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

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

Thesis (Ph. D.)--University of New South Wales, 2007. / Title from caption (viewed on May 7, 2008).

O câncer do colo do útero ocasiona 7% dos óbitos por câncer na população feminina brasileira, com taxa de incidência estimada em 15,33/100 mil, sendo a infecção pelo papilomavírus humano (HPV) causa necessária. O rastreamento com citologia do esfregaço cervical convencional é ação escolhida no Brasil e em outros países para prevenir e controlar o câncer, através da detecção precoce das lesões pré-neoplásicas. Porém, apresenta falhas na cobertura, com desigualdade de acesso, e na qualidade, devido aos problemas nas etapas operacionais, desde a coleta na unidade até a interpretação dos resultados. O rastreamento é oportunístico, e uma alternativa organizada pode ser a utilização de cadastros populacionais, como os disponíveis pela Estratégia Saúde da Família (ESF). A efetividade do rastreamento pode ser aumentada com a incorporação de novas técnicas, destacando-se aquelas de biologia molecular, que testam o HPV nas mulheres, em acréscimo ao rastreamento com citologia ou a substituindo integralmente. O conhecimento acerca dos fatores relacionados à infecção vem sendo ampliado para compreender por que mulheres que se encontram em risco semelhante de transmissão apresentam infecção e outras, não. A inexistência de um sistema de vigilância dos tipos circulantes de HPV prejudica a avaliação do cenário atual, inclusive no que se refere à recente implantação da vacinação contra o HPV no calendário básico de vacinação. Neste estudo, o objetivo foi estimar a prevalência de infecção pelo HPV no grupo de mulheres estudadas, relacionando estes achados aos fatores relacionados à infecção, que podem auxiliar na identificação de mulheres mais vulneráveis à infecção e à presença de lesões pré-neoplásicas. 2062 mulheres da periferia de Juiz de Fora, após serem convocadas para rastreamento, participaram do estudo, desenvolvido em duas unidades com a ESF, tendo respondido a um questionário padronizado, submetidas ao exame citológico cervical convencional e testadas para o HPV com o teste cobas 4800 (Roche). A adesão das mulheres convocadas foi semelhante àquela observada em estudos de outros delineamentos, os quais podem contribuir para o seu entendimento. Foram calculadas estimativas de prevalência de infecção pelo HPV segundo características selecionadas. A prevalência global de infecção pelo HPV foi 12,61%. A análise multivariada por regressão de Poisson com variância robusta mostrou associação estatisticamente significativa para ser solteira, consumir bebida alcoólica e ter três ou mais parceiros sexuais ao longo da vida, com razões de prevalência ajustadas de 1,40, 1,44 e 1,35, respectivamente. A prevalência de lesões precursoras do câncer do colo do útero foi 7,27%. A qualidade do esfregaço, avaliada pela representatividade dos epitélios coletados, mostrou que há variação da prevalência de lesões pré-neoplásicas mediante qualidade do esfregaço, porém, não há variação nos resultados do teste HPV mediante a representatividade celular, achado que consistente com uma sensibilidade maior e possivelmente um melhor valor preditivo positivo. A testagem do HPV mostrou ser útil, especialmente entre mulheres com resultado de citologia normal. / Cervical cancer is the cause of 7% of the deaths among Brazilian women, with a estimated incidence rate in 15,33/100 thousand for all over the country, and HPV infection is a necessary cause for this disease. The screening with conventional cervical cytology is the strategy chosen in Brazil and other countries to prevent and control cervix cancer, through early detection of precancerous lesions. However, the screening has failures in the coverage, inequality of access, and quality, attributable to operational aspects in the several steps, since the collection of smears in the health facilities until results interpretation. The screening is also opportunistic, and to use population databases of Health Family Strategy can be an organized alternative. The screening effectiveness can be increased by incorporation of new techniques, especially those related to molecular biology, which provide HPV test for women, that can improve the screening or replace it. Knowledge about risk factors for infection is necessary to understand why some women groups who live under similar conditions are more vulnerable to infection than others. There is not a surveillance system for circulating types of HPV, which harms the evaluation of the circumstances, even more now in relation to recent implantation of HPV vaccination in the public basic calendar. In this study, our goal was to estimate the prevalence of HPV infection in women of a certain population, correlating the results to the risk factors for HPV infection, and evaluate the results in a quality assay perspective. 2062 women living in a suburban area in Juiz de Fora, Minas Gerais, participated, responding to a questionnaire, submitted to conventional cervical cytology and cobas 4800 HPV test (Roche) for DNA detection. The entrance of women invited was lower than expected, so studies of other designs can contribute to its understanding. HPV infection prevalence estimates were calculated according to selected features. The overall prevalence of HPV infection was 12,61%. Multivariate analysis using Poisson regression with robust variance showed statistically significant association with being single, consuming alcohol and having three or more sexual partners over a lifetime, with adjusted prevalence ratio 1,40, 1,44 e 1,35, respectively. The prevalence of precursor lesions of cervical cancer was 7,27%. The quality of the smear, evaluated by the representativeness of the collected epithelia showed that there is variation in the prevalence of precancerous lesions by quality of smears; however, there is no variation in HPV test results by cell representation, a finding that is consistent with a higher sensitivity and a better positive predictive value. Testing HPV proved to be useful, especially among women with normal cytology results.

