L’évolution de la denture des vertébrés est un sujet majeur et des plus intéressants en biologie développementale évolutive (évo-dévo). Dans ce domaine, la souris Mus musculus est traditionnellement l’animal modèle utilisé. La denture de la souris inclus quatre incisives à croissance continue et douze molaires présentant une organisation caractéristique de leurs cuspides. Le réseau moléculaire régulant le développement de ces deux types de dents est très spécifique.La cascade ERK-MAPK est impliquée lors de différentes étapes du développement dentaire. Une étude comparée du phénotype des molaires de souris mutantes pour des gènes activés à différents points de la cascade a démontré l’existence d’un phénotype caractéristique, à savoir la présence d’une dent surnuméraire en position mésiale des rangées de molaires, ainsi que la présence d’anomalie dans le nombre et la forme de certaines cuspides. Parmi ces caractères présents chez les mutants, certains rappellent des caractères ancestraux présents uniquement chez des rongeurs fossiles. Ceci appuie le rôle que la cascade ERK-MAPK a pu jouer au cours de l’évolution de la denture chez les rongeurs. En travaillant sur une lignée transgénique sur-exprimant un inhibiteur de cette cascade, j’ai pu perfectionner notre connaissance du rôle des gènes de la famille Fgf dans les processus de mise en place des centres de signalisation dentaire et de minéralisation de l’émail.Si l’on considère les incisives à croissance continue, la denture de la souris est dynamique à l’échelle de la vie d’un individu. Réalisant un suivi des incisives supérieures d’une cohorte de souris au cours de leur vieillissement, j’ai pu préciser la chronologie d’apparition de défauts liés au vieillissement. Ces défauts apparaissent sur les incisives à partir de six mois, et le plus fréquent est le développement d’un sillon visible sur l’émail de l’incisive. J’ai enfin utilisé des techniques de séquençage de nouvelle génération (NGS) pour comprendre les bases moléculaires du vieillissement des niches de cellules souches des incisives. Ce faisant, j’ai détecté des changements dans les profils d’expression de gènes régulant le maintien des cellules souches, la prolifération cellulaire et le métabolisme. La présence d’un sillon apparaît corrélée à une forte réponse immunitaire détectée dans les tissus dentaires, ce qui constitue une perspective d’étude majeure dans le but d’achever la caractérisation du vieillissement des cellules souches dentaires. / The evolution of the vertebrate dentition is among the most exciting topics in the evo-devo field, with particular attention being drawn to the mouse model. The mouse dentition includes four ever-growing incisors and twelve molars with a specific cusp pattern. Incisors and molars develop according to a tightly regulated molecular network.The ERK-MAPK cascade is involved at various stages of tooth development. Molar tooth phenotype comparisons in mutant mice for genes acting at various levels of the cascade highlighted a dental phenotype signature, which consists in the presence of a supernumerary tooth and shared cusp pattern defects. Some of these recall characters present in fossil rodents, supporting the ERK-MAPK as a good candidate to explain some evolutionary trends of the rodent dentition. By working on a mouse line over-expressing one of this pathway inhibitor in the oral epithelium, I perfect our understanding of Fgf gene role in specifying signaling center formation at the right stage, and in achieving correct mineralization.When considering evergrowing incisors, mouse dentition is also dynamic at the lifetime scale. I monitored the ageing process of the mouse upper incisors, and provided a chronology of occurrence of the variety of age-related defects display. These defects are set up from the six months on, the most frequent abnormality being the presence of an enamel groove along the surface of the incisor. Using Next Generation Sequencing technologies, I detected transcriptomic changes in the stem cell niches affecting cell proliferation and metabolism, as well as the stem cell niche functioning. The correlation found between the groove occurrence and a large immune response in dental tissues expands our concern for dental stem cell ageing.

