INTRODUÇÃO: O papilomavírus humano (HPV) encontra-se amplamente distribuído na população mundial. Apesar da grande maioria das infecções serem transientes, assintomáticas e passíveis de regressão espontânea, a infecção persistente por tipos de alto risco de HPV é necessária para o desenvolvimento de neoplasias intraepiteliais cervicais. Uma vez que apenas uma pequena parcela das infecções progride à lesões malignas após um longo período desde o diagnóstico inicial de lesões precursoras, tem-se iniciado a busca por fatores que possam influenciar na progressão ou na eliminação destas manifestações iniciais. A variabilidade genética viral tem sido apontada como um dos fatores que interagem neste processo. Embora virtualmente todos os tumores da cérvice uterina apresentem o DNA viral, neoplasias em outros sítios anatômicos têm sido apenas em parte correlacionadas com a presença viral, sendo o HPV proposto como um dos agentes causadores de tumores em sítios de cabeça e pecoço. MÉTODOS: Espécimens clínicos de tumores de cabeça e pescoço, fixados em formalina e contidos em parafina (FFPE), provenientes do Instituto do Câncer do Estado de São Paulo (n=79) e da Santa Casa de Misericórida de São Paulo (n=94), tiveram seu DNA extraído, seguido de diagnóstico e genotipagem de HPV pela metodologia de Inno-LiPA. Análises de linhagens moleculares foram realizadas nas amostras HPV-16 positivas. Análise imunohistoquímica de P16INK4a foi realizada em todas as amostras. RESULTADOS: A presença do DNA viral foi encontrada em 24,1% (19/79) dentre a série de tumores provenientes do ICESP, sendo a cavidade oral o sítio em que foi observada a maior proporção de DNA viral (27,1%), enquanto que 13,8% (13/94) dentre os espécimens provenientes da Santa Casa apresentaram-se positivos para HPV, sendo a cavidade oral o sítio em que foi observada a maior proporção do DNA viral (18,1%). O HPV-16 foi o tipo mais prevalente, detectado em 73,4% das amostras HPV positivas provenientes do ICESP e 61,5% das amostras provenientes da Santa Casa. Independente da Instituição, as amostras foram alocadas no clado das linhagens Asiático-Americana e Europeia em 50%, cada uma, entre os 18 tumores HPV-16 positivos em que as análises de linhagem foram possíveis. Não foi observada, nestas séries, correlação entre a superexpressão de P16INK4a e a presença do DNA viral. CONCLUSÃO: Nas amostras analisadas, o DNA de HPV foi detectado em 18,5% dos 173 espécimens. O HPV-16 foi o tipo mais prevalente. Isolados da linhagem Europeia e da linhagem Asiatico-Americano foram detectados em 50% dos casos, cada uma, dentre as amostras HPV-16 analisadas por este estudo / INTRODUCTION: Human papillomaviruses (HPV) are widely distributed worldwide. Although the majority of infections are usually transient, asymptomatic and frequently regress spontaneously, persistent infections by high-risk HPVs are necessary for the development of cervical intraepithelial neoplasia. Once only a small proportion of infections progress to malignant lesions after a long period of time since the initial diagnosis of precursor lesions, the search for factors that might influence the progression or clearence of these early manifestations are currently under way. Viral genetic variability has been proposed as one of the factors interacting in this process. Although virtually all cervix tumors present the viral DNA, neoplasias from other anatomical sites have been only in part correlated with viral presence, and HPV has been proposed as one causative agent in tumors from head and neck sites. METHODS: Clinical specimens of formalin-fixed paraffin embedded head and neck tumors, provided by the Cancer Institute of São Paulo (n=79) and also by the Santa Casa de Misericórdia de São Paulo (n=94), were submitted to DNA extraction and further HPV diagnostic and genotyping by the Inno-LiPA methodology. Molecular lineages analyses were performed in all HPV-16 positive samples. P16INK4a immunohistochemical analyses were conducted in all samples. RESULTS: HPV DNA was detected among 24.1% (19/79) of samples provided by ICESP, tumors from oral cavity presented the highest viral positivity (27.1%), whereas 13,8% (13/94) of the samples from Santa Casa presented HPV DNA, tumors from the oral cavity also presented the highest HPV positivity with 18.1% of viral DNA presence. HPV-16 was the most prevalent type detected in 73.4% and 61.5% of HPV positive ICESP and Santa Casa samples, respectively. Irrespective of the Institution, samples submitted to lineage analyzes were allocated in the Asiatic-American and European phylogenetic branches in 50%, each one, among the 18 tumors HPV-16 positive for which lineage analysis was possible. No correlation between P16INK4a overexpression and HPV DNA presence was observed. CONCLUSION: In this study, HPV DNA was detected in 18.5% among 173 head and neck tumor specimens. HPV-16 was the most prevalent type. The European and the Asiatic-American lineage were detected in 50% of the cases, each one, among the cases HPV-16 positive analyzed

Orientador: Regina Helena Garcia Martins / Resumo: Mercuri G. Incidência e Prevalência da Papilomatose Laríngea no Estado de São Paulo [dissertação]. Botucatu, SP: Faculdade de Medicina de Botucatu, Universidade Estadual Paulista; 2019. Introdução: A papilomatose respiratória recorrente ou papilomatose laríngea recorrente é a neoplasia benigna da laringe causada pelo Papiloma Vírus Humano, caracterizando-se pela presença de lesões proliferativas exofíticas recorrentes. A ausência de tratamento curativo, os custos relacionados ao diagnóstico, prevenção e tratamento das doenças causadas pelo HPV apresentam importante impacto econômico em todo mundo. No entanto, a falta de dados epidemiológicos nacionais, quanto à incidência e prevalência da papilomatose laríngea no Brasil não nos permite analisar o comportamento da doença em nossa população, bem como os benefícios da vacinação. Objetivo: Estimar a incidência e a prevalência da Papilomatose Laríngea no Estado de São Paulo. Métodos: Estudo populacional que objetivou levantar todos os casos diagnosticados de Papilomatose Laríngea do Estado de São Paulo, considerando os principais centros de diagnóstico desta enfermidade. Foram mapeados e selecionados os principais serviços de residência e especialização em Otorrinolaringologia do Estado de São Paulo, a partir dos dados fornecidos pela Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial (ABORL-CCF). Através do preenchimento de um questionário obtivemos os dados para estimar a prevalência e a incidência mínimas p... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mercuri G. Incidence and Prevalence of Laryngeal Papillomatosis in São Paulo State [thesis]. Botucatu, SP: Faculty of Medicine of Botucatu, Universidade Estadual Paulista; 2019. Introduction: Recurrent respiratory papillomatosis or recurrent laryngeal papillomatosis is a benign laryngeal neoplasm caused by Human Papilloma Virus, characterized by the presence of recurrent exophytic proliferative lesions. Without curative treatment, costs related to the diagnosis, prevention, and treatment of HPV-related diseases have a significant economic impact worldwide. However, the lack of national epidemiological data on the incidence and prevalence of laryngeal papillomatosis in Brazil does not allow us to analyze the behavior of the disease in our population, as well as the benefits of vaccination. Objective: To estimate the incidence and prevalence of Laryngeal Papillomatosis in the State of São Paulo. Methods: A population study was conducted aiming to raise all the diagnosed cases of Laryngeal Papillomatosis of the State of São Paulo, considering the main centers of diagnosis of this disease. The main services of residence and specialization in Otorhinolaryngology of the State of São Paulo were mapped and selected, based on the data provided by the Brazilian Association of Otorhinolaryngology and Cervical-Facial Surgery (ABORL-CCF). By completing a questionnaire we obtained the data to estimate the minimum prevalence and incidence for the State in 2017. Results: The questionnaire wa... (Complete abstract click electronic access below) / Mestre

Tam On Yi Ann. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 138-152). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.VI / ABBREVIATIONS --- p.VIII / LIST OF FIGURES --- p.X / LIST OF TABLES --- p.XI / CONTENTS --- p.XII / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- History of human papillomavirus --- p.2 / Chapter 1.2 --- Biology of human papillomavirus --- p.4 / Chapter 1.2.1 --- Classification --- p.4 / Chapter 1.2.2 --- Genome structure --- p.5 / Chapter 1.2.3 --- Properties of gene products --- p.6 / Chapter 1.2.3.1 --- El gene --- p.6 / Chapter 1.2.3.2 --- E2 gene --- p.7 / Chapter 1.2.3.3 --- E4 gene --- p.7 / Chapter 1.2.3.4 --- E5 gene --- p.7 / Chapter 1.2.3.5 --- E6 gene --- p.7 / Chapter 1.2.3.6 --- E7 gene --- p.8 / Chapter 1.2.3.7 --- LI and L2 genes --- p.9 / Chapter 1.2.4 --- Latent and lytic life cycle --- p.9 / Chapter 1.2.5 --- Host specificity --- p.10 / Chapter 1.2.6 --- Site of infection --- p.11 / Chapter 1.2.7 --- Clinical manifestations --- p.11 / Chapter 1.2.8 --- Mode of infection --- p.12 / Chapter 1.2.9 --- Detection method --- p.13 / Chapter 1.2.9.1 --- DNA hybridization --- p.13 / Chapter 1.2.9.2 --- DNA amplification methods --- p.15 / Chapter 1.2.9.3 --- Hybrid capture assay --- p.16 / Chapter 1.2.9.4 --- Other DNA detection methods --- p.17 / Chapter 1.2.9.5 --- Serology --- p.18 / Chapter 1.3 --- Biology of cervical intraepithelial neoplasia and cervical cancer --- p.19 / Chapter 1.3.1 --- Grading of severity of cervical neoplasia --- p.20 / Chapter 1.3.2 --- Treatment of cervical intraepithelial lesions --- p.22 / Chapter 1.3.3 --- Prognosis after treatment --- p.22 / Chapter 1.4 --- Epidemiology of cervical cancer --- p.23 / Chapter 1.4.1 --- Global burden of disease --- p.23 / Chapter 1.4.2 --- Local burden of disease --- p.23 / Chapter 1.4.2.1 --- Incidence --- p.23 / Chapter 1.4.2.2 --- Mortality --- p.24 / Chapter 1.4.2.3 --- Age distribution --- p.24 / Chapter 1.4.2.4 --- Trends of incidence and mortality --- p.25 / Chapter 1.4.2.5 --- Morbidity --- p.25 / Chapter 1.4.2.6 --- International comparison --- p.25 / Chapter 1.5 --- Aetiology and risk factors --- p.26 / Chapter 1.5.1 --- Human papillomavirus infection --- p.26 / Chapter 1.5.2 --- Number of sexual partners --- p.26 / Chapter 1.5.3 --- Age of first sexual intercourse --- p.27 / Chapter 1.5.4 --- Presence of other sexually-transmitted diseases --- p.28 / Chapter 1.5.5 --- Cigarette smoking --- p.29 / Chapter 1.5.6 --- Diet --- p.30 / Chapter 1.5.7 --- Oral contraceptives --- p.30 / Chapter 1.5.8 --- Parity --- p.31 / Chapter 1.5.9 --- Age --- p.32 / Chapter 1.5.10 --- Socio-economic status --- p.32 / Chapter 1.6 --- Malignant transformation of human papillomavirus infection --- p.33 / Chapter 1.7 --- Primary prevention of cervical cancer - vaccine for human papillomavirus --- p.38 / Chapter 1.7.1 --- Classification of vaccine for human papillomavirus --- p.38 / Chapter 1.7.2 --- Human papillomavirus vaccination combined with human papillomavirus screening --- p.39 / Chapter 1.8 --- Secondary prevention of cervical cancer --- p.40 / Chapter 1.8.1 --- Cytology screening --- p.40 / Chapter 1.8.2 --- Detection of human papillomavirus --- p.41 / Chapter 1.9 --- Human papillomavirus and cervical cancer --- p.43 / Chapter 1.9.1 --- Risk association between cervical cancer and human papillomavirus infection --- p.43 / Chapter 1.9.2 --- World-wide prevalence of human papillomavirus types in cervical cancer --- p.43 / Chapter 1.9.3 --- Human papillomavirus prevalence in China and Hong Kong --- p.44 / Chapter Chapter Two: --- Materials and Methods --- p.49 / Chapter 2.1 --- Ethics approval --- p.50 / Chapter 2.2 --- Sample management --- p.50 / Chapter 2.2.1 --- Sample collection --- p.50 / Chapter 2.2.2 --- Sample storage and labelling --- p.50 / Chapter 2.3 --- DNA extraction --- p.51 / Chapter 2.3.1 --- Physical extraction 226}0ؤ heating --- p.51 / Chapter 2.3.2 --- Chemical extraction - Qiagen kit extraction --- p.51 / Chapter 2.4 --- Polymerase chain reaction --- p.53 / Chapter 2.4.1 --- Controls for polymerase chain reaction --- p.53 / Chapter 2.4.2 --- Beta-globin polymerase chain reaction --- p.53 / Chapter 2.4.3 --- HPV 52-specific human papillomavirus polymerase chain reaction --- p.56 / Chapter 2.4.4 --- Consensus human papillomavirus L1 open-reading frame polymerase chain reaction --- p.57 / Chapter 2.4.4.1 --- GP5+/6+ polymerase chain reaction --- p.57 / Chapter 2.4.4.2 --- MY09/11 polymerase chain reaction --- p.60 / Chapter 2.4.4.3 --- PGMY09/11 polymerase chain reaction --- p.63 / Chapter 2.5 --- Genotyping of human papillomavirus --- p.65 / Chapter 2.5.1 --- Restriction fragment length polymorphism --- p.65 / Chapter 2.5.2 --- Reverse line-blot hybridization --- p.67 / Chapter 2.6 --- Sequencing --- p.69 / Chapter 2.6.1 --- Sequencing for HPV genotyping --- p.69 / Chapter 2.6.2 --- Sequencing of HPV 52 E6 and E7 genes --- p.69 / Chapter 2.7 --- Statistical analysis --- p.70 / Chapter Chapter Three --- Study I 226}0ؤ Comparison of Three HPV DNA Detection Methods --- p.71 / Chapter 3.1 --- Objective --- p.72 / Chapter 3.2 --- Study plan --- p.72 / Chapter 3.3 --- Results --- p.74 / Chapter 3.3.1 --- Study population --- p.74 / Chapter 3.3.2 --- Optimisation of polymerase chain reactions --- p.74 / Chapter 3.3.3 --- Method 1: GP5+/6+ PCR followed by cycle sequencing --- p.76 / Chapter 3.3.4 --- Method 2: MY09/11 PCR followed by restriction fragment length polymorphism --- p.76 / Chapter 3.3.5 --- Method 3: PGMY09/11 PCR followed by reverse line-blot hybridization --- p.77 / Chapter 3.3.6 --- Prevalence and genotype distribution of human papillomavirus infection in cervical cancer patients --- p.81 / Chapter 3.3.7 --- Detection of multiple infections --- p.81 / Chapter 3.3.8 --- Sensitivity of the detection methods --- p.82 / Chapter 3.3.9 --- Comparison of prevalence rates of human papillomavirus genotypes --- p.82 / Chapter 3.3.10 --- Comparison of genotype distribution in Hong Kong cervical cancer patients with other geographic regions --- p.83 / Chapter 3.3.11 --- Follow-up investigation of GP5+/6+ primer binding site in HPV 52 --- p.84 / Chapter 3.4 --- Discussion --- p.91 / Chapter Chapter Four --- Study II - Post-treatment Follow-up Study on Patients with High-grade Cervical Lesions --- p.95 / Chapter 4.1 --- Objective --- p.96 / Chapter 4.2 --- Study plan --- p.96 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Study population --- p.97 / Chapter 4.3.2 --- The prevalence and genotype distribution of human papillomavirus infection before treatment --- p.98 / Chapter 4.3.3 --- Persistent human papillomavirus infection --- p.99 / Chapter 4.3.4 --- Risk-factors associated with persistent human papillomavirus infection --- p.99 / Chapter 4.3.4.1 --- Excision margin status --- p.99 / Chapter 4.3.4.2 --- Multiple human papillomavirus infections --- p.99 / Chapter 4.4 --- Discussion --- p.108 / Chapter 4.4.1 --- Prevalence and genotype distribution of human papillomavirus in high-grade cervical neoplasia --- p.108 / Chapter 4.4.2 --- Risk factors for cervical intraepithelial neoplasia recurrence --- p.110 / Chapter Chapter Five --- Study III - Investigation of Human Papillomavirus 52 Sequence Variation --- p.115 / Chapter 5.1 --- Objective --- p.116 / Chapter 5.2 --- Study plan --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.3.1 --- Study population --- p.117 / Chapter 5.3.2 --- Nucleotide sequence variations --- p.119 / Chapter 5.3.2.1 --- Human papillomavirus 52 E6 open-reading frame --- p.119 / Chapter 5.3.2.2 --- Human papillomavirus 52 E7 open-reading frame --- p.123 / Chapter 5.3.2.3 --- Comparison of nucleotide sequence variations in HPV 52 E6 and E7 open-reading frame --- p.128 / Chapter 5.4 --- Discussion --- p.134 / References --- p.137

Introdução: O câncer de colo uterino é o quarto câncer mais comum no sexo feminino e um importante problema mundial de saúde pública. Recentemente, o uso de biomarcadores para melhorar a sensibilidade e especificidade do rastreamento e diagnóstico desta patologia passou a ter maior importância. Dentre estes, P16 e Ki67 passaram a ser largamente utilizados em imunohistoquímica de amostras preservadas em parafina. Entretanto, não se sabe qual a influência do tempo e temperatura de armazenamento de amostras previamente cortadas. Objetivo: O presente estudo tem por finalidade avaliar a influência do tempo de preparo e temperatura de armazenamento na imunorreatividade para P16 e Ki67 de cortes de amostras cervicais. Métodos: Amostras de blocos de parafina de câncer de colo uterino foram seccionadas e montadas em lâminas, de maneira seriada, no período de 9, 6, 3, 1 mês e tempo zero, sendo armazenadas em -20°C, 4°C e temperatura ambiente (TA). Todas as amostras então foram processadas ao mesmo tempo por imunohistoquímica para detecção de P16 e Ki67, sendo realizada leitura da mesma região do tumor nas diferentes condições. Resultados: Dos 10 casos de câncer de colo uterino, foram analisadas 75 regiões para P16 e Ki67 nas diferentes condições. A expressão de P16 e Ki67 não variou de maneira significativa ao longo do tempo nas diferentes condições de temperatura de armazenamento. Por exemplo, os cortes de 9 meses apresentaram a seguinte expressão quando armazenados a -20°C, 4°C e TA, respectivamente [mediana (p25-75)]: marcador P16 - 200 (160-300), 200 (180-300) e 200 (170-300), o que não foi estatisticamente diferente do corte em tempo zero, 200 (200-300), P=0,210; marcador Ki 67 - 210 (160-270), 210 (160-270) e 210 (145-270), o que também não foi estatisticamente diferente do corte em tempo zero, 240 (180-270), P=0,651. Conclusão: Não há influência significativa do tempo de preparo e temperatura de armazenamento de lâminas com material já cortado para a realização de imunohistoquímica posteriormente, no período de até 9 meses, para P16 e Ki67. Isto permite que, ao processarmos lâminas para HE e/ou outros marcadores, podemos reservar lâminas para posterior processamento com P16 e Ki67 sem prejuízo à imunorreatividade. / Introduction: Cervical cancer is the fourth most common cancer in women and a major public health problem in the world. Recently, the use of biomarkers to improve the sensitivity and specificity of the screening and diagnosis of this pathology has become more important. Among these, P16 and Ki67 became widely used in immunohistochemistry of samples preserved in paraffin. However, the influence of storage time and temperature of previously cut samples is not known. Aim: The aim of this study was to evaluate the influence of preparation time and storage temperature on the immunoreactivity for P16 and Ki67 of cervical specimens. Methods: Cervical cancer paraffin blocks were sectioned and mounted onto glass slides in 9, 6, 3, 1 month and zero time and stored at -20°C, 4°C and room temperature (RT). All slides were then processed at the same time by immunohistochemistry for the detection of P16 and Ki67, and the same tumor region was read under the different conditions. Results: Of the 10 cases of cervical cancer, 75 regions were analyzed for P16 and Ki67 under different conditions. Expression of P16 and Ki67 did not vary significantly over time at different storage temperature conditions. For example, the 9-month slides showed the following expression when stored at -20°C, 4°C and RT, respectively [median (p25-75)]: P16 - 200 (160-300), 200 (180-300) and 200 (170-300), which was not statistically different from zero time cut, 200 (200-300), P = 0.210; Ki 67 - 270 (160-270), 210 (160-270) and 210 (145-270), which was also not statistically different from zero-time cutoff, 240 (180-270), P = 0.651. Conclusion: There is no significant influence of the preparation time and storage temperature of slides of cervical cancer to be processed by immunohistochemistry later, in the period of up to 9 months, for P16 and Ki67. This allows, when processing slides for HE and / or other markers, we can reserve slides for further processing with P16 and Ki67 without impairing immunoreactivity.

