Diversas alterações genéticas estão associadas à patogênese do carcinoma epidermoide (CE), a neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do AKT, envolvida em diferentes fenômenos celulares. Este projeto teve como objetivo estudar a expressão da proteína pAKT em linhagens celulares de carcinomas epidermoides de cabeça e pescoço, de forma a verificar possíveis alterações na expressão dessas moléculas em células de CE tratadas com Curcumina e Celecoxib. Foram utilizadas duas linhagens celulares de CE de cabeça e pescoço e uma linhagem de queratinócitos que foram divididas em quatro grupos: a. grupo controle não tratado; b. células tratadas com Curcumina, c. células tratadas com Celecoxib d. células tratadas com Curcumina e Celecoxib. A proliferação celular foi monitorada através do teste de viabilidade celular. A análise da expressão da proteína foi realizada através da técnica do Western Blot. Desta forma, foi possível entender em maior profundidade a ação do Celecoxib, da Curcumina e sua associação na via do AKT em carcinomas epidermoides de cabeça e pescoço. A associação de Curcumina com Celecoxib foi o tratamento que apresentou melhores resultados na redução da viabilidade celular. A linhagem SCC9 não apresentou expressão de pAKT após o tratamento com Curcumina e com a associação desta com o Celecoxib. A linhagem HaCat apresentou maior estabilidade na expressão de pAKT nos dois tempos de tratamento no grupo tratado com a associação das substâncias. A combinaçãoo da Curcumina com o Celecoxib mostrou resultados satisfatórios e promissores. / Several genetic changes are associated with the squamous cell carcinoma\'s (SCC) pathogenesis, the most common malignant head and neck tumor. Some of these changes compromise proteins that belong to the AKT signaling pathway, involved in different cellular\'s phenomena. The aim of this project was to study the expression of pAKT in head and neck SCC cell lines in order to investigate possible alterations in these molecules\'s expression in cells treated with Curcumin and Celecoxib. Two head and neck SCC cell lines and a line of keratinocytes was divided into four groups: a. control group: untreated; b. cells treated with Curcumin, c. cells treated with Celecoxib d. cells treated with Curcumin and Celecoxib. Cell proliferation was monitored by cell viability test. Analysis of the protein\'s expression was performed using the Western blot technique. Then was possible to understand the action of Curcumin, Celecoxib and its association in the AKT pathway in head and neck SCC. Curcumin\'s association with Celecoxib was the treatment that showed best results in cell viability reduction. The SCC9 cell line not presented pAKT expression after the treatment with Curcumin and its association with Celecoxib. The cell line HaCat showed greater stability in pAkt expression during the treatment period with the association of substances. The association of Curcumin and Celecoxib showed satisfactory and promising results.

Introdução: A queilite glandular (QG) é uma doença inflamatória rara, de causa desconhecida, que afeta as glândulas salivares menores, principalmente do lábio inferior. Há secreção de saliva espessa através das glândulas salivares alteradas, causando desconforto ao doente. O quadro clínico consiste em graus variáveis de macroqueilia, acompanhada pela presença de ostíolos dilatados de glândulas salivares menores no vermilião. À expressão, há saída de material espesso e mucóide, que geralmente adere ao lábio acometido. A palpação cuidadosa pode revelar a presença de áreas nodulares endurecidas, que raramente podem supurar e drenar material purulento. O lábio inferior é o mais frequentemente acometido, e a doença tende a ser crônica e de difícil manejo. As opções terapêuticas incluem uso de corticóides tópicos ou intralesionais, antibioticoterapia oral, fotoproteção e cirurgia. A queilite glandular é considerada por alguns autores como uma condição pré-maligna, com alguns relatos de desenvolvimento de carcinoma epidermóide no lábio acometido. Aquaporinas são proteínas transmembrana que possuem a capacidade de transportar água entre os meios extra e intracelular. Desempenham, dessa maneira, papel importante na homeostase corporal. São proteínas amplamente distribuídas pelos tecidos humanos, existindo mais publicações sobre sua importância na fisiopatologia renal e do sistema nervoso central, até o momento. Os estudos sobre o papel das aquaporinas nas glândulas salivares são escassos, muitos realizados apenas em animais, não em humanos. Na queilite glandular, há alteração clínica evidente da viscosidade da saliva, o que é uma das queixas mais importantes. A alteração da expressão das aquaporinas na queilite glandular poderia corroborar o achado clínico-patológico de composição da saliva nesses doentes. Objetivos: Apresentar os achados clínicos e histopatológicos de 22 doentes com diagnóstico de queilite glandular, revisar os aspectos histopatológicos, assim como as terapias utilizadas e estudar a expressão das aquaporinas nas glândulas salivares menores labiais dos doentes em relação a controles de glândulas normais. Método: Foram analisados 22 doentes com diagnóstico de QG, acompanhados no Ambulatório de Estomatologia da Divisão de Dermatologia do HCFMUSP. Todos foram avaliados quanto aos dados clínicos e demográficos. Dez dos 22 doentes foram submetidos a tratamento cirúrgico, que consistiu na vermilionectomia do lábio inferior, seguida da dissecção das glândulas salivares menores, sendo o material analisado histopatologicamente. Em sete doentes foi realizada a análise da expressão das aquaporinas nas glândulas salivares menores labiais, comparando-os a glândulas salivares normais, através da técnica de imuno-histoquímica. Resultados: Os achados clínicos e histopatológicos evidenciaram maior prevalência de QG em indivíduos de pele clara. Observaram-se graus variáveis de sialadenite crônica e alterações epiteliais nos doentes submetidos a biopsias ou a tratamento cirúrgico. Dos 22 doentes, 3 apresentaram focos de carcinoma epidermóide no lábio inferior. A análise da expressão das aquaporinas nas glândulas salivares menores labiais mostrou positividade para AQP1, AQP2, AQP4, AQP5 e AQP8, em intensidades e localizações diversas em relação aos casos controles. Conclusões: Na queilite glandular, há aumento da chance de desenvolvimento de carcinoma epidermóide do lábio. A doença parece ter origem tanto a partir de causas exógenas, como a exposição aos raios ultravioleta, como causas endógenas, fato sugerido pelo encontro de alteração na expressão das aquaporinas nas glândulas salivares menores labiais dos doentes. Na literatura, algumas aquaporinas ainda não haviam sido detectadas por imuno-histoquímica na glândula salivar menor labial humana / Background: Cheilitis glandularis (CG) is a condition of unknown cause which thick saliva is secreted from swollen minor salivary glands from the lips. The condition is considered rare; there are few published case series, and most reports refer to single cases. The clinical picture of CG consists of variable degrees of macrocheilia accompanied by the presence of red, dilated ostia of minor salivary glands on the vermilion area. A thick, mucoid material can be obtained from these ostia by manual expression. This viscous saliva often sticks to the vermilion causing discomfort to the patient. Changes occur more frequently on the lower lip. The condition tends to be chronic and difficult to manage. Treatments vary from topical or intra lesional steroids, oral antibiotics, sun protection and surgery. Cheilitis glandularis is frequently regarded as a \"premalignant\" condition, with a few reported cases of development of squamous cell carcinoma. Aquaporins are small membrane proteins that exhibit channel activity specific for water and small solutes. They are considered essencial for corporal homeostasis, and are widely expressed through human tissues. Until now, most aquaporins studies are based on renal and nervous system fisiopathology, with few studies on situations involving salivary glands, such as Sjögrens disease. Some of them are performed over murine models, not human salivary glands. Objectives: In cheilitis glandularis, there is clinical evidence of thick saliva, which is one of the biggest complains of affected patients. We have diagnosed CG at our Oral Diseases Clinic in what seems to be a higher frequency than usually reported. This has prompted us to study these patients and present our results. Most CG reports are about isolated cases. We performed immunohistochemistry analysis to indicate aquaporins expression in human minor salivary glands affected by CG, since there is clinically thick saliva and its production is related to the activity of aquaporin channels. Methods: Data from 22 patients with diagnosis of cheilitis glandularis, from Oral Diseases Clinic of HC-FMUSP, were reviewed. Ten of 22 patients were submitted to surgical treatment, which was the surgical removal of the lower lip. We performed, on seven patients, analysis of aquaporins expression in minor labial salivary glands, in comparison to healthy tissues, through immunohistochemistry studies. RESULTS: Clinical findings showed higher prevalence of CG on fair skinned patients. Histopathological exams revealed variable degrees of chronic sialoadenitis and epithelial changes in patients submitted to surgical treatment or biopsy. Three of them have developed epidermoid carcinoma of lower lip. Analysis of aquaporins expression showed positivity for AQP1, AQP2, AQP4, AQP5 e AQP8, that differs from normal minor labial salivary glands. Conclusions: In CG, the chance of develop labial epidermoid carcinoma is increased. It seems that exogenous and endogenous components are related to the etiology of CG, as we found different expression of aquaporins in affected glands

Introdução: O câncer de cabeça e pescoço vem aumentando sua incidência, sendo o quinto tipo de câncer mais comum e a sexta causa de morte por câncer no mundo, portanto é causa importante de morbimortalidade da população. O entendimento dos fatores de risco como tabagismo, etilismo e infecção pelo papilomavirus (HPV) é de fundamental importância, tanto para ações de prevenção e controle da doença, bem como avaliação de fatores prognósticos e terapêuticos. Objetivos: O presente estudo visa analisar as características clínicas, buscar o principal sítio anatômico tumoral e avaliar os fatores prognósticos entre os pacientes tabagistas ou não, e portadores ou não do HPV, que foram tratados cirurgicamente. Casuística e Métodos: Foram analisadas as variáveis clínicas e prognósticas dos grupos de pacientes tabagistas, não tabagistas, HPV negativo e HPV positivo. A amplificação do DNA viral, detecção e genotipagem do HPV se deu através da extração do DNA total a partir de blocos parafinados. Resultados: A amostra total foi composta por 399 pacientes, sendo que 266 eram tabagistas, 33 não tabagistas, e desses últimos, cinco eram HPV positivo. Houve diferença estatística em relação sexo, sendo que para o tabagista o predomínio foi do sexo masculino e para o HPV positivo foi o feminino. O paciente tabagista apresentou-se mais jovem (média 57,9 anos) que o não tabagista (média 64,1 anos). O sítio anatômico mais comum para os tabagistas foi a laringe e para o HPV positivo foi a cavidade oral e orofaringe. O tempo livre de doença foi de 63,6 meses para os tabagistas, 31,3 meses para os não tabagistas e 29,3 meses para os HPV positivo, havendo diferença quanto às curvas de sobrevidas livre da doença dos fumantes e não fumantes. Dos pacientes que foram á óbito pela neoplasia, o paciente HPV positivo foi o que apresentou menor tempo de sobrevida. Não houve diferença estatística entre os grupos quanto às curvas de sobrevidas global e da doença. Discussão: Apesar da literatura mostrar que o paciente não tabagista e o paciente HPV positivo geralmente serem mais jovens e apresentarem melhor prognóstico do que os típicos pacientes tabagistas, isso não foi observado nesse estudo, pois o paciente não tabagista apresentou-se com mais idade ao diagnóstico da doença e com pior prognóstico que o tabagista, e o paciente HPV positivo apresentouse com menor tempo até o óbito pela doença quando comparado ao paciente tabagista. Conclusões: O câncer nos pacientes tabagistas acometeu mais o sexo masculino, localizou-se principalmente na laringe e apresentou melhor prognóstico quando comparado aos não tabagistas. O câncer nos pacientes HPV positivo acometeu mais o sexo feminino e localização tumoral foi principalmente a cavidade oral e a orofaringe. / Introduction: Head and Neck cancer has been increasing its incidence, and it is the fifth most common type of cancer and the sixth leading cause of cancer death in the world, so it is an important cause of morbidity and mortality of the population. Understanding the risk factors such as smoking, alcohol use and papillomaviruses (HPV) infection is of fundamental importance, both for prevention and control of disease like for assessment of prognostic and therapeutic factors. Objectives: This study aims to analyze the clinical, seek the main site anatomical of the tumor and evaluate the prognostic factors among smokers or not, with the presence or absence of the HPV, which were surgically treated. Methods: The variables clinical and prognostic were analyzed in the groups of smokers, nonsmokers, HPV negative and HPV positive. The amplification of viral DNA, detection and genotyping of the HPV were made through the extraction of total DNA from paraffin blocks. Results: The total sample consisted of 399 patients, 266 were smokers, 33 nonsmokers, and this latter group had five patients HPV positive. There was statistical difference regarding sex, for smokers the prevalence was male and for HPV positive was female. The smoker patient was younger (mean 57.9 years) than the non-smoker (mean 64.1 years). The most common anatomic site for the smokers was the larynx and for the HPV positive was the oral cavity and oropharynx. The diseasefree interval was 63.6 months for smokers, 31.3 months for nonsmokers and 29.3 months for HPV positive, with difference between free disease survival curves of the smokers and nonsmokers. Of the patients who died by disease, HPV positive patient showed the shorter survival. There was no statistical difference between the groups in terms of overall survival curves and of the disease. Discussion: Although the literature show that the nonsmoker patient and HPV positive patients are generally younger and present a better prognosis than the typical smokers, this was not observed in this study because the nonsmoker patient presented with more age at diagnosis of disease and a worse prognosis than the smoker, and the HPV positive patient presented with shorter survival of the disease when compared to smoking patients. Conclusions: The cancer in smokers affected more males, was located mainly in the larynx and showed better prognosis when compared to nonsmokers. The cancer in patients positive HPV affected more females and tumor location was mainly oral cavity and oropharynx.

Orientador: Luiz Paulo Kowalski / Taxto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T18:08:30Z (GMT). No. of bitstreams: 1 Jardim_JuscelinodeFreitas_M.pdf: 8723200 bytes, checksum: c5a9b07e52853342905f7f5f7df0ed12 (MD5) Previous issue date: 2014 / Resumo: O carcinoma de células escamosas (CEC) é a neoplasia maligna mais frequente na cavidade bucal, correspondendo a quase 95% destas lesões, e cerca de 38% dos tumores malignos de cabeça e pescoço. Mais de 50% dos portadores deste tipo de tumor apresentam estágio avançado da doença no momento do diagnóstico, fator que reflete em baixas taxas de sobrevidas em 5 anos. Fatores histopatológicos como invasões perineural e vascular têm sido relacionadas com recorrência e baixas taxas de sobrevida. Ainda, pacientes com mesmo sítio de acometimento e histologia tumorais semelhantes podem ter comportamentos biológicos distintos, frente a isso, a importância da busca por biomarcadores para predizer prognóstico e risco estratificado. O propósito deste estudo consistiu em avaliar o significado clínico e prognóstico dos fenômenos de invasão perineural (IP) e invasão vascular (IV) bem como da imunoexpressão de Mcm-2, Ciclina D1, Ki-67 e p53 em CECs de língua e assoalho em estádio clínico avançado. Foi realizado um levantamento retrospectivo de pacientes com CEC de língua e assoalho de boca em estádio clínico avançado, tratados previamente por cirurgia no departamento de cirurgia de cabeça e pescoço do AC Camargo Cancer Center entre os anos de 1998 e 2009. De 142 casos elegíveis para o estudo, 88 blocos de parafina foram resgatados do departamento de Patologia da mesma instituição e reações de imunoístoquímica foram realizadas para os marcadores já descritos anteriormente. Todas as lâminas passaram por um processo de digitalização através do equipamento Aperio System (Vista, CA, USA). Quantificações das marcações foram obtidas através do software Imagescope (Aperio System, USA) e testes estatísticos com nível de significância de 5% foram tomados para acessar correlações com prognóstico. Os resultados mostraram que tanto IP (p< 0,001) como IV (p= 0,01) influenciaram negativamente a sobrevida em 5 anos dos pacientes. Outros fatores como tamanho tumoral (estádio T) (p=0,003), estádio N+ (p= 0,002) e ruptura de cápsula linfonodal (p< 0,001) também obtiveram impacto em predizer sobrevida. Com relação aos marcadores de proliferação celular, altas taxas de Mcm-2 (p<0,001) e Ciclina D1 (p = 0,005) tiveram relação direta com taxas de sobrevida global menores que 5 anos, enquanto p53 e Ki-67 não alcançaram significância. Mcm-2 também foi altamente relacionado com recorrência (p=0,025), enquanto Ciclina D1 e p53 foram correlacionados com estádio N. Em conclusão, o aumento na expressão de Mcm-2 e Ciclina D1 exibem importante correlação com prognóstico e sobrevida dos pacientes, assim como fatores histopatológicos como invasões perineural / Abstract: Oral squamous cell carcinoma (OSCC) is the most common malignancy in the oral cavity, accounting for almost 95% of these injuries, and about 38% of malignant tumors of the head and neck. More than 50% of all patients have advanced disease at the time of diagnosis, factor that reflects in low survival rates at 5 years. Histopathological factors such as vascular and perineural invasion have been associated with low rates of recurrence and survival. Moreover, patients with the same site and similar histology may have different biological behaviors, due to their differing biological characteristics and therefore exist an interest in the identification of biomarkers that could be used in the clinical practice in order to better prognosticate and risk-stratify patients. The aim of this study was to evaluate the prognostic significance of the perineural invasion (PNI) and lymphovascular invasion (LVI) as well as the expression of Mcm-2, Cyclin D1, Ki-67 and p53 in advanced stage OSCC. A retrospective review of patients with OSCC of the tongue and floor of mouth in advanced clinical stage, treated by surgery in the Department of head and neck of the AC Camargo Cancer Center between the years 1998 and 2009 was conducted. Of 142 eligible cases, 88 paraffin-embedded tissue were rescued from the Department of Pathology of the same institution and immunohistochemistry reactions were performed for markers previously described. All slides have gone digitalized through the equipment Aperio System (Vista, CA, USA). Quantifications were performed from Imagescope software (Aperio System, USA) and statistical tests with significance level of 5% were taken to access correlations with prognosis. Our results showed that both PNI (p<0.001) as LVI (p=0.01) had negatively influence on overall survival of the patients. Other factors as T stage (p=0.003), positive lymph node (p=0.002) and extracapsular nodal spread (p<0.001) also had prognostic impact to predict poor survival. In relation to the proliferative markers, we found that high expression of Mcm-2 (p<0.001) and Cyclin D1 (p=0.005) had strong association with low rates of overall survival, whereas p53 and Ki-67 did not reach significance with this parameter. Mcm-2 was also correlated with recurrence (p=0.025). Cyclin D1 and p53 were significance with the N stage. In conclusion, the increasing expression of Mcm-2 and Cyclin D1 showed important correlation with prognosis and survival as well as histopathological features, such as PNI / Mestrado / Estomatologia / Mestre em Estomatopatologia

Orientador: Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T23:45:07Z (GMT). No. of bitstreams: 1 Costa_ErickaFrancislaineDiasCosta_M.pdf: 2497812 bytes, checksum: e6a45e68bd7bd5e6338b3a0c6353c3d4 (MD5) Previous issue date: 2014 / Resumo: A cisplatina (CDDP) associada à radioterapia (RT) é utilizada no tratamento de portadores de carcinoma de células escamosas de cabeça e pescoço (CCECP). Já é conhecido que tanto a resposta ao tratamento como seus efeitos colaterais variam de indivíduo para indivíduo. Uma possível explicação para o fato pode ser a variabilidade genética no metabolismo da CDDP. O objetivo deste estudo é verificar se as habilidades herdadas para induzir apoptose de células danificadas, mediadas pelas enzimas P53, CASP3 e CASP9, alteram os efeitos colaterais, a concentração de CDDP urinária e a taxa de resposta à terapêutica em pacientes com CCECP. Foram avaliados, de forma prospectiva, 90 pacientes consecutivos com CCECP do Ambulatório de Oncologia Clínica do Hospital de Clínicas da UNICAMP, que receberam CDDP associada à RT como tratamento neoadjuvante, definitivo ou paliativo da doença. Os genótipos dos polimorfismos P53 Arg72Pro, CASP9 A-1263G, CASP9 C-712T e CASP3 A-928G foram analisados por meio da reação em cadeia da polimerase (PCR) e digestão enzimática em DNA de sangue periférico. Os efeitos colaterais ao tratamento foram graduados por meio de questionário e exames laboratoriais, de acordo com os critérios do National Cancer Institute. As toxicidades auditiva e renal foram avaliadas, respectivamente, por meio de audiometria tonal, clearance de creatinina estimado e taxa de filtração glomerular (TFG) com EDTA-51Cr, realizados antes e após o tratamento. As dosagens urinárias da CDDP foram realizadas por cromatografia líquida de alta eficiência. A resposta ao tratamento foi avaliada por tomografia computadorizada de pescoço, de acordo com os critérios Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) e por tomografia computadorizada por emissão de fóton único (SPECT-TC). O significado estatístico das diferenças entre grupos foi calculado pelo teste da probabilidade exata de Fisher ou qui-quadrado. A regressão logística múltipla foi feita para obter a razão das chances, e a ANOVA por transformação em postos foi realizada para medidas repetidas. O genótipo P53 72ProPro esteve associado com maior ocorrência de vômitos dos graus 2, 3 e 4 e os genótipos CASP3 -928 AA e AG estiveram associados com menor perda auditiva unilateral e menor nefrotoxicidade após terapêutica em nossos casos. Concluímos que os polimorfismos P53 Arg72Pro e CASP3 A-928G modulam os efeitos colaterais do tratamento de pacientes com CCECP com CDDP e RT, como vômitos, acuidade auditiva e nefrotoxicidade. Acreditamos que estes resultados podem contribuir para definir o tratamento personalizado futuro de pacientes com CCECP / Abstract: Cisplatin (CDDP) associated with radiotherapy (RT) is used in treatment of patients with squamous cell head and neck carcinoma (HNSCC). It is well known that both response to treatment and side effects vary among individuals. A possible explanation for this may be genetic variability in CDDP metabolism. The aim of this study was to assess if inherited ability of inducing damaged cells to apoptosis, mediated by P53, CASP3 and CASP9 enzymes, change side effects, urinary concentration of CDDP, and rate of response to therapy in HNSCC patients. We evaluated prospectively, 90 HNSCC patients of Outpatient Oncology Clinic of UNICAMP¿s Clinical Hospital, who received CDDP associated to RT as neoadjuvant, definitive or palliative treatment. Genotypes of P53 Arg72Pro, CASP9 A-1263G, CASP9 C-712T and CASP3 A-928G, polymorphisms were analyzed by polymerase chain reaction (PCR) and restriction enzyme digestion of peripheral blood DNA. Treatment side effects were ranked by questionnaire and laboratory tests, according to criteria of National Cancer Institute. Hearing and renal toxicities were assessed using, respectively, audiometry, estimated creatinine clearance and EDTA-51Cr glomerular filtration rate (GFR), measured before and after treatment. CDDP Urinary dosages were measured by high performance liquid chromatography. Treatment response was assessed by computed tomography of neck, according to Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) and by Single Photon Emission Computed Tomography (SPECT-CT). Statistical significance of differences between groups was calculated by Fisher's exact probability test or chi-square. Multiple logistic regression was performed to obtain odds ratio, and ANOVA with rank-transform method was performed for repeated measures. P53 72ProPro genotype was associated with vomiting of grades 2, 3 and 4 and CASP3 -928 AA and AG genotypes were associated with lower unilateral hearing loss and lower nephrotoxicity after therapy in our cases. We conclude that P53 Arg72Pro and CASP3 A-928G polymorphisms modulate the side effects, such as vomiting, hearing thresholds decrease and nephrotoxicity, in HNSCC treated with CDDP and RT. We believe these results may contribute to definition of future personalized treatment of HNSCC patients / Mestrado / Clinica Medica / Mestra em Clínica Médica

Orientador: Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T03:53:51Z (GMT). No. of bitstreams: 1 Nogueira_GuilhermeAugustodaSilva_M.pdf: 2742030 bytes, checksum: f23fb1e053f1302891b66643feebad7b (MD5) Previous issue date: 2014 / Resumo: A cisplatina (CDDP) associada à radioterapia (RT) é utilizada no tratamento de portadores de células escamosas de cabeça e pescoço (CCECP). Já é conhecido que tanto a resposta ao tratamento como seus efeitos colaterais variam de indivíduo para indivíduo. Uma possível explicação para o fato pode ser a variabilidade genética no metabolismo da CDDP. O objetivo deste estudo foi o de verificar se habilidades herdadas no reparo de lesões do DNA, mediadas pelas enzimas MLH1, MSH2, MSH3, e EXO1, alteram os efeitos terapêuticos, colaterais e a concentração de CDDP urinária em pacientes com CCECP. Foram avaliados, de forma prospectiva, 90 pacientes consecutivos com CCECP do Hospital de Clínicas da UNICAMP, que receberam CDDP associada à RT como tratamento neoadjuvante, definitivo ou paliativo da doença. Os genótipos dos polimorfismos MLH1 G-93A, MSH2 IVS1+9G>C, MSH3 Ala1045Thr, EXO1 Pro757Leu e EXO1 Glu589Lys foram analisados por meio da reação em cadeia da polimerase (PCR) e digestão enzimática ou PCR em tempo real em DNA de sangue periférico. A resposta ao tratamento foi avaliada por tomografia computadorizada do pescoço, de acordo com os critérios Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1). Os efeitos colaterais ao tratamento foram graduados por meio de questionário e exames laboratoriais, de acordo com os critérios do National Cancer Institute 4.0. As toxicidades renal e auditiva foram avaliadas por meio do clearance de creatinina estimado, da taxa de filtração glomerular com EDTA-51Cr e de audiometria tonal, respectivamente, medidas antes e após o tratamento. As dosagens urinárias da CDDP foram realizadas por cromatografia líquida de alta eficiência. O significado estatístico das diferenças entre grupos foi calculado pelo teste da probabilidade exata de Fisher ou qui-quadrado e regressão logística múltipla. O genótipo MLH1 GG+GA esteve associado com menor ocorrência de náuseas. O genótipo MSH2 CC esteve associado com melhora da acuidade auditiva e tendência a menor excreção de CDDP na urina. Os genótipos MSH3 AlaAla+AlaThr e AlaAla estiveram associados com maior ototoxicidade e maior nefrotoxicidade, e piora da acuidade auditiva, respectivamente. O genótipo EXO1 ProLeu esteve associado com maior ototoxicidade e piora da acuidade aditiva. Os genótipos EXO1 GluLys+LysLys e LysLys estiveram associados com menor resposta completa, e piora da acuidade auditiva e tendência a nefrotoxicidade, respectivamente. Concluímos que os referidos polimorfismos, relacionados a anormalidades herdadas no reparo de DNA, podem alterar a taxa de resposta, os efeitos colaterais e a eliminação urinária de CDDP em pacientes com CCECP tratados com CDDP e RT. Acreditamos que estes resultados possam contribuir para o tratamento personalizado futuro de pacientes com o tumor / Abstract: Cisplatin (CDDP) associated with radiotherapy (RT) is used in treatment of patients with head and neck squamous cell carcinoma (HNSCC). It is well known that both response to treatment and side effects vary among individuals. A possible explanation for this may be the genetic variability in metabolism of CDDP. The aim of this study was to acess if inherited ability to repair DNA damage, mediated by MLH1, MSH2, MSH3 and EXO1 enzymes change the therapeutic side effects and urinary concentration of CDDP in HNSCC patients. We evaluated prospectively, 90 consecutive HNSCC patients of UNICAMP¿s Hospital, who received CDDP-associated RT as neoadjuvant, definitive or palliative treatment. Genotypes of MLH1 G-93A, MSH2 IVS1+9G>C, MSH3 Ala1045Thr, EXO1 P757L and EXO1 K589E polymorphisms were analyzed by polymerase chain reaction (PCR) and restriction enzyme digestion or real-time PCR in DNA of peripheral blood. Treatment response was assessed by computed tomography of the neck, according to Response Evaluation Criteria in Solid Tumors 1.1. Treatment side effects were ranked through questionnaire and laboratory tests, according to the National Cancer Institute 4.0. Renal and hearing toxicities were assessed using, respectively, estimated creatinine clearance and glomerular filtration 51Cr-EDTA, and audiometry, measured before and after treatment. Urinary doses of CDDP were performed by high performance liquid chromatography. Statistical significance of differences between groups was calculated by Fisher's exact probability test or chi-square, and multiple logistic regression. MLH1 GG+GA genotype was associated with lower incidence of nausea. MSH2 CC genotype was associated with improvement of hearing acuity and tendency of lower CDDP urinary excretion. MSH3 AlaAla+AlaThr and AlaAla genotypes were associated with high ototoxicity and high nephrotoxicity, and worsening of hearing acuity, respectively. EXO1 ProLeu genotype was associated with high ototoxicity and worsening of hearing acuity. EXO1 GluLys+LysLys and LysLys genotypes were associated with lower complete response, and worsening of hearing acuity and and tendency to high nephrotoxicity, respectively. We conclude that the referred polymorphisms, related to inherited abnormalities in DNA repair, may change rate of complete response, side effects, and CDDP urinary concentration in patients with HNSCC treated with CDDP and RT. We believe that these results may contribute to the future personalized treatment of HNSCC patients / Mestrado / Fisiopatologia Médica / Mestre em Ciências

Abstract Basement membranes (BM) underlie epithelia and endothelia and surround many tissues. In cutaneous BM epithelial cells are attached to the stroma via multiprotein complexes called hemidesmosomes (HD). Collagen XVII and integrin α6β4 are components of HD and they bind to laminin 332, a component of anchoring filaments, extracellularly. The main interest of this study is the function of collagen XVII and its interactions with these proteins. What is known about the function of collagen XVII is mostly derived from its role as an adhesive component in cutaneous HD. Here we demonstrate for the first time that collagen XVII is expressed by podocytes in the human and murine glomerulus and that mutant mice lacking collagen XVII in addition to small size, blisters and diffuse hair loss, also have deficient glomerular development and a high mortality rate. We also show for the first time at the protein level that collagen XVII is expressed, and probably has a functional interaction with laminin 332, in normal colon epithelia. We demonstrate that collagen XVII is expressed by the invasive cells of human colorectal carcinoma (CRC) samples and its immunostaining is increased in metastasis in CRC. The higher proportion of collagen XVII positive tumor cells correlates with decreased disease-free survival and cancer-specific survival times and we also suggest a functional interaction between collagen XVII and laminin 332 in CRC. Previous studies have suggested that collagen XVII participates in keratinocyte migration by affecting the correlation of HD disassembly and assembly, its expression is increased in squamous cell carcinoma (SCC) and it may have a role in cell adhesion and migration in SCC carcinogenesis. Here we demonstrate upregulated collagen XVII, integrin β4 and laminin γ2 expression in actinic keratosis, Bowen’s disease and SCC. The expression of collagen XVII was increased with a high degree of variation, especially in samples taken from areas where SCC is particularly invasive. We also demonstrate in the SCC-25 cell line that lack of collagen XVII or integrin β4 severely disrupts the adhesion, migration and invasivity of these cells. Taken together, in this study we show that collagen XVII is needed for normal glomerular development, is expressed in normal colon epithelia and participates in CRC and SCC carcinogenesis together with laminin 332 and integrin β4. / Tiivistelmä Tyvikalvot sijaitsevat epiteelin ja endoteelin alla ja ympäröivät monia kudoksia. Ihon tyvikalvossa epiteelisoluja alla olevaan verinahkaan kiinnittää rakenne, jota kutsutaan hemidesmosomiksi (HD). Kollageeni XVII ja integriin α6β4 ovat HD:n rakenneproteiineja. Ne kiinnittyvät solun ulkopuolella laminiin 332 nimiseen proteiiniin, joka muodostaa ankkurifilamentit. Kollageeni XVII ilmentyminen ja toiminta yhdessä näiden kahden proteiinin kanssa on tämän tutkimuksen keskeisin kohde. Valtaosa tutkimuksista, jotka käsittelevät kollageeni XVII:ää, koskevat sen toimintaa ihon keratinosyyteissä. Tässä tutkimuksessa osoitimme ensi kertaa, että hiiren ja ihmisen munuaiskerästen podosyyttisolut ilmentävät kollageeni XVII. Geenimanipuloidut hiiret, joilta kollageeni XVII oli poistettu, olivat pieniä, kehittivät rakkuloita ja karvattomuutta, niillä oli korkea kuolleisuus ja niiden munuaiskerästen kehitys oli häiriintynyt. Kollageeni XVII esiintymistä proteiinitasolla, sekä mahdollista toiminnallista yhteyttä laminiin 332:een, ei aiemmin ole osoitettu paksusuolen epiteelissä. Havaitsimme, että paksu- ja peräsuolen adenokarsinooman (CRC) invasiivinen solukko ilmentää kollageeni XVII:ää, kollageeni XVII esiintyminen on merkittävän voimakasta CRC:n metastasoinnin yhteydessä ja lisääntynyt kollageeni XVII esiintyminen lyhentää syöpävapaata aikaa ja heikentää syöpäspesifistä selviytymistä. Myös CRC:ssä kollageeni XVII toiminta voi liittyä laminiini 332:een. Aiempien tutkimusten mukaan kollageeni XVII osallistuu keratinosyyttien migratioon vaikuttamalla toimivien HD:ien määrään. Sen määrän on havaittu olevan korkeampi okasolusyövässä (SCC) ja sen on ehdotettu osallistuvan syöpäsolujen adheesioon ja migraatioon SCC:n kehittyessä. Me osoitimme kohonneen kollageeni XVII, integriini β4 ja laminiini γ2 ilmenemisen aktiinisessa keratoosissa, Bowenin taudissa sekä SCC:ssä. Kollageeni XVII määrä oli korkea, mutta vaihteli paljon, sekä hiiren että ihmisen invasiivisilla SCC alueilla. Havaitsimme myös SCC-25 solulinjalla, että kollageeni XVII tai integriini β4 puutos häiritsee vakavasti solujen adheesiota, migraatiota ja invaasiota. Yhteenvetona tässä työssä osoitimme, että kollageeni XVII:ää tarvitaan munuaiskerästen kehittymisessä, sitä esiintyy paksusuolen epiteelissä, ja että kollageeni XVII osallistuu CRC:n ja SCC:n kehittymiseen yhdessä integriini β4:n ja laminiini 332:n kanssa.

En 2008, le génome d’un nouveau virus a été caractérisé au sein d’un cancer cutané rare survenant préférentiellement chez l’immunodéprimé, le carcinome de Merkel. Ce nouveau virus appartenait à la famille des Polyomaviridae qui comprend des virus dont le caractère cancérigène chez l’animal est avéré depuis plus de 50 ans. Dénommé Polyomavirus de Merkel puisqu’il semblait lié à la survenue du cancer du même nom, il constituait le premier Polyomavirus impliqué de manière consistante dans un cancer humain. Cette implication reposant sur une étude unique limitée à 10 cas, l’objectif de notre travail de thèse était de confirmer le rôle étiologique du Polyomavirus de Merkel dans le carcinome de Merkel.Nous avons montré que le génome du Polyomavirus de Merkel était présent dans les trois quarts des cas de carcinome de Merkel, mais également que le virus infecte la population générale de manière quasi-ubiquitaire et de nombreux tissus en dehors de la peau. Les faits que chez les sujets atteints de carcinome de Merkel, l’ADN viral soit présent à des taux décelables de manière chronique dans différents tissus et que les titres d’anticorps sériques spécifiques du virus soient élevés suggèrent que ces sujets développent une infection chronique active. Celle-ci pourrait faciliter la survenue de mutations et d’intégrations de l’ADN viral qui sont spécifiquement associées aux carcinomes de Merkel. Ces modifications secondaires du génome viral aboutissent à la production d’oncoprotéines virales par les cellules tumorales, mais à l’abolition des capacités réplicatives donc lytiques du virus et constitueraient ainsi le support de la transformation tumorale. / Nucleotidic sequences defining the genome of a new virus, the Merkel Cell Polyomavirus, has been discovered in 2008 in Merkel cell carcinoma, a rare form of cutaneous cancer developing mostly in immunosupressed individuals. Whereas this new virus belongs to the Polyomaviridae family, which includes known oncogenic viruses in animals, it was the first study consistently implicating a Polyomavirus in human cancer. Because scientific arguments were only based on a ten-case-single report, the primary goal of our work was to confirm the role of the Merkel Cell Polyomavirus in Merkel Cell Carcinoma.Our work demonstrated that Merkel Cell Polyomavirus DNA was indeed present in three quarters of Merkel Cell Carcinoma cases, but also that Merkel Cell Polyomavirus was a near ubiquitous virus infecting various tissues among healthy individuals. Nonetheless, viral DNA is chronically detected in various tissues from Merkel Cell Carcinoma cases, which harbour elevated seric titters of specific antibodies. Those facts suggest that Merkel Cell Polyomavirus develop an active and chronic infection that could favour genomic mutation and integration events specifically associated to Merkel Cell Carcinoma. Those modifications, inducing both expression of truncated viral oncoproteins and abolishment of cell lysis mediated by viral replication, may support cell transformation.

The human skin provides us with an excellent protective barrier and possesses a remarkable ability of constant renewal. Basal cell carcinoma is the most common type of skin cancer. The aim of this project was to verify results from an earlier study investigating the molecular differences between basal cell carcinoma (BCC) and basal cells of normal human epidermis. In that study microdissection of cell populations from BCC and basal cells of normal epidermis respectively was performed in five cases of confirmed BCC. Following RNA extraction and amplification, a gene expression analysis was performed using a 46 k human cDNA microarray. Comparison of expression profiles showed a differential expression of approximately 300 genes in BCC. An upregulation of signaling pathways previously known to be of importance in BCC development could be observed, as well as a downregulation of differentiation markers, MHC class II molecules, and proteins active in scavenging of oxygen radicals. We wanted to confirm these findings for a number of selected genes, using real time PCR. The focal point of this project was microdissection of cells from BCC and subsequent isolation of RNA. Microdissection based methods offer a possibility of selecting well defined cell populations for further analysis by using a focused laser beam. Initially tests in order to optimize the method were also performed, concerning the dehydration process and choice of slides used in microdissection. Isolation of RNA may, as we experienced, be associated with problems due to destruction of RNA by degrading enzymes.

Abstract In the kidney, Wnt is involved in ureteric bud branching and nephrogenesis. Wnt mutation may lead to specific developmental dysfunctions and diseases. As part of this thesis, I show that Wnt11 is expressed in the renal tubules, except for the ureteric epitheliums, and I examine the function of Wnt11 in renal tubule organization using the new C57Bl6 Wnt11-/- mouse model. Convoluted and dilated tubules were observed in the Wnt11 mutated kidneys that may cause glomerular cysts and kidney dysfunction. More specifically, a lack of Wnt11 in the kidney reduced Six2, Hoxd1, and Hox10 expression, which may have contributed to the anomalies in the kidney tubular system. Embryogenesis and carcinogenesis share molecular characteristics. Gene expression changes take place during development to meet the demands of the tissue formation, but ectopic expression of embryonic genes by deletion, SNPs, or epigenetic modification in adult may lead to cancer. The research carried out as part of this thesis identified genes that were differentially expressed in both induced metanephric mesenchymes (MM) and human ccRCC. Gene silencing of Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr, and Itgb2 mediated by siRNA inhibited the migration, viability and invasion capacity of Renca cells (RCC). Furthermore, by using the novel 3D in vitro MM-Renca co-culture setup, the result revealed that downregulation of Bnip3, Cav1 or Gsn in Renca cells partly rescued the RCC-mediated inhibition epithelial tubules formation during nephrogenesis. Altogether, the data demonstrates that renal ontogenesis control genes play a role both in normal kidney development and in kidney cancer. The 3D RCC-MM chimera organoids developed as part of the research may also serve as a novel model for kidney cancer study. / Tiivistelmä Wnt-viestit ohjaavat munuaisen kehityksen yhteydessä sekä virtsanjohtimen että munuaiskeräsen kasvua. Virhe Wnt-proteiinia tuottavassa geenissä johtaa puolestaan vakavaan kehityshäiriöön ja syöpään, jos geeni aktivoituu aikuisvaiheessa. Tässä väitöskirjassa osoitetaan, että Wnt11-geeni osallistuu alkion munuaisen epiteeliputkiston kehityksen säätelyyn. Wnt11-signaalin tehtävää tutkittiin munuaisen putkiston rakenteen synnyssä poistogeenisen C57Bl6-hiirimallin avulla. Wnt11-puutos häiritsi virtsan kokoojatiehyiden rakenteiden kehitystä. Tämä on mahdollinen syy siihen, että Wnt11-poistogeenisen hiiren munuaiseen kehittyy hiusverisuonikerästen laajentumia. Munuaisen toiminta on myös heikentynyt verrokkiin verrattuna. Munuaisen epiteeliputkien kehitykseen osallistuvien geenien Six1, Hoxd1 ja Hox10 aktiivisuus oli niin ikään heikentynyt Wnt11-signaloinnin puutoksen vuoksi. Nämä seikat voivat olla puutoksen aiheuttamien kudosrakenteellisten muutosten taustalla. Solun kasvusäätely on keskeinen tekijä sekä alkion että syövän kehityksessä. Prosessien taustalla ovat myös samankaltaiset molekyylit. Wnt-solusignaalit esimerkiksi säätelevät normaalin kehityksen aikana solujen itsesäätelyä, solujen jakautumista, solujen vaeltamista ja solujen invaasiota kudoksiin ja osallistuvat syövän syntyyn niiden viestinnän häiriintyessä. Kehitystä säätelevien geenien tuotteet ohjaavat solujen jakautumista, mikä on edellytys kudosrakenteen synnylle. Jos nämä geenit syystä tai toisesta aktivoituvat uudelleen aikuisvaiheen aikana, se voi johtaa syöpäkasvaimen syntyyn. Tässä työssä verrattiin keskenään munuaisen elinkehitykseen ja ihmisen syövän kasvuun osallistuvia geenejä. siRNA-välitteinen hiljennys geeneille Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr ja Itgb2 inhiboi Renca-solujen migraatiota, elinkykyä ja invaasiota. Lisäksi tulokset paljastivat, että käytettäessä uutta 3D in vitro MM Renca -kasvatusmenetelmää Bnip3-, Cav1- tai Gsn-geenien hiljennys Renca-soluissa osittain pelastaa RCC-välitteisen inhibition epiteeliputkissa nefrogeneesin aikana. Kaiken kaikkiaan työssä seulottiin uusin tavoin sekä ihmisen munuaissyöpään liittyviä geenejä että kehitettiin lisäksi uusia niin kutsuttuja kokeellisia funktionaalisia organoidimenetelmiä. Työn tulokset tarjoavat uuden strategian, jolla geenien osuutta munuaissyöpään voidaan tutkia seikkaperäisesti yhdessä 3D-kasvatusmenetelmien kanssa