En conséquence de l'extension de l’utilisation des nanoparticules dans différents secteurs industriels, le nombre de travailleurs potentiellement exposés ne cesse de croître, sans parfaitement connaître les propriétés toxicologiques de ces matériaux. Étant donné que les nanoparticules peuvent se trouver en suspension dans l’atmosphère professionnelle, l'inhalation représente une voie d'exposition professionnelle majeure. De ce fait, l’évaluation des risques liés à l’exposition aux nanomatériaux requiert d’entreprendre des études de toxicologie sur des modèles cellulaires des voies aériennes. Dans ce manuscrit, les réponses cellulaires et moléculaires des macrophages alvéolaires de rat (NR8383) exposés à des nanoparticules d’oxydes métalliques : ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 et TiO2-NRCWE001, ont été étudiées, en combinant des analyses toxicologiques classiques (caractérisation des nanoparticules par microscopie électronique à transmission et par diffusion dynamique de la lumière, évaluation de la cytotoxicité par tests WST-1 et libération de LDH); et de criblage moléculaire à haut débit (analyses de transcriptomique et de protéomique). Des cellules NR8383 ont été exposées aux nanoparticules ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 et TiO2-NRCWE001 pendant 24 h ce qui a permis de déterminer une dose sub-toxique pour chaque nanoparticule à laquelle les macrophages ont été exposés pour l’analyse moléculaire. Quatre heures suite à l’exposition des cellules aux nanoparticules, de nombreux gènes et protéines étaient différentiellement exprimés. Le stress oxydant était la réponse biologique adverse suite à l’exposition des cellules aux nanoparticules composées de zinc. En revanche, l’inflammation était la principale voie activée dans les cellules exposées à la forme anatase et rutile des nanoparticules de TiO2. En conclusion, cette étude expose les « empreintes biologiques » des deux groupes de nanoparticules d’intérêt. Enfin, notre étude combinée à des travaux antérieurs de la littérature pourraient aussi être profitables pour valider les biomarqueurs d’exposition et d’effets aux nanomatériaux suggérés afin de prédire les effets biologiques adverses. / As a consequence of the extension of the use of nanoparticles in different industrial sectors, the number of potentially exposed workers continues to grow, without fully knowing the toxicological properties of these materials. Since nanoparticles can be aerosolized in the occupational atmosphere, inhalation is the major occupational exposure route. For this reason, risk assessment of exposure to nanomaterials requires toxicology studies to be conducted on cellular models of the airways. In this manuscript, the cellular and molecular responses of rat alveolar macrophages (NR8383) exposed to metallic oxide nanoparticles: ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 and TiO2-NRCWE001, were studied, combining conventional toxicological analyzes (characterization of nanoparticles by transmission electron microscopy and dynamic light scattering, evaluation of cytotoxicity by WST-1 assays and LDH release); and high throughput molecular screening (transcriptomic and proteomic analyzes). NR8383 cells were exposed to the ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 and TiO2-NRCWE001 nanoparticles for 24 h which allowed for the determination of a sub-toxic dose for each nanoparticle to which the macrophages were exposed for molecular analysis. Four hours after exposure NR8383 to nanoparticles, many genes and proteins were differentially expressed. Oxidative stress was the adverse biological response following exposure of cells to nanoparticles composed of zinc. In contrast, inflammation was the main activated pathway in cells exposed to the anatase and rutile form of TiO2 nanoparticles. In conclusion, this study exposes the "biological fingerprints" of the two groups of nanoparticles of interest. Finally, our study, combined with previous literature studies, could also be beneficial in validating biomarkers of exposure and effects of nanomaterials suggested in order to predict adverse biological effects.

