Doenças envolvendo órgãos endodermicamente derivados afetam milhares de pessoas no mundo. O sucesso da terapia celular para o tratamento de doenças de órgãos oriundos da mesoderme e ectoderme gera ótimas perspectivas para o uso do mesmo em tratamento de doenças de órgãos de origem endodérmica (tireóide, pulmões, fígado, vesícula biliar e pâncreas). Particularmente, no fígado, ainda que sejam atribuídas propriedades regenerativas em muitas lesões o mecanismo de reparação é insuficiente, tornando o transplante hepático à única opção definitiva. Dentre as células-tronco embrionárias, as células de linhagens hepáticas fetais podem estabelecer-se como fonte importante para a terapia celular em indivíduos com doenças hepáticas, pois possuem um alto índice de diferenciação de hepatócitos e células do ducto biliar in vitro. Evidências apontam a progranulina como um fator de crescimento de grande habilidade para a indução de proliferação celular, uma vez que está envolvida no desenvolvimento embrionário e neonatal. Sendo assim, neste trabalho foram utilizados embriões de ratos Fischer 344 com idades gestacionais 12,5; 13,5; 14,5; 15,5; 16;5 para caracterizar o papel do novo fator de crescimento progranulina na hepatogênese. Foram realizadas análises histológicas, de microscopia eletrônica de transmissão e imuno-histoquímicas nos embriões. Houve expressão de progranulina e Oct-4 (marcador de célula tronco indiferenciada) principalmente nas idades gestacionais de 13,5 a 16,6 dias e 12,5 a 16,5 dias para PCNA (marcador de proliferação celular). Dessa forma acredita-se que, a progranulina atua nos processos de proliferação celular e diferenciação das células-tronco do broto hepático, podendo ser usada como um fator de diferenciação em culturas in vitro visando à diferenciação de células-tronco do broto hepático em hepatócitos funcionais para a terapia celular. / Diseases involving endodermal-derivated organs affect thousands of people all over the world. The cell therapy success for treating diseases of organs originated from mesoderm and ectoderm generates excellent perspectives for its use in treating diseases of organs of endodermal origin (thyroid, lungs, liver, gallbladder and pancreas). Particularly in the liver, although regenerative proprieties are attributed in many lesions, the repairing mechanism is insufficient, making the hepatic transplant the only definitive option. Among the embryo stem cells, the fetal hepatic lineage cells may establish themselves as important sources for cell therapy in individuals with hepatic diseases, since they have high ability in hepatocytes and billiar duct cells differentiation in vitro. However, for the use of hepatic lineage embryo stem cells as a bi-potential source of differentiation, it is necessary the establishment of efficient proliferation methods in this type of cells. Evidences point to progranuline as a growth factor with high capability for the induction of cell proliferation, since it is involved in the embryo and neo-natal development. Thus, this study used embryos of Fischer 344 rats with gestational ages 12.5, 13.5, 14.5, 15.5 and 16.5 to characterize the role of the new growth factor progranuline in hepatogenesis. We conducted histological, transmission electron microscopy and immunohistochemical in embryos. There was expression of progranuline and Oct-4 (undifferentiated stem cell marker), especially at gestational ages 13.5 to 16.5 days and 12.5 to 16.5 days for PCNA (proliferation marker). So it is believed that progranuline acts on cell proliferation and differentiation of stem cells from the liver bud, which can be used as a differentiating factor in order to cultures in vitro differentiation of stem cells from the liver bud into functional hepatocytes for cell therapy.

