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FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / O cÃncer oral representa um sÃrio problema de saÃde pÃblica mundial. Entre os tumores deste sÃtio anatÃmico, os carcinomas de cÃlulas escamosas orais (CCEO) respondem por atà 94% do total. Os mecanismos moleculares envolvidos na gÃnese e desenvolvimento tumoral ainda nÃo estÃo completamente elucidados. Algumas evidÃncias tÃm sugerido a participaÃÃo viral neste processo. AlÃm disso, estes tumores ainda carecem de marcadores confiÃveis para determinar o perfil de agressividade. Neste contexto, o presente estudo teve como objetivo avaliar a expressÃo das proteÃnas p53, E-caderina, COX-2, p16, MLH1 e MYC numa sÃrie de CCEO, considerando tambÃm a marcaÃÃo citoplasmÃtica eventualmente observada para as Ãltimas trÃs proteÃnas, confrontando os resultados entre elas e com as caracterÃsticas demogrÃficas e clÃnico-patolÃgicas. AlÃm de avaliar a prevalÃncia do PapilomavÃrus Humano (HPV) e do VÃrus Epstein-Barr (EBV) na amostra e comparÃ-las com a expressÃo das referidas proteÃnas. Materiais e MÃtodos â Cem espÃcimes de CCEO, fixados em formalina e incluÃdos em blocos de parafina, foram submetidos à imunohistoquÃmica para a detecÃÃo das referidas proteÃnas e à hibridaÃÃo in situ para detecaÃà de HPV e EBV. Resultados â Foi observada associaÃÃo referente à perda de expressÃo concomitante de p16 e MLH1 (p=0,029) e na coexpressÃo de p53 e COX-2 (p=0,045). Ademais, foi verificado que a COX-2 e o MYC nuclear estavam relacionados com a marcaÃÃo citoplasmÃtica de MLH1 (p=0,060 e p=0,018; respectivamente). A anÃlise combinada dos marcadores revelou cinco grupos principais de expressÃo alterada que eram constituÃdos, em sua maioria, de tumores mais agressivos, principalmente o grupo MLH1(-)/COX-2(+)/p16(-). Os casos com marcaÃÃo citoplasmÃtica para p16, MLH1 e/ou MYC foram mais frequentes em tumores avanÃados (p=0,009) e naqueles com metÃstases em linfonodos (p=0,001). Trinta e um casos demonstraram marcaÃÃo para HPV em tecido tumoral. O EBV nÃo foi detectado em nenhum dos casos investigados, nem no tecido tumoral nem no epitÃlio nÃo neoplÃsico. O grupo HPV(+) exibiu elevada positividade para o p16 nuclear (p=0,029) e MYC cytoplasmÃtico (p=0,039), tambÃm uma maior perda de expressÃo nuclear de MLH1 (p=0,031). Houve ainda uma tendÃncia referente ao aumento da positividade de COX-2 no grupo infectado (p=0,084). ConclusÃes â As significÃncias verificadas entre p16 e MLH1 sugerem que a ausÃncia do membro do sistema de reparo de encaixe (MMR) tambÃm favoreÃa a ocorrÃncia de mutaÃÃes no gene p16, culminando na inativaÃÃo deste supressor tumoral. As associaÃÃes de COX-2 e MYC com o MLH1 de expressÃo citoplasmÃtica suscitam um mecanismo de bloqueio de entrada de MLH1 no nÃcleo. A anÃlise combinada das proteÃnas, bem como, a marcaÃÃo citoplasmÃtica de p16, MLH1 e MYC, podem representar indicadores Ãteis na avaliaÃÃo do perfil de agressividade e, provavelmente, de prognÃstico em CCEO. Acerca dos vÃrus, nossos achados sugerem que o HPV esteja envolvido em uma importante parcela de casos de CCEO e que possa promover a expressÃo de p16 nuclear, MYC citoplasmÃtico e COX-2, e suprimir a expressÃo nuclear de MLH1. Quanto ao EBV, nÃo foram detectados EBERs (EBV-encoded small RNAs) na amostra. / The oral cancer represents a serious world public health problem. The oral squamous cell carcinomas (OSCC) account for up to 94% of the tumors of this anatomic site. The molecular mechanisms involved in the genesis and progression are still not well elucidated. Some evidences have suggested the involvement of viruses in this process. Also, these tumors still lack of reliable markers to determine the aggressiveness profile. In this context, the aim of the present study was to evaluate the expression of the proteins p53, E-cadherin, COX-2, p16, MLH1 and MYC in a serie of OSCC, including the cytoplasmic staining eventually observed for the latter three proteins, confronting the results between them and with demographic and clinico-pathological features. Besides evaluating the prevalence of Human Papillomavirus (HPV) and Epstein-Barr virus (EBV) in the sample and compare them with the expression of the referred proteins. Materials and Methods â One hundred formalin-fixed paraffin-embedded OSCC specimens were submitted to immunohistochemistry for detection of the referred proteins, and to in situ hybridization for HPV and EBV detection. Results â OSCC was associated with a concomitant