Introdução: Os linfomas não-Hodgkin de células T periféricas (LCTP) são neoplasias raras caracterizadas pela proliferação monoclonal de linfócitos T maduros. Correspondem a 15% das malignidades linfoides e têm distribuição geográfica peculiar. Compreendem 22 entidades clínico-patológicas distintas, heterogêneas do ponto de vista clínico-epidemiológico, morfológico, fenotípico e molecular. O grupo de apresentação predominantemente nodal compreende as variantes histológicas LGCA/ALK+, LGCA/ALK-, LCTA e LCTP/SOE. Sua terapêutica se baseia em poliquimioterápicos à base de antraciclina e consolidação com transplante de células tronco hematopoiéticas autólogas (TCTH). Com exceção do LGCA/ALK+, apresentam sobrevida global em cinco anos de 30% a 40%. Devido aos desfechos desfavoráveis, estudos de perfil de expressão gênica e de metilação de genes supressores tumorais têm emergido nos últimos anos para refinar o diagnóstico destas neoplasias, melhorar o conhecimento fisiopatológico e o prognóstico e estabelecer possíveis alvos terapêuticos. Estudos preliminares com LCTP nodais indicam valor prognóstico favorável da hiperexpressão de genes de padrão inflamatório NFkB1 e IKBkB e desfavorável nos casos de supraregulação de genes de padrão proliferativo como CCNA2, TOP2A e CHEK1 e do fenótipo metilado de p15INK4b e p16INK4a. Objetivo:Avaliar o impacto da expressão relativa dos genes CCNA2, TOP2A, CHEK1, NFkB1, IKBkB e VEGF1 e da metilação dos genes p15 e p16 em população brasileira com LCTP nodais tratados com quimioterapia CHOP-símile para os desfechos de sobrevida global, sobrevida livre de progressão e sobrevida livre de doença. Métodos: A expressão gênica foi avaliada por qtPCR de amostras fixadas em formol e incluídas em parafina de 63 pacientes. A mediana de expressão dos genes foi comparada com variáveis clínicas e desfechos. PCR qualitativa metilação-específico foi usada avaliar a metilação de p15 e p16. Resultados: Com mediana de seguimento de vinte meses, as SG, SLP e SLD foram, respectivamente, 45,6%, 34,3% e 63,0% e a resposta completa de 46,0%. Em análise multivariada, ECOG >= 2 e a hiperexpressão do gene CCNA2 foram associadas à pior SG em cinco anos, nos LCTP nodais (p=0,008 e p=0,002). Em análise univariada os genes CCNA2, TOP2A e CHEK1 foram associados ao pior prognóstico nos LCTP/SOE e melhor nos LGCA/ALK-. A hiperexpressão do gene VEGF1 se associou ao pior prognóstico no LGCA/ALK- e LCTA. Metilação de p15INK4b não foi encontrada nos LGCA/ALK+ e em análise multivariada foi associada a pior SLP em 5 anos nos LCTP não-ALK+ (HR: 9,88; p=0,03). O painel gênico testado não apresentou poder para discriminar as diferentes variantes histopatólogicas de LCTP nodais, porém demonstrou-se associação direta entre a intensidade de mediana de expressão desses genes e agressividade biológica nesse grupo heterogêneo de neoplasias. O significado prognóstico de imunoexpressão da proteína ALK sofreu influência das variáveis constituintes do IPI nessa coorte. Conclusão: A hiperexpressao dos genes CCNA2, TOP2A e CHEK1 foram associadas a prognóstico desfavorável nos LCTP/SOE e favorável nos LGCA/ALK-. Fenótipo hipermetilado de p15INK4b não foi evento observado em LGCA/ALK+, porém foi associado a pior prognóstico nos LCTP nodais não-ALK+ / Background: Peripheral T-cell non-Hodgkin\'s lymphomas (PTCL) are rare tumors characterized by monoclonal proliferation of mature T lymphocytes; they correspond to 15% of lymphoid malignancies and have specific geographic distribution. PTCL comprise 22 distinct clinicopathologic entities, heterogeneous from the clinical and epidemiological perspective, as well as morphologic, phenotypic and molecular. The group of presentation predominantly nodal comprises the histological variants ALCL/ALK+, ALCL/ALK-, AITL and PTCL/NOS. Its treatment is based on polychemotherapy with anthracycline and consolidation with autologous hematopoietic stem cell transplantation (ASCT). With the exception of ALCL/ALK+, these tumors show overall survival at 5 years from 30% to 40%. Due to unfavorable outcomes, gene expression profile studies, as well as studies of methylation of tumor suppressor genes, have emerged in recent years in order to refine the pathological diagnosis of these cancers, improve the pathophysiological knowledge and prognosis, and establish possible therapeutic targets. Preliminary studies with nodal PTCLs indicate favorable prognostic