Doenças envolvendo órgãos endodermicamente derivados afetam milhares de pessoas no mundo. O sucesso da terapia celular para o tratamento de doenças de órgãos oriundos da mesoderme e ectoderme gera ótimas perspectivas para o uso do mesmo em tratamento de doenças de órgãos de origem endodérmica (tireóide, pulmões, fígado, vesícula biliar e pâncreas). Particularmente, no fígado, ainda que sejam atribuídas propriedades regenerativas em muitas lesões o mecanismo de reparação é insuficiente, tornando o transplante hepático à única opção definitiva. Dentre as células-tronco embrionárias, as células de linhagens hepáticas fetais podem estabelecer-se como fonte importante para a terapia celular em indivíduos com doenças hepáticas, pois possuem um alto índice de diferenciação de hepatócitos e células do ducto biliar in vitro. Evidências apontam a progranulina como um fator de crescimento de grande habilidade para a indução de proliferação celular, uma vez que está envolvida no desenvolvimento embrionário e neonatal. Sendo assim, neste trabalho foram utilizados embriões de ratos Fischer 344 com idades gestacionais 12,5; 13,5; 14,5; 15,5; 16;5 para caracterizar o papel do novo fator de crescimento progranulina na hepatogênese. Foram realizadas análises histológicas, de microscopia eletrônica de transmissão e imuno-histoquímicas nos embriões. Houve expressão de progranulina e Oct-4 (marcador de célula tronco indiferenciada) principalmente nas idades gestacionais de 13,5 a 16,6 dias e 12,5 a 16,5 dias para PCNA (marcador de proliferação celular). Dessa forma acredita-se que, a progranulina atua nos processos de proliferação celular e diferenciação das células-tronco do broto hepático, podendo ser usada como um fator de diferenciação em culturas in vitro visando à diferenciação de células-tronco do broto hepático em hepatócitos funcionais para a terapia celular. / Diseases involving endodermal-derivated organs affect thousands of people all over the world. The cell therapy success for treating diseases of organs originated from mesoderm and ectoderm generates excellent perspectives for its use in treating diseases of organs of endodermal origin (thyroid, lungs, liver, gallbladder and pancreas). Particularly in the liver, although regenerative proprieties are attributed in many lesions, the repairing mechanism is insufficient, making the hepatic transplant the only definitive option. Among the embryo stem cells, the fetal hepatic lineage cells may establish themselves as important sources for cell therapy in individuals with hepatic diseases, since they have high ability in hepatocytes and billiar duct cells differentiation in vitro. However, for the use of hepatic lineage embryo stem cells as a bi-potential source of differentiation, it is necessary the establishment of efficient proliferation methods in this type of cells. Evidences point to progranuline as a growth factor with high capability for the induction of cell proliferation, since it is involved in the embryo and neo-natal development. Thus, this study used embryos of Fischer 344 rats with gestational ages 12.5, 13.5, 14.5, 15.5 and 16.5 to characterize the role of the new growth factor progranuline in hepatogenesis. We conducted histological, transmission electron microscopy and immunohistochemical in embryos. There was expression of progranuline and Oct-4 (undifferentiated stem cell marker), especially at gestational ages 13.5 to 16.5 days and 12.5 to 16.5 days for PCNA (proliferation marker). So it is believed that progranuline acts on cell proliferation and differentiation of stem cells from the liver bud, which can be used as a differentiating factor in order to cultures in vitro differentiation of stem cells from the liver bud into functional hepatocytes for cell therapy.

Les dégénérescences lobaires fronto-temporale (DLFT) sont des démences neurodégénératives rares. 30-50% des DLFT a une cause génétique, la plupart sont des mutations des gènes C9orf72 et progranuline (GRN). L'objectif de la thèse a été d'élargir le spectre mutationnel et phénotypique des mutations GRN. Nous avons identifié les premières délétions partielles du gène GRN chez des patients avec progranulinémie baisse (la progranulinémie est abaissée en cas de mutation), mais sans mutation détectée par séquençage. Nous avons contribué à élargir le spectre clinique de la maladie en décrivant un phénotype d'atrophie corticale postérieure et des lésions de la substance blanche cérébrale chez des patients GRN, caractéristique évocatrice de cette forme génétique. Enfin, nous avons étudié la phase présymptomatique de la maladie, alors que se développent les premiers essais thérapeutiques, par une approche longitudinale avec IRM et TEP-FDG. Le métabolisme cérébral est réduit dans le lobe temporal latéral gauche 20 ans avant l'apparition des symptômes et, après 20 mois, dans les régions frontales et l'épaisseur corticale dans les régions temporales gauche. Le lobe temporal latéral pourrait être donc l'"épicentre " de la maladie, et le processus lésionnel pourrait, secondairement, progresser vers les régions frontales. J'ai également contribué à définir les phénotypes associés aux mutations de gènes plus rares de DLFT/DLFT-SLA. TARDBP est associé à un large spectre phénotypique; TBK1 est caractérisé par une démence sémantique ou aphasie non fluent associés à l'atteinte de la corne antérieure. Cette étude importante souligne le rôle de ces mutations dans le spectre clinique des DLFT. / Frontotemporal lobar degeneration (FTLD) are rare neurodegenerative dementias. 