Yeung Sze-wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references. / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.vii / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-1 / Chapter 1.1 --- Anatomy of the Cervix --- p.1-1 / Chapter 1.2 --- Histology --- p.1-1 / Chapter 1.2.1 --- Squamous Epithelium --- p.1-1 / Chapter 1.2.2 --- The Endocervical Epithelium --- p.1-3 / Chapter 1.2.3 --- The Squamo-columnar Junction --- p.1-4 / Chapter 1.2.3.1 --- The Embryology --- p.1-4 / Chapter 1.2.3.2 --- Definition --- p.1-4 / Chapter 1.3 --- Human Papillomaviruses (HPVs) --- p.1-6 / Chapter 1.3.1 --- Structure of the Viruses --- p.1-6 / Chapter 1.3.2 --- The Nomenclature --- p.1-7 / Chapter 1.3.3 --- HPVs Genomic Structure and Properties of Gene Products --- p.1-7 / Chapter 1.3.4 --- Target Tissues --- p.1-8 / Chapter 1.3.5 --- Role of HPVs in the Carcinogenesis of Lesions --- p.1-9 / Chapter 1.3.6 --- Risk Groups of HPVs --- p.1-10 / Chapter 1.4 --- Pathology --- p.1-11 / Chapter 1.4.1 --- Macroscopic Features --- p.1-11 / Chapter 1.4.2 --- Symptoms and Diagnosis --- p.1-12 / Chapter 1.4.3 --- Histopathology --- p.1-13 / Chapter 1.4.3.1 --- Histopathological Grading of Cervical Intraepithelial Neoplasia --- p.1-19 / Chapter 1.4.3.2 --- Staging of Cervical Cancer --- p.1-24 / Chapter 1.5 --- Epidemiology of Cervical Intraepithelial Neoplasia and Cervical Cancer --- p.1-27 / Chapter 1.5.1 --- Descriptive Epidemiology --- p.1-28 / Chapter 1.5.2 --- Risk Factors --- p.1-30 / Chapter 1.6 --- Human Papillomavirus Type 16 --- p.1-42 / Chapter 1.6.1 --- Role of HPV16 in CIN and Cervical Carcinoma --- p.1-42 / Chapter 1.6.2 --- Viral Load of HPV 16 in CIN --- p.1-43 / Chapter 1.6.3 --- HPV 16 Viral Load as a Screening Tool --- p.1-46 / Chapter 1.7 --- Quantitation of HPV 16 --- p.1-48 / Chapter 1.7.1 --- Methods in Viral Quantification --- p.1-48 / Chapter 1.7.2 --- Selection of Methodology --- p.1-51 / Chapter 1.7.3 --- Correlation of HPV 16 Viral Loading with Severity of Cervical Lesions --- p.1-54 / Chapter CHAPTER 2 --- AIMS OF STUDY --- p.2-1 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.3-1 / Chapter 3.1 --- Materials --- p.3-1 / Chapter 3.1.1 --- Patients and Specimens --- p.3-1 / Chapter 3.2 --- Methods --- p.3-3 / Chapter 3.2.1 --- DNA Extraction --- p.3-3 / Chapter 3.2.2 --- Polymerase Chain Reaction --- p.3-7 / Chapter 3.2.3 --- Gel Electrophoresis --- p.3-8 / Chapter 3.2.4 --- Real-time Quantitation Polymerase Chain Reaction --- p.3-11 / Chapter 3.2.5 --- Statistical Analysis --- p.3-15 / Chapter CHAPTER 4 --- RESULTS --- p.4-1 / Chapter 4.1 --- Grading of Cervical Smears --- p.4-1 / Chapter 4.2 --- Incidence of HPV 16 Detected in Cervical Smears --- p.4-2 / Chapter 4.2.1 --- Detection of HPV 16 in Women for Routine Pap Smear --- p.4-2 / Chapter 4.2.2 --- Detection of HPV 16 in Women for Colposcopic Examination --- p.4-5 / Chapter 4.3 --- Quantification of HPV 16 by Real-time PCR --- p.4-5 / Chapter 4.3.1 --- Range of Detection --- p.4-10 / Chapter 4.3.2 --- Standard Curve --- p.4-12 / Chapter 4.3.3 --- Reproducibility of Quantitative Real-time PCR --- p.4-17 / Chapter 4.3.4 --- Sensitivity of Quantitative Real-time PCR --- p.4-17 / Chapter 4.3.5 --- Detection and Quantification of HPV 16 E6/7 Genes in HPV16 Positive Cervical Scrapes --- p.4-21 / Chapter 4.4 --- Comparison of HPV 16 Copy Number Detected among Three Lesion Groups --- p.4-22 / Chapter 4.5 --- Clinical Analysis --- p.4-27 / Chapter 4.6 --- HPV 16 DNA Copy Number in Lesion Groups --- p.4-28 / Chapter CHAPTER 5 --- DISCUSSION --- p.5-1 / Chapter 5.1 --- Selection of Material (Scrapes) --- p.5-1 / Chapter 5.2 --- Detection of HPV 16 in Cervical Scrapes --- p.5-3 / Chapter 5.2.1 --- Selection of HPV Type --- p.5-3 / Chapter 5.2.2 --- Techniques in Detecting HPV Viral Load --- p.5-3 / Chapter 5.2.2.1 --- Advantages of Quantitative Real-time PCR --- p.5-6 / Chapter 5.2.2.2 --- Parameters Affecting the Performance of Real-time PCR --- p.5-8 / Chapter 5.2.3 --- Selection of Detection Sites --- p.5-9 / Chapter 5.2.4 --- Standard Curve Establishment --- p.5-10 / Chapter 5.3 --- Comparison between Real-time PCR and Traditional PCR --- p.5-12 / Chapter 5.4 --- Role of HPV Viral Load in Cervical Neoplasm --- p.5-13 / Chapter 5.5 --- HPV Infection in Hong Kong Chinese Women --- p.5-17 / Chapter 5.6 --- Clinical Significance of HPV 16 Viral Load Detected in Cervical Neoplasm --- p.5-18 / Chapter 5.7 --- Future Prospect --- p.5-20 / Chapter CHAPTER 6 --- CONCLUSION --- p.6-1 / REFERENCES --- p.R-I

Resumo: O papilomavirus humano (HPV) tem sido associado ao desenvolvimento do cancer de cabeca e pescoco. Entretanto, seu papel na carcinogenese bucal nao e bem definido. O proposito deste estudo foi investigar a prevalencia do HPV em carcinoma epidermoide de assoalho bucal e correlaciona-la com variaveis clinico-patologicas e fatores de risco, bem como verificar sua influencia na sobrevida dos pacientes estudados. A presenca do HPV foi avaliada atraves da nested PCR (nPCR) (GP5+/GP6+ e MY11/MY09) em 29 amostras parafinadas de carcinoma epidermoide de assoalho bucal. O virus foi detectado em 17.2% (5 de 29) das amostras estudadas, tendo maior prevalencia em lesoes de pacientes naotabagistas com mais de 60 anos de idade. Das amostras positivas para o HPV, 100% apresentaram-se em pacientes do sexo masculino com lesoes classificadas clinicamente em estagio III ou IV, geralmente com o diagnostico histologico de carcinoma epidermoide moderadamente diferenciado. No entanto, nao houve significancia estatistica entre as variaveis analisadas, incluindo a sobrevida. A baixa prevalencia do HPV sugere que esse virus nao participa isoladamente no desenvolvimento dos carcinomas epidermoides de assoalho bucal. / Abstract: The human papillomavirus (HPV) has been associated with the development of head and neck cancers. However, its role in oral carcinogenesis is not well defined. The aim of this study was to investigate the prevalence of HPV in mouth floor squamous cell carcinoma and correlate its presence with clinicopathologic variables and risk factors, as well as to verify its influence in the patients'survival. The HPV presence was evaluated by nested PCR (nPCR) (GP5+/GP6+ and MY11/MY09) in 29 paraffin-embedded specimens of mouth floor squamous cell carcinoma. HPV DNA was detected in 17.2% (5 of 29) of the specimens and its higher prevalence was higher in nonsmoking patients over the age of 60 years. From the HPV-DNA-positive specimens, 100% were detected in men and tumors clinically classified as stage III and IV lesions, being most of them moderately differentiated. However, no statistically significant difference was observed among the analyzed variables, including patients' survival. The low incidence of HPV DNA suggests that this virus does not participate isolatedly in the development of mouth floor squamous cell carcinoma. / Orientador: Glauco Issamu Miyahara / Coorientador: José Fernando Garcia / Banca: Marília Heffer Cantisano / Banca: Fábio Daumas Nunes / Mestre