value of overexpression of inflammatory pattern genes like NFkB1 e IKBkB, and unfavorable in the case of proliferative genes as CCNA2, TOP2A, CHEK1 and the methylated phenotype of suppressor genes p15INK4b e p16INK4a. Objectives: Assess the impact of relative expression of genes CCNA2, TOP2A, CHEK1, NFkB, IKBkB and VEGF1 and the methylation of the genes p15 and p16 in Brazilian population with nodal PTCLs treated with CHOP-like chemotherapy for the outcomes of overall survival, progression-free survival and disease-free survival. Methods: Gene expression was assessed by qtPCR of paraffin samples of 63 patients. The median gene expression was compared with clinical variables and outcomes. Qualitative methylation-specific PCR was used to assess the methylation of p15 and p16. Results: With a segment median of 20 months, the OS, PFS and DFS were, respectively, 45,6%, 34,3% and 63,0% and the complete response (CR) was 46,0%. In multivariate analysis, ECOG >= 2 and overexpression of the gene CCNA2 were associated with worse OS at 5 years in nodal PTCLs (p = 0,008 and p = 0,002). In univariate analysis, the genes CCNA2, TOP2A and CHEK1 were associated with worse prognosis in PTCL/NOS and better in ALCL/ALK negative. The overexpression of the gene VEGF1 was associated with worse prognosis in the variants AITL and ALCL/ALK negative. Methylation of the gene p15INK4b was not found in ALCL/ALK+ group, and in multivariate analysis was associated with worse 5-years PFS in the group of PTCL non-ALK+ (HR: 9,88 and p = 0,03). The gene panel tested showed no power to discriminate the different histopathology of nodal PTCL, but it showed a direct association between the median intensity of expression of these genes and biological aggressiveness in this heterogeneous group of neoplasms. The prognostic significance of immunostaining of ALK protein was influenced by IPI constituents variables in this cohort. Conclusion: The overexpression of genes CCNA2, TOP2A e CHEK1 were associated with poor prognosis in PTCL/NOS and favorable in ALCL/ALK negative. Hypermethylated phenotype of gene p15INK4b was not an observed event in ALCL/ALK+, but it was associated with poor prognosis in nodal PTCL non-ALK+

Introdução: Os linfomas não-Hodgkin de células T periféricas (LCTP) são neoplasias raras caracterizadas pela proliferação monoclonal de linfócitos T maduros. Correspondem a 15% das malignidades linfoides e têm distribuição geográfica peculiar. Compreendem 22 entidades clínico-patológicas distintas, heterogêneas do ponto de vista clínico-epidemiológico, morfológico, fenotípico e molecular. O grupo de apresentação predominantemente nodal compreende as variantes histológicas LGCA/ALK+, LGCA/ALK-, LCTA e LCTP/SOE. Sua terapêutica se baseia em poliquimioterápicos à base de antraciclina e consolidação com transplante de células tronco hematopoiéticas autólogas (TCTH). Com exceção do LGCA/ALK+, apresentam sobrevida global em cinco anos de 30% a 40%. Devido aos desfechos desfavoráveis, estudos de perfil de expressão gênica e de metilação de genes supressores tumorais têm emergido nos últimos anos para refinar o diagnóstico destas neoplasias, melhorar o conhecimento fisiopatológico e o prognóstico e estabelecer possíveis alvos terapêuticos. Estudos preliminares com LCTP nodais indicam valor prognóstico favorável da hiperexpressão de genes de padrão inflamatório NFkB1 e IKBkB e desfavorável nos casos de supraregulação de genes de padrão proliferativo como CCNA2, TOP2A e CHEK1 e do fenótipo metilado de p15INK4b e p16INK4a. Objetivo:Avaliar o impacto da expressão relativa dos genes CCNA2, TOP2A, CHEK1, NFkB1, IKBkB e VEGF1 e da metilação dos genes p15 e p16 em população brasileira com LCTP nodais tratados com quimioterapia CHOP-símile para os desfechos de sobrevida global, sobrevida livre de progressão e sobrevida livre de doença. Métodos: A expressão gênica foi avaliada por qtPCR de amostras fixadas em formol e incluídas em parafina de 63 pacientes. A mediana de expressão dos genes foi comparada com variáveis clínicas e desfechos. PCR qualitativa metilação-específico foi usada avaliar a metilação de p15 e p16. Resultados: Com mediana de seguimento de vinte meses, as SG, SLP e SLD foram, respectivamente, 45,6%, 34,3% e 63,0% e a resposta completa de 46,0%. Em análise multivariada, ECOG >= 2 e a hiperexpressão do gene CCNA2 foram associadas à pior SG em cinco anos, nos LCTP nodais (p=0,008 