30-50% of FTLD has a genetic cause, most are mutations in C9orf72 and in progranulin gene (GRN). The aim of the thesis was to expand the mutational and phenotypic spectrum of GRN mutations. We identified the first partial deletions of GRN gene in patients with low plasmatic progranulin (the plasmatic progranulin is low in case of mutation), but without mutation detected by sequencing. We contributed to expand the clinical spectrum of the disease by describing a posterior cortical atrophy phenotype and lesions of the cerebral white matter in GRN patients, evocative feature of this genetic form. Finally, we studied the presymptomatic stage of the disease, while the first clinical trials develop, for a longitudinal approach with MRI and FDG-PET. The cerebral metabolism is reduced in the left temporal lobe 20 years before clinical onset and, after 20 months, the metabolism is reduced in the frontal regions and the cortical thickness in the left temporal regions. The lateral temporal lobe could thus be the "epicenter" of the disease, and the lesional process could secondarily progress towards the frontal regions. I also contributed to define the phenotypes associated with rare gene mutations in FTLD/FTLD-ALS. TARDBP is associated with a wide phenotypic spectrum; TBK1 is characterized by semantic dementia or not fluent aphasia associated with involvement of the anterior horn. This important study highlights the role of these mutations in the clinical spectrum of FTLD.

Le terme dégénérescence lobaire frontotemporale ou FTLD définit un groupe hétérogène de maladies neurodégénératives caractérisé par des troubles du langage, du comportement et/ou moteurs qui résultent principalement d’une dégénérescence du cortex frontal et temporal. Cette hétérogénéité tant au niveau clinique, génétique que neuropathologique rend cette pathologie très complexe et il existe aujourd’hui un véritable problème de diagnostic différentiel des FTLD. Le diagnostic final des FTLD repose ainsi sur l’examen neuropathologique, la nature des lésions observées et leurs constituants moléculaires. La caractérisation de ces lésions a permis d’établir une classification des FTLD qui ne cesse d’évoluer avec la découverte de nouveaux acteurs moléculaires. À l’instar de nombreuses maladies neurodégénératives, les FTLD sont caractérisées par la présence de protéines agrégées dans les régions cérébrales affectées. Cependant, contrairement à la maladie d’Alzheimer (MA), ces agrégats ne sont pas toujours constitués des mêmes protéines. Ainsi, approximativement 40% des cas de FTLD présentent des agrégats composés de protéines Tau hyper et anormalement phosphorylées, et forment le groupe FTLD-Tau. Lorsqu’aucune pathologie Tau n’est détectée, les patients présentent généralement des inclusions neuronales cytoplasmiques ou intranucléaires immunoréactives pour la protéine TDP-43 (transactive response DNA binding protein 43), et constituent la sous-classe FTLD-TDP. Plus rarement, la protéine FUS (Fused in Sarcoma, FTLD-FUS) ou des protéines liées au système ubiquitine protéasome peuvent également s’agréger (FTLD-UPS). La génétique représente également une composante majeure des FTLD avec 10 à 15% des cas correspondant à des formes héréditaires dominantes. Les premières mutations furent découvertes sur le gène MAPT. Le gène de la progranuline (GRN) fut ensuite identifié comme fréquemment associé aux FTLD. Plus récemment, une répétition anormale d’héxanucléotides GGGGCC au sein du gène C9ORF72 (chromosome 9 open reading frame 72) a été montrée comme étant responsable d’un grand nombre de cas familiaux de FTLD. De manière moins fréquente, d’autres gènes tels que VCP (valosin containing protein) ou CHMP2B (charged multivesicular body protein 2B) peuvent aussi être associés à des cas familiaux de FTLD. Des années avant la découverte des principaux acteurs moléculaires des FTLD, des études ont décrit une perte partielle ou totale des protéines Tau physiologiques dans le tissu cérébral. A l’origine, ce phénomène fut observé dans un groupe de démences appelées DLDH pour démences sans signe histopathologique distinctif (plus tard appelé FTLD-ni pour no inclusion). En 2006, la majorité de ces cas a été