Sonic hedgehog (Shh) is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. When cells receive the Shh signal a cascade of effects begin which in turn regulate downstream target genes. The genes controlled by Sonic hedgehog provide messages instructing cells how to differentiate or when to divide. Disruption of the hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. Patched, the receptor for Shh, acts as a tumour suppressor and is mutated in naevoid basal cell carcinoma syndrome (NBCCS). NBCCS patients display a susceptibility to tumour formation, particularly for basal cell carcinoma (BCC). The discovery of Patched mutations in sporadic BCCs and other tumour types further highlights the importance of this pathway to human cancer. The identification of genes regulated by hedgehog is crucial for understanding how disruption of this pathway leads to neoplastic transformation. It is assumed that the abnormal expression of such genes plays a large role in directing cells to divide at inappropriate times. Only a small number of genes controlled by Shh have been described in vertebrate tissues. In the work presented in this thesis a Sonic hedgehog responsive embryonic mouse cell line, C3H/10T1/2, was used as a model system for hedgehog target gene discovery. Known downstream target genes were profiled to determine their induction kinetics, building up a body of knowledge on the response to Shh for this cell type. During this work, it was discovered that C3H/10T1/2 cells do not become fully competent to respond to Shh stimulation until the cells reach a critical density, a factor that had to be taken into account when determining timepoints of interest for further investigation. Several techniques were employed to identify genes that show expression changes between Shh stimulated and control cells. In one of these techniques, RNA from cell cultures activated with Shh was used to interrogate cDNA microarrays, and this provided many insights into the downstream transcriptional consequences of hedgehog stimulation. Microarrays consist of thousands of spots of DNA of known sequence gridded onto glass slides. Experiments using this technology allow the expression level of thousands of genes to be measured simultaneously. Independent stimulation methods combined with northern blotting were used to investigate individual genes of interest, allowing genuine targets to be confirmed and false positives eliminated. This resulted in the identification of eleven target genes. Seven of these are induced by Sonic hedgehog (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (Gilz), Brain factor 2 (Bf2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (Igf2), Peripheral myelin protein 22 (Pmp22), Lim and SH3 Protein 1 (Lasp1)), and four are repressed (Secreted frizzled related proteins 1 and 2 (Sfrp1 and Sfrp2), Macrophage inflammatory protein-1 gamma (Mip-1?), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh. The new target genes have a diverse range of functions, and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. The identification of novel Sonic hedgehog responsive genes provides candidates whose abnormal expression may be decisive in initiating tumour formation and future studies will investigate their role in development and disease. It is expected that such findings will provide vital clues to the aetiology of various human cancers, and that an understanding of their roles may ultimately provide greater opportunities in the future design of anti-tumour therapies.

Sonic hedgehog (Shh) is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. When cells receive the Shh signal a cascade of effects begin which in turn regulate downstream target genes. The genes controlled by Sonic hedgehog provide messages instructing cells how to differentiate or when to divide. Disruption of the hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. Patched, the receptor for Shh, acts as a tumour suppressor and is mutated in naevoid basal cell carcinoma syndrome (NBCCS). NBCCS patients display a susceptibility to tumour formation, particularly for basal cell carcinoma (BCC). The discovery of Patched mutations in sporadic BCCs and other tumour types further highlights the importance of this pathway to human cancer. The identification of genes regulated by hedgehog is crucial for understanding how disruption of this pathway leads to neoplastic transformation. It is assumed that the abnormal expression of such genes plays a large role in directing cells to divide at inappropriate times. Only a small number of genes controlled by Shh have been described in vertebrate tissues. In the work presented in this thesis a Sonic hedgehog responsive embryonic mouse cell line, C3H/10T1/2, was used as a model system for hedgehog target gene discovery. Known downstream target genes were profiled to determine their induction kinetics, building up a body of knowledge on the response to Shh for this cell type. During this work, it was discovered that C3H/10T1/2 cells do not become fully competent to respond to Shh stimulation until the cells reach a critical density, a factor that had to be taken into account when determining timepoints of interest for further investigation. Several techniques were employed to identify genes that show expression changes between Shh stimulated and control cells. In one of these techniques, RNA from cell cultures activated with Shh was used to interrogate cDNA microarrays, and this provided many insights into the downstream transcriptional consequences of hedgehog stimulation. Microarrays consist of thousands of spots of DNA of known sequence gridded onto glass slides. Experiments using this technology allow the expression level of thousands of genes to be measured simultaneously. Independent stimulation methods combined with northern blotting were used to investigate individual genes of interest, allowing genuine targets to be confirmed and false positives eliminated. This resulted in the identification of eleven target genes. Seven of these are induced by Sonic hedgehog (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (Gilz), Brain factor 2 (Bf2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (Igf2), Peripheral myelin protein 22 (Pmp22), Lim and SH3 Protein 1 (Lasp1)), and four are repressed (Secreted frizzled related proteins 1 and 2 (Sfrp1 and Sfrp2), Macrophage inflammatory protein-1 gamma (Mip-1?), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh. The new target genes have a diverse range of functions, and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. The identification of novel Sonic hedgehog responsive genes provides candidates whose abnormal expression may be decisive in initiating tumour formation and future studies will investigate their role in development and disease. It is expected that such findings will provide vital clues to the aetiology of various human cancers, and that an understanding of their roles may ultimately provide greater opportunities in the future design of anti-tumour therapies.