O câncer do colo do útero ocasiona 7% dos óbitos por câncer na população feminina brasileira, com taxa de incidência estimada em 15,33/100 mil, sendo a infecção pelo papilomavírus humano (HPV) causa necessária. O rastreamento com citologia do esfregaço cervical convencional é ação escolhida no Brasil e em outros países para prevenir e controlar o câncer, através da detecção precoce das lesões pré-neoplásicas. Porém, apresenta falhas na cobertura, com desigualdade de acesso, e na qualidade, devido aos problemas nas etapas operacionais, desde a coleta na unidade até a interpretação dos resultados. O rastreamento é oportunístico, e uma alternativa organizada pode ser a utilização de cadastros populacionais, como os disponíveis pela Estratégia Saúde da Família (ESF). A efetividade do rastreamento pode ser aumentada com a incorporação de novas técnicas, destacando-se aquelas de biologia molecular, que testam o HPV nas mulheres, em acréscimo ao rastreamento com citologia ou a substituindo integralmente. O conhecimento acerca dos fatores relacionados à infecção vem sendo ampliado para compreender por que mulheres que se encontram em risco semelhante de transmissão apresentam infecção e outras, não. A inexistência de um sistema de vigilância dos tipos circulantes de HPV prejudica a avaliação do cenário atual, inclusive no que se refere à recente implantação da vacinação contra o HPV no calendário básico de vacinação. Neste estudo, o objetivo foi estimar a prevalência de infecção pelo HPV no grupo de mulheres estudadas, relacionando estes achados aos fatores relacionados à infecção, que podem auxiliar na identificação de mulheres mais vulneráveis à infecção e à presença de lesões pré-neoplásicas. 2062 mulheres da periferia de Juiz de Fora, após serem convocadas para rastreamento, participaram do estudo, desenvolvido em duas unidades com a ESF, tendo respondido a um questionário padronizado, submetidas ao exame citológico cervical convencional e testadas para o HPV com o teste cobas 4800 (Roche). A adesão das mulheres convocadas foi semelhante àquela observada em estudos de outros delineamentos, os quais podem contribuir para o seu entendimento. Foram calculadas estimativas de prevalência de infecção pelo HPV segundo características selecionadas. A prevalência global de infecção pelo HPV foi 12,61%. A análise multivariada por regressão de Poisson com variância robusta mostrou associação estatisticamente significativa para ser solteira, consumir bebida alcoólica e ter três ou mais parceiros sexuais ao longo da vida, com razões de prevalência ajustadas de 1,40, 1,44 e 1,35, respectivamente. A prevalência de lesões precursoras do câncer do colo do útero foi 7,27%. A qualidade do esfregaço, avaliada pela representatividade dos epitélios coletados, mostrou que há variação da prevalência de lesões pré-neoplásicas mediante qualidade do esfregaço, porém, não há variação nos resultados do teste HPV mediante a representatividade celular, achado que consistente com uma sensibilidade maior e possivelmente um melhor valor preditivo positivo. A testagem do HPV mostrou ser útil, especialmente entre mulheres com resultado de citologia normal. / Cervical cancer is the cause of 7% of the deaths among Brazilian women, with a estimated incidence rate in 15,33/100 thousand for all over the country, and HPV infection is a necessary cause for this disease. The screening with conventional cervical cytology is the strategy chosen in Brazil and other countries to prevent and control cervix cancer, through early detection of precancerous lesions. However, the screening has failures in the coverage, inequality of access, and quality, attributable to operational aspects in the several steps, since the collection of smears in the health facilities until results interpretation. The screening is also opportunistic, and to use population databases of Health Family Strategy can be an organized alternative. The screening effectiveness can be increased by incorporation of new techniques, especially those related to molecular biology, which provide HPV test for women, that can improve the screening or replace it. Knowledge about risk factors for infection is necessary to understand why some women groups who live under similar conditions are more vulnerable to infection than others. There is not a surveillance system for circulating types of HPV, which harms the evaluation of the circumstances, even more now in relation to recent implantation of HPV vaccination in the public basic calendar. In this study, our goal was to estimate the prevalence of HPV infection in women of a certain population, correlating the results to the risk factors for HPV infection, and evaluate the results in a quality assay perspective. 