lack of expression of p16 and MLH1 (p=0.029) and coexpression of p53 and COX-2 (p=0.045). Additionally, COX-2 and nuclear MYC were found to be related to exclusively cytoplasmic staining of MLH1 (p=0.060 and p=0.018, respectively). The combination analyses of the markers revealed five main groups of altered protein expression, which were mostly of the more aggressive tumors, mainly the MLH1(-)/COX-2(+)/p16(-) group. The cases with cytoplasmic staining for p16, MLH1 and/or MYC were more frequent in advanced tumors (p=0.009) and in those with lymph node metastasis (p=0.001). Thirty-one cases showed staining for HPV in tumor tissue. The EBV was not detected in any case investigated, neither in the tumor tissue nor in the non-neoplastic epithelium. The HPV(+) group demonstrated high positivity for nuclear p16 (p=0,029) and cytoplasmic MYC (p=0,039), and an increase of the lack of MLH1 nuclear expression (p=0,031). There was also a trend related to the increase of the COX-2 positivity in the HPV(+) group (p=0,084). Conclusions â The significance between p16 and MLH1 suggests that the lack of this member of mismatch repair system also favors the occurrence of mutations in the p16 gene, culminating in inactivation of this tumor suppressor. The associations of COX-2 and MYC with cytoplasmic MLH1 suggest a blocking mechanism for the entry of MLH1 into the nucleus. The combined analyses of the proteins investigated, as well as the cytoplasmic staining of p16, MLH1 and MYC, may be useful in the evaluation of the aggressive profile and probably prognosis of OSCC. Regarding the viruses, our findings suggest that the HPV is involved in an important portion of OSCC cases and that may promote the expression of the nuclear p16, cytoplasmic MYC and COX-2, and suppress the nuclear expression of MLH1. About EBV, it was not detected the EBV-encoded small RNAs (EBERs) in the sample.

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo. / A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin.

The incidence of human papillomavirus (HPV) -associated oropharyngeal tumors is steadily increasing therefore, information about the prevalence of oral HPV and its risk factors is very important for future screening and early diagnosis of the disease. This thesis addresses three topics. The first topic is to evaluate the prevalence of oral HPV in a healthy population and to investigate risk factors for oral HPV infection, given that these data are almost completely absent in Central Europe. A statistically significantly higher rate of positivity (8.8%) of oral HPV infection was found in the group of older unvaccinated probands than in younger partially vaccinated volunteers (2.0%). The seropositivity rate of anamnestic HPV antibodies was comparable in both groups. None of the analyzed risk factors were significantly associated with oral HPV positivity. The second topic of the thesis is the dynamics of HPV specific antibodies in patients with head and neck cancer and their prognostic significance. In patients with cervical cancer, a decrease in HPV E6 / E7-specific antibodies is associated with a better prognosis. Another goal of the dissertation was to assess the importance of anamnestic antibodies and antibodies against oncoproteins E6 and E7 in long-term follow-up 2-14 years after the end of...

Angesichts der nach wie vor hohen Mortalität und Morbidität des Bronchialkarzinoms ist die Entwicklung geeigneter Methoden zur früheren Diagnostik eine wichtige Notwendigkeit, um die geringe durchschnittliche 5-Jahres-Überlebensrate von 15% – 18% zu steigern. Unter diesem Gesichtspunkt wurde in der vorliegenden Arbeit das Atemkondensat von Patienten mit Bronchialkarzinom als nicht-invasiv und kostengünstig zu gewinnendes Medium auf das Vorliegen eines potentiellen Screeningmarkers – dem methylierten Tumorsuppressor-Gen p16 – untersucht. Dazu wurde ein Versuchsablauf entwickelt, bei dem trotz des geringen DNA-Gehaltes im Atemkondensat p16-Methylierungen nachgewiesen werden konnten. Die letztendlich etablierte Methode war eine methylierungsspezifische nested-PCR mit anschließendem Restriktionsverdau durch das Restriktionsenzym BstUI. Des Weiteren erfolgte die Untersuchung von in Paraffin eingebetteten Tumorpräparaten der Patienten. In der anschließenden statistischen Auswertung wurde der Einfluss von verschiedenen Faktoren wie COPD-Grad, Tumorlage, Tumorart, Nikotinabusus und stattgehabte Chemo- oder Strahlentherapie auf den Methylierungsstatus des p16-Gens analysiert.