Le cil primaire (CP) est une organelle présente chez l’Homme dans la grande majorité des cellules. Lors du développement le CP est d’une importance capitale, puisqu’il contrôle les voies de signalisation comme Hedgehog ou Wnt. Certaines pathologies génétiques affectant spécifiquement le CP, engendrent une obésité. Au cours de ma thèse je me suis intéressé à l’évolution du CP au cours de l’adipogenèse des cellules souches mésenchymateuses humaines. Les résultats que nous avons obtenus indiquent que le cil est présent dans les cellules indifférenciées, qu’il subit une élongation importante suite à l’induction de la différenciation, suivi d’une diminution de sa taille et fini par disparaitre dans les adipocytes. L’élongation de la taille du cil ne semble pas affecter la localisation des protéines qui lui sont associées comme Kif3-A ou Smoothened, une protéine importante de la voie Hedgehog. Néanmoins, il apparait que la voie de signalisation Hedgehog est inhibée après trois jours de différenciation et que les cellules ont développé une résistance à Sonic Hedgehog. La déacétylase de la tubuline acétylée HDAC6 est apparue comme étant une bonne cible puisque son expression augmente au cours de la différenciation et qu’elle est décrite pour être responsable de la perte du cil pendant la mitose. Les données que nous avons obtenues ont permis de montrer que l’inhibition, ou la surexpression d’HDAC6 au cours de l’adipogenèse engendrent une inhibition de l’élongation du cil associée à une forte inhibition de la différenciation adipocytaire. Ces résultats permettront, à terme de mieux comprendre les liens entre le cil primaire et la différenciation adipocytaire. / The primary cilium (PC) is an organelle present in almost all cell types of the organism. During development, the PC plays an important function by driving signaling pathways such as Hedgehog or Wnt. Some genetic syndromes affecting specifically the PC are associated with obesity. My project has consisted to analyze the evolution of the PC during adipocyte differentiation of human mesenchymal stems cells. Our results indicate that the PC is present in undifferentiated cells, then it undergoes a strong elongation at the beginning of the differentiation followed by a decreased of its size, and disappears in differentiated cells. This increase in the cilium size does not affect the localization of its associated proteins such as KIF3-A and Smoothened an important protein of the Hedgehog signaling pathway. However, this pathway is inhibited after three days of differentiation and cells have developed a Sonic Hedgehog resistance. The tubulin deacetylase HDAC6 appeared as a good target because its expression increases during differentiation and it is known to be responsible for the loss of the cilium during mitosis. Our data show that an inhibition or an overexpression of HDAC6 lead to a decrease in the cilium elongation associated with an inhibition of adipocyte differentiation. These results will ultimately lead to a better understanding of the connections between the PC and adipocyte differentiation.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-04-02T13:47:38Z No. of bitstreams: 1 Rosane Borges Dias - Estudo do perfil... 2014.pdf: 2418992 bytes, checksum: 9a4f389957bc3f98114112321eee18b4 (MD5) / Made available in DSpace on 2014-04-02T13:47:38Z (GMT). No. of bitstreams: 1 Rosane Borges Dias - Estudo do perfil... 2014.pdf: 2418992 bytes, checksum: 9a4f389957bc3f98114112321eee18b4 (MD5) Previous issue date: 2014 / Universidade Federal da Bahia. SAlvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / A displasia epitelial oral (DEO) é um aspecto histológico descrito em lesões potencialmente malignas, cujos mecanismos relacionados a patogênese e potencial de transformação são pouco conhecidos. Sabendo-se que a via de sinalização Sonic Hedgehog(SHH)tem participação no desenvolvimento do carcinoma escamocelular de boca(CEB) e que as proteínas relacionadas a esta via de sinalização estão envolvidas nos mecanismos biológicos relacionados a iniciação e progressão de tumores humanos. O objetivo deste trabalho foi estudar a expressão das proteínas da via de sinalização SHH em DEO, a fim de contribuir para o conhecimento do perfil biológico desta lesão. Material e Métodos: As amostras de DEO foram revisadas e classificadas de acordo com o risco de malignidade descrito por Kujan et al.(2006) e OMS(2005). Vinte e cinco casos de DEO foram submetidos a reação imuno-histoquímica para as proteínas SHH, PTCH1, SUFU, HHIP, GLI1 e Ciclina D1(CCND1)utilizando o sistema polimérico AdvanceTM(Dako). Para fins comparativos, cinco casos de hiperqueratose, oito de hiperplasia fibrosa inflamatória(HFI) e quatro de mucosas não-neoplásica(MNN)também foram avaliados.Todos os casos foram analisados de acordo com os parâmetros semiquantitativos descritos por Gurgel et al.