2062 women living in a suburban area in Juiz de Fora, Minas Gerais, participated, responding to a questionnaire, submitted to conventional cervical cytology and cobas 4800 HPV test (Roche) for DNA detection. The entrance of women invited was lower than expected, so studies of other designs can contribute to its understanding. HPV infection prevalence estimates were calculated according to selected features. The overall prevalence of HPV infection was 12,61%. Multivariate analysis using Poisson regression with robust variance showed statistically significant association with being single, consuming alcohol and having three or more sexual partners over a lifetime, with adjusted prevalence ratio 1,40, 1,44 e 1,35, respectively. The prevalence of precursor lesions of cervical cancer was 7,27%. The quality of the smear, evaluated by the representativeness of the collected epithelia showed that there is variation in the prevalence of precancerous lesions by quality of smears; however, there is no variation in HPV test results by cell representation, a finding that is consistent with a higher sensitivity and a better positive predictive value. Testing HPV proved to be useful, especially among women with normal cytology results.

Bakgrund: Humant Papillomvirus (HPV) orsakar livmoderhalscancer. Årligen drabbas 800 kvinnor och 300 män av HPV-relaterad cancer i Sverige. Både pojkar och flickor får HPV- vaccin enligt nationella vaccinationsprogrammet. Vaccin tillsammans med gynekologisk cellprovtagning kan utrota HPV-orsakad cancer. Tidigare forskning visade att kunskapsläget kring HPV varierade globalt. Syfte: Att undersöka unga kvinnors kunskap om HPV och inställning till prevention och gynekologisk cellprovtagning i Sverige.Metod: Kvantitativ tvärsnittsstudie med webbenkät. Undersökt grupp var kvinnor i åldrarna 23-29 år i Sverige. Totalt 160 enkäter analyserades. Resultat: Resultatet visade goda kunskaper hos unga kvinnor avseende smittväg, prevention och att HPV orsakar livmoderhalscancer. Kunskaperna var sämre avseende HPV och könssjukdomar, att HPV kan orsaka annan cancer samt att HPV kan drabba män. Mer än hälften av deltagarna var vaccinerade mot HPV. Samtliga hade en positiv inställning till gynekologisk cellprovtagning och visste att det utförs för att upptäcka cellförändringar. Majoriteten av deltagarna hade gått på gynekologisk cellprovtagning.Slutsats: Kunskapen var generellt god hos deltagarna. Det fanns en positiv inställning till gynekologisk cellprovtagning. I utbildningsgrupperna kunde enstaka större skillnader i kunskap ses men generellt var skillnaderna små. Information om HPV kan tillgängliggöras i väntrummen på barnmorskemottagningar och ungdomsmottagningar samt i kallelsen till gynekologisk cellprovtagning. / Background: Human Papillomavirus (HPV) causes cervical cancer. Approximately 800 women and 300 men are diagnosed with HPV-related cancer annually in Sweden. Both boys and girls receive the HPV-vaccine according to the national program. Vaccines and pap tests can together exterminate all cancers caused by HPV. Research showed that knowledge about HPV is varying globally. Method: Quantitative cross-sectional study with web based surveys. The target group consisted of women aged 23-29 in Sweden. A total of 160 surveys were analyzed.Aim: To examine knowledge about HPV and attitudes towards prevention and pap testing amongst young Swedish women. Results: Young women had gratifying knowledge about the route of infection, prevention and that HPV causes cervical cancer. There was less knowledge about HPV and STIs, that HPV causes other sorts of cancers and that HPV can affect men. More than half of the participants were vaccinated. All participants had a positive attitude towards pap testing and knew that it is performed to locate pre cancerous cervical lesions. The majority of the participants had undergone a pap test. Conclusion: The knowledge was overall good in the participants. There was an entirely positive attitude towards pap testing. Only a few significant differences in knowledge were found affected by level of education, generally the differences were only slight. Information about HPV can be made available in the waiting rooms in midwife and youth clinics, and in the pap test summoning letter.

