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The Hedgehog protein (Hh) is a morphogen that is necessary for cell survival, growth and patterning in flies and mammals. In germline cells, alterations in the Hh signaling pathway can result in developmental disorders; in somatic cells, misregulation of the Hh signaling pathway can result in cancer. Most components of the signaling pathway were identified by genetic screens in Drosophila that were later found to be conserved in mammals. In the presence of the Hh signal, multiple Hh signaling components interact to mediate the induction of Hh target genes. In flies, Cubitus Interruptus (Ci) is the singular transcription factor of the pathway that is regulated by multiple upstream components of the pathway including Costal2 (Cos2). Cos2 is a scaffold protein that can both positively and negatively regulate Hh signaling by binding to Ci and various kinases such as Fused (Fu). We disrupted the binding of Cos2 to Fu using a physiological expressed genomic Costal2 transgene (gCosΔFu) and found that Fu must bind to Cos2 to promote efficient processing and activation of full-length Ci (Ci-155). Fu was thought to activate Ci-155 by phosphorylating Cos2 at sites S931 and S572, but we found that gCosS931A and gCosS572A did not reduce Ci activity in the fly wing disc. Instead, we hypothesize that Fu could directly phosphorylate Ci-155 or another unknown protein. To investigate if another protein was involved we developed a Hh sensitized genetic screen. We obtained multiple “hits” from the genetic screen but we did not find an obvious candidate that could be a substrate for Fu. Instead, we identified Mago Nashi and Srp54 which we found to be involved with the post-transcriptional regulation of ci RNA. We confirmed the existence of ci isoforms A and B and found that knockdown of Mago Nashi, resulted in an altered splicing pattern while knockdown of Srp54 reduced ci RNA levels. Mago Nashi inhibition and intronless Ci reduced Ci-155 protein levels, which suggests efficient splicing is necessary for normal Ci-155 levels. Furthermore, we found that reduced Ci-155 levels only affected Ci activity in sub-optimal Hh signaling conditions. In order to further dissect the mechanism Ci processing, activation and stabilization, we used physiologically expressed genomic Ci (gCi) and CRISPR Ci variants (crCi). First we examined Ci-S849A, which prevents Ci processing and we found that in the absence of processing, Ci-155 levels are uniformly high throughout the wing disc. Cos2 and PKA are necessary for Ci processing but we wanted to know if they had an additional role in Ci silencing We found that Cos2 but not PKA can silence and stabilize Ci-155 in the absence of processing. Activated Fu in the Ci-S849A wing disc highly activated and destabilized Ci-155, which was similar to Hh signaling at the AP Border. To test if Ci is the direct target of Fu, we are testing physiologically expressed Ci with point mutations and deletions that are near the Suppressor of Fused (Su(fu)) binding site to examine whether they are unresponsive to activated Fu. Su(fu) binds to Ci-155 to stabilize Ci- 155 levels and inhibit Ci activity, but the mechanism is not well understood. We developed Ci transgenes that have altered Su(fu) binding to determine if Su(fu) inhibits Ci by cytoplasmic anchoring, co-repressor recruitment, or by blocking a co-activator.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 188-204).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 10, 2008). Includes bibliographical references.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.146

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.124-150

Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

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.