(2008)e os dados foram analisados usando Graph Pad Prism5.01. Resultados: Vinte e um(84%)DEO foram classificados como lesões de baixo risco e 4(16%)como alto risco. A proteína SHH foi predominante no citoplasma(n =14, sendo 56%) e a positividade de PTCH1 foi observada em membrana e citoplasma (n=23, 92%). As proteínas HHIP e SUFU foram observadas principalmente no citoplasma das células epiteliais e foram positivas em 17 (68%) e 11 (44%) das DEO, respectivamente. GLI1 foi positivo em 21 (84%), principalmente localizada nos núcleos das células epiteliais (n=12; 70,6%). A proteína CCND1 foi exclusivamente no núcleo 19(76%).Em DEO, os escores predominantes para SHH, PTCH1, HHIP, SUFU, GLI1 e CCND1 foram: 2+ (57,14%), 3+ (73,91%), 3+ (58,82%), 2+ e 3+ (36,36%), 3+ (90,48 %), 1+(47,37 %), respectivamente. Os casos de DEO exibiram maior expressão de HHIP(p=0,02) e GLI1(p=0,00), em comparação com hiperqueratose. Além disso, níveis elevados de PTCH1 (p=0,05), HHIP (p= 0,01) e CCND1 (p=0,00) foram observados em DEO em comparação com HFI. Ainda, uma maior expressão de PTCH(p=0,01) foi observada nos casos de DEO quando comparados com MNN. Conclusões: Os nossos resultados sugerem que a via SHH pode participar da patogênese da DEO e reforçam a relação desta cascata sinalizadora na carcinogênese oral. / Oral epithelial dysplasia (OED) is a histological aspect described in premalignant lesions and the mechanisms related to the pathogenesis and malignant progression are poorly understood. It is known that Sonic Hedgehog (SHH) signaling pathway participates in the development of oral squamous cell carcinoma, and proteins related to this cascade are involved in biological mechanisms related to the initiation and progression of human tumors. The aim of this study was to investigate SHH signaling molecules in OED, in order to contribute to the knowledge of the biological profile of this lesion. Methods: Samples of OED were reviewed and classified according to the risk of malignancy described by Kujan et al. (2006) and WHO (2005). Twenty five cases of DEO were to submitted to immunohistochemical reactions for SHH, PTCH1, SUFU, HHIP, GLI1 and Cyclin D1 (CCND1) proteins using AdvanceTM polymer system (Dako). For comparative purposes, five cases of hyperkeratosis, eight inflammatory fibrous hyperplasia (IFH) and four non- neoplastic (NNM) mucosa were also evaluated. All OED were analyzed according to the semi-quantitative parameters described by Gurgel et al. (2008) and the data was analyzed using Graph Pad Prism 5.01. Results: Twenty- one (84%) OED were classified as low risk lesions and 4 (16%) as high risk. SHH protein was predominant in cytoplasm (n=14; 56%) and PTCH1 positivity was described in both membrane and cytoplasm (n=23; 92%). HHIP and SUFU proteins were observed in cytoplasm of epithelial cells and were positive in 17 (68%) and 11 (44%) OED, respectively. GLI1 protein was positive in 21 (84%), mainly located in the cells nuclei (n=12; 70.6%). CCND1 protein was exclusively in the nucleus 19 (76%). The predominant scores for SHH, PTCH1, HHIP, SUFU, GLI1 and CCND1 were: +2 (57.14%), +3 (73.91%), +3 (58.82%), +2 and +3 (36.36%), +3 (90.48%), +1 (47.37%), respectively. OED sample had higher levels of HHIP (p=0.02) and GLI1 (p=0.00) as compared to hyperkeratosis. Furthermore, high levels of PTCH (p=0.05), HHIP (p=0.01) and CCND1 (p=0.00) were observed in OED as compared to IFH OED. Also, a high level of PTCH (p= 0.01) was observed in OED as compared to NNM. Conclusions: Our results suggest that the SHH pathway may participate in the pathogenesis of DEO and corroborates to the relationship of this signaling cascade in oral carcinogenesis.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-03-27T17:37:40Z No. of bitstreams: 1 Ludmila de Faro Valverde Via hedgehog....pdf: 1435521 bytes, checksum: 2442c32fdacd9e814b9e8a6035eb3bdc (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-03-27T17:37:59Z (GMT) No. of bitstreams: 1 Ludmila de Faro Valverde Via hedgehog....pdf: 1435521 bytes, checksum: 2442c32fdacd9e814b9e8a6035eb3bdc (MD5) / Made available in DSpace on 2015-03-27T17:37:59Z (GMT). No. of bitstreams: 1 Ludmila de Faro Valverde Via hedgehog....pdf: 1435521 bytes, checksum: 2442c32fdacd9e814b9e8a6035eb3bdc (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO/OBJETIVO: O Carcinoma Escamocelular de Boca (CEB) corresponde a mais de 95% dos casos de câncer diagnosticados na cavidade bucal e consiste numa neoplasia invasiva e agressiva. Sabendo-se que a via Hedgehog (HH) está envolvida na patogênese de diversos tumores, o presente trabalho propôs-se a avaliar a expressão de componentes desta via em CEB, associando a expressão destas moléculas com aspectos clínicos, angiogênese, graus de diferenciação tumoral, potencial proliferativo e macrófagos CD163+. MATERIAL E MÉTODOS: Vinte e oito casos de CEB, 9 casos de margens tumorais (MAT) e 4 casos de mucosa bucal não neoplásica (MNN) foram submetidos à reação imuno-histoquímica para as proteínas MCM3, SHH, IHH, GLI1, CD163 e CD105 utilizando o sistema polimérico AdvanceTM. A co-localização das proteínas IHH/CD163 e GLI1/CD105 foi avaliada através de dupla marcação imuno-histoquímica. As análises das proteínas MCM3, SHH, IHH e GLI1 foram realizadas em 5 áreas coincidentes de cada caso, de acordo com os parâmetros semi-quantitativos descritos por Gurgel et al. (2008). A densidade de macrófagos (DM) e microdensidade vascular (MDV) foram mensuradas considerando-se a população destas células e vasos neoformados em 5 áreas e os resultados expressos