A key effect of human papillomavirus (HPV) infection is to disrupt the normal cell cycle in order to subvert the cellular DNA replication machinery. Morphologically, condylomata induced by high and low risk HPV types cannot be distinguished and many studies have shown that the pattern of viral gene expression is similar in condylomata caused by both high risk and low risk HPV types. Detailed morphological study of cell cycle protein expression has not previously been performed on condylomata infected with low risk HPV types. The findings presented suggest that the mechanisms employed by low risk HPV6 or HPV11 to subvert cellular functions in condylomata acuminata are similar to those employed by high risk HPVs, with the exception of cyclin D1 and p53 protein over-expression. The differences in p53 expression and cyclin D1 expression seen between high and low risk HPV infection, reflect the known differences between high and low risk types and are in agreement with the known differences between high risk and low risk E6 and E7 proteins. PHK transduction studies demonstrated HPV E6 and E7 induce changes in cell cycle protein expression and that there are differences in cell cycle abrogation between HPV6 and HPV16. Disruption of the p53-MDM2 interaction can lead to activation of the p53 pathway. HPV infected lesions almost always contain wild-type p53. The binding of HPV E6 to p53, and its subsequent targeting for degradation, prevents activation of the p53 pathway in HPV infected cells. Cells over expressing HPV genes were treated with Nutlin-3, a MDM2-small molecule antagonist. The findings presented suggest treatment with Nutlin-3 induces cell cycle arrest in cells expressing HPV16 E7 and HPV6 E6 and HPV6 E7. This suggests a potential role for Nutlin-3 in the treatment of HPV infected cells.

Os papilomavírus são pequenos vírus de DNA dupla-fita, não envelopados, mucoepiteliotrópicos, capazes de infectar inúmeros vertebrados superiores de maneira espécie-específica. A infecção por estes vírus está associada a uma série de desordens proliferativas que levam desde de a formação de verrugas comuns até a do carcinoma invasivo. Aproximadamente 200 tipos de papilomavírus humano (HPVs) foram identificados, sendo que cerca de 40 deles infectam o trato genital. Dentre estes, os chamados HPVs de alto-risco estão associados etiologicamente ao carcinoma de colo de útero, enquanto que os de baixo-risco estão relacionados às lesões epiteliais benignas. A infecção por HPVs de alto-risco é muito comum, no entanto, a maioria destas é transitória e somente uma pequena proporção de mulheres desenvolvem o carcinoma. Entretanto, algumas mulheres são incapazes de eliminar esta infecção, levando a persistência viral e o conseqüente desenvolvimento da neoplasia. Para que a infecção pelo HPV persista é necessário um mecanismo de escape ao sistema imune do hospedeiro. O mecanismo de escape à resposta imune inata parece ser característico da infecção pelo HPV, pois o ciclo infeccioso deste vírus não promove inflamação. A infecção por HPV promove a liberação de citocinas, tal como o fator de necrose tumoral (TNF). Esta citocina possui um potente efeito citostático em queratinócitos normais e imortalizados com HPV, enquanto que em queratinóctos imortalizados com HPV18 este efeito não é observado. Do mesmo modo, observamos que a expressão do oncogene E6 de HPV16 ou 18 é suficiente para promover resistência ao efeito antiproliferativo do TNF em culturas em monocamada e organotípica. A expressão aumentada e contínua destes ocogenes é sabidamente o principal evento favorável ao desenvolvimento do câncer de colo de útero. Estas proteínas são essenciais na indução da transformação celular, visto que interferem na regulação do ciclo celular e apoptose. O produto dos genes E6 e E7 se liga ao produto dos genes supressores de tumor p53 e pRb, respectivamente, levando a sua degradação pela via de proteólise