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Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-02-14T18:23:05Z No. of bitstreams: 1 Gonzalez, ACO.pdf: 3789736 bytes, checksum: 1fbb6dad4c1a43846555898698d711fa (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-03-23T12:50:19Z (GMT) No. of bitstreams: 1 Gonzalez, ACO.pdf: 3789736 bytes, checksum: 1fbb6dad4c1a43846555898698d711fa (MD5) / Made available in DSpace on 2017-03-23T12:50:19Z (GMT). No. of bitstreams: 1 Gonzalez, ACO.pdf: 3789736 bytes, checksum: 1fbb6dad4c1a43846555898698d711fa (MD5) / Os processos imunobiológicos que regulam a biossíntese e o remodelamento da matriz extracelular na cicatrização de ferimentos fotobiomodulados e no contexto tumoral foram contemplados no presente trabalho. Na primeira parte, foram descritas as fases da cicatrização integrantes do processo de reparo tecidual cutâneo, com ênfase no papel biológico desempenhado por diferentes células e suas moléculas sinalizadoras. Na segunda seção, foi realizado estudo experimental, duplo cego a fim de avaliar o efeito do laser de baixa potência na fase tardia da cicatrização, em especial em relação à angiogênese, células do sistema imune e a via de sinalização Hedgehog. Quarenta ratos foram alocados randomicamente em dois grupos experimentais, controle e tratado com laser diodo semicondutor de AsGaAl (670nm). Após indução de ferimento cutâneo padronizado, os espécimes foram eutanasiados em 14, 21, 28 e 35 dias. As secções teciduais foram submetidas à coloração com hematoxilina-eosina e à técnica de imuno-histoquímica com os anticorpos anti CD31, NG2, actina alfa de músculo liso, CD8, CD68, Ptch, Ihh e Gli-2. Os níveis séricos das citocinas IL-2, IL-4, IL-10 e IFNɣ foram avaliados através da técnica de citometria de fluxo. Todos os dados histomorfométricos foram submetidos à análise estatística e o nível de significância adotado foi p<0,05. Observou-se que a neoangiogênese persistiu, pois houve presença de CD31+(p=0,016), NG2+ e alfa actina de músculo liso (p=0,025) em ambos os grupos, sendo que no grupo tratado com laser ocorreu mais cedo. No grupo laser, aos 21 dias houve decréscimo de células CD68+ (p=0,032) e aumento de CD8+ (p=0,038). Os níveis sanguíneos de IL-2 e IL-10 foram indetectáveis em ambos os grupos experimentais, ao passo que o perfil de IL-4 e IFNɣ não foi significativo (p>0,05). Na fase do remodelamento, a sinalização da via Hedgehog pareceu estar ativada, haja vista a presença da expressão fenotípica de algumas proteínas desta via, embora, a expressão de Ihh e Ptch parece não ter sido fotobiomodulada, pois não houve diferenças estatisticamente significativas entre os grupos experimentais. A terceira parte do trabalho abordou o processo de transformação neoplásica através de um estudo comparativo de lesões pré-cancerizáveis de mucosa oral e carcinoma escamocelular, no qual foi investigada a presença de algumas proteínas da via Hedgehog em dezesseis casos humanos. Observou-se significativo aumento progressivo da expressão de todas as proteínas da via Hedgehog, tanto no epitélio quanto na lâmina própria, quando se comparou as secções de mucosa oral normal, displasia e carcinoma (p<0,05). Através da realização do primeiro estudo, conclui-se que o laser de baixa potência foi capaz de modular a fase do remodelamento da matriz extracelular durante a cicatrização experimental, uma vez que os tecidos fotobiomodulados apresentaram alterações mais precoces em relação ao grupo controle e os eventos que ocorrem no reparo tecidual parecem ser cíclicos e continuam se desenvolvendo no tecido, independente do aspecto clínico da lesão que evidencia o seu fechamento e completa cicatrização. Em relação ao estudo realizado em mucosa oral humana, observou-se que a ativação da via Hedgehog pareceu influenciar na transformação neoplásica, pois foi demonstrado aumento progressivo de células Hedgehog positivas nas lesões pré-cancerizáveis presentes no mesmo indivíduo. / This study evaluated the immunological processes that regulate the biosynthesis and extracellular matrix remodeling in the context of photobiomodulated wound healing and tumors. Initially, the stages of the skin tissue repair process were described, with emphasis on the biological role played by different cells and their signaling molecules. Subsequently, a double-blind experimental study was conducted, in order to assess the effect of low power laser in the late stages of healing, with emphasis on