Orientador: Albina Messias de Almeida Milani Altemani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T21:50:24Z (GMT). No. of bitstreams: 1 Costa_AnaFlaviadeMattos_M.pdf: 3298163 bytes, checksum: 3fb6838b3da137877a94a25afd98a7e4 (MD5) Previous issue date: 2009 / Resumo: Para investigar se carcinomas salivares com e sem diferenciação mioepitelial poderiam apresentar diferenças em relação ao nível de angiogênese, nós comparamos a vascularização tumoral entre os carcinomas adenóide cístico (31 casos) e epitelial mioepitelial (14 casos) versos carcinoma mucoepidermóide (37 casos). A expressão da peroxiredoxina I foi também estudada para verificar a relação potencial entre o metabolismo celular e a densidade microvascular. A densidade microvascular para o CD34 e CD105 foi significantemente menor em carcinomas com diferenciação mioepitelial. Porém, nenhuma relação foi encontrada entre a angiogênese e a quantidade de células mioepiteliaís. A forte expressão de peroxiredoxina I foi encontrada em 73.7% de carcinomas mucoepidermóides enquanto que 85.1% dos carcinomas com diferenciação mioepitelial apresentaram fraca expressão. Em conclusão, carcinomas com diferenciação mioepitelial, independente da quantidade de células mioepiteliaís, estão associados com significante baixa densidade microvascular. As razões para essa baixa atividade angiogênica ainda deve ser esclarecida, mas pode ser relacionada com as características metabólicas das células cancerosas. / Abstract: To investigate whether salivary carcinomas with and without myoepithelial differentiation could present differences regarding degree of angiogenesis, we compared tumor vascularization between adenoid cystic (31 cases) and epithelial- myoepithelial carcinomas (14) versus mucoepidermoid (37) carcinoma. The expression of peroxiredoxin I was also studied to verify the potential relationship between cellular metabolism and microvascular density. Microvascular density for CD34 and CD105 were significantly lower in carcinomas with myoepithelial differentiation. However, no correlation was found between degree of angiogenesis and amounts of myoepithelial cells. High-grade peroxiredoxin I expression was found in 73.7% of mucoepidermoid carcinomas whereas 85.1 of carcinomas with myoepithelial differentiation presented low-grade expression. In conclusion, carcinomas with myoepithelial differentiation, regardless of the amounts of myoepithelial cells, are associated to a significantly lower vascular density. The reasons for this lower angiogenic activity remain to be determined, but could be related to metabolic characteristics of the cancer cells. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

A thesis on Head and neck cancer focusing on dose planning, salivary gland carcinoma and Single nucleotide polymorphism. For dose planning PET/CT (Positron emissions tomography/computed tomography) with tracer gave more precise information in comparison dose planning with CT. More primary tumours and metastases were found with the acetate tracer than with glucose tracer. Acetate PET/CT also showed larger volume of tumours attributed to lipid metabolism. In a retrospective study salivary gland cancer 5-year overall survival (OS) was 53 %. Salivary gland carcinoma consists of many histopathological groups, the two largest groups being mucoepidermoid carcinoma (MEC) and adenoid cystic carcinoma (ASCC). For ACC, having the best 5-year OS, it was 70 percent. Facial palsy, advanced stage disease, lymph node metastases worsened prognosis. ACC and polymorphous low grade carcinoma (PLGA) expressed c-myc and cyclin D1 to a larger extent than MEC. In squamous cell carcinoma of the head and neck we examined the occurrence of Single Nucleotide polymorphism, SNP. We found that the SNPs in male and female patients differed from each other. In male patients the SNPs were associated with immune response while in female patients the association was to SNPs concerning inflammation. This means that different pathways were engaged in cancer development for men and women. We also found that the SNPs in patients were different from those expressed in the healthy controls.