e p=0,002). Em análise univariada os genes CCNA2, TOP2A e CHEK1 foram associados ao pior prognóstico nos LCTP/SOE e melhor nos LGCA/ALK-. A hiperexpressão do gene VEGF1 se associou ao pior prognóstico no LGCA/ALK- e LCTA. Metilação de p15INK4b não foi encontrada nos LGCA/ALK+ e em análise multivariada foi associada a pior SLP em 5 anos nos LCTP não-ALK+ (HR: 9,88; p=0,03). O painel gênico testado não apresentou poder para discriminar as diferentes variantes histopatólogicas de LCTP nodais, porém demonstrou-se associação direta entre a intensidade de mediana de expressão desses genes e agressividade biológica nesse grupo heterogêneo de neoplasias. O significado prognóstico de imunoexpressão da proteína ALK sofreu influência das variáveis constituintes do IPI nessa coorte. Conclusão: A hiperexpressao dos genes CCNA2, TOP2A e CHEK1 foram associadas a prognóstico desfavorável nos LCTP/SOE e favorável nos LGCA/ALK-. Fenótipo hipermetilado de p15INK4b não foi evento observado em LGCA/ALK+, porém foi associado a pior prognóstico nos LCTP nodais não-ALK+ / Background: Peripheral T-cell non-Hodgkin\'s lymphomas (PTCL) are rare tumors characterized by monoclonal proliferation of mature T lymphocytes; they correspond to 15% of lymphoid malignancies and have specific geographic distribution. PTCL comprise 22 distinct clinicopathologic entities, heterogeneous from the clinical and epidemiological perspective, as well as morphologic, phenotypic and molecular. The group of presentation predominantly nodal comprises the histological variants ALCL/ALK+, ALCL/ALK-, AITL and PTCL/NOS. Its treatment is based on polychemotherapy with anthracycline and consolidation with autologous hematopoietic stem cell transplantation (ASCT). With the exception of ALCL/ALK+, these tumors show overall survival at 5 years from 30% to 40%. Due to unfavorable outcomes, gene expression profile studies, as well as studies of methylation of tumor suppressor genes, have emerged in recent years in order to refine the pathological diagnosis of these cancers, improve the pathophysiological knowledge and prognosis, and establish possible therapeutic targets. Preliminary studies with nodal PTCLs indicate favorable prognostic value of overexpression of inflammatory pattern genes like NFkB1 e IKBkB, and unfavorable in the case of proliferative genes as CCNA2, TOP2A, CHEK1 and the methylated phenotype of suppressor genes p15INK4b e p16INK4a. Objectives: Assess the impact of relative expression of genes CCNA2, TOP2A, CHEK1, NFkB, IKBkB and VEGF1 and the methylation of the genes p15 and p16 in Brazilian population with nodal PTCLs treated with CHOP-like chemotherapy for the outcomes of overall survival, progression-free survival and disease-free survival. Methods: Gene expression was assessed by qtPCR of paraffin samples of 63 patients. The median gene expression was compared with clinical variables and outcomes. Qualitative methylation-specific PCR was used to assess the methylation of p15 and p16. Results: With a segment median of 20 months, the OS, PFS and DFS were, respectively, 45,6%, 34,3% and 63,0% and the complete response (CR) was 46,0%. In multivariate analysis, ECOG >= 2 and overexpression of the gene CCNA2 were associated with worse OS at 5 years in nodal PTCLs (p = 0,008 and p = 0,002). In univariate analysis, the genes CCNA2, TOP2A and CHEK1 were associated with worse prognosis in PTCL/NOS and better in ALCL/ALK negative. The overexpression of the gene VEGF1 was associated with worse prognosis in the variants AITL and ALCL/ALK negative. Methylation of the gene p15INK4b was not found in ALCL/ALK+ group, and in multivariate analysis was associated with worse 5-years PFS in the group of PTCL non-ALK+ (HR: 9,88 and p = 0,03). The gene panel tested showed no power to discriminate the different histopathology of nodal PTCL, but it showed a direct association between the median intensity of expression of these genes and biological aggressiveness in this heterogeneous group of neoplasms. The prognostic significance of immunostaining of ALK protein was influenced by IPI constituents variables in this cohort. Conclusion: The overexpression of genes CCNA2, TOP2A e CHEK1 were associated with poor prognosis in PTCL/NOS and favorable in ALCL/ALK negative. Hypermethylated phenotype of gene p15INK4b was not an observed event in ALCL/ALK+, but it was associated with poor prognosis in nodal PTCL non-ALK+