Luk, Chun Shui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 174-189). / Abstract also in Chinese. / Declaration --- p.I / Acknowledgements --- p.II / Funding Support --- p.IV / Abstract of thesis entitled --- p.V / 論文摘要 --- p.VII / Abbreviations --- p.IX / Table of Contents --- p.XIII / List of Figures --- p.XVIII / List of Tables --- p.XX / List of Appendix --- p.XXI / Chapter Chapter One - --- Literature Review --- p.1 / Chapter 1.1 --- History of Knowledge on Human Papillomavirus --- p.1 / Chapter 1.2 --- Virology of Human Papillomavirus --- p.2 / Chapter 1.2.1 --- Taxonomic Classification of Human Papillomavirus --- p.2 / Chapter 1.2.2 --- Morphology of Human Papillomavirus --- p.3 / Chapter 1.2.3 --- The Viral Genome --- p.3 / Chapter 1.2.4 --- The Viral Gene Products --- p.5 / Chapter 1.2.4.1 --- E1 and E2 Proteins --- p.5 / Chapter 1.2.4.2 --- E4 Protein --- p.6 / Chapter 1.2.4.3 --- "E5，E6, E7 Proteins" --- p.7 / Chapter 1.2.4.4 --- L1 and L2 Proteins --- p.8 / Chapter 1.3 --- Evolution of Human Papillomavirus --- p.9 / Chapter 1.3.1 --- Rates of Evolution --- p.11 / Chapter 1.3.2 --- Co-evolution Between Human Papillomavirus and Human --- p.11 / Chapter 1.4 --- Human Papillomavirus Infection and Disease --- p.13 / Chapter 1.4.1 --- Human Papillomavirus and Cervical Cancer --- p.13 / Chapter 1.4.1.1 --- Disease Burden of Cervical Cancer --- p.13 / Chapter 1.4.1.2 --- Epidemiology of Cervical Cancer --- p.14 / Chapter 1.4.1.3 --- Distribution of HPV types in Cervical Precancerous Lesions --- p.14 / Chapter 1.4.2 --- Human Papillomavirus and Non-cervical Diseases --- p.15 / Chapter 1.5 --- Human Papillomavirus Type 58 --- p.15 / Chapter 1.5.1 --- Biology of Human Papillomavirus Type 58 --- p.15 / Chapter 1.5.2 --- Epidemiology of Human Papillomavirus Type 58 Infections --- p.16 / Chapter Chapter Two - --- Background and Objectives of Study --- p.17 / Chapter 2.1 --- Background of study --- p.17 / Chapter 2.1.1 --- The Need for Research on HPV58 --- p.17 / Chapter 2.1.2 --- Intratypic Classification System for HPV --- p.17 / Chapter 2.2 --- Implication and Impact of Study --- p.19 / Chapter 2.2.1 --- Implication on HPV Virology --- p.19 / Chapter 2.2.2 --- HPV58 Classification --- p.19 / Chapter 2.2.3 --- Improvement on in the Detection of HPV58 --- p.20 / Chapter 2.2.4 --- Implication on Vaccine Development --- p.20 / Chapter 2.3 --- Objectives of Study --- p.21 / Chapter 2.3.1 --- To Generate a Database for Intratypic Variation of Different Gene Regions of HPV58 --- p.21 / Chapter 2.3.2 --- To Study the Variability of Seven Gene Regions of HPV58 --- p.21 / Chapter 2.3.3 --- To Study the Geographical Distribution of HPV58 Variants --- p.22 / Chapter 2.3.4 --- To Study the Phylogeny of HPV58 --- p.22 / Chapter 2.3.5 --- To Develop an Intratypic Classification System for HPV58 --- p.22 / Chapter 2.3.6 --- To Predict the Effectiveness of Commonly Used Primers on the Detection of HPV58 --- p.22 / Chapter Chapter Three - --- Materials and Methods --- p.24 / Chapter 3.1 --- Overall Study Design --- p.24 / Chapter 3.2 --- Study Population --- p.25 / Chapter 3.3 --- Sample Processing and Storage --- p.25 / Chapter 3.4 --- Primer Design --- p.26 / Chapter 3.5 --- Specimen Quality Assessment and Sample Selection --- p.30 / Chapter 3.6 --- Amplification of Gene Region --- p.30 / Chapter 3.7 --- Agarose Gel Electrophoresis --- p.34 / Chapter 3.8 --- Sequencing Reaction --- p.34 / Chapter 3.8.1 --- Purification of PCR Product --- p.34 / Chapter 3.8.2 --- Sequencing Reaction --- p.35 / Chapter 3.8.3 --- Purification of Fluorescence-labelled Product --- p.35 / Chapter 3.8.4 --- Sequence Identification --- p.35 / Chapter 3.9 --- Sequence Analysis --- p.36 / Chapter 3.9.1 --- Sequence Editing --- p.36 / Chapter 3.9.2 --- Criteria for Confirming the identity of HPV58 --- p.36 / Chapter 3.9.3 --- Identification of Variants --- p.38 / Chapter 3.9.4 --- Identification of Conserved and Variable Regions --- p.39 / Chapter 3.9.5 --- Phylogenetic Analysis --- p.40 / Chapter 3.9.5.1 --- Construction of Maximum Likelihood Tree --- p.40 / Chapter 3.9.5.2 --- Bootstrap Analysis --- p.41 / Chapter 3.9.5.3 --- Bayesian Phylogenetic Analysis --- p.42 / Chapter 3.9.5.4 --- Non-synonymous to Synonymous Substitution Rate Ratio (dN/dS) --- p.42 / Chapter 3.9.6 --- Evaluation of Performance of Commonly Used Primers --- p.43 / Chapter Chapter Four - --- Results --- p.44 / Chapter 4.1 --- Specimen Quality Assessment and HPV58 Confirmation --- p.44 / Chapter 4.2 --- HPV58 Genome Variability --- p.44 / Chapter 4.2.1 --- E6 Open Reading Frame --- p.45 / Chapter 4.2.2 --- E7 Open Reading Frame --- p.51 / Chapter 4.2.3 --- E2 Open Reading Frame --- p.56 / Chapter 4.2.4 --- E4 Open Reading Frame --- p.61 / Chapter 4.2.5 --- E5 Open Reading Frame --- p.66 / Chapter 4.2.6 --- L1 Open Reading Frame --- p.71 / Chapter 4.2.7 --- Long Control Region --- p.88 / Chapter 4.2.8 --- Whole HPV genome --- p.94 / Chapter 4.3 --- Evaluation of Commonly Used Primers --- p.99 / Chapter 4.3.1 --- PGMY09/11 Primers --- p.99 / Chapter 4.3.2 --- MY09/11 Primers --- p.99 / Chapter 4.3.3 --- GP5+/6+ Primers --- p.100 / Chapter 4.3.4 --- SPF Primers --- p.100 / Chapter 4.3.5 --- L1F/L1R Primers --- p.101 / Chapter Chapter Five - --- Discussion --- p.111 / Chapter 5.1 --- Overall Variation of HPV58 Genome --- p.111 / Chapter 5.2 --- Variability of Each Gene Region --- p.114 / Chapter 5.2.1 --- E6 Open Reading Frame --- p.115 / Chapter 5.2.2 --- E7 Open Reading Frame --- p.116 / Chapter 5.2.3 --- E2 Open Reading Frame --- p.117 / Chapter 5.2.4 --- E4 Open Reading Frame --- p.118 / Chapter 5.2.5 --- E5 Open Reading Frame --- p.119 / Chapter 5.2.6 --- L1 Open Reading Frame --- p.120 / Chapter 5.2.7 --- Long Control Region --- p.121 / Chapter 5.3 --- Phylogenetics of HPV58 --- p.122 / Chapter 5.3.1 --- Natural Selection Pressure --- p.122 / Chapter 5.3.2 --- HPV58 Lineage Using the L1 Gene --- p.124 / Chapter 5.3.3 --- Methods for Lineage Identification --- p.125 / Chapter 5.3.4 --- Geographical Distribution of the Four Lineages --- p.126 / Chapter 5.3.5 --- Recombination --- p.127 / Chapter 5.4 --- Evaluation of Commonly Used Primers --- p.128 / Chapter 5.5 --- Limitations of the Current Study --- p.129 / Chapter 5.6 --- Future Studies --- p.130 / Appendix --- p.133 / References --- p.174