em cel/mm² e vasos/mm². A análise estatística foi realizada utilizando GraphPad Prism versão 6.03. RESULTADOS: Todos os casos de CEB foram positivos para a proteína MCM3, em citoplasma e núcleo de células do parênquima tumoral, sendo o escore 4+ predominante (n=19; 67,85%). Em parênquima tumoral, a proteína SHH estava presente no citoplasma de células neoplásicas e foi positiva em 25 casos (89,28%) de CEB, sendo o escore 4+ predominante (n=17; 68%). Vinte e dois casos (78,57%) apresentaram marcação citoplasmática da proteína IHH no parênquima tumoral, predominando os escores 4+ (n=7; 31,82%) e 3+ (n=7; 31,82%). A proteína GLI1 apresentou marcação citoplasmática e nuclear em 24 (85,72%) casos de CEB, predominando, no parênquima do tumor, o escore 4+ (n=15; 62,5%). Vinte e três casos de CEB (82,14%) apresentaram imunoexpressão de CD163, enquanto 24 (85,72%) exibiram positividade para a proteína CD105. Foi observada uma tendência de maior imunoexpressão de SHH e IHH nos grupos com alto perfil proliferativo. Foi observada uma tendência de maior expressão das proteínas MCM3 (p=0.11), SHH (p=0.21) e IHH (p=0.07) em tumores menos diferenciados. Uma forte correlação positiva foi observada entre a DM e a MDV em CEBs e correlação positiva perfeita em MATs. Quando comparado à MAT e MNN, os casos de CEB apresentaram maior imunoexpressão de MCM3 (p=0.0003), SHH (p=0.01), IHH (p=0.39), GLI1 (p=0.03), DM (p=0.0002) e MDV (p=0.0002). CONCLUSÕES: Nossos resultados sugerem a participação da via HH em CEBs, através de uma sinalização autócrina e parácrina e com participação de ambos ligantes, SHH e IHH, os quais parecem contribuir para a proliferação e diferenciação do CEB. Demonstramos que células endoteliais também exibem positividade para componentes da via HH e que essas moléculas podem contribuir na angiogênese tumoral, ao mesmo tempo que macrófagos CD163+ expressam IHH, ligante da via, e estão associados com a neo-vascularização tumoral. Em adição, nossos achados sugerem que a proteína MCM3 pode despontar como um bom marcador de proliferação celular em CEB. / INTRODUCTION/OBJETIVE: The Oral Squamous Cell Carcinoma (OSCC) accounts for over 95% of all cancers diagnosed in the oral cavity and it consists on an invasive and aggressive type of tumor. The Hedgehog pathway (HH) has been involved in the pathogenesis of different tumors. The aim of this study was to evaluate the components of the HH pathway in OSCC, correlating the results with clinical aspects, angiogenesis, tumor differentiation, proliferative potential and macrophages CD163+. MATERIAL AND METHODS: Twenty-eight cases of OSCC, 9 cases of tumor margins (TM) and 4 cases of non-neoplastic oral mucosa (NNM) were submitted to immunohistochemical reaction for MCM3, SHH, IHH, GLI1, CD163 and CD105 proteins using the AdvanceTM polymer system. Co-localization for IHH/GLI1 and CD163/CD105 proteins was evaluated using double staining method. The analysis of MCM3, SHH, IHH and GLI1 proteins were conducted in 5-matching areas and data described using the semi-quantitative parameters described by Gurgel et al. (2008). The density of macrophages (MD) and microvessel density (MVD) were measured considering the population of these cells and newly formed vessels in 5-matching areas and the results expressed in cells/mm² and vessels/mm², respectively. Statistical analysis were performed using GraphPad Prism v. 6.03. RESULTS: All cases of OSCC were positive for MCM3 protein on the cytoplasm and nucleus of tumor cells, and 4+ was the main score (n= 19; 67.85%). In tumor cells, the SHH protein was observed in the cytoplasm and it was positive in 25 OSCC cases (89.28%), with a 4+ score predominant (n= 17; 68%). Twenty-two cases (78.57%) presented with cytoplasmic IHH protein in tumor cells and scores 4+ (n=7; 31.82%) and 3+ (n=7; 31.82%) were more common. The GLI1 protein was positive in 24 OSCC cases (85.72%) and detected in tumor cells on cytoplasmic and nuclear patterns, with score 4+ (n=15; 62.5%). Positive staining for CD163 and CD105 were described in 23 (82.14%) and 24 OSCC, respectively. In groups with high proliferative profile, we observed a larger amount of SHH and IHH proteins. In addition, on OSCC with a lower differentiation, we observed a tendency to increased levels of MCM3 (p=0.11), SHH (p=0.21) and IHH (p=0.07) proteins. A strong and a perfect positive correlation was observed between MD and the MVD in OSCC and TMs, respectively. A higher expression of MCM3 (p=0.0003), SHH (p=0.01), IHH (p=0.39), GLI1 (p=0.03), MD (p=0.0002) and MVD (p=0.0002) were founded in OSCC by comparing to TM and NNM. CONCLUSIONS: Our results suggest that the HH pathway may participate in OSCC through an autocrine and paracrine mechanisms, with parcipation of both ligands, SHH and IHH. These ligands seem to contribute to the proliferation and differentiation of the OSCC. We demonstrate that endothelial cells also display positivity for HH pathway components, which may contribute to the tumor angiogenesis. Moreover, CD163+ macrophages are positives for IHH ligand and highly associated with tumor-vessels. Additionally, our findings suggest that MCM3 protein could emerge as a good marker for cell proliferation in OSCC.