angiogenesis, immune cells and Hedgehog signaling. Forty rats were allocated randomly in two groups; control and those treated with laser diode of GaAlAs (670 nm). After induction of standardized skin wounds, the specimens were euthanized at 14, 21, 28 and 35 days. Tissue sections were subjected to hematoxylin-eosin and immunohistochemical techniques with antibodies to CD31, NG2, smooth muscle alpha actin, CD8, CD68, Ptch, Gli-2 and Ihh. Serum levels of the cytokines IL-2, IL-4, IL-10 and IFNɣ were assessed by flow cytometry. All histomorphometric data were statistically analyzed and significance level was at p<0,05. We observed that neoangiogenesis persisted, because of the presence of CD31+ (p=0,016), NG2+ and smooth muscle alpha actin (p=0,025) in both groups. The laser-treated group responded earlier. By day 21, the laser-treated group had decreased CD68+ cells (p=0,032) and increased CD8+ (p=0,038).Blood levels of IL-2 and IL-10 were undectable in both experimental groups, whereas IL-4 and IFNɣ profile was not significant (p>0,05). At the remodeling stage, the Hedgehog signaling pathway seemed to be activated, given the presence of phenotypic expression of some of its proteins, although the expression of Ihh and Ptch did not seem to have been photobiomodulated because there were no statistically significant differences between the experimental groups. The third part of our study dealt with neoplastic transformation process, through a comparative study of pre-cancerous lesions of the oral mucosa and squamous cell carcinoma, in which we investigated the presence of some proteins of the Hedgehog pathway in sixteen human cases. There was a significant progressive increase in the expression of all proteins in the Hedgehog pathway, both in the epithelium and lamina propria, when comparing lesions of normal mucosa, dysplasia and carcinoma (p<0,05). We concluded that laser therapy was able to modulate the remodeling phase because laser treated tissues showed modifications earlier than control group and the events that occur in tissue repair appear to be cyclical and continue to remodel the tissue, independent of the clinical aspects of the lesion such as its closure and completed healing. Regarding the study of human oral mucosa, it was observed that Hedgehog pathway activation seems to influence the neoplastic transformation. Progressive increase of Hedgehog positive cells was observed in precancerous lesions present in the same individual.

Submitted by Programa de Pós-Graduação em Odontologia Saúde (mestrodo@ufba.br) on 2018-09-05T14:56:42Z No. of bitstreams: 1 Tese Final REBECA.pdf: 4431734 bytes, checksum: 07c03f057f035f7cfc830f968ac47ee6 (MD5) / Approved for entry into archive by Edvaldo Souza (edvaldosouza@ufba.br) on 2018-09-12T20:57:33Z (GMT) No. of bitstreams: 1 Tese Final REBECA.pdf: 4431734 bytes, checksum: 07c03f057f035f7cfc830f968ac47ee6 (MD5) / Made available in DSpace on 2018-09-12T20:57:34Z (GMT). No. of bitstreams: 1 Tese Final REBECA.pdf: 4431734 bytes, checksum: 07c03f057f035f7cfc830f968ac47ee6 (MD5) / INTRODUÇÃO/OBJETIVO: A via Hedgehog (HH) está ativada no Carcinoma Escamocelular de Boca (CEB) e pode estar relacionada a transição epitelialmesenquimal (TEM) e a proliferação celular neste tumor. Diante disso, o presente trabalho teve como objetivo avaliar a TEM e potencial proliferativo em CEBs com a via HH ativada. MATERIAL E MÉTODOS: Vinte e três casos de CEBs GLI1 positivos foram submetidos à reação imuno-histoquímica para as proteínas Snail, Slug, Ncaderina, E-caderina, β-catenina e MCM3. As análises da imunoexpressão foram realizadas em front de invasão e ilhas tumorais e os dados clínico-patológicos foram avaliados e comparados. RESULTADOS: No front de invasão, os casos de CEBs apresentaram expressão positiva para Snail, Slug e MCM3 no núcleo das células tumorais e observou-se perda de expressão membranar e citoplasmática de E-caderina e β-catenina. A expressão positiva para N-caderina foi observada em 31,78% dos casos. Imunoexpressão de GLI1 foi associada com a perda de expressão de E-caderina (p<0,001), β-catenina membranar (p<0,001) e β-catenina citoplasmática (p=0,02). Nas ilhas tumorais, observou-se expressão nuclear GLI1, Snail, Slug e MCM3. E-caderina e β-catenina exibiram marcação positiva membranar restabelecida. Observou-se uma correlação positiva e estatisticamente significante entre GLI1 e Snail (p=0,05), Ecaderina (p=0,01) e β-catenina citoplasmática (p=0,04). A expressão