子宮頸癌是女性的主要癌症殺手，而人類乳頭瘤病毒 (HPV) 則是子宮頸癌形成的必要條件之一。HPV16型及HPV18型是全球最普遍的高危型HPV；而另一方面，HPV52及HPV58兩型在東亞地區的流行程度比世界其他地區為高。 / 過往有科學研究顯示HPV病毒載量的高低是引致高度癌前病變的重要決定因素，也有研究指出病毒載量與病變的嚴重程度成正比例，但同時亦有研究指兩者並無關係。HPV基因組可以兩種物理形態存在：游離型及整合型。HPV的E2基因可對E6及E7致癌基因產生重要的調節作用，而當HPV病毒與宿主染色體整合後，可使E2基因斷裂，因而令控制E6及E7致癌基因表達的負反饋基制失效。 / 本研究假設高病毒載量及由HPV基因組整合所造成的E2基因斷裂，並非引致子宮頸癌的僅一途徑。本研究分析了在不同程度的子宮頸細胞病變下，HPV16型、18型、52型及58型的病毒載量及基因整合情況。其中，有關HPV16型的研究部份更深入地探討了E6/7 mRNA的轉錄水平、E2和LCR的序列變異及E2結合位點的甲基化情況，最終希望能找出除了病毒基因整合之外的另一種致癌機理。 / 本研究的結果顯示，在不同HPV型所引致的子宮頸細胞病變中，病毒載體及病變程度之間的關係也存有差異；而根據管家基因的數量來為細胞DNA標準化，對準確分析不同程度子宮頸細胞病變的實驗結果至關重要。本研究的一項重要發現是部份侵襲性癌細胞只含有游離型HPV基因組；而在只含游離HPV基因組的侵襲性子宮頸癌樣本中，有三種E6/E7 mRNA的抄錄本水平與只含整合型基因組的樣本相若，反映在只含游離型HPV基因組的侵襲性子宮頸癌樣本中，E6/ E7 mRNA的表達量亦有上調。最重要的是，此表達量的上調並非由基因整合或E2基因斷裂所引致。 / 在只含有游離型病毒基因組的侵襲性子宮頸癌樣本中，E6及E7致癌基因表達上調的另一種機理，很可能是HPV16啟動區內E2結合位點上的CpG位點出現甲基化。這項觀察解釋及支持了當E2蛋白因結合位點甲基化而失去對E6及E7基因轉錄的抑制功能時，E6及E7致癌蛋白仍能保持高水平，而兩種蛋白產生協同作用，令細胞轉型及出現癌變。總結之言，本實驗也肯定了HPV整合並非導致子宮頸癌形成的唯一機理。 / Cervical cancer is a major cause of cancer-related death in women worldwide. Human papillomavirus (HPV) is essential, though not sufficient, to cause cervical cancer. HPV16 and HPV18 are the most prevalent high-risk types worldwide, whereas, HPV52 and HPV58 also show a notable higher prevalence in East Asia than in other parts of the world. / Studies have suggested that HPV viral load is an important determinant for the development of high-grade lesions. While some studies observed a positive correlation between viral load and disease severity, others have reported no association. The HPV genome can exist in two physical forms, episomal or integrated. The E2 gene, encoded by HPV has an important role in the regulation of E6 and E7 viral oncogenes. When HPV integrates into the host chromosome, it may result in disruption of the E2 gene thereby its control on the expression of the E6 and E7. / The hypothesis for this study was that high viral load and disruption of E2 gene associated with integration of HPV into the host genome was not the only pathway leading to cervical cancer development. In this study, the viral load and integration profile for HPV types 16, 18, 52 and 58 among different severity of cervical lesions were analyzed. Further detailed studies were performed on HPV16 with emphases on E6/E7 mRNA transcript levels, E2 and LCR sequence variation and the methylation status of two E2 binding sites. The ultimate aim was to determine what other alternative mechanisms exist apart from viral integration to drive the oncogenicity of HPV that lead to the development of cervical cancer. / The results showed that the relationship between viral load and disease varied between different HPV types and that normalization of cellular DNA input using a housekeeping gene was crucial for accurate interpretation among different cervical lesion grades. A key finding from this study was that a substantial proportion of invasive cervical carcinomas were found to contain the purely episomal form of the HPV genome. The levels of the three E6/E7 mRNA transcripts species in invasive cervical carcinomas containing the pure episomal form of the viral genome were found to be similar to those with pure integrated forms. This observation suggested that invasive cervical carcinoma samples containing the episomal form of the HPV genome were also mediated by the up-regulated E6/E7 mRNA expression. More importantly, this up-regulation in E6/E7 mRNA expression did not depend on integration and disruption of the E2 gene. / The alternative mechanism that up-regulated of the expression of E6 and E7 oncogene found in invasive cervical carcinoma samples harbouring the episomal form of the viral genome was likely to be a consequence of methylation of CpG sites in the two E2 binding sites at the promoter region of HPV16. This observation explained and supported that the repressive role of E2 on E6 and E7 transcriptional regulation was abolished due to methylation of the E2 binding sites, and that a sustained level of the E6 and E7 oncoproteins was maintained, working in synergy in cell transformation and in carcinogenesis. These observations confirmed the hypothesis that HPV integration was not the only mechanism leading to the development of cervical cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Lai Ken Jo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 233-248). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.I / Abstract of thesis --- p.IV / 論文摘要 --- p.VII / Publications --- p.IX / Contents --- p.X / Figures --- p.XV / Tables --- p.XVIII / Abbreviations --- p.XIX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cervical Cancer --- p.2 / Chapter 1.1.1 --- Cervical Cytology Screening --- p.3 / Chapter 1.1.2 --- Classification System for Cervical Squamous Cell Dysplasia --- p.4 / Chapter 1.1.3 --- Histological Grading of Cervical Lesions --- p.6 / Chapter 1.1.4 --- Development of Cervical Cancer --- p.6 / Chapter 1.2 --- Structure of HPV --- p.7 / Chapter 1.1.1 --- HPV Genome Organization --- p.8 / Chapter 1.1.2 --- The E1 Protein --- p.10 / Chapter 1.1.3 --- The E2 Protein --- p.10 / Chapter 1.1.4 --- The E4 Protein --- p.13 / Chapter 1.1.5 --- The E5 Protein --- p.13 / Chapter 1.1.6 --- The E6 Protein --- p.14 / Chapter 1.1.7 --- The E7 Protein --- p.14 / Chapter 1.1.8 --- The L1 Protein --- p.15 / Chapter 1.1.9 --- The L2 Protein --- p.16 / Chapter 1.1.10 --- The Long Control Region --- p.17 / Chapter 1.3 --- HPV and Cervical Cancer --- p.19 / Chapter 1.3.1 --- HPV is an Etiological Cause of Cervical Cancer --- p.19 / Chapter 1.3.2 --- Establishment of HPV Infection --- p.20 / Chapter 1.3.3 --- Regulation and Control of HPV Viral Gene Transcription --- p.23 / Chapter 1.3.4 --- Viral Oncogene Expression by Alternative RNA Splicing --- p.23 / Chapter 1.3.5 --- DNA Methylation in Viral Oncogene Expression --- p.24 / Chapter 1.3.6 --- The Roles of