O carcinoma mucoepidermóide (CME) é o tumor mais comum entre as neoplasias malignas de glândula salivar. Recentemente, uma rara população de células com características de multipotência, autorrenovação e potencial tumorigênico, denominadas como células tronco tumorais (CTT), foi descrita no CME. As CTT são resistentes as terapias atuais, e têm sido consideradas responsáveis pela recorrência e metástase, levando a um pior prognóstico para o paciente. A descoberta de que as CTT do CME superexpressam componentes da via de sinalização mTOR, levantou a hipótese que os pacientes poderiam ser beneficiados com o uso inibidores de mTOR como terapia para eliminação das CTT. O objetivo desse estudo foi avaliar o potencial uso de inibidores de mTOR como terapia para o CME com foco em CTT, assim como, investigar o funcionamento da via de sinalização mTOR e os efeitos moleculares do tratamento com inibidores dessa via nas CTT do CME. Foi realizada imuno-histoquímica para p-mTOR e p-S6K-1 em casos de pacientes diagnosticados com CME, os resultados foram correlacionados com os dados clínicos dos pacientes e também foi realizada dupla marcação por imunofluorescência para ALDH/p-mTOR. Estudos in vitro foram realizados em 3 linhagens de CME (UM-HMC-1, -3A, -3B) e com inibidores da via de sinalização mTOR. Após exposição aos inibidores, realizou-se ensaios de western blot (proteínas da via mTOR e BMI-1), citometria de fluxo para ALDH/CD44; salisfera; e apoptose, esse último comparando com quimioterápicos utilizados atualmente. Adicionalmente, foi utilizado o silenciamento genético de mTOR para confirmar os resultados obtidos com inibidores químicos. Por fim, foram realizados ensaios in vivo com as células silenciadas e com o inibidor de mTOR tensirolimo. Os resultados evidenciaram que a via de sinalização mTOR está ativa no CME, é correlacionada com pior prognóstico clínico e está superexpressa nas CTT. O tratamento com inibidores da via mTOR levaram a diminuição da fração de CTT, devido a perda de autorrenovação e apoptose das CTT. A apoptose, junto a diminuição de p-AKT revelada por western blot, sugeriram que esteja ocorrendo inibição de mTORC2 nas CTT, um importante componente na eficácia do tratamento com inibição de mTOR no câncer. Além disso, também houve redução de vasos sanguíneos, nichos das CTT, e diminuição do crescimento tumoral com uso de inibidores ou silenciando mTOR in vivo. Coletivamente, os resultados mostraram que a inibição de mTOR foi capaz de agir nas CTT por mecanismos diretos (indução de apoptose e diminuição da autorrenovação) e indiretamente através da redução de angiogênese, sugerindo que o uso de inibidores de mTOR no tratamento do CME é uma estratégia molecular eficiente para a redução de CTT, e uma potencial terapia adjuvante. / Mucoepidermoid carcinoma (MEC) is the most common tumor among malignant salivary gland neoplasms. Recently, a rare population of multipotent, self-renewing, and tumorigenic cells, termed cancer stem cells (CSC), was described in MEC. CSC are resistant to current therapies, and have been considered responsible for recurrence and metastasis, leading to worse patient prognosis. The discovery that CSC from MEC overexpressed the mTOR signaling pathway raised the hypothesis that use of mTOR inhibitors as therapy for CSC elimination could benefit patients. The objective of this study was to evaluate the potential use of mTOR inhibitors as a therapy for MEC targeting CTT, as well to investigate the functioning of mTOR signaling pathway and the molecular effects of treatment with mTOR inhibitors in MEC CSC. Immunohistochemistry was performed for p-mTOR and p-S6K-1 in paraffin samples from patients diagnosed with MEC and results were correlated with clinical data, in addition, co-immunoflorescence was performed for ALDH/p-mTOR. In vitro studies were performed with 3 MEC cell lines (UM-HMC-1, -3A, -3B) and with inhibitors of the mTOR signaling pathway. After exposure to inhibitors the following assays were performed: western blot (mTOR signaling pathway proteins and BMI-1), flow cytometry for ALDH/CD44, salispheres, and apoptosis, the latter comparing with currently chemotherapeutic agents used as treatment for cancer patients. In addition, mTOR genetically silent was used to confirm results with chemical inhibitors. Finally, in vivo assays were performed with knockdown cells and with mTOR inhibitor temsirolimus treatment. Results showed that mTOR signaling pathway is active in MEC, it is correlated with worse clinical prognosis and it is overexpressed in CSC. Treatment with inhibitors of mTOR signaling pathway led to a decrease in CSC fraction, caused by loss of self-renewal and apoptosis. Apoptosis, together with the decrease in p-AKT revealed by western blot, suggest that inhibition of mTORC2, an important compound in the efficacy of mTOR inhibition for cancer treatment, is occurring. In addition, there was reduction of blood vessels, CSC niches, and decreased tumor growth using mTOR inhibitors or silencing mTOR. Collectively, results showed that inhibition of mTOR was able to act in CSC by direct mechanisms (induction of apoptosis, decreased self-renewal) and indirectly through reduction of angiogenesis, suggesting that the use of mTOR inhibitors in treatment of MEC is an efficient molecular strategy to reduce fraction of CSC, and a potential therapy.