dependente de ubiquitina. As bases moleculares desta resistência ao TNF ainda são pouco conhecidas. Neste estudo, comparamos o efeito desta citocina em queratinócitos normais e que expressam E6 ou E7. Observamos através de cDNA Microarray a expressão de um grupo de genes, entre eles TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 e MMP9, com expressão diferencial entre as células sensíveis e as resistentes ao TNF. / Papillomaviruses are small, non-enveloped, epitheliotropic, double-stranded DNA viruses that infect mucosal and cutaneous epithelia in a wide variety of higher vertebrates in a species-specific manner. Papillomavirus infections are associated to a series of proliferative disorders that range from common warts to invasive carcinomas. Almost 200 types of human papillomaviruses (HPVs) have been identified and approximately 40 of them infect the genital tract. Only the so-called high-risk HPV types mediate human carcinogenesis, whereas the low-risk HPVs have been linked to benign epithelial lesions. High-risk genital HPV infection is very common, and the majority of individuals clear their infection with time. However, a proportion of women cannot effectively clear the virus, and the persistence of a high-risk HPV is the major risk factor for the development of anogenital malignancies. To persist, HPV must escape the host immune system. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections, since the infectious cycle is one in which viral replication and virion release is not associated with inflammation. Furthermore, HPV infections promote cytokine release, as tumor necrosis factor-alpha (TNF). This cytokine has a potent cytostatic effect on normal and HPV16 immortalized keratinocytes, while it does not affect HPV18 immortalized keratinocytes proliferation. In addition, we have observed that expression of HPV 16 or 18 E7 oncogene is sufficient to overcome TNF antiproliferative effect in monolayer and organotypic cell cultures. The increased and sustained expression of HPV oncogenes, E6 and E7, is the main contributor to the development of cervical cancer. Both E6 and E7 proteins are essential to induce and maintain cellular transformation, due to their interference with cell-cycle and apoptosis regulation. The most manifest function of the E6 protein is to promote the degradation of p53, while E7 is known to bind to and promote the proteasomal degradation of the retinoblastoma tumor suppressor gene product, pRb, and its family members. The molecular basis of TNF resistance is not well understood. In this study we compared the effect of TNF between normal and HPV16 E6 or E7 expressing keratinocytes. We observed by cDNA Microarray the differential expression of a common set of genes in TNF-sensitive cell lines, including TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 and MMP9, that differs from those modulated in TNF-resistant cells.

Ho, Ching Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 142-160). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENTS --- p.II / ABSTRACT (ENGLISH VERSION) --- p.IV / ABSTRACT (CHINESE VERSION) --- p.VII / TABLE OF CONTENTS --- p.IX / LIST OF FUGURES --- p.XII / LIST OF TABLES --- p.XIII / LIST OF ABBREVIATIONS --- p.XV / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Biology of human papillomavirus --- p.2 / Chapter 1.1.1 --- History --- p.2 / Chapter 1.1.2 --- Classification --- p.3 / Chapter 1.1.3 --- Genome structure --- p.5 / Chapter 1.1.4 --- Life cycle --- p.9 / Chapter 1.2 --- Epidemiology of cervical cancer --- p.10 / Chapter 1.2.1 --- Cervical intraepithelial neoplasia and cervical cancer --- p.10 / Chapter 1.2.2 --- Spectrum of cervical neoplasia --- p.13 / Chapter 1.2.3 --- Incidence of cervical cancer --- p.15 / Chapter 1.2.4 --- Screening programme --- p.16 / Chapter 1.3 --- Risk factors for cervical cancer --- p.17 / Chapter 1.4 --- Oncogenic HPV infection --- p.20 / Chapter 