de β-catenina membranar estava relacionada com presença de metástase em linfonodos e estadiamento clínico e GLI1 apresentou associação com o estadiamento clínico. CONCLUSÕES: A via HH pode atuar na regulação da expressão de fenótipo mesenquimal. A perda de expressão de E-caderina e β-catenina está relacionada a região de front, enquanto que a adesão celular se encontra reestabelecida em ilhas tumorais, de forma independente de MCM3. O padrão de expressão de N-caderina e Slug não refletem de forma significante o processo de TEM em CEBs com a via HH ativada. / INTRODUCTION/OBJECTIVE: The Hedgehog (HH) pathway is activated in the Oral Squamous Cell Carcinoma (OSCC) and may be related to the epithelialmesenchymal transition (EMT) and cellular proliferation in this tumor. The aim of the present study was to evaluate the EMT and the cell proliferative potential in OSCC with the activated HH pathway. MATERIAL AND METHODS: Twenty-three positive GLI1 OSCC cases were submitted to the immunohistochemical reaction for Snail, Slug, N-cadherin, E-cadherin, β-catenin and MCM3 proteins. Immunoexpression analyzes were performed in front of invasion and tumor nests and the clinical and pathological data were evaluated and compared. RESULTS: At the invasive front, OSCC cases showed positive expression for Snail, Slug and MCM3 in the nucleus of tumor cells and the loss of membrane and cytoplasmic expression of E-cadherin and β-catenin were observed. Positive expression for N-cadherin was observed in 31.78% of the cases. Immunoexpression of GLI1 was associated with loss of E-cadherin expression (p <0.001), membrane β-catenin (p <0.001) and cytoplasmic β-catenin (p = 0.02). In the tumor nests, it observes nuclear expression GLI1, Snail, Slug and MCM3. E-cadherin and β-catenin showed re-established membrane positive expression There was a positive and statistically significant correlation between GLI1, Snail (p = 0.05), Ecadherin (p = 0.01) and cytoplasmic β-catenin (p = 0.04). The expression of β-catenin membrane was related to the presence of matastasis in lymph nodes and to clinical staging and GLI1 was associated with clinical staging. CONCLUSIONS: The HH pathway may act to regulate the expression of mesenchymal phenotype. The loss of Ecadherin and β-catenin expression is related to the front region, while cell adhesion is re-established in tumor nests independently of MCM3. The expression pattern of Ncadherin and Slug do not significantly reflect the TEM process in positive GLI1 OSCC

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-01-05T16:59:19Z No. of bitstreams: 1 GeissyLainnyDeLimaAraujo_DISSERT.pdf: 3295602 bytes, checksum: 5e99ad6700c5552b3b1ad3651c7bce8c (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-01-08T18:10:33Z (GMT) No. of bitstreams: 1 GeissyLainnyDeLimaAraujo_DISSERT.pdf: 3295602 bytes, checksum: 5e99ad6700c5552b3b1ad3651c7bce8c (MD5) / Made available in DSpace on 2016-01-08T18:10:33Z (GMT). No. of bitstreams: 1 GeissyLainnyDeLimaAraujo_DISSERT.pdf: 3295602 bytes, checksum: 5e99ad6700c5552b3b1ad3651c7bce8c (MD5) Previous issue date: 2014-10-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / O Sonic Hedgehog (Shh) ? um morf?geno com importantes a??es no sistema nervoso central (SNC) em desenvolvimento, assim como na vida adulta em quadros de les?o tecidual e processos tumorig?nicos. A rela??o da sua via de sinaliza??o com prolifera??o, diferencia??o e sobreviv?ncia celular ? amplamente estudada em regi?es ventrais do SNC. No entanto, o papel da sinaliza??o por Shh em egi?es dorsais, como o telenc?falo dorsal, origem do c?rtex cerebral, n?o est? bem documentada. A partir do cultivo de c?lulas de roedores retiradas do telenc?falo dorsal em desenvolvimento, observamos a influ?ncia do Shh sobre a prolifera??o e diferencia??o das c?lulas-tronco neurais. Utilizando v?deo-microscopia de tempo intervalado, podemos avaliar o tempo de ciclo celular, tamanho de c?lulas progenitoras antes da divis?o celular e tipo de divis?o sofrida pelas c?lulas na presen?a ou na aus?ncia de sinaliza??o por Shh. Verificamos um aumento do n?mero de c?lulas em estado proliferativo assim como um aumento de c?lulas reativas para o marcador astrocit?rio GFAP com o tratamento com Shh. Em contrapartida, ap?s bloqueio da sinaliza??o por Shh, observamos um menor n?mero de c?lulas em estado proliferativo, desacelera??o do ciclo celular, aumento da morte celular e redu??o da astrogliog?nese. Por fim, com intuito de avaliar a