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:19:15Z (GMT). No. of bitstreams: 1 XavierFerrucio_JulianaMartins_D.pdf: 4222784 bytes, checksum: e2845997ddb128f2d29a2cabc667da17 (MD5) Previous issue date: 2015 / Resumo: A hematopoiese, processo pelo qual a célula-tronco hematopoiética (CTH) dá origem a todas as células do sangue, é regulada através do seu contato com diversos tipos celulares que compõem o estroma da medula óssea, mantendo o balanço entre autorrenovação e diferenciação das CTHs, processos também regulados por vias de sinalização como a via Hedgehog e proteínas reguladoras de citoesqueleto como RhoGTPases. Sendo assim, os objetivos gerais do trabalho foram investigar a via Hedgehog na medula óssea de pacientes com síndromes mielodisplásica (SMD) e a função da ARHGAP21 na autorrenovação das CTH. Através de imunohistoquímica de bióspias de medula óssea de pacientes com SMD em comparação com anemia megaloblástica (usada como controle) observamos o aumento de células marcadas para os ligantes Sonic e Dessert Hedgehog e células positivas para c-Kit nas amostras de SMD. Em seguida, analisamos a expressão gênica dos membros da via Hedgehog em células totais de medula óssea de pacientes SMD e doadores normais. Não houve diferença na expressão de Patched (PTCH) em SMD em comparação com doadores saudáveis, porém quando as amostras são classificadas de acordo com a WHO 2008, observamos aumento significativo de PTCH nos pacientes com <5% de blastos na medula óssea (AR, ARSA e CRDM). As expressões tanto de GLI1 como de SUFU foram reduzidas nos pacientes SMD e no grupo de pacientes com ARSA, CRDM e CRDU quando comparados aos doadores normais. A expressão de SUFU também foi reduzida em grupo de pacientes SMD com > 5% de blastos na medula óssea (AREB-1 e 2). Observamos aumento de 7 vezes na expressão de SMO no grupo de SMD e de 14 vezes no grupo AREB-1 e 2 em comparação com doadores normais. Análises de sobrevida de Cox demonstraram que o aumento na expressão de SMO e a redução na expressão de PTCH são fatores independentes na sobrevida global dos pacientes com SMD. Além disso, confirmamos o aumento da ativação da via Hedgehog em células CD34+ de pacientes com SMD através do aumento da expressão dos alvos da via: GLI1, BMI1 e Nanog. Nossos dados indicam que a via Hedgehog está desregulada na medula óssea dos pacientes com SMD e o possível envolvimento dessa via na progressão da doença. Através do modelo murino Arhgap21+/- estudamos a importância dessa proteína na hematopoiese normal e observamos que esses animais apresentam redução nas diferenciações eritroide e megacariocítica juntamente com aumento da mobilização mielóide. Observamos ainda o aumento na frequência das CTHs de curta e longa duração na medula óssea dos heterozigotos. Esse aumento foi responsável pela maior recuperação no número de neutrófilos após indução de estresse hematopoiético nos animais Arhgap21+/-. Durante o transplante seriado de células de medula óssea observamos menor reconstituição após 4 semanas, juntamente com menor reconstituição a longo prazo nos animais que receberam células dos heterozigotos, sugerindo menor autorrenovação das CTHs nas células com redução da Arhgap21. O nicho medular dos animais Arhgap21+/- demonstrou redução no suporte inicial da hematopoiese o que foi relacionado à maior produção de ROS após irradição subletal dos heterozigotos. Juntos, esses resultados indicam que a ARHGAP21 tem importante papel na hematopoiese normal, pois participa de processos como a diferenciação, mobilização e autorrenovação das CTHs / Abstract: Hematopoiesis is a process by which hematopoietic stem cell (HSC) gives rise to all blood cells and it is regulated by HSC contact with different cell types that compose the bone marrow stroma and maintain the balance between HSC self-renewal and differentiation, processes that are also regulated by signaling pathways such as the Hedgehog and regulatory proteins of the cytoskeleton as RhoGTPases. The overall aims of this work were to investigate the Hedgehog pathway in myelodysplastic syndrome (MDS) bone marrow and the ARHGAP21 role in hematopoiesis. Immunohistochemistry assays revealed that MDS bone marrow biopsies showed increased Sonic and Dessert Hedgehog together with c-kit positive stained cells compared to megaloblastic anemia (used as control).We also analyzed gene expression of the Hedgehog pathway members in total cells from MDS bone marrow and healthy donors. There was no difference in Patched (PTCH) expression in MDS compared to healthy donors, however, when MDS samples were classified according to WHO 2008, we observed a significant increase in PTCH1 expression in MDS patients <5% bone marrow blast (RCUD, RCMD, ARSA). GLI1 and SUFU expressions were significantly reduced in the MDS patients and in the group of patients with RCUD, RCMD, ARSA, when compared to healthy donors. SUFU expression was also decreased in MDS patients > 5% bone marrow blast (RAEB-1 and RAEB-2) compared to controls. We also observed 7-fold increase of SMO transcripts in the MDS group and 14-fold increase in RAEB-1 and RAEB-2. Cox survival analyses showed that increased SMO and decreased PTCH expression were considered as independent factors influencing the survival of these patients. We also confirmed the enhanced Hedgehog activation in CD34+ cells from MDS patients through increased expression of the pathway targets: GLI1, BMI1 and Nanog. Together, our data indicate that the Hedgehog pathway is deregulated in MDS bone marrow and the possible role of that pathway in disease progression. Arhgap21+/- murine model study showed the