Background: Oral Tongue Squamous Cell Carcinoma (OTSCC) is the most frequent and aggressive carcinoma in the head and neck region. Its incidence has increased during the last decades, especially in young patients (≤40 years) mainly female. These young patients have either not been exposed to the traditional risk factors for this disease, or have a much reduced duration of exposure than the typical OTSCC patient. The reasons behind this increasing incidence remain unknown. The aims of this thesis were to analyse the presence and possible role of human papillomavirus (HPV) in oral tongue cancer in correlation with its surrogate marker p16 and its receptor syndecan-1. Other aims were to evaluate expression of EMT (epithelial to mesenchymal transition) - related markers, such as E-cadherin, β-catenin, CK5 and CK19, and to address the potential predictive role of podoplanin in the loco-regional metastatic process. Clinical parameters including age, sex, geographical distribution, relapse, tumour staging and grading were also investigated for a possible correlation with biomarker expression and prediction of survival rate and therapeutic strategy. Materials and methods: More than one hundred samples of OTSCC coming from two University Hospitals of two different countries (Sweden and Italy) were analysed. HPV presence was evaluated by in situ hybridisation for detection of the high-risk HPV 16 and indirectly by immunohistochemistry (IHC) of its surrogate marker p16. Expression of the HPV receptor syndecan-1 and the EMT biomarkers E-cadherin, β-catenin, CK5, CK19 were also evaluated by immunohistochemistry. Samples were scored using a quick score (QS), taking both number and intensity of cells stained into account. Podoplanin expression was investigated at both protein and RNA level. Results: Tumour size and lymph node metastasis correlated to both overall and disease-free survival. Despite variable expression of the syndecan-1 receptor, HPV 16 was not detected in any sample analysed, excluding a possible association with p16, which was expressed in 33% of the cases. All EMT-related markers were commonly expressed in tongue cancer. Data showed E-cadherin to be an independent prognostic factor with higher expression associated with poor overall survival. Notably, E-cadherin, β-catenin and CK5 directly correlated to each other. Multivariate analysis of clinical data demonstrated that age of the patient is an independent prognostic factor with younger patients showing a worse survival rate. Patients younger than 40 years also showed significantly higher expression of podoplanin. Data for geographic distribution revealed a difference in expression of E-cadherin between Swedish and Italian patients. Conclusions: In contrast to SCC of the base of the tongue and the tonsil, HPV is not present in OTSCC, excluding HPV infection as a risk factor. Higher levels of E-cadherin and young age is associated with poor survival in OTSCC patients. The different frequency of EMT markers seen between Swedish and Italian patients suggests an important role for the environment and the geographical area in the onset of different molecular patterns of OTSCC.

This paper argues that the cumulative causation processes between wealth and power risk leading to an escalation of wealth inequality. We confirm with new survey data for the Eurozone Piketty's conclusions that wealth is highly concentrated and that this inequality is perpetuated through dynastic wealth. This leads to an ever-concentrating ability to shape economic and political institutions. While neoclassical economics has a blind spot where power is concerned, we discuss how heterodox approaches have attempted to conceptualize this structural power which influences the framework of economic activity. Finally, we discuss three concrete channels through which the unequal distribution of private assets may affect power relations and economic activity.