reclassée en tant que FTLD-U (présence de lésions immunoréactives pour l’ubiquitine). En revanche, aucune étude ne s’est intéressée à cette perte de Tau depuis celle de Zhukareva et collègues en 2003. Au regard des récentes avancées sur la compréhension de la base moléculaire et génétique des FTLD, la pertinence de cette perte de Tau reste ainsi encore à déterminer. Dans ce contexte, ce travail de recherche a pour principal objectif d’étudier l’expression des protéines Tau au sein du tissu cérébral d’individus sains ou atteints de différents troubles neurodégénératifs (MA, FTLD-Tau, FTLD-TDP-GRN, FTLD-TDP-C9ORF72, FTLD-TDP et FTLD-FUS sporadiques) en utilisant la technique d’immunoempreinte. De manière remarquable, nous avons mis en évidence une réduction significative de Tau, et ce, spécifiquement chez les patients FTLD-TDP-GRN. Ainsi, nos résultats démontrent que ces cas, appelés FTLD-TDP-GRNltau (pour low Tau protein level), caractérisés par une altération synaptique et une astrogliose très importante, pourraient constituer un groupe distinct dans la classification des FTLD [...] / FTLD is a clinical syndrome mainly characterized by progressive deterioration in behavior, personality and/or language resulting from progressive frontal and temporal degeneration. In addition, movement disorder can also be frequently observed. Given this phenotype variability, FTLD clinical diagnosis remains difficult and uneasy to establish with certainty.The final diagnosis relies on neuropathological examination of the brain, the characteristics of these brain lesions and their molecular basis. Indeed, as many neurodegenerative diseases, FTLD are characterized by the presence of protein aggregates in the affected brain regions. However, in contrast to the well-characterized nature of protein inclusions in Alzheimer’s disease (AD), proteinaceous aggregates in FTLD can be composed of different proteins. Thus, approximatively 40% of FTLD cases display aggregates made of abnormally and hyperphosphorylated Tau proteins and constitute the FTLD-Tau subclass. However, most of FTLD brains are negative for Tau inclusions and exhibit neuronal cytoplasmic and/or nuclear inclusions immunoreactive for transactive response DNA binding protein 43 (TDP-43) and constitute the FTLD-TDP subclass). To a lesser extent, another protein called FUS (Fused in Sarcoma protein) is found in aggregates that are Tau and TDP-43 negative. This subclass is thus named FTLD-FUS. Finally, inclusions negative for Tau, TDP-43 or FUS are observed in rare cases of FTLD and associated with ubiquitin-proteasome system related proteins (FTLD-UPS).Gene mutations also play an important role in FTLD with 30 to 50% of patients reporting a positive family history of FTD and 10 to 15% of patients corresponding to dominantly inherited form. Firstly described are the MAPT mutations. Mutations in the progranulin gene GRN were then found to be the most frequent mutations associated with FTLD. More recently, two studies demonstrated that expanded hexanucleotide GGGGCC repeats in a noncoding region of the chromosome 9 open reading frame 72 (C9ORF72) gene was responsible for a large proportion of FTLD. Less frequently mutations in the valosin containing protein (VCP) gene or charged multivesicular body protein 2B (CHMP2B) gene are also found associated with FTLD.Prior to the discovery of the main molecular actors of FTLD, studies described a partial or total loss of soluble or physiological Tau protein expression in both grey and white matter. This loss of Tau was originally found in a subset of dementia called DLDH for Dementia Lacking Distinctive Histopathology (renamed later FTLD-ni for FTLD with no inclusion). In 2006, most of these cases were reclassified as FTLD-U (presenting with ubiquitin positive inclusions). However, additional investigation with specific regards to this loss of Tau expression has not been reported since Zhukareva et al. in 2003. With the progress in genetics and neuropathology of FTLD, the question of whether this reduction of Tau expression is seldom remains ill-defined.This work takes place in this context whose primary goal was to investigate human brain Tau protein expression in Control, AD, FTLD-Tau, FTLD-TDP-GRN, FTLD-TDP-C9ORF72, sporadic FTLD-TDP and sporadic FTLD-FUS brains using western blot analysis. Remarkably, we demonstrated a huge reduction of all six human brain Tau isoforms only in a subset of FTLD-TDP brains with mutation on the GRN gene. Thus, our data clearly suggest that these specific cases, referred to as FTLD-TDP-GRNltau (for low levels of Tau protein), could be part of the current classification as a distinct entity with more severe synaptic dysfunction and astrogliosis. Beside this, we also performed a comparative proteomic study between the different FTLD sub-classes in order to find common physiopathological mechanisms.