E6 and E7 Protein in Cervical Carcinogenesis --- p.26 / Chapter Chapter 2 --- Controversies and Hypothesis --- p.33 / Chapter 2.1 --- Controversies in Mechanism of Cervical Carcinogenesis --- p.34 / Chapter 2.1.1 --- Viral Integration and Risk of Cervical Cancer Development --- p.34 / Chapter 2.1.2 --- Viral Load and Risk of Cervical Cancer Development --- p.35 / Chapter 2.2 --- Hypothesis of Study --- p.37 / Chapter 2.2.1 --- Study Design --- p.38 / Chapter Chapter 3 --- Materials and Methods --- p.41 / Chapter 3.1 --- Patient Recruitment and Sample Preparation --- p.42 / Chapter 3.1.1 --- Study subject recruitment --- p.42 / Chapter 3.1.2 --- Collection of cytology samples --- p.43 / Chapter 3.1.3 --- Collection of cervical biopsy samples --- p.44 / Chapter 3.2 --- Nucleic Acid Extraction and Preparation --- p.44 / Chapter 3.2.1 --- Extraction of DNA from cervical cytology samples --- p.44 / Chapter 3.2.2 --- Extraction of DNA from cervical biopsy samples --- p.45 / Chapter 3.2.3 --- Extraction of RNA from cervical cytology samples --- p.45 / Chapter 3.2.4 --- Extraction of RNA from cervical biopsy samples --- p.46 / Chapter 3.3 --- Detection and Genotyping of Human Papillomavirus --- p.46 / Chapter 3.4 --- Determination of Viral Load using Real-Time Polymerase Chain Reaction --- p.47 / Chapter 3.4.1 --- Optimization of HPV16, 18, 52 and 58 E7 real-time PCR --- p.48 / Chapter 3.4.2 --- Optimization of housekeeping gene real-time PCR --- p.50 / Chapter 3.4.3 --- Determination of HPV16, 18, 52 and 58 viral load --- p.50 / Chapter 3.5 --- Determination of HPV Genome Physical Status --- p.53 / Chapter 3.5.1 --- HPV E2 gene primer design --- p.53 / Chapter 3.5.2 --- Optimization of HPV16, 18, 52 and 58 E2 Real-time PCR --- p.56 / Chapter 3.5.3 --- Determination of the HPV genome physical status --- p.59 / Chapter 3.6 --- Evaluation of Housekeeping Genes for Normalization of Viral Gene Expression --- p.62 / Chapter 3.6.1 --- Optimization of housekeeping gene real-time PCR --- p.62 / Chapter 3.6.2 --- Quantitation of RNA and DNase treatment --- p.66 / Chapter 3.6.3 --- cDNA synthesis from the extracted RNA --- p.67 / Chapter 3.6.4 --- Detection of five housekeeping gene levels from cervical cytology samples by real-time PCR --- p.67 / Chapter 3.6.5 --- Data analyses --- p.68 / Chapter 3.7 --- Quantitation of HPV16 mRNA Transcripts --- p.69 / Chapter 3.7.1 --- Preparation of RNA from CaSki cells --- p.69 / Chapter 3.7.2 --- Amplification of mRNA transcripts from CaSki cells --- p.69 / Chapter 3.7.3 --- Amplification of artificial mRNA transcript E6*II --- p.73 / Chapter 3.7.4 --- Gel purification of mRNA transcript amplicons --- p.73 / Chapter 3.7.5 --- Cloning of E6 mRNA transcripts --- p.74 / Chapter 3.7.6 --- Confirmation of the mRNA transcript inserts --- p.74 / Chapter 3.8 --- Quantitation HPV16 E6 mRNA Transcript Levels Using Real-Time PCR --- p.79 / Chapter 3.8.1 --- mRNA transcript primer and probe design --- p.79 / Chapter 3.8.2 --- Optimization of real-time PCR for the detection of mRNA transcripts --- p.82 / Chapter 3.8.3 --- Determination of mRNA transcript levels from invasive carcinomas --- p.83 / Chapter 3.8.4 --- Normalization of mRNA transcript expression with a housekeeping gene --- p.84 / Chapter 3.9 --- Sequence Variation of the HPV16 E2 and Long Control Region --- p.84 / Chapter 3.9.1 --- Identification of sequence variation of the E2 gene --- p.84 / Chapter 3.9.2 --- Identification of sequence variation of the long control region --- p.87 / Chapter 3.1 --- Detection of Methylation Status of E2BS1 and E2BS2 on the LCR using Pyrosequencing --- p.87 / Chapter 3.10.1 --- Bisulfite DNA conversion --- p.87 / Chapter 3.10.2 --- Amplification of E2 binding site regions on the LCR --- p.88 / Chapter 3.10.3 --- Purification of PCR product prior to pyrosequencing --- p.92 / Chapter 3.10.4 --- Quantitation of methylation using pyrosequencing --- p.92 / Chapter Chapter 4 --- Results --- p.93 / Chapter Hypothesis 1 --- p.94 / Chapter Results of Study Part: 1 --- p.95 / Chapter 4.1 --- Human Papillomavirus Type 16 Viral Load and Genome Physical Status --- p.96 / Chapter 4.1.1 --- E7 viral load --- p.96 / Chapter 4.1.2 --- Viral genome physical status --- p.100 / Chapter 4.1.3 --- E2 disruption site --- p.105 / Chapter 4.2 --- Human Papillomavirus Type 18 Viral Load and Genome Physical Status --- p.107 / Chapter 4.2.1 --- E7 viral load --- p.107 / Chapter 4.2.2 --- Viral genome physical status --- p.110 / Chapter 4.2.3 --- E2 disruption site --- p.113 / Chapter 4.2.4 --- Infection status --- p.116 / Chapter 4.2.5 --- Adeno/adenosquamous carcinoma versus squamous cell carcinoma --- p.119 / Chapter 4.3 --- Human Papillomvirus Type 52 Viral Load and Genome Physical Status --- p.120 / Chapter 4.3.1 --- E7 viral load --- p.120 / Chapter 4.3.2 --- Viral genome physical status --- p.123 / Chapter 4.3.3 --- E2 disruption site --- p.126 / Chapter 4.3.4 --- Infection status --- p.129 / Chapter 4.4 --- Human Papillomavirus Type 58 Viral Load and Genome Physical Status --- p.131 / Chapter 4.4.1 --- E7 viral load --- p.131 / Chapter 4.4.2 --- Viral genome physical status --- p.133 / Chapter 4.4.3 --- E2 disruption site --- p.134 / Chapter 4.4.4 --- Infection status --- p.137 / Chapter 4.5 --- Summary of Study Part 1: --- p.140 / Chapter Hypothesis 2 --- p.141 / Chapter Results of Study Part 2: --- p.142 / Chapter 4.6 --- Housekeeping Gene mRNA Expression Level --- p.143 / Chapter 4.6.1 --- Expression levels across different grades of cervical lesion --- p.143 / Chapter 4.6.2 --- Expression stability of housekeeping genes --- p.145 / Chapter 4.7 --- Summary of Study Part 2: --- p.149 / Chapter Results of Study Part: 3 --- p.150 / Chapter 4.8 --- HPV16 mRNA Transcript Expression Level --- p.151 / Chapter 4.8.1 --- HPV16 viral genome physical status --- p.151 / Chapter 4.8.2 --- HPV16 E2 disruption site --- p.151 / Chapter 4.8.3 --- Expression level of E6/E7 mRNA transcripts --- p.155 / Chapter 4.8.4 --- Expression level of E6/E7 mRNA transcripts and viral genome physical status --- p.157 / Chapter 4.8.5 --- Expression level of E6/E7 mRNA transcripts and E2 gene disruption status --- p.161 / Chapter 4.9 --- Summary of Study Part 3: --- p.163 / Chapter Hypothesis 3 --- p.165 / Chapter Results of Study Part 4: --- p.166 / Chapter 4.1 --- HPV 16 E2 Gene Sequence Variation --- p.167 / Chapter 4.10.1 --- Sequence variation of