L’objectif était de mieux définir les relations entre l’exposition à certaines fibres minérales et les anomalies génétiques somatiques associées à la transformation tumorale de cellules de l’appareil respiratoire. Deux études indépendantes ont été conduites à partir du modèle murin Nf2+/- de mésothéliome malin (MM) développé dans le laboratoire, exposé par inoculation intrapéritonéale à des fibres d’amiante crocidolite (souris abs-Nf2+/- et abs-Nf2+/+) et à des fibres céramiques réfractaires (FCR) (souris ceram-Nf2+/-).Ce choix a été fondé selon une stratégie raisonnée à partir de la connaissance des anomalies génétiques observées dans le MM. La validité de ce modèle a été vérifiée en comparant les MM murins et humains. Des MM ont été induits par le crocidolite chez des souris abs-Nf2+/- et abs-Nf2+/+. Les souris abs-Nf2+/- ont significativement développé plus de MM que les souris abs-Nf2+/+. Les caractéristiques histologiques des MM murins sont analogues aux MM humains, avec des altérations génétiques similaires, en terme de fréquence et de mécanismes d’inactivation (mutations ponctuelles pour le gène TP53, délétions pour les gènes NF2 et P16/CDKN2A). Ce modèle murin a été ensuite utilisé pour évaluer la toxicité de FCR. Les souris ceram-Nf2+/- ont développé des MM similaires aux MM humains du point de vue histologique et moléculaire. Ainsi, le profil des altérations génétiques des MM murins ceram-Nf2+/- est semblable à celui des MM murins abs-Nf2+/- et abs-Nf2+/+, et humains. Les cellules mésotheliales des souris exposées aux FCR et à l’amiante semblent suivre les mêmes voies de transformation néoplasique. Une étude à la recherche d’altérations génétiques de ces 3 gènes et des gènes Ki-ras et EGFr fréquemment mutés dans le cancer broncho-pulmonaire (CBP) a été conduite à partir de cas de CBP sélectionnés (50 sujets exposés à l’amiante (E+) et 50 sujets non exposés (E-) appariés sur l’âge, le sexe, le type histologique et le tabagisme) provenant d’une série de cas opérés. A l’instar du MM, l’analyse du gène P16/CDKN2A suggère que le mécanisme d’inactivation pourrait être différent chez les sujets E+ (par délétion) par rapport aux sujets E- (par hyperméthylation du promoteur), indépendamment de l’âge et du tabagisme. Deux SNP (rs12947788 et rs12951053) du gène TP53 sont plus fréquemment retrouvés chez les sujets E+. En revanche, aucune différence significative n’est retrouvée pour les autres gènes entre ces 2 groupes. Ainsi, les mutations du gène NF2 dans le MM seraient plus liées à une spécificité cellulaire et à une fonction particulière de la protéine nf2 dans ces cellules / Résumé en anglais non communiqué

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-10-01T14:53:33Z No. of bitstreams: 2 Tese - Luciana Ximenes Salustiano - 2013.pdf: 3049558 bytes, checksum: b1f84cfd9b4978f7ecf5dd1b5da12e3d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-01T15:33:57Z (GMT) No. of bitstreams: 2 Tese - Luciana Ximenes Salustiano - 2013.pdf: 3049558 bytes, checksum: b1f84cfd9b4978f7ecf5dd1b5da12e3d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-01T15:33:57Z (GMT). No. of bitstreams: 2 Tese - Luciana Ximenes Salustiano - 2013.pdf: 3049558 bytes, checksum: b1f84cfd9b4978f7ecf5dd1b5da12e3d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-12-17 / Retinoblastoma (RB) is the malignant neoplasm of embryonic neural retinal cells and is the most common intraocular tumor of childhood. Early diagnosis associated with modern methods of treatment resulted in increased survival rate of patients with retinoblastoma. However, in those cases whose diagnosis is delayed and in untreated cases, the disease is invariably fatal. The knowledge of the molecular and histopathological features of retinoblastomas can influence the choice of treatment and benefit the prognosis of these tumors. Thus, the objectives of this study are to evaluate the main histopathological and molecular characteristics of retinoblastomas, including the evaluation of p16, Ki67 and VEGF protein expression, as well as the potential role of such molecules as prognostic markers in the retinoblastomas. Fifty-seven cases of RB were evaluated in enucleated eyes at Hospital Araújo Jorge, in Goiânia, in the period of 1998 to 2011. Clinical data were collected from the respective medical files and the cases were reviewed for analysis of histopathological aspects. The expression of Ki67, p16 and VEGF was assessed by immunohistochemistry, using specific antibodies and a detection system associated with polymers. The results were analyzed by descriptive statistics and categorical variables by chi-square test and Fisher exact test, when necessary. Ki67 overexpression was observed in 28% of cases, characterizing the retinoblastomas with high rate of cellular proliferation, while the p16 overexpression was observed in 42% of retinoblastomas evaluated. Significant