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:22:54Z No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:23:08Z (GMT) No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) / Made available in DSpace on 2015-05-12T13:23:08Z (GMT). No. of bitstreams: 1 Manuela Torres Andion Vidal Estudo...2015.pdf: 4528048 bytes, checksum: dbdb598bed619445f96ad9d571f8324e (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O adenoma pleomórfico (AP), o carcinoma mucoepidermóide (CME) e o carcinoma adenóide cístico (CAC) representam tumores frequentes em glândula salivar. A via de sinalização Sonic Hedgehog (Hh) e o Transdutor de sinal e ativador da transcrição 3 (STAT3) desempenham funções importantes na proliferação celular, favorecendo o desenvolvimento tumoral e a proteína MCM3 tem sido considerada uma nova classe de marcadores de proliferação celular. Portanto, o presente trabalho propõe-se a estudar componentes da via Hh, bem como o STAT3 e o MCM3 em neoplasias de glândula salivar, na tentativa de adicionar informações sobre as características biológicas dessas neoplasias. Foram utilizados 9 casos de AP, 17 casos de CAC e 20 casos de CME e, por meio da técnica imunoistoquímica, realizou-se a detecção das seguintes proteínas: SHH, GLI1, SUFU, HHIP, STAT3 e MCM3. No AP, observou-se alta expressão citoplasmática de SHH e SUFU, e baixa expressão de STAT3 e MCM3. No CAC, observou-se alta expressão de GLI1, HHIP e STAT3 e baixa expressão de SHH, SUFU e MCM3. No CME, observou-se alta expressão de SHH, GLI1, SUFU e HHIP e baixa expressão de STAT3 e MCM3. Quando comparado entre os tipos tumorais, observou-se diferença estatisticamente significante para expressão de SHH (p=0.0064), STAT3 (p=0.0003) e MCM3 (p=0.0257). Ao comparar a marcação parenquimal e estromal, observou-se maior expressão em parênquima para todos os tumores (p<0.05). Em glândula salivar normal, observou-se marcação citoplasmática das células ductais para SHH, GLI1, SUFU e HHIP, uma discreta marcação citoplasmática para STAT3 e ausência de marcação para MCM3. Não foi observada correlação entre a via Hh e STAT3 nos tumores de glândula salivar (p>0.05). Os resultados desse trabalho apontam para uma possível participação da via Hh na morfogênese e no desenvolvimento do AP, CAC e CME, assim como a participação do STAT3 no desenvolvimento do CAC. Em adição, o MCM3 não parece ser um bom marcador de proliferação para tumores de glândula salivar. Sugerimos que novos estudos sejam realizados visando compreender o mecanismo pelo qual a via Hh e o STAT3 promovem o crescimento e progressão desses tumores e identificar inibidores dessas vias. / The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC) and the adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands. The Sonic Hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The MCM3 protein has been considered as a novel class of cell proliferation markers. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 and MCM3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC and 20 cases of MEC. Using immunohistochemistry, were investigated: SHH, GLI1, Sufu, HHIP, STAT3 and MCM3. In PA, there was high expression of cytoplasmic SHH and Sufu, and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP and STAT3 and low expression of SHH, SUFU and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p=0.0064), STAT3 (p=0.0003) and MCM3 (p=0.0257) when all tumors were compared. And a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p<0.05). In normal salivary gland, ductal segment showed imunolabeling for SHH, GLI1, SUFU and HHIP, a discrete cytoplasmic labeling for STAT3 and MCM3 was negative. No correlation was observed between the Hh pathway and STAT3 in salivary gland tumors (p>0.05). The findings suggest a possible role of Hh pathway in the morphogenesis and development of AP, CAC and CME, as well as the participation of STAT3 in the development of ACC. In addition, the MCM3 did not seem to be a good marker of proliferation for salivary gland tumors. It is important that further studies be conducted to understand the mechanism by which the Hh pathway and the STAT3 promote the growth and progression of these tumors and inhibitors of these pathways might be identified.