E2 gene --- p.167 / Chapter 4.10.2 --- Sequence variation and viral genome physical status --- p.168 / Chapter 4.10.3 --- Sequence variation in the E2 binding sites --- p.169 / Chapter 4.10.4 --- Sequence variations of E2 in HPV16 cancer derived cell lines --- p.170 / Chapter 4.11 --- HPV16 Long Control Region Sequence Variation --- p.174 / Chapter 4.11.1 --- Sequence variation of LCR --- p.174 / Chapter 4.11.2 --- Sequence variation and viral genome physical status --- p.175 / Chapter 4.11.3 --- Sequence variation in E2 binding sites --- p.176 / Chapter 4.11.4 --- Sequence variation of LCR in HPV16 cancer derived cell lines --- p.176 / Chapter 4.12 --- Summary of Study Part 4: --- p.183 / Chapter Hypothesis 4 --- p.185 / Chapter 4.13 --- Methylation Status of E2 Binding Sites --- p.187 / Chapter 4.13.1 --- Proportion methylation in E2 binding sites --- p.187 / Chapter 4.13.2 --- Methylation in invasive carcinomas according to the viral genome physical status --- p.191 / Chapter 4.14 --- Summary of Study Part 5: --- p.195 / Chapter Chapter 5 --- Discussion --- p.196 / Chapter 5.1 --- Viral Load --- p.197 / Chapter 5.2 --- Viral Integration --- p.200 / Chapter 5.2.1 --- HPV16 Viral Load and Physical Status --- p.201 / Chapter 5.2.2 --- HPV18 Viral Load and Physical Status --- p.204 / Chapter 5.2.3 --- HPV52 Viral Load and Physical Status --- p.207 / Chapter 5.2.4 --- HPV58 Viral Load and Physical Status --- p.210 / Chapter 5.2.5 --- Viral Load and Physical Status Summary --- p.214 / Chapter 5.3 --- HPV16 E6/E7 mRNA Transcript and Genome Physical Status --- p.215 / Chapter 5.4 --- HPV16 E2 Sequence Variation and Genome Physical Status --- p.218 / Chapter 5.5 --- HPV16 LCR Sequence Variation and Genome Physical Status --- p.222 / Chapter 5.6 --- Methylation of HPV16 E2 Binding Sites and Genome Physical Status --- p.225 / Chapter 5.7 --- Conclusions --- p.230 / Chapter 5.8 --- Implication of Current Findings and Future Work --- p.231 / References --- p.233

The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation. / In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein. / Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.

Introdução: O câncer de colo uterino é o quarto câncer mais comum no sexo feminino e um importante problema mundial de saúde pública. Recentemente, o uso de biomarcadores para melhorar a sensibilidade e especificidade do rastreamento e diagnóstico desta patologia passou a ter maior importância. Dentre estes, P16 e Ki67 passaram a ser largamente utilizados em imunohistoquímica de amostras preservadas em parafina. Entretanto, não se sabe qual a influência do tempo e temperatura de armazenamento de amostras previamente cortadas. Objetivo: O presente estudo tem por finalidade avaliar a influência do tempo de preparo e temperatura de armazenamento na imunorreatividade para P16 e Ki67 de cortes de amostras cervicais. Métodos: Amostras de blocos de parafina de câncer de colo uterino foram seccionadas e montadas em lâminas, de maneira seriada, no período de 9, 6, 3, 1 mês e tempo zero, sendo armazenadas em -20°C, 4°C e temperatura ambiente (TA). Todas as amostras então foram processadas ao mesmo tempo por imunohistoquímica para detecção de P16 e Ki67, sendo realizada leitura da mesma região do tumor nas diferentes condições. Resultados: Dos 10 casos de câncer de colo uterino, foram analisadas 75 regiões para P16 e Ki67 nas diferentes condições. A expressão de P16 e Ki67 não variou de maneira significativa ao longo do tempo nas diferentes condições de temperatura de armazenamento. Por exemplo, os cortes de 9 meses apresentaram a seguinte expressão quando armazenados a -20°C, 4°C e TA, respectivamente [mediana (p25-75)]: marcador P16 - 200 (160-300), 200 (180-300) e 200 (170-300), o que não foi estatisticamente diferente do corte em tempo zero, 200 (200-300), P=0,210; marcador Ki 67 - 210 (160-270), 210 (160-270) e 210 (145-270), o que também não foi estatisticamente diferente do corte em tempo zero, 240 (180-270), P=0,651. Conclusão: Não há influência significativa do tempo de preparo e temperatura de armazenamento de lâminas com material já cortado para a realização de imunohistoquímica posteriormente, no período de até 9 meses, para P16 e Ki67. Isto permite que, ao processarmos lâminas para HE e/ou outros marcadores, podemos reservar lâminas para posterior processamento com P16 e Ki67 sem prejuízo à imunorreatividade. / Introduction: Cervical cancer is the fourth most common cancer in women and a major public health problem in the world. Recently, the use of biomarkers to improve the sensitivity and specificity of the screening and diagnosis of this pathology has become more important. Among these, P16 and Ki67 became widely used in immunohistochemistry of samples preserved in paraffin. However, the influence of storage time and temperature of previously cut samples is not known. Aim: The aim of this study was to evaluate the influence of preparation time and storage temperature on the immunoreactivity for P16 and Ki67 of cervical specimens. Methods: Cervical cancer paraffin blocks were sectioned and mounted onto glass slides in 9, 6, 3, 1 month and zero time and stored at -20°C, 4°C and room temperature (RT). All slides were then processed at the same time by immunohistochemistry for the detection of P16 and Ki67, and the same tumor region was read under the different conditions. Results: Of the 10 cases of cervical cancer, 75 regions were analyzed for P16 and Ki67 under different conditions. Expression of P16 and Ki67 did not vary significantly over time at different storage temperature conditions. For example, the 9-month slides showed the following expression when stored at -20°C, 4°C and RT, respectively [median (p25-75)]: P16 - 200 (160-300), 200 (180-300) and 200 (170-300), which was not statistically different from zero time cut, 200 (200-300), P = 0.210; Ki 67 - 270 (160-270), 210 (160-270) and 210 (145-270), which was also not statistically different from zero-time cutoff, 240 (180-270), P = 0.651. Conclusion: There is no significant influence of the preparation time and storage temperature of slides of cervical cancer to be processed by immunohistochemistry later, in the period of up to 9 months, for P16 and Ki67. This allows, when processing slides for HE and / or other markers, we can reserve slides for further processing with P16 and Ki67 without impairing immunoreactivity.