle. Le cartilage ostéoarthritique humain a été obtenu de 25 patients subissant un remplacement total du genou et classé en différents niveaux de dégradation. Les niveaux d'expression de l'ARNm de CD105, CD166, Notch 1, Sox9, Acan, Col II A1 et Col I A1 ont été mesurés au jour 0, au jour 14 (2 semaines in vitro) et au jour 35 (après chondrogénèse). Les cellules de toutes les classes d'OA ont augmenté de façon significative les marqueurs MPC de l'ARNm avec expression in vitro. Les cellules proliférées ont exprimées des marqueurs spécifiques aux MPC: CD105, CD166, CD73, CD90, Notch – 1 and Nucleostemin. La chondrogénèse induit une diminution de l'ARNm de CD105, de Notch 1 et de Sox9 seulement dans l'OA légère et modérée. Cependant, seules les pastilles modérées dérivées d 'OA ont révélé des signes de cartilage hyaline élevé - collagène II et faible expression de fibrocartilage - collagène I à la fois au niveau de l’ARNm et de la protéine. Une nouvelle conclusion émerge de nos données et confirme les différences dans les marqueurs MPC entre les différents niveaux de dégradation. Seules les cellules dérivées d 'OA modérées ont été capables de former une matrice hyaline composée de protéoglycanes et de collagène II avec le niveau faible en collagène I fibrocartilagineux. Nos résultats montrent que les CPM provenant d’un cartilage d’un niveau de dégradation modéré ont un fort potentiel d'auto-réparation. / Osteoarthritis (OA) is a degenerative disease with a remarkable impact on quality of life. Yet no fully appropriate pharmacological intervention, biologic therapy or procedure stops the progressive degradation of the OA joint. Herein, we explore mesenchymal stem cells (MSCs)—multipotent precursors of cells that can be isolated from different grades of OA cartilage. We hypothesize that mesenchymal progenitors cells (MPC), could emerge as a potential therapy. Human osteoarthritic cartilage were obtained and scored (according to the OARSI) from 25 patients undergoing total knee replacement. mRNA expression levels of CD105, CD166, Notch 1, Sox9, Acan, Col II A1 and Col I A1 were measured at day 0, day 14 (2 weeks in vitro) and day 35 (after chondrogenesis). Cells from all OA grades significantly increased MPC markers mRNA with in vitro expression. Proliferated cells expressed MPC specific antigens: CD105, CD166, CD73, CD90, Notch – 1 and Nucleostemin. The chondrogenesis induced decrease in CD105, Notch 1 and Sox9 mRNA only in mild and moderate OA. Yet, only moderate OA – derived pellets revealed high hyaline cartilage marker – collagen II and low fibrocartilage marker – Collagen I expression at both mRNA and protein level. A novel finding emerges from our data and confirms differences in MPC markers between OA grades. Only moderate – OA derived cells were able to form hyaline – like matrix composed of proteoglycans and collagen II with law level of fibrocartilaginous collagen I. Further studies that investigate the mechanistic effects of chondrogenic progenitor populations associated with aging and the progression of OA are crucial to our understanding of the clinical relevance of these cells for use in cartilage repair therapies.

A causa mais frequente de surdez de herança autossômica recessiva são as mutações no lócus DFNB1, onde estão os genes GJB2 e GJB6. Dentre os indivíduos com deficiência auditiva associada a esse lócus, 10% a 50% apresentam uma única mutação recessiva no gene GJB2, frequência muito superior à esperada em função da frequência de heterozigotos na população geral. Apesar de alguns desses casos terem sido elucidados após a identificação de grandes deleções no gene GJB6 ou nas suas proximidades, a existência de muitos indivíduos com uma única mutação patogênica no gene GJB2 sugere que a haplo-insuficiência nesse gene possa interagir com outras mutações no mesmo gene, no gene GJB6 vizinho, ou até em outros genes. O objetivo desse estudo foi identificar novos alelos patogênicos, novas proteínas e novos genes que interagem com o lócus DFNB1, do ponto de vista molecular e celular, e que possam ser responsáveis por surdez de herança autossômica recessiva. Desse modo, pretendemos contribuir para o esclarecimento da patogênese da surdez de herança autossômica recessiva. Nesse trabalho, três tipos de estudos foram realizados, com metodologias próprias. Na primeira parte, buscamos identificar novos alelos patogênicos no lócus DFNB1 que poderiam ser responsáveis por surdez quando presentes em heterozigose composta com outros alelos patogênicos nos genes GJB2 e GJB6. Foi realizada a análise do DNA de 16 pacientes surdos portadores de uma única mutação patogênica em um desses dois genes por meio: (i) do sequenciamento das regiões codificadora, promotora e doadora de splicing (intron 1) do gene GJB2, (ii) da triagem de uma deleção de 200 kb localizada a 130 kb da proximidade distal da região 5\' do gene GJB6 e (iii) da pesquisa de variações no número de cópias de um ou mais exons dos genes GJB2, GJB6, GJB3 e WFS1 por MLPA (Multiplex Ligation-dependent Probe Amplification). Detectamos uma segunda mutação provavelmente patogênica em dois dos 16 pacientes heterozigotos: em um deles, a mutação p.L76P (c.C227T) foi identificada na região de código do gene GJB2 e foi por nós descrita pela primeira vez; no segundo caso, uma duplicação (0,4-1,2Kb) que inclui a região de código do gene GJB2 foi detectada, também inédita na literatura. Na segunda parte, tivemos como objetivo obter um modelo experimental para estudos funcionais in vitro da proteína codificada pelo gene GJB2, a conexina 26, em seu local de expressão que são as células de suporte do órgão de Corti. Padronizamos o cultivo in vitro