influencia do Shh in vivo, n?s injetamos f?rmacos agonista (Purmorfamina) e antagonista (Ciclopamina) da via de sinaliza??o dessa prote?na em diferentes per?odos da gesta??o de roedores. Ao avaliar os animais na vida p?s-natal, observamos um aumento no n?mero de progenitores gliais gerados com o tratamento com Purmorfamina na subst?ncia branca, enquanto na subst?ncia cinzenta n?o parece haver altera??o dessa popula??o em ambos os tratamentos. Al?m disso, a popula??o de c?lulas astrocit?rias, evidenciada por marcadores espec?ficos, parece estar alterada com a manipula??o da sinaliza??o por Shh. Em conjunto, nossos dados sugerem que a Shh est? presente no telenc?falo dorsal em per?odos precoces do desenvolvimento e influencia a gera??o, sobreviv?ncia e prolifera??o de progenitores e c?lulas gliais.

This thesis describes the work that I have carried out in the Laboratory of Neurophysiolgy at the Université Libre de Bruxelles, under the supervision of Prof. Serge Schiffmann, in collaboration with Prof. Stéphane Schurmans of Université of Liège.The work is divided in two distinct but related projects and the results section is thus divided into two main chapters. The results described are presented in the form of two manuscripts, the first chapter is named “Ciliary phosphoinositides regulation by INPP5E controls Shh signaling by allowing trafficking of Gpr161 in neural stem cells primary cilium”.The second is named “Regulation of phosphoinositides ciliary levels controls trafficking and ciliogenesis in ependymal cells”.Since both manuscripts are comprehensive regarding the results, and methods, these are inserted as such into the thesis.An expanded introduction to the field, placing the results into context, precedes these two chapters. An extended discussion section follows each chapter; it presents some elements of discussion not included in the manuscripts, the implications of the results and the scope for further research. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

The Hedgehog (HH) signalling pathway and its primary transducer, GLI2, regulate cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells. To further assess the role of HH signalling during mouse embryonic stem (mES) cell differentiation, we studied the effects of GLI2 overexpression during mES cell differentiation. GLI2 overexpression resulted in temporal enhancement of cardiac progenitor genes, Mef2c and Nkx2-5, along with enhancement of Tbx5, Myhc6, and Myhc7 in day 6 differentiating mES cells. Mass spectrometric analysis of proteins that immunoprecipitate with GLI2 determined that GLI2 forms a complex with BRG1 during mES cell differentiation. Furthermore, modulation of HH signalling during P19 EC cell differentiation followed by chromatin immunoprecipitation with an anti-BRG1 antibody determined that HH signalling regulates BRG1 enrichment on Mef2c. Therefore, HH signalling accelerates cardiac progenitor gene expression during mES cell differentiation potentially by recruiting a chromatin remodelling factor to at least one cardiac progenitor gene.

Proper central nervous system development is critical for survival and depends on complex intracellular and extracellular signaling to regulate neural progenitor cell growth and differentiation; however, the mechanisms that mediate molecular crosstalk between pathways during neurogenesis are not fully understood. Here, we explored the integration of the Hedgehog (Hh) signaling pathway with the two critical developmental pathways, Receptor Tyrosine Kinase (RTK) and Notch signaling, in the growth and maintenance of neural progenitors in the developing neuroretina. We found combined and sustained RTK and Hh signaling was sufficient to establish long-term retinal progenitor cell (RPC) cultures and these cells maintained neurogenic and gliogenic, but not retinogenic, competence in vitro and in vivo. In addition, we identified crosstalk between Notch and Hh signaling, where Notch is required for Hh-mediated proliferation and Gli protein accumulation, and gain-of-function of Notch is sufficient to extend the window of Hh responsiveness in a subset of Müller glia. Both Hh-RPC monolayer establishment and Notch mediated Hh-responsiveness required Gli2. Taken together, we identified molecular cross-communication between the Hh pathway and two major pathways, Notch and RTK, during retinogenesis, advancing our understanding of mechanisms that influence Hh to control neural progenitor growth.