Le cancer colorectal (CCR) est un problème majeur de santé publique. Les nouvelles stratégies thérapeutiques incluant les anticorps anti-EGFR ou anti-VEGF en association avec les chimiothérapies ont amélioré la survie des patients présentant un CCR métastatique. Nous avons évalué le rôle de nouveaux biomarqueurs pour individualiser les patients porteurs d’un CCR métastatique qui peuvent bénéficier d’un traitement par thérapies ciblées. Dans la première partie, nous avons démontré pour la première fois la valeur de pSTAT3 (STAT3 phosphorylé) comme biomarqueur prédictif pour sélectionner les populations porteuses d’un CCR métastatique qui pourraient bénéficier d’un traitement par anti-EGFR. Dans ce cadre nous avons publié les résultats de la valeur prédictive et pronostique de pSTAT3 chez 94 patients porteurs d’un CCR métastatique traités par cétuximab en 2ème ligne de traitement (Dobi et al, 2012).La probabilité d’avoir une réponse objective était de 13% chez les patients avec l’expression de STAT3 comparé à 41% chez les patients pSTAT3 négatif (p=0.02). Nous avons montré également l’impact négatif de pSTAT3 sur la durée de la survie sans progression (PFS) et de la survie globale (OS) des patients présentant un CCR métastatique. Ce résultat montre l’intérêt de STAT3 comme biomarqueur prédictif de traitement par anti-EGFR. Dans la deuxième partie, nous avons évalué l’impact en survie des facteurs d’angiogenèse complexe comme angiopoetin-2, syndecan-1 (CD138), FGL2. Dans ce cadre nous avons étudié une cohorte exploratoire à Besançon de patients porteurs d’un CCR métastatique, traitée par chimiothérapie cytotoxique et bévacizumab (anti-VEGF). Après la détermination des seuils de valeurs de chaque biomarqueurs dans le sérum de patients utilisant les courbes ROC, nous avons évalué l’impact négatif de score élevé d’angiopoetin-2 et CD138 en survie de ces patients. Nous avons également démontré l’impact négatif de la combinaison de 3 marqueurs combinés en termes d’OS et de PFS dans une cohorte de validation incluant 244 patients de Besançon, Tours et Dijon. / Despite the progress made in the management of metastatic colorectal cancer (mCRC), this disease remains a major health problem. Targeted therapies (anti-EGFR and anti-VEGF therapies) have improved the clinical outcomes achieved by conventional chemotherapy regimens. Half of the patients with K-RAS wild type colo-rectal cancer doen’t benefit from adding anti-EGFR to standard chemotherapy regimen. We decided to assess the clinical outcomes of anti-EGFR containing chemotherapy regimens in 94 patients with mCRC according to phosphorylated STAT3 (pSTAT) status. The probability of achieving an objective reponse was 13% among patients with positive nuclear expression of pSTAT3 compared with 41% for patients displaying pSTAT3 négative tumors (p=0.02). In a multivariate logistic regression model, high-grade skin rash, wild-type K-RAS status, and negative pSTAT3 status significantly improved time to progression (TTP) and overal survival (OS). These results underscore the impact of pSTAT3 on the clinical efficacy of anti-EGFR-containing chemotherapy regimens and support the prospective assessment of this biomarker. Inhibition of pSTAT3 in mCRCs might be a promising strategy to target chemotherapy-resistant cancer cells and should be assessed in association with anti-EGFR monoclonal antibodies. The second goal of our study was the identification of angiopoetin-2, syndecan1 (CD 138) and FGL2 as prognostic biomarquers of metastatic colorectal cancer patients. Plasma angiopoetin-2, FGL2 et CD 138 levels were mesured in 10 healthy volunteers, 13 patients with locally advanced CRC et 51 mCRC, received bevacizumab containing chemotherapy. Angiopoetin-2 and CD138 levels were significantly elevated in patients with mCRC compared with healthy controls. Amongst patients with mCRC, low pre-therapeutic plasma angiopoetin-2 and CD 138 levels were associated with a prolonged median progression-free survival (PFS) (p<0.01 and p=0.03 respectively), and overall survival (p<0.01 and p=0.02 respectively). PFS and OS was prolonged in patients with out or with only one detectable biomarker compared with patients with two or three high biomarker levels (p<0.01).