importance of this protein in normal hematopoiesis as these animals showed alterations in erythroid and megakariocyte differentiation along with increased myeloid mobilization. We also observed increased short and long term HSC frequency in the heterozygous bone marrow. This increase was responsible for enhanced neutrophil reconstitution during induced hematopoietic stress in Arhgap21+/-. Serial bone marrow transplantation assay showed lower chimerism in peripheral blood 4 weeks after the transplant together with lower long term reconstitution in animals who received Arhgap21+/- bone marrow, indicating decreased HSC self-renew from heterozygous cells. Bone marrow niche of Arhgap21+/- animals showed a reduction in initial support hematopoiesis and this result was correlated with the increased ROS production from Arhgap21+/- bone marrow after sublethally irradiation. Taken together, our data indicate that ARHGAP21 has an important role in hematopoiesis regulating process such as differentiation, mobilization and self-renewal of HSC / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutora em Fisiopatologia Médica

Au cours des dernières années, des progrès significatifs ont été réalisés dans la prévention des épisodes de démyélinisation qui surviennent au cours de la sclérose en plaques, mais beaucoup moins dans le domaine de la régénération de la myéline détruite. Notre équipe a précédemment démontré que la protéine Sonic Hedgehog (Shh) est un régulateur positif des progéniteurs oligodendrocytaires favorisant la réparation de la myéline dans un modèle de démyélinisation induite par injection focale de lysolécithine dans le corps calleux chez la souris. Cependant, les mécanismes moléculaires mis en jeu restent encore largement méconnus. Mon projet de thèse consiste à explorer le rôle d'un co-récepteur de Shh, Boc, une glycoprotéine reliée aux molécules d'adhésion cellulaire (CAM) et fortement induite à l’issue d’une démyélinisation. Dans un premier temps, nous avons focalisé nos investigations sur la caractérisation du mutant Boc knockout au cours de la myélinisation développementale. Nos résultats révèlent des activités région et temps spécifiques de Boc sur les mécanismes de prolifération et de maturation des progéniteurs oligodendrocytaires. De plus, les souris Boc knockout présentent un phénotype d’hypermyélinisation qui apparaît au cours des premières semaines post-natales et persiste à l’âge adulte.Dans un second temps, nous avons déterminé l'effet de l'absence de Boc sur les processus de démyélinisation et de réparation de la myéline. En utilisant le modèle d’injection focale de lysolécithine dans le corps calleux, nous avons démontré un retard dans la maturation des progéniteurs oligodendrocytaires et une réduction de la régression des lésions chez les souris dépourvues de Boc.Nos résultats indiquent ainsi un rôle positif de Boc dans le processus de régénération faisant suite à une démyélinisation. Par ailleurs, l’absence de Boc diminue la réaction gliale inflammatoire étroitement associée au mécanisme de régénération de la myéline. L’ensemble de nos résultats indique une activité différente du récepteur au cours des processus de myélinisation et de remyélinisation du système nerveux central. Ces données originales contribuent à la caractérisation d’une composante de la signalisation Hedgehog impliquée dans le processus régénératif associé aux pathologies démyélinisantes. Ces résultats seront importants à considérer dans le cadre de l’approche thérapeutique des maladies démyélinisantes. / During the last years, significant progress have been made in preventing demyelinating events that occur during multiple sclerosis, but much less in the field of regeneration of the lost myelin. Our team has previously demonstrated that the Sonic Hedgehog (Shh) protein is a positive regulator of oligodendrocyte progenitors enhancing myelin repair in a demyelination model relying on the focal injection of lysolecithin into the corpus callosum. However, the molecular mechanisms that are involved are still largely unknown. My thesis project was aimed at exploring the role of a co-receptor of Shh, Boc, a glycoprotein linked to the cell adhesion molecules (CAM) and strongly induced during myelin repair. First, we focused our investigations on the characterization of the Boc knockout mutant during developmental myelination. Our results reveal region and time specific activities for Boc in the mechanisms of proliferation and maturation of oligodendrocyte progenitors. In addition, Boc knockout mice exhibit hypermyelination that occurs in the firstpostnatal weeks and persists until adulthood In a second step, we determined the effect of the absence of Boc in the processes of demyelination and myelin repair. Using the model of focal injection of lysolecithin into the corpus callosum, we demonstrated a delay in oligodendrocyte maturation and a decrease in lesion regression in the Boc knockout mice. Thus, our results indicate a positive role for Boc in the regeneration process following demyelination.Furthermore, the absence of Boc decreases the inflammatory glial reaction closely associated with myelin repair. Altogether, our results indicate a different activity of the receptor during the processes of myelination and remyelination in the central nervous system. These original data contribute to the characterization of a component of the Hedgehog signaling pathway involved in the regenerative process associated with demyelinating pathologies.Those results will be important to consider in the context of the therapeutic approach of the demyelinating diseases.