de células progenitoras do órgão de Corti de camundongos e de cobaias e conseguimos obter a diferenciação in vitro das otoesferas dos camundongos em células que expressam marcadores de células ciliadas (Miosina VIIa e Jagged2) e de células de suporte (p27kip e Jagged1). Por fim, na terceira parte, buscamos por proteínas que interagem com a conexina 26 por meio de ensaios de precipitação por afinidade. Para isso, produzimos clones recombinantes de uma proteína de fusão GST-Cx26 e de uma proteína controle (GST), e realizamos sua expressão in vitro em bactérias E.coli B21. Ensaios de precipitação por afinidade entre a proteína de fusão GST-Cx26 ou GST sozinha e proteínas extraídas de cérebro ou fígado de camundongos foram realizados em diferentes condições. A identificação e a análise das proteínas presentes em bandas de SDS-PAGE, obtidas no ensaio de precipitação com a proteína de fusão GST-Cx26 e ausentes no ensaio com a GST, foram realizadas por espectrometria de massas. Identificamos um total de 49 proteínas candidatas a interagirem com a região C-terminal da Cx26. Realizamos diversas análises in silico e em literatura específica e após exclusão de candidatas por: (i) redundância de representação no ensaio GST-Cx26, (ii) diferença entre a massa molecular esperada e a obtida, (iii) precipitação inespecífica e (iv) localização subcelular incompatível com a conexina 26, selecionamos um total de 22 proteínas candidatas a interagirem com a região C-terminal da conexina 26, para estudos futuros. A confimação da interação entre essas 22 proteínas e a conexina 26 é desejável por meio de estudos de co-localização e imuno-coprecipitação / The most frequent causes of nonsyndromic recessive hearing loss are mutations in locus DFNB1, in the GJB2 and GJB6 genes. Among the individuals with hearing loss with mutations in this locus, 10% to 50% present a single recessive mutation in the GJB2 gene, frequency much higher than expected taking into account the frequency of heterozygotes in the general population. Although some of these cases have been elucidated after the identification of large deletions in GJB6 or its surrounding regions, the existence of many individuals with a single pathogenic mutation in the GJB2 gene suggests that haplo-insufficiency of this gene may interact with other types of mutations in the same gene, in the neighbor gene GJB6, or even in other genes. The aim of this study was to identify new pathogenic alleles, proteins and genes that interact with the locus DFNB1, from the molecular and cellular perspective, and that may be responsible for autosomal recessive deafness. Thus, we aimed to contribute to the understanding of the pathogenesis of autosomal recessive deafness. In this work, three different types of studies were performed, each one with a particular methodology. In the first part, we searched for new pathogenic alleles in the locus DFNB1 that could be responsible for deafness, when present in compound heterozygosis with other pathogenic alleles in GJB2 and GJB6 genes. We performed DNA analysis in samples from 16 deaf patients, carriers of a single pathogenic mutation in one of these two genes by: (i) sequencing the coding, promoter and splice donor (intron 1) regions of the GJB2 gene, (ii) screening for a deletion of 200 kb located 130 kb upstream from GJB6 gene and (iii) investigating copy number variations in of one or more exons of the genes GJB2, GJB6, GJB3 and WFS1 by MLPA (Multiplex Ligation-dependent Probe Amplification). We detected a second mutation, probably pathogenic, in two of the 16 heterozygous patients: in one case, the p.L76P (c.C227T) mutation was identified in the coding region of the GJB2 gene and was firstly described by us; in the second case, a novel duplication (0.4 - 1.2 Mb) that includes the coding region of the GJB2 gene was detected. In the second part, our objective was to obtain an experimental model for in vitro functional studies of the protein encoded by the GJB2 gene, connexin 26, in its site of expression, that is, in the supporting cells of the organ of Corti. We standardized the culturing of guinea pigs and mice progenitor cells of organ of Corti. We were also able to induce differentiation of mice\'s otospheres into cells that express markers of hair (myosin VIIa and Jagged2) and supporting cells (p27kip and Jagged1). Finally, we searched for connexin 26 interacting proteins by pull-down assays. Recombinant clones expressing a fusion protein GST-Cx26 and a control protein (GST) were produced, so that in vitro expression in E. coli B21 could be performed. Pull-down experiments, perfomed with fusion protein GST-Cx26 or GST alone, and with proteins from mice brain or liver extracts were done under several different conditions. The identification and analysis of proteins present in SDS-PAGE bands in experiments performed with the fusion protein GST-Cx26, and absent in the GST assay, were performed by mass spectrometry. We identified a total of 49 candidate proteins for interaction with the C-terminal region of Cx26. In silico analyses performed in several databases and search in the literature allowed exclusion of candidates by: (i) redundancy of representation in the GST-Cx26 experiments; (ii) discrepancy between the expected and the obtained molecular weight; (iii) nonspecific precipitation and (iv) subcellular localization incompatible with connexin 26 localization. Summing up, we selected a total of 22 candidate proteins to interact with the C-terminal region of connexin 26. Confirmation of the interaction between these proteins and connexin 26 is planned to be performed by co-localization studies and by immuno-coprecipitation