1.4.1 --- Risk association --- p.21 / Chapter 1.4.2 --- Geographical distribution --- p.23 / Chapter 1.4.3 --- Age distribution --- p.24 / Chapter 1.4.4 --- Oncogenic property of HPV --- p.25 / Chapter 1.4.5 --- Sequence variation --- p.28 / Chapter 1.5 --- Prevention by vaccination --- p.30 / Chapter 1.6 --- Objectives --- p.31 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.33 / Chapter 2.1 --- HPV type and prevalence distribution --- p.34 / Chapter 2.1.1 --- Study population --- p.34 / Chapter 2.1.2 --- Specimen and epidemiological data collection --- p.34 / Chapter 2.1.3 --- DNA extraction --- p.35 / Chapter 2.1.4 --- PCR amplification of DNA --- p.36 / Chapter 2.1.4.1 --- PCR for Beta-globin --- p.36 / Chapter 2.1.4.2 --- PCR for HPV DNA --- p.37 / Chapter 2.1.5 --- HPV typing by reverse line-blot hybridization --- p.39 / Chapter 2.1.6 --- Statistical method --- p.40 / Chapter 2.2 --- HPV 52 SEQUENCE VARIATION --- p.43 / Chapter 2.2.1 --- Study population --- p.43 / Chapter 2.2.2 --- Specimen processing --- p.43 / Chapter 2.2.3 --- DNA extraction --- p.44 / Chapter 2.2.4 --- PCR amplification for sequencing --- p.45 / Chapter 2.2.4.1 --- Optimization of gene-specific PCR --- p.45 / Chapter 2.2.4.2 --- Validation of type-specificity of gene-specific PCR --- p.46 / Chapter 2.2.4.3 --- PCR for HPV52 E6 and E7 --- p.46 / Chapter 2.2.4.4 --- PCR for LI gene --- p.47 / Chapter 2.2.4.5 --- PCR for long control region (LCR) --- p.48 / Chapter 2.2.5 --- Purification of PCR products --- p.49 / Chapter 2.2.6 --- Sequencing --- p.50 / Chapter 2.2.6.1 --- Preparation of template --- p.50 / Chapter 2.2.6.2 --- Purification of template --- p.50 / Chapter 2.2.6.3 --- Sequencer and data analysis --- p.51 / Chapter 2.2.7 --- Statistical methods --- p.51 / Chapter CHAPTER 3: --- RESULTS --- p.54 / Chapter 3.1 --- HPV TYPE AND PREVALENCE DISTRIBUTION --- p.55 / Chapter 3.1.1 --- Study population --- p.55 / Chapter 3.1.2 --- HPV prevalence --- p.59 / Chapter 3.1.2.1 --- Prevalence for HPV infection --- p.59 / Chapter 3.1.2.2 --- HPV age-specific prevalence --- p.68 / Chapter 3.1.3 --- Epidemiological risk profile --- p.73 / Chapter 3.1.3.1 --- Age-adjusted analyses --- p.73 / Chapter 3.1.3.2 --- Multivariate analyses --- p.76 / Chapter 3.2 --- HPV52 SEQUENCE VARIATION --- p.79 / Chapter 3.2.1 --- Study population --- p.79 / Chapter 3.2.2 --- Sequence variability of HPV52 --- p.79 / Chapter 3.2.3 --- HPV52 --- p.82 / Chapter 3.2.3.1 --- Sequence variation of E6 ORF --- p.82 / Chapter 3.2.3.2. --- HPV52 E6 variants and risk for cervical neoplasia --- p.86 / Chapter 3.2.4 --- HPV52 E7 --- p.89 / Chapter 3.2.4.1 --- Sequence variation of E7 ORF --- p.89 / Chapter 3.2.4.2 --- HPV52 E7 variants and risk for cervical neoplasia --- p.93 / Chapter 3.2.5 --- HPV52 LI --- p.95 / Chapter 3.2.5.1 --- Sequence variation of LI ORF --- p.95 / Chapter 3.2.5.2 --- HPV52 LI variants and risk for cervical neoplasia --- p.100 / Chapter 3.2.6 --- HPV52 long control region (LCR) --- p.104 / Chapter 3.2.6.1 --- Sequence variation of LCR --- p.104 / Chapter 3.2.6.2 --- HPV52 LCR variants and risk for cervical neoplasia --- p.110 / Chapter CHAPTER 4: --- DISCUSSION --- p.113 / Chapter 4.1 --- HPV PREVALENCE AND TYPE DISTRIBUTION --- p.114 / Chapter 4.1.1 --- HPV prevalence --- p.114 / Chapter 4.1.2 --- Age-specific prevalence --- p.116 / Chapter 4.1.3 --- Epidemiological risk profile --- p.121 / Chapter 4.1.4 --- Conclusions --- p.126 / Chapter 4.2 --- HPV 52 SEQUENCE VARIATION --- p.127 / Chapter 4.2.1 --- Sequence variability of HPV52 --- p.127 / Chapter 4.2.2 --- Sequence variation of E6 gene --- p.129 / Chapter 4.2.3 --- Sequence variation of E7 gene --- p.132 / Chapter 4.2.4 --- Sequence variation of LI gene --- p.134 / Chapter 4.2.5 --- Sequence variation of LCR --- p.135 / Chapter 4.2.6 --- Conclusions --- p.139 / Chapter 4.3 --- FURTHER STUDIES --- p.140 / REFERENCES --- p.142 / APPENDIXES --- p.A