Le médulloblastome est la plus fréquente des tumeurs pédiatriques malignes du cerveau et est divisé en quatre sous-groupes moléculaires. Le groupe Sonic Hedgehog (SHH), caractérisé par l’activation de la voie SHH, présente une surexpression du facteur de transcription basique hélice-boucle-hélice Atoh1. Atoh1 est essentiel pour le développement du cervelet et plus spécifiquement pour la formation des précurseurs des cellules granulaires, qui sont aussi la cellule d’origine du médulloblastome SHH. Dans le médulloblastome SHH, Atoh1 agit comme un facteur pro-tumoral en collaboration avec la voie SHH. De plus, l’inhibition des niveaux protéiques d’Atoh1 inhibe la prolifération tumorale in vitro et in vivo. Etant donnée l’importance de la protéine Atoh1 dans la formation du médulloblastome SHH, Atoh1 pourrait être une cible thérapeutique potentielle pour traiter le médulloblastome SHH. Cependant, les mécanismes de régulation d’Atoh1 sont encore mal connus.Les modifications post-traductionnelles sont connues pour réguler les niveaux protéiques dans les cellules. Plusieurs types de modifications régulent la dégradation protéique et elles incluent l’ubiquitination et la phosphorylation. Nous avons décidé de nous concentrer dans un premier temps sur le rôle potentiel de la phosphorylation d’Atoh1 sur sa fonction et régulation durant le développement du cervelet et la formation de médulloblastome.Par une analyse de spectrométrie de masse, nous avons identifié douze sites de phosphorylation sur la protéine Atoh1. Parmi ces douze sites, seulement deux, la sérine 328 (S328) et la sérine 339 (S339), sont importantes pour la stabilité et la fonction de la protéine. En effet, les deux mutants de phosphorylation spécifiques de ces deux sites, Atoh1-S328A et Atoh1-S339A, ont une demi-vie plus longue ainsi qu’une activité transcriptionnelle augmentée dans les précurseurs des cellules granulaires par rapport à la forme sauvage d’Atoh1. Nous avons ensuite réalisé une purification d’affinité en tandem différentielle suivie par une analyse par la technologie d’identification protéique multidimensionnelle (MudPIT) pour définir les partenaires phospho-spécifiques d’Atoh1. Nous avons découvert que l’ubiquitine ligase E3 Huwe1 est responsable de la dégradation d’Atoh1 de manière phospho-dépendante dans les progéniteurs des cellules granulaires. Nous avons aussi montré que SHH protège Atoh1 de la dégradation médiée par Huwe1 par l’intermédiaire des phosphatases de la famille des PP2A. De manière importante, ce mécanisme de régulation d’Atoh1 est nécessaire au bon développement du cervelet. De plus, dans le contexte tumoral, un faible niveau d’ARNm d’HUWE1 est associé à une mauvaise survie chez les patients ayant un medulloblastome SHH.Au vu de ces résultats, nous souhaitons étudier plus en détail le rôle de la phosphorylation d’Atoh1 ainsi que la contribution de ce nouveau mécanisme dans le médulloblastome. Nous souhaiterions ainsi exploiter nos résultats pour développer de nouvelles stratégies thérapeutiques dans le médulloblastome SHH. / Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is divided in four subgroups by gene profiling. The well-known subgroup harboring an activation of the Sonic Hedgehog (SHH) pathway shows an upregulation of the proneural basic helix-loop-helix transcription factor Atoh1. Atoh1 is essential for cerebellum development and more specifically for the formation of the granule neuronal progenitors (GNPs), which are the cells of origin of SHH induced MB. In tumoral context, Atoh1 acts as a pro-tumor factor in cooperation with SHH pathway. In addition, the inhibition of Atoh1 protein level prevents MB proliferation in vitro and in vivo. Thus, given the strong implication of Atoh1 protein in MB formation, Atoh1 seems to be a potential therapeutic target to treat SHH MB. However, up to date, mechanisms underlying its regulation remain to be elucidated. Posttranslational modifications are known to regulate protein levels in cells. Several modifications regulate protein turnover including the two most prominent, ubiquitination and phosphorylation. We decided to focus primarily our study on a potential role of Atoh1’s phosphorylation on its function and regulation both during cerebellar development and MB genesis. Using mass spectrometry analysis, we identified twelve phosphorylation sites on Atoh1 protein. Among them, only two, the serine 328 (S328) and serine 339 (S339), were critical for Atoh1 stability and function. The two single phospho-deficient mutants, Atoh1-S328A and Atoh1-S339A, displayed a longer half-life and increased transcriptional activity in granule neuron progenitors when compared to the wild-type form of Atoh1. Next, we employed differential tandem affinity purification followed by Multidimensional Protein Identification Technology (MudPIT) analysis to define Atoh1 phospho-specific partners. We uncovered that the E3 ubiquitin ligase Huwe1 is responsible for Atoh1 degradation in a phospho-dependent manner in granule neuron progenitors. We also discovered that SHH protects Atoh1 against its degradation mediated by Huwe1 through the phosphatases of PP2A family. Importantly, this machinery is required for proper cerebellar development and we highlighted that low levels of HUWE1 are associated with a poor prognosis in patient harboring a SHH medulloblastoma.Given this data, we wish to dissect the role of Atoh1 phosphorylation, and the contribution of this new pathway in MB. We anticipate exploiting our findings to develop new therapeutic strategies in SHH MB.