Au cours des maladies démyélinisantes du système nerveux central (SNC), les gaines de myéline qui entourent les axones et contribuent à la rapidité de la conduction nerveuse, mais aussi les oligodendrocytes qui synthétisent la myéline sont détruits. C’est le cas dans la plus commune de ces pathologies, la sclérose en plaques, qui se caractérise par des épisodes inflammatoires et démyélinisants récurrents évoluant après quelques années vers une forme progressive secondaire. A l’heure actuelle, les traitements disponibles pour les patients sont essentiellement de type immunomodulateurs. Ces molécules sont efficaces pour réduire la fréquence des épisodes démyélinisants, mais elles sont dépourvues d’activité sur la forme progressive de la maladie. Juste avant que ne débute mon travail de thèse, les voies de signalisation respectivement induites par les protéines Hedgehog et les stéroïdes de type androgènes se sont révélées être des régulateurs positifs de la réparation de la myéline vraisemblablement en utilisant des mécanismes différents. A partir de ces découvertes, nous avons émis l’hypothèse que la modulation pharmacologique simultanée des deux voies pourrait être intéressante dans une perspective thérapeutique. En utilisant des cultures de cellules gliales mixtes ou en administrant ces modulateurs à des souriceaux nouveau-nés, nous avons d’abord montré l’existence d’une interaction fonctionnelle entre les deux voies au cours du développement physiologique de la myéline à la période post-natale précoce. De façon intéressante, le blocage de la signalisation Hedgehog est requis pour que les androgènes puissent jouer leur rôle dans le processus de myélinisation. Les mécanismes moléculaires impliqués dans la communication entre les deux voies sont apparemment indépendants de la régulation de la transcription du principal effecteur de la signalisation Hedgehog (Gli1), ainsi que de celle du récepteur nucléaire des androgènes (AR). Par ailleurs, l’administration de ces mêmes modulateurs à des animaux ayant subi une démyélinisation du SNC nous a permis de mettre en évidence un effet synergique des deux voies sur la régénération de la myéline, la résolution de l’inflammation et la récupération fonctionnelle. Ces résultats seront importants à considérer dans le contexte d’une nouvelle approche thérapeutique des maladies démyélinisantes. / During demyelinating diseases of the central nervous system (CNS), the myelin sheaths that surround the axons and contribute to nervous conduction velocity, but also the oligodendrocytes that synthesize myelin are lost. This is particularly true in multiple sclerosis, the most common of those pathologies, which is characterized by recurrent inflammatory and demyelinating attacks evolving after several years into a secondary progressive form. Presently, the treatment of patients mostly relies on the use of immunomodulators, which are successful in decreasing the frequency of the attacks. However, these molecules lead to poor results in the progressive form of the disease. Just before the beginning of my PhD, the Hedgehog and androgen signaling pathways have been identified as positive regulators of myelin repair likely by using different mechanisms. On the basis of these findings, we hypothesized that a combination therapy using pharmacological modulators of each of these pathways could be interesting from a therapeutic point of view. By using primary mixed glial cell cultures and in vivo administration of the modulators to early postnatal mice, we have shown that the Hedgehog and androgen signaling pathways functionally interact during the physiological development of myelin at the early postnatal period. Importantly, the blockade of the Hedgehog signaling is required to allow androgen to play their role in the myelination process. The molecular mechanisms implicated in the pathway crosstalk do not involve the transcriptional regulation of the main effector of Hedgehog signaling (Gli1) or the nuclear androgen receptor (AR). Moreover, the same modulators administered into demyelinated animals led us to demonstrate a synergetic effect both on myelin repair, inflammation resolution and functional recovery. These results should be considered in the context of a novel therapeutic approach of demyelinating diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22640号 / 医博第4623号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 近藤 玄, 教授 斎藤 通紀, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM