Orientador: Marilia de Freitas Calmon / Coorientador: Paula Rahal / Coorientador: Caroline Measso do Bonfim / Banca: Carolina Colombelli Pacca / Banca: Ricardo Barros Mariutti / Resumo: Condiloma acuminado (CA) ou verrugas genitais são lesões proliferativas benignas epidérmicas ou mucosas. O condiloma é causado pela infecção pelo papilomavírus humano (HPV), principalmente os tipos de baixo-risco 6 e 11, mas também pode ocorrer coinfecção com os HPVs de alto-risco. As variantes dos HPVs são definidas como sequências virais que compartilham identidade na sequência nucleotídica do gene L1 maior que 98%. Baseado nesse critério, linhagens de variantes de HPV6 e 11 têm sido estudadas e ainda há uma tentativa de correlacionar essas variações genéticas com os resultados clínicos diferenciais da infecção. Dessa forma, o objetivo do estudo foi detectar as variantes e alterações nucleotídicas presentes em E6 do HPV dos tipos 6 e 11 presentes em amostras de condiloma acuminado, correlacionar a presença de HPV com os dados clínico-patológicos das pacientes e determinar as relações filogenéticas das variantes encontradas com variantes de outras partes do mundo. A região E6 de 32 amostras de condiloma acuminado foi sequenciada, sendo dessas 25 positivas para HPV6 e sete para HPV11. Entre as amostras HPV6, 12 foram identificadas como a variante HPV6a, apresentando a mutação nucleotídica G474A, sendo uma delas também apresentando a mutação T369G no genoma. As 13 pacientes restantes foram positivas para a variante HPV6vc, não apresentando alterações nucleotídicas. Na análise das amostras HPV11, observou-se que todas as pacientes mostraram as mutações T137C e C380T. Além disso... / Abstract: Condyloma acuminatum (CA) or genital warts are benign proliferative lesions that can be epidermal or mucous. Condyloma is caused by infection with human papillomavirus (HPV), mainly the low-risk types 6 and 11, but can also occur coinfection with high-risk HPVs. Human papillomaviruses have the capacity to infect the skin, oral and genital mucosa, and induce benign or malignant proliferative lesions. The variants of HPVs are defined as viral sequences that share identity in the nucleotide sequence of the L1 gene greater than 98%. Based on this criterion, HPV6 and 11 variants lineages have been studied and there is still an attempt to correlate these genetic variants with different clinical findings of infection. In this way, the aim of this study was to detect variants and nucleotide alterations presents in E6 region of HPV types 6 and 11 detected in condyloma acuminatum samples, to correlate the HPV presence with the clinical-pathological data of the patients and to determine phylogenetic relations of variants found with variants of others places of the world. The E6 region of 32 samples of condyloma were sequenced, being 25 positive to HPV6 and seven diagnosed HPV11. Twelve samples were identified as the HPV6a variant, and presented the mutation G474A, and one of these also showed the mutation T369G. The others 13 patients were positive to HPV6vc without nucleotide alterations. In the analysis of the seven HPV11 samples, it was observed that all patients showed the mutations ... / Mestre

Du Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 70-89). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.IV / PUBLICATIONS --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATIONS --- p.VIII / CONTENTS --- p.X / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- Laryngeal carcinoma and HPV --- p.1 / Chapter 1.2 --- HPV --- p.2 / Chapter 1.3 --- Human papillomavirus E6 protein --- p.6 / Chapter 1.3.1 --- Transformation by HPV E6 --- p.7 / Chapter 1.3.2 --- Inhibition of apoptosis by E6 --- p.8 / Chapter 1.3.3 --- Alteration of gene transcription --- p.11 / Chapter 1.3.4 --- E6 interation with other proteins --- p.12 / Chapter 1.3.5 --- E6 as a therapeutic target --- p.14 / Chapter 1.4 --- HPV E7 protein --- p.15 / Chapter 1.4.1 --- Regulation of viral life cycle by HPV E7 --- p.16 / Chapter 1.4.2 --- Degradation of retinoblastoma tumor suppressor by HPV E7 --- p.18 / Chapter 1.4.3 --- Inhibition of p53 by HPV E7 --- p.22 / Chapter 1.4.4 --- Interaction with other proteins by HPV E7 --- p.24 / Chapter 1.5 --- Objective --- p.26 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Materials for cDNA and RNA manipulation --- p.28 / Chapter 2.1.2 --- Culture media and transfection reagents --- p.28 / Chapter 2.1.3 --- Antibodies --- p.29 / Chapter 2.1.4 --- Materials for protein manipulation --- p.29 / Chapter 2.1.5 --- Kits --- p.30 / Chapter 2.1.6 --- Instrumentation --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Plasmid construction --- p.32 / Chapter 2.2.1.1 --- DNA preparation --- p.34 / Chapter 2.2.1.2 --- DNA ligation --- p.34 / Chapter 2.2.1.3 --- Transformation of competent E. coli --- p.35 / Chapter 2.2.2 --- Mini preparation --- p.35 / Chapter 2.2.3 --- Clone selection and confirmation --- p.37 / Chapter 2.2.4 --- Sequencing gel electrophoresis --- p.37 / Chapter 2.2.5 --- Cell culture and cytokine treatment --- p.39 / Chapter 2.2.6 --- Plasmid transfection --- p.39 / Chapter 2.2.7 --- Confirming construction of stable cell lines by RT-PCR --- p.40 / Chapter 2.2.7.1 --- Total cellular RNA extraction --- p.40 / Chapter 2.2.7.2 --- First strand cDNA synthesis --- p.41 / Chapter 2.2.7.3 --- Polymerase chain reaction (PCR) --- p.41 / Chapter 2.2.8 --- Fluorescence microscopy and imaging --- p.43 / Chapter 2.2.9 --- DNA fragmentation assay --- p.44 / Chapter 2.2.10 --- Protein detection --- p.46 / Chapter 2.2.10.1 --- Preparation of protein extract --- p.46 / Chapter 2.2.10.2 --- SDS-PAGE electrophoresis and protein transfer --- p.47 / Chapter 2.2.10.3 --- Immunoblotting analysis --- p.47 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE: --- RESULTS --- p.49 / Chapter 3.1 --- Plasmid construction --- p.49 / Chapter 3.2 --- Expression of HPV16 viral oncogenes in transfected UMSCC12 --- p.51 / Chapter 3.3 --- HPV16 E6 and E7 protect apoptosis induced by TNF-alpha and CHX --- p.53 / Chapter 3.4 --- Detection of apoptosis with fluorescence staining --- p.55 / Chapter 3.5 --- Regulation of the expression of apoptosis-associated proteins by E6 and E7 oncoproteins --- p.57 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.59 / Chapter CHAPTER FIVE: --- CONCLUSION AND FUTURE PERSPECTIVE --- p.68 / REFERENCES --- p.70 / APPENDIX DNA SEQUENCING RESULTS --- p.90

Li Chun-bong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 147-169). / Abstracts in English and Chinese. / Abstract --- p.i / Declaration --- p.vi / Acknowledgments --- p.vii / Table of Contents --- p.xi / List of Figures --- p.xii / List of Tables --- p.xvi / Abbreviations --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION AND LITERATURE REVIEWS / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Carcinoma of the cervix --- p.6 / Chapter 1.2.1 --- Squamous carcinoma --- p.6 / Chapter 1.2.2 --- Adenosquamous carcinoma --- p.7 / Chapter 1.2.3 --- Adenocarcinoma --- p.8 / Chapter 1.3 --- Molecular biology of Human papillomavirus --- p.9 / Chapter 1.3.1 --- Genome structure and organization of HPV --- p.9 / Chapter 1.3.2 --- Expression of papillomavirus genes --- p.11 / Chapter 1.3.3 --- Taxonomy of HPV --- p.20 / Chapter 1.4 --- Diagnostic techniques in HPV detection --- p.23 / Chapter 1.4.1 --- Southern blot analysis --- p.23 / Chapter 1.4.2 --- Dot blot analysis --- p.25 / Chapter 1.4.3 --- In situ hybridization --- p.26 / Chapter 1.4.4 --- Hybird Capture System --- p.28 / Chapter 1.4.5 --- Polymerase Chain Reaction --- p.30 / Chapter 1.5 --- Human papillomavirus in cervical carcinoma --- p.33 / Chapter 1.5.1 --- Prevalence --- p.33 / Chapter 1.5.2 --- Transmission --- p.37 / Chapter 1.5.3 --- Risk Factors --- p.39 / Chapter CHAPTER2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.44 / Chapter 2.1.1 --- Chemicals and regents --- p.44 / Chapter 2.1.2 --- Specimens collection --- p.48 / Chapter 2.2 --- Methods --- p.49 / Chapter 2.2.1 --- Summary of methodology --- p.50 / Chapter 2.2.2 --- DNA extraction from fresh and paraffin embedded tissues --- p.51 / Chapter 2.2.3 --- Polymerase Chain Reaction using HPV Consensus Primer MY09/11 --- p.55 / Chapter 2.2.3.1 --- Template for PCR --- p.55 / Chapter 2.2.3.2 --- PCR amplification --- p.55 / Chapter 2.2.3.3 --- PCR product analysis --- p.56 / Chapter 2.2.4 --- DNA sequencing --- p.57 / Chapter 2.2.4.1 --- DNA sequencing reaction for ALFexpress DNA automatic sequencing --- p.57 / Chapter 2.2.4.2 --- ABI comparative PCR sequencing --- p.59 / Chapter 2.2.4.3 --- DNA sequence analysis --- p.60 / Chapter 2.2.5 --- Restriction Fragment Length Polymorphism --- p.61 / Chapter 2.2.5.1 --- Template preparation --- p.61 / Chapter 2.2.5.2 --- Restriction enzyme digestion --- p.62 / Chapter 2.2.5.3 --- Agarose gel electrophoresis analysis --- p.62 / Chapter 2.2.6 --- HPV Type Specific PCR --- p.63 / Chapter 2.2.6.1 --- Preparation of positive control DNA --- p.63 / Chapter 2.2.6.2 --- Preparation of HPV 52 and HPV 58 type specific PCR --- p.63 / Chapter 2.2.6.3 --- PCR primer design --- p.66 / Chapter 2.2.6.4 --- PCR amplification --- p.68 / Chapter 2.2.7 --- Polymerase Chain Reaction using HPV Consensus Primer GP5+/6+ --- p.71 / Chapter 2.2.7.1 --- Template for PCR --- p.71 / Chapter 2.2.7.2 --- PCR amplification --- p.71 / Chapter 2.2.7.3 --- PCR product analysis --- p.72 / Chapter 2.2.8 --- Statistical analysis --- p.72 / Chapter CHAPTER3 --- RESULTS / Chapter 3.1 --- Histology review of tumor specimens --- p.73 / Chapter 3.2 --- Polymerase chain reaction of HPV consensus primer MY09/11 --- p.76 / Chapter 3.3 --- DNA sequencing reaction --- p.81 / Chapter 3.4 --- Restriction fragment length polymorphism --- p.86 / Chapter 3.5 --- HPV type specific polymerase chain reaction --- p.90 / Chapter 3.6 --- Polymerase chain reaction of HPV consensus primer GP5+/6+ --- p.109 / Chapter 3.7 --- "Correlations of HPV prevalence, geographical variation, histology and age of the cervical cancer patients" --- p.112 / Chapter CHAPTER4 --- DISCUSSION / Chapter 4.1 --- Prevalence of HPV infection in cervical cancer in China --- p.118 / Chapter 4.2 --- DNA extraction and detection methods --- p.131 / Chapter 4.3 --- Intratype variation of HPV --- p.141 / Chapter CHAPTER5 --- CONCLUSION AND FUTURE PERSPECTIVE --- p.143 / REFERENCES --- p.147 / RELEVANT PUBLICATIONS --- p.170

Orientador: Paula Rahal / Banca: Laura Sichero / Banca: João Manuel Grisi Candeias / Banca: Paola Jocelan Scarin Provazzi / Banca: Fátima Pereira de Souza / Resumo: O carcinoma de pênis (CEP) é um tumor epitelial invasivo raro com alta morbidade decorrente da própria doença e/ou de seu tratamento. O perfil socioeconômico e cultural dos pacientes dificulta o diagnóstico precoce, tratament o e seguimento dos enfermos. Pacientes sem tratamento geralmente morrem dentro de dois anos após diagnóstico devido à proliferação celular regional ou a metástases distantes. Não há padrão nos sistemas de estadiamento e nas condutas clínicas, resultando em dificuldades na abordagem terapêutica. Comparado ao câncer cervical, poucos estudos moleculares em CEP foram realizados. O presente projeto teve como objetivo geral identificar genes diferencialmente expressos em tecidos penianos tumorais e normais e o possível papel do vírus do papiloma humano (HPV) no desenvolvimento de CEP. Diferenças na expressão gênica entre tecidos tumorais e normais foram verificadas pela metodologia de RaSH (rapid subctration hybridization), que selecionou 5 genes possivelmente rel acionados à carcinogênese ( KIAA1033, NAMPT, RPL6, CDKN2A e ANXA1) com expressão alterada em tecidos tumorais. A validação das alterações de expressão destes genes foi realizada por polymerase chain reaction (reação em cadeia da polimerase, PCR) em tempo real. Tanto os genes como as proteínas de ANXA1 como CDKN2A apresentaram aumento de expressão em pacientes com presença de HPV de alto risco em comparação com tecidos negativos para HPV. Por imunohistoquímica foi possível estabelecer correlação entre alterações na expressão da proteína anexina A1 e a presença de HPV de alto risco. Pacientes portadores de CEP provenientes de duas regiões (São Paulo e Pará) foram avaliados para a presença de HPV por PCR convencional. Amostras positivas foram genotipadas por hib ridização... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Penile carcinoma is a rare epithelial tumor with high morbidity due to own disease and/or treatment. The socio -economic and cultural profile of the patients hampers early diagnosis, treatment and follow-up. Without treat ment patients usually dies within two years after first diagnosis due to regional cellular proliferation or distant metastasis. There is no standard in staging systems or in clinical procedures, resulting in difficulties in the therapeutic approach. Compar ed to cervical cancer, there are few molecular studies about this tumor. The present work aimed to identify differentially expressed genes in tumor and normal penile tissues and the role of human papillomavirus (HPV) in the development of this tumor. Differences in gene expression between tumoral and normal tissues were accessed by RaSH (rapid subctration hybridization) methodology, which selected five possibly carcinogenesis related genes (KIAA1033, NAMPT, RPL6, CDKN2A and ANXA1) with altered expression in tumor tissues. The validation of gene expression changes was performed by real time polymerase chain reaction (PCR). The ANXA1 and CDKN2A, both genes and proteins, showed superexpression in patients with high-risk HPV compared to HPV negative tissues. Immunohistochemical assays established a correlation between alterations in the superexpression of annexin A1 and the presence of high risk HPV. Penile cancer harboring patients from two regions (São Paulo and Pará) was assessed for HPV presence by conventional PCR. Positive samples were genotyped by reverse hybridization-based line probe assay (INNO -LiPA). The overall HPV prevalence in single or multiple infections was significantly greater in Pará (81.67%) than in São Paulo (64.10%). A wilder range of genotypes were found in Pará, suggesting that the genotypes circulating in the population of Pará... (Complete abstract click electronic access below) / Doutor

Orientador: Kellen Cristine Tjioe / Resumo: Objetivo: Avaliar a expressão do p16Ink4a por imunohistoquímica e a presença do HPV16 pela Real time PCR em tecido fresco, plasma e saliva de pacientes com leucoplasia bucal (LB) e hiperplasia fibrosa inflamatória (HFI). Material e métodos: Foram incluídos 67 pacientes com o diagnóstico de LB e 44 pacientes com diagnóstico de HFI no estudo. Foram coletados dados sociodemográficos, clinicopatológicos, amostras de tecido fresco, sangue e saliva, que foram armazenados em um freezer a -80o C para realização de análise molecular. Também foi utilizado o tecido parafinizado para realização da imunohistoquímica para a p16Ink4a. As amostras de materiais biológicos obtidos dos pacientes foram submetidas à detecção do DNA do HPV-16 pela Real time PCR. O tecido parafinizado dos mesmos pacientes foram utilizados para avaliar a expressão da p16Ink4a pela imunohistoquímica. Resultados: Dos 67 pacientes incluídos de LB no estudo, 55,2% eram do sexo masculino com uma média de idade de 57,1 anos. Os pacientes idosos (>45 anos) compuseram 86,6% da amostra. As localizações anatômicas mais acometidas por LB foram a mucosa jugal (35,8%) e língua (20,9%). Sobre o tabagismo, 71,8% dos pacientes eram fumantes, onde a maioria (47,8%) foi classificada como tabagista leve. Em relação ao consumo de álcool, 47,4% eram alcoolistas, sendo a sua maioria classificada como alcoolista leve (59,3%). O grupo HFI foi composto por 54,5% pacientes do sexo masculino com uma média de idade de 57,3 anos. 90,9% dos paci... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 142-165). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Made available in DSpace on 2015-03-03T11:52:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2000Bitstream added on 2015-03-03T12:06:22Z : No. of bitstreams: 1 000800314_20160307.pdf: 286328 bytes, checksum: 0f0b5c545f16ee37737e2e8c5a43a17c (MD5) Bitstreams deleted on 2016-03-07T11:06:17Z: 000800314_20160307.pdf,. Added 1 bitstream(s) on 2016-03-07T11:07:08Z : No. of bitstreams: 1 000800314.pdf: 1268218 bytes, checksum: f4729461b52712f8f52075ab13040415 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O líquen plano caracteriza-se como uma doença inflamatória crônica mucocutânea relativamente comum na população. Possui etiologia incerta, sendo possivelmente associado a fatores genéticos, psicológicos e infecciosos, dentre os quais o último vem ocupando um maior destaque devido a uma possível correlação com o vírus do papiloma humano (HPV) e com o Epstein-Barr vírus (EBV). O HPV possui alguns tipos considerados oncogênicos associados ao câncer de colo de útero e fortemente associado ao carcinoma espinocelular (CEC) de orofaringe. O EBV pertence à família herpesvirus humano e está relacionado com o carcinoma nasofaríngeo, linfoma de Burkitt e linfoma não-Hodgkin e sua possível relação com o CEC vem sendo estudada. O objetivo deste estudo foi detectar a presença do DNA do HPV e do EBV em amostras de tecido fresco, plasma sanguíneo, saliva e células esfoliadas orais, extraídas de um grupo pareado por sexo e idade de pacientes portadores de líquen plano bucal (LPB) e de um grupo de pacientes sem lesões de LPB, além de correlacionar as variáveis epidemiológicas dos grupos estudados com a presença viral e verificar se as fontes materiais testadas por este estudo são fontes viáveis para detecção do HPV e do EBV. Foram avaliados 24 pacientes portadores de LPB (Grupo caso) e 17 pacientes sem lesões de LPB (Grupo controle). A extração de DNA das amostras foi realizada após confirmar a presença e integridade do DNA. Os resultados obtidos foram submetidos à análise estatística (Teste exato de Fisher e Teste do Qui-Quadrado Mantel-Haenszel, ambos, com nível de significância de 5%). A nPCR foi utilizada para detecção do HPV e do EBV. Obteve-se a positividade viral para o HPV em 41,7% das amostras teciduais, em 12,5% das amostras de células esfoliadas e em nenhuma amostra de plasma sanguíneo e saliva dos pacientes do Grupo / Lichen planus is characterized as a chronic inflammatory mucocutaneous disease relatively common in the population. It has uncertain etiology, possibly associated with genetic, psychological and infectious factors. The infectious factor has excelled due the possible correlation of lichen planus with human papilloma virus (HPV) and Epstein-Barr virus (EBV). HPV has some types considered oncogenic, associated with cervical cancer and strongly associated with squamous cell carcinoma (SCC) of oropharynx. EBV belongs to human herpesvirus family and is associated with nasopharyngeal carcinoma, Burkitt's lymphoma and non-Hodgkin lymphoma. Its possible relation to SCC has been studied. The aim of this study was to detect the presence of the DNA of the HPV and EBV in fresh tissue samples, blood plasma, saliva and oral exfoliated cells extracted from a group of patients with oral lichen planus (OLP) paired by age and gender and from a group of patients without OLP lesions, and also correlate the epidemiological variables of the studied groups with the viral presence and verify that the source materials tested in this study are viable for HPV and EBV detection. It was evaluated 24 patients with OLP (Case group) and 17 patients without OLP lesions (Control group). DNA extraction of samples was performed after confirming the presence and integrity of DNA. The results were subjected to statistical analysis (Fisher's exact test and Mantel-Haenszel 19 chi-square test, both with a significance level of 5%). The nPCR was used to detect the presence of HPV and EBV. Was obtained the viral positivity for HPV in 41.7% of tissue samples and in 12.5% of exfoliated cells samples. No samples of blood plasma and saliva were positive in the Case group. On the other hand, the Control group / FAPESP: 11/05499-8

Orientador: Rogério Saad Hossne / Coorientador: Sidney Roberto Nadal / Banca: Fábio Vieira Teixeira / Banca: Maria Aparecida C. Arruda Henry / Resumo: O Papiloma Vírus Humano (HPV), é considerado um problema mundial de saúde pública, sendo a doença sexualmente transmissível mais prevalente. Guarda uma relação direta com o risco e a incidência do câncer do canal anal. Seu diagnóstico, tratamento e seguimento são de extrema importância. Neste sentido o escovado do canal anal tem um papel fundamental no rastreamento e seguimento das lesões HPV induzidas e consequente evolução para o câncer anal. Determinar a ocorrência de lesão HPV induzida em mulheres que participam dos programas de prevenção do câncer de colo uterino nas Unidades Básicas de Saúde (UBS) no município de Botucatu. Trata-se de um estudo transversal observacional que teve 228 mulheres submetidas ao escovado do canal anal a fim de estabelecer a ocorrência de lesão HPV induzida e suas correlações com dados sociais e comportamentais. Os 11 casos que apresentaram alteração de ASCUS e LSIL no escovado do canal anal traziam relação com estado civil, baixa escolaridade, não prática do sexo seguro, e a prática do sexo anal / Abstract: Human Papillomavirus (HPV) has been a world concern in Public Health, and it is the most prevalent sexually transmitted disease. It has a direct association with the risk and incidence of cancer in the anal canal. Its diagnosis, treatment and follow-up are extremely important. Using this approach, the smear of the anal canal has a crucial role in the screening and follow up of HPV-induced lesions and in the resulting development of anal cancer. To determine the occurrence of HPVinduced lesions in women who attended programs of uterine cervix cancer prevention in Basic Health Units (BHU) in Botucatu city. It is a cross sectional observational study, in which 228 women underwent brushing of the anal canal in order to establish the occurrence of HPV-induced lesion and its correlation with social and behavioral data. The 11 cases which had ASCUS and LSIL changes in the smear of the anal canal were associated with marital status, low education level, practice of unsafe intercourse and anal intercourse / Mestre

Orientador: Glauco Issamu Miyahara / Coorientador: Daniel Galera Bernabé / Coorientadora: Kellen Cristine Tjioe / Banca: Maria José Hitomi Nagata / Banca: Luciana Estevam Simonato de Oliveira / Resumo: Objetivo: Avaliar a presença do HPV-16 em tecido fresco, saliva e plasma sanguíneo de pacientes com leucoplasia bucal pela real time PCR na região noroeste do estado de São Paulo, Brasil. Pacientes e métodos: Trinta e sete pacientes com diagnóstico de leucoplasia bucal foram incluídos no estudo. Destes, foram obtidos dados sociodemográficos, clinicopatológicos, estilo de vida e amostras de tecido fresco, sangue e saliva que foram armazenados a -80ºC para posterior análise molecular. Os materiais obtidos destes pacientes foram submetidos à detecção do DNA viral pela técnica da real time PCR com sonda específica para o HPV-16. Resultados: Dos 37 pacientes incluídos no estudo, 64,8% eram homens e a idade variou de 25 a 82 anos, com uma média de 58,72 anos. Dezesseis pacientes (43,2%) eram idosos e 43,2%, adultos de meia idade, e apenas 13,6%, adultos jovens. A maioria dos pacientes era fumante (72,9%), sendo que 16,3% eram ex-fumantes e 10,8%, não fumantes. Da mesma forma, a maioria (62,2%) era etilista, 21,6%, ex-etilistas e 16,2%, não-etilistas. Vinte e sete por cento das lesões apresentaram algum grau de displasia epitelial. A detecção do HPV-16 pela PCR em tempo real não foi positiva para nenhuma amostra, resultando em um índice de 0% de detecção. Conclusão: O HPV-16 não foi identificado na população estudada. No entanto, outros subtipos do HPV de baixo e alto risco podem estar associados à ocorrência de leucoplasia bucal nesta população, o que requer novas investigações. Es... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Objective: To evaluate the prevalence of HPV-16 DNA detection in fresh tissue, saliva and blood plasma from patients with oral leukoplakia by the real time PCR in the northwest region of the São Paulo state, Brazil. Patients and methods: Thirty-seven patients diagnosed with oral leukoplakia were included in the study. Sociodemographic, clinicopathologic and lifestyle data, fresh tissue, saliva and blood plasma samples were collected. Biologic material was stored at -80ºC and then submitted to viral DNA detection by the real time PCR technique with a probe specific for HPV-16. Results: Of the 37 patients included in the study, 64.8% were men, and the age ranged from 25 to 82 years, with a mean of 58.72. Sixteen patients (43.2%) were elderly, 43.2% were middle-aged adults, and only 13.6% were young adults. Most patients were smokers (72.9%), 16.3% were former smokers, and 10.8% were non-smokers. Most patients (62.2%) were current drinkers, 21.6% were ex-drinkers and 16.2% were non-drinkers. Twenty seven percent of the lesions presented some degree of dysplasia. HPV-16 detection by real-time PCR was not positive for any sample, resulting in a 0% detection rate. Conclusion: The HPV-16 was not identified in the population studied. However, other low and high-risk HPV subtypes might be associated to the occurrence of oral leukoplakia in this population, which requires further investigations. Broader epidemiological studies are required to clarify the geographic variability in the p... (Complete abstract click electronic access below) / Mestre

Orientador: Paula Rahal / Coorientador: Laura Sichero / Banca: Enrique Mário Boccardo Pierulivo / Banca: Márcia Guimarães da Silva / Banca: Alessandra Vidotto / Banca: Cíntia Bittar / Resumo: A papilomatose respiratória recorrente (PRR) é uma doença caracterizada pela formação de papilomas benignos no trato respiratório superior. A infecção pelo Papilomavírus Humano (HPV) é a principal causa da doença, principalmente os tipos 6 e 11, que são considerados de baixo risco oncogênico. A região promotora LCR (long control region) contêm elementos cis-reguladores para fatores transcricionais (FTs) celulares e virais que controlam a expressão gênica precoce e a replicação viral. Alterações nucleotídicas dentro da LCR podem sobrepor sítios de ligação de FTs e influenciar a afinidade de ligação, a atividade transcricional e o curso clínico das doenças associadas á infecções por HPV. Os objetivos deste trabalho foram caracterizar as variantes moleculares de HPV entre os indivíduos com diagnóstico de PRR e analisar o impacto da variabilidade nucleotídica da LCR sob a transcrição viral. O sequenciamento da LCR de amostras HPV-6 positivas revelou cinco variantes genômicas não descritas anteriormente. Por meio de análise computacional, observou-se que as alterações nucleotídicas detectadas sobrepõem potenciais sítios de ligação para alguns fatores de transcrição. A variante HPV-6vc foi a mais prevalente dentre as amostras analisadas (69,2%), sendo mais prevalente nos casos de papilomatose juvenil. Com relação à atividade transcricional, a variante HPV-6vc é mais ativa que a variante molecular HPV-6a. Além disso, observou-se que outras alterações observadas influenciaram na atividade transcricional que foi indiretamente medida por ensaios de luciferase. Vale ressaltar que este é o primeiro trabalho que descreve as diferenças de atividade do promotor entre variantes naturais de HPV-6. Esta pesquisa é de extrema relevância, pois fornece informações importantes sobre a função biológica de variabilidade genômica intratípica de HPV-6 / Abstract: Recurrent respiratory papillomatosis (RRP) is a disease characterized by the formation of benign papillomas in the upper respiratory tract. Human Papillomavirus infection (HPV) is the main cause of the disease, particularly types 6 and 11 which are considered low-risk oncogenic HPV. The promoter region LCR (Long Control Region) contains cis-regulatory elements for cellular and viral transcription factors (TF) that control viral early gene expression and replication. Nucleotide alterations within the LCR may overlap TFs elements and impact upon the binding affinity, the transcriptional activity and ultimately on the clinical outcome associated to HPV infections. Our aim was to characterize molecular variants of HPV among individuals diagnosed with RRP and to analyze the impact of LCR nucleotide divergence upon viral early transcription. LCR sequencing of the HPV-6 positive samples revealed five genomic variants not previously described. Through computational analysis, we found that nucleotide changes detected overlapps potential binding sites for several transcription factors. HPV-6vc-related variant was the most prevalent among the samples analyzed (69.2%) and more prevalent in cases of juvenile papillomatosis. Concerning transcriptional activity, HPV-6vc-related variant is more active than HPV-6a molecular variant. Further, we observed that other alterations observed strongly impacts transcriptional activity indirectly measured by luciferase assays. To our knowledge, this is the first report describing differences in promoter activity among naturally occurring variants of HPV-6. Research in this area is anticipated to provide important information concerning the biological significance of HPV-6 intratype genomic variability / Doutor