associations were observed between the overexpression of these proteins and histopathological characteristics of worse prognosis, including invasion of the optic nerve, choroid, sclera and anterior chamber of the eye, as well as to a lower degree of cellular differentiation. VEGF expression was extensively observed in all retinoblastoma cases, independent of the histopathological aspects of the tumors. The associations observed in this study between tumor aggressive histopathological aspects and Ki67 and p16 overexpression, indicate the use of these markers in determining the prognosis of retinoblastomas. On the other hand, the high angiogenic potential of retinoblastomas, translated by diffuse VEGF expression in all cases analyzed in this study, raises new investigations on the use of anti-angiogenic drugs in the treatment of retinoblastomas. / O retinoblastoma (RB) é o tumor maligno das células neurais embrionárias da retina e consiste no tumor intraocular mais comum da infância. O diagnóstico precoce associado a modernos métodos de tratamento resultou em aumento da taxa da sobrevida de pacientes com retinoblastoma. No entanto, nos casos cujo diagnóstico é tardio e nos casos não tratados, a doença é invariavelmente fatal. O conhecimento das características histopatológicas e moleculares dos retinoblastomas pode direcionar a escolha do tratamento e beneficiar o prognóstico desses tumores. Este estudo objetiva avaliar as principais características histopatológicas e moleculares dos retinoblastomas, incluindo a avaliação da expressão das proteínas p16, Ki67 e VEGF e o potencial papel prognóstico desses marcadores que são anticorpos envolvidos na carcinogênese, angiogênese e proliferação celular. Cinquenta e sete casos de RB foram avaliados em olhos enucleados no Hospital Araújo Jorge, em Goiânia, no período de 1998 a 2011. Os dados clínicos foram coletados dos respectivos prontuários e todos os casos foram revistos para análise dos aspectos histopatológicos. A expressão de Ki67, p16 e VEGF foi avaliada por imunoistoquímica, utilizando anticorpos específicos e sistema de detecção associado a polímeros. Os resultados foram analisados por estatística descritiva e as variáveis categóricas pelo teste do qui-quadrado e teste exato de Fisher, quando necessário. A superexpressão de Ki67 foi observada em 28% dos casos, caracterizando os retinoblastomas com alto índice de proliferação celular, enquanto a superexpressão de p16 foi observada em 42% dos retinoblastomas avaliados. Associações significativas foram detectadas entre a superexpressão dessas proteínas e as características histopatológicas de pior prognóstico, incluindo invasão do nervo óptico, esclera, coroide e câmara anterior do olho, bem como ao menor grau de diferenciação celular. A expressão de VEGF foi observada de forma difusa em todos os casos analisados, independente dos aspectos histopatológicos dos tumores. As associações observadas neste estudo, entre os aspectos histopatológicos de maior agressividade tumoral e a superexpressão de Ki67 e p16, indicam que o uso destes marcadores pode influir na determinação do prognóstico dos retinoblastomas. Por outro lado, o alto potencial angiogênico dos retinoblastomas, traduzido pela expressão difusa do VEGF em todos os casos analisados neste estudo, suscita novas investigações sobre o uso de medicamentos anti-angiogênicos no tratamento dos retinoblastomas.

Nas últimas décadas, é intensa a procura de uma explicação genética sobre a origem, crescimento e progressão do melanoma cutâneo. A tentativa de encontrar uma ligação direta entre as mutações gênicas e a origem da doença tem sido o objetivo dos pesquisadores dedicados ao estudo dessa neoplasia. Diversos métodos são utilizados na busca de uma avaliação prognóstica para a progressão do melanoma, citando-se, entre eles, a pesquisa do linfonodo sentinela, a imunoistoquímica, as técnicas moleculares e a técnica de microarray. A necessidade de estabelecer um método, com excelente sensibilidade e especificidade, tem levado os pesquisadores a buscarem melhores evidências. É importante para esses estudos a obtenção de dados confiáveis sobre as técnicas, progressão e sobrevida livre de doença. Por meio da imunoistoquímica, técnica relativamente simples e de baixo custo, a expressão da proteína p16 pode ser analisada e correlacionada com o prognóstico da doença. No melanoma cutâneo, a expressão da proteína diminui, à medida que aumenta sua agressividade, ou seja, é forte nos nevos e melanomas in situ, e fraca ou ausente nos melanomas metastáticos. Em alguns estudos, a comparação com outros marcadores é analisada. A finalidade deste estudo é fazer uma revisão da literatura internacional sobre o uso da proteína p16 como fator prognóstico para o melanoma, bem como avaliar a importância das alterações do gene p16INK4a, co-responsáveis pela gênese e evolução do melanoma.