Made available in DSpace on 2014-12-17T15:32:22Z (GMT). No. of bitstreams: 1 LucileideCO_DISSERT.pdf: 2248988 bytes, checksum: f9aafad24354c13fb349a052f5b90d8e (MD5) Previous issue date: 2012-06-27 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The expression of glucose transporter protein 1 (GLUT-1), as well the angiogenesis has been associated to clinical behavior and aggressiveness in tumors of various origin. It is believed that the expression of this protein denotes metabolic demand of the tumor cells and, thus its influence upon the formation of new blood vessels. Pleomorphic adenoma (PA) and the adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC) represent, respectively, the most commom benign and malignant tumors of salivary glands. The aim of this study was to analyze and compare the immunohistochemical expression of GLUT-1 and its correlation with angiogenesis in cases of PAs, ACCs and MECs considering their histological grades. The sample consisted of 20 PAs, 20 ACCs and 10 MECs. The cases were analyzed and classified according to their histological grades. The expression of GLUT-1 was evaluated in the parenchyma lesions, establishing the percentage of immunopositive cells, according to the following scores: 0 (no cell immunomarked), 1 (up to 25% of tumor cells immunostained), 2 (25 - 50% of tumor cells immunostained) and 3 (more than 50% of tumor cells immunostained). The angiogenic index was analyzed by counting the microvessels immunostained by anti-CD34 antibody, in 5 fields (200X). The analysis of the expression of GLUT-1 in tumor parenchyma showed statistically significant differences between benign and malignant groups (p = 0.022). The average number of microvessels in PAs was 40.4, 21.2 in ACCs and 66.5 in MECs, with significant differences between groups (p <0.001). When compared to the expression of GLUT-1 and angiogenic index as a whole, there was no significant correlation between the number of microvessels and the expression of GLUT-1 (r = 0.211, p = 0.141). In conclusion, the results of this study suggest not only that differences in biological behavior between PAs, ACCs and MECs may be associated to the expression of GLUT-1, but also that benign and malignant salivary gland present differences in the average number of microvessels, with higher levels considered more aggressive tumors. Furthermore, the number of newly formed microvessels can be independent of the metabolic demand of the tumor cells / A express?o da prote?na transportadora de glicose tipo 1 (GLUT-1), bem como a angiog?nese, t?m sido relacionadas ao comportamento cl?nico e agressividade em neoplasias de origem diversas. Acredita-se que a express?o desta prote?na denote a demanda metab?lica das c?lulas tumorais e, assim, a sua influ?ncia na forma??o de novos vasos sanguineos. O adenoma pleom?rfico (AP) e o carcinoma adenoide c?stico (CAC) e carcinoma mucoepiderm?ide (CME) representam, respectivamente, a neoplasia benigna e as malignas mais frequentes das gl?ndulas salivares. O prop?sito deste estudo foi comparar a express?o imuno-histoqu?mica da GLUT-1, bem como correlacionar com a angiog?nese em casos de APs, CACs e CMEs levando em considera??o suas grada??es histol?gicas. A amostra foi composta por 20 APs, 20 CACs e 10 CMEs os quais foram classificados de acordo com os graus histol?gicos apresentados. A express?o da GLUT-1 foi avaliada no par?nquima das les?es, estabelecendo-se o percentual de c?lulas imunopositivas, de acordo com os escores: 0 (nenhuma c?lula imunomarcada), 1 (at? 25% das c?lulas tumorais imunomarcadas), 2 (de 25-50% das c?lulas tumorais imunomarcadas) e 3 (mais de 50% das c?lulas tumorais imunomarcadas). O ?ndice angiog?nico foi analisado por meio da contagem de microvasos imunomarcados pelo anticorpo anti-CD34, em 5 campos (200x). A an?lise da express?o da GLUT-1 revelou diferen?as estatisticamente significativas entre os grupos benignos e malignos (p = 0,022). O n?mero m?dio de microvasos foi de 40,4 em APs, 21,2 em CACs e 66,5 em CMEs, com diferen?as significativas entre os grupos (p < 0,001). Quando comparadas a express?o da GLUT-1 com o ?ndice angiog?nico em conjunto, n?o foi evidenciada correla??o significativa entre a quantidade de microvasos e a express?o da GLUT-1 (r = 0,211; p = 0,141). Os resultados do presente estudo sugerem que as diferen?as no comportamento biol?gico entre APs, CACs e CMEs podem estar relacionadas ? express?o da GLUT-1 e que tumores benignos e malignos de gl?ndulas salivares exibem diferen?as no n?mero m?dio de microvasos, com maiores ?ndices nos tumores considerados mais agressivos. Al?m disto, o n?mero de microvasos neoformados pode ser independente da demanda metab?lica das c?lulas tumorais

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