Hedgehog (Hh) est un morphogène secrété qui contrôle la croissance et la différentiation cellulaire chez les métazoaires. La dérégulation de son activité entraine des maladies développementales et de nombreux cancers chez l’adulte. Chez la drosophile, la transduction du signal Hh est initiée par la fixation de Hh sur son récepteur Patched (Ptc), conduisant à la stabilisation de la protéine membranaire Smoothened (Smo) et à l’activation du complexe de transduction composé de 5 protéines : les kinases Fused (Fu), PKA, GprK2, la kinésine Costal 2 (Cos2), et le facteur de transcription Cubitus Interruptus (Ci). Ma thèse a porté sur l’étude de la régulation et des interactions moléculaires entre les composants du complexe de transduction. Par des approches complémentaires, j’ai montré qu’en absence d’Hh, les protéines PKA et Fu interagissent du côté C-terminal de Ci, alors que la présence d’Hh induit leur relocalisation vers le domaine N-terminal de Ci. J’ai pu prouver que l’élément déclencheur de ce remaniement protéique est Smo. En présence d’Hh, Smo s’incorpore dans le complexe de transduction, conduisant à l’activation et au déplacement de Fu vers la région N-terminale de Ci. Ce remaniement entraine la phosphorylation et l’activation de Ci. Ma thèse révèle l’importance des changements de conformation au sein du complexe de transduction de la voie Hh. Le mécanisme de transduction étant conservé entre invertébrés et invertébrés, mon doctorat apporte des éléments de recherche pour mieux comprendre le fonctionnement normal et pathologique des cellules. / Hedgehog (Hh) is a secreted morphogen that controls growth and differentiation in both vertebrates and invertebrates. The dysregulation of its activity leads to severe developmental defects, and the onset of cancer in adults. In Drosophila, the Hh signal transduction is initiated by the binding of Hh to its receptor Patched (Ptc). This induces the stabilization of the transmembrane protein Smoothened (Smo) and the subsenquent activation of a transduction complex consisting of 5 proteins: the kinases Fused (Fu), PKA and Gprk2, the kinesin Costal2 (Cos2), and the transcription factor of the pathway Cubitus Interruptus (Ci). The aim of my thesis was to study the regulation and molecular interactions between the different components of the transduction complex. Thanks to complementary techniques, I have shown that in absence of Hh the proteins Fu and PKA interact in C-terminal part of Ci, whereas on the presence of Hh induces their relocalization toward the N-terminal domain of Ci. I have proved that the trigger element of this moving is Smo. In presence of Hh, Smo goes into transduction complex, allowing the activation and the moving of Fu toward N-terminal domain of Ci. This relocalization is responsible of Ci phosphorylation and activation. My thesis reveals the importance of conformational changes inside the transduction complex of Hh pathway. As the mechanism of transduction is conserved between species, my PhD provides research elements in order to better understand the normal and abnormal functioning of cells.

Hedgehog (Hh) shape up development by playing important role in signaling, and thereby controlling growth and pattern formation. It is for this reason that their spatial distribution is tightly regulated. The 19kDa active form of Hh is modified with a palmitate at its N-terminal and with cholesterol at its C-terminal. This dually lipid modified form of Hh act as a morphogen, and is also referred to as HhNp (Mann and Beachy, 2004). In most cases, they are released from producing cells and spread into adjacent non-expressing cells within the tissue, where it activates target gene expression in a concentration-dependent manner. In Drosophila, Lipophorin (Lpp) particles carry these lipid-modified forms of Hh and play a role in long range signaling in the developing wing disc. Further, these particles circulate throughout the larvae in the hemolymph to distribute nutrients mostly in the form of lipids to different tissues of the animal. Thus, Lpp plays important role in metabolism and development. Hh as a morphogen plays a very important role in development and patterning of embryo and imaginal discs in Drosophila. We wanted to understand the role of Hh in overall development of Drosophila. In my thesis work, I discovered a new form of Hh that is systemically circulating in the 3rd instar larva of Drosophila. I show that imaginal tissues do not produce this form of circulating Hh. Our experiments strongly suggest that systemic Hh can travel from one tissue to another, a feature that was previously unknown. I also show that it could rescue the growth of the imaginal disc, implying its ability to influence cell proliferation. Since the concentration of systemic Hh is low it fails to up regulate the target genes. I characterized fat body as a target of systemically circulating Hh. I clearly demonstrate that fat body transcribes most of the components of Hh signaling pathway except Hh. Further, Hh accumulates in the fat body during late 3rd instar larvae. That makes the fat body an ideal target of systemic Hh. This could shed light in understanding the role of Hh in overall development of Drosophila melanogaster that includes tissue-based interaction.