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

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

The Papanicolaou Smear is a screening test which detects premalignant lesions of the uterine cervix. By treating these lesions, cervical cancer can be evaded. In 1988, a cytological diagnosis which communicated a state of uncertainty in the atypicality of cervical cells was first created in the Bethesda Cytology Classification scheme. This diagnosis is now known as atypical squamous cells of undetermined significance (ASC-US) and still little is known about its natural history. / This paper analyzes the results of a longitudinal study incorporating repeated regular measurements of viral and cytological endpoints as well as lifestyle and behavioural aspects, to understand the natural history of an ASC-US Pap smear and identify determinants of ASC-US acquisition and clearance. / Overall, the median duration of ASC-US is short, and is dependent on the definition of clearance since most lesions regress to normal. The factors most predictive of ASC-US acquisition but not clearance relate to HPV infection.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

O Lúpus eritematoso sistêmico (LES) é uma doença auto-imune, mais prevalente em mulheres, com marcante aumento da sobrevida nos últimos anos, principalmente relacionado ao avanço do ratamento com terapia imunossupressora / citotóxica. Este estudo visa identificar a prevalência de alterações em esfregaços cervicais nesse grupo de pacientes. Participaram do estudo 80 pacientes do grupo com LES e 101 pacientes do grupo controle. Observou-se que a prevalência de alterações no grupo de paciente com LES foi de 31,3% contra 5,0% do grupo controle (p = 0,000). Não foram encontradas associações estatisticamente significativas entre a presença de alteração no esfregaço cervical e as variáveis: história de DST, sexarca com idade ≤ 17 anos, parceiros múltiplos (≥ 4), história de HPV prévio, uso de ACO, tabagismo, estrato social ou com droga imunossupressora específica. Encontrou-se associação estatisticamente significativa apenas para a variável grupo, tendo paciente pertencente ao grupo LES chance 8,2 vezes maior (IC 95% = 2,7 24,9) de ter alterações no esfregaço cervical que uma paciente do grupo controle. Tais dados demonstram que pacientes com LES em terapia imunossupressora apresentam mais alterações em exames preventivos ginecológicos, merecendo atenção especial quanto à prevenção do câncer de colo do útero. / Systemic Lupus Erythematosus (SLE) is an autoimmune disease with higher prevalence over women. There has been a significant increase in survival time over the years resulting from the improvement of citotoxic and immunosuppressant therapy. This study aims at identifying the prevalence of altered results for cervical smears in this group of patients. 80 patients of the SLE group and 101 patients of the control group were part of the study. The prevalence of alterations in the SLE group was 31.3%, and 5% in the control group (p=0.000). No statistically significant association was found between the presence of alterations in pap smears and the variables STD history, first sexual intercourse at age ≤ 17, multiple partners (≥ 4), previous history of HPV, use of oral contraceptives, tobacco smoking, social class and cancer presence in the family. Statistically significant association was found only in the group variable; patients in the SLE group had a 8.2 higher chance (IC 95% = 2.7 24.9) of showing alterations in the pap smears than those in the control group. These numbers show that SLE patients in immunosuppressant therapy have more alterations in pap smear exams, deserving special attention with regards to cervical cancer prevention.

Made available in DSpace on 2014-06-11T19:25:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-11-24Bitstream added on 2014-06-13T19:32:20Z : No. of bitstreams: 1 simonato_le_me_araca.pdf: 1776800 bytes, checksum: 3a9572e5560385e74fdc0951de2ba7e7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / 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. / 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.

O Lúpus eritematoso sistêmico (LES) é uma doença auto-imune, mais prevalente em mulheres, com marcante aumento da sobrevida nos últimos anos, principalmente relacionado ao avanço do ratamento com terapia imunossupressora / citotóxica. Este estudo visa identificar a prevalência de alterações em esfregaços cervicais nesse grupo de pacientes. Participaram do estudo 80 pacientes do grupo com LES e 101 pacientes do grupo controle. Observou-se que a prevalência de alterações no grupo de paciente com LES foi de 31,3% contra 5,0% do grupo controle (p = 0,000). Não foram encontradas associações estatisticamente significativas entre a presença de alteração no esfregaço cervical e as variáveis: história de DST, sexarca com idade ≤ 17 anos, parceiros múltiplos (≥ 4), história de HPV prévio, uso de ACO, tabagismo, estrato social ou com droga imunossupressora específica. Encontrou-se associação estatisticamente significativa apenas para a variável grupo, tendo paciente pertencente ao grupo LES chance 8,2 vezes maior (IC 95% = 2,7 24,9) de ter alterações no esfregaço cervical que uma paciente do grupo controle. Tais dados demonstram que pacientes com LES em terapia imunossupressora apresentam mais alterações em exames preventivos ginecológicos, merecendo atenção especial quanto à prevenção do câncer de colo do útero. / Systemic Lupus Erythematosus (SLE) is an autoimmune disease with higher prevalence over women. There has been a significant increase in survival time over the years resulting from the improvement of citotoxic and immunosuppressant therapy. This study aims at identifying the prevalence of altered results for cervical smears in this group of patients. 80 patients of the SLE group and 101 patients of the control group were part of the study. The prevalence of alterations in the SLE group was 31.3%, and 5% in the control group (p=0.000). No statistically significant association was found between the presence of alterations in pap smears and the variables STD history, first sexual intercourse at age ≤ 17, multiple partners (≥ 4), previous history of HPV, use of oral contraceptives, tobacco smoking, social class and cancer presence in the family. Statistically significant association was found only in the group variable; patients in the SLE group had a 8.2 higher chance (IC 95% = 2.7 24.9) of showing alterations in the pap smears than those in the control group. These numbers show that SLE patients in immunosuppressant therapy have more alterations in pap smear exams, deserving special attention with regards to cervical cancer prevention.

INTRODUÇÃO: Co-infecção HPV / HIV altera história natural das infecções por HPV, aumentando o risco de verrugas e neoplasias malignas do trato ano-genital. Há, no entanto, escassez de estudos de coorte envolvendo HPV no pênis dessa população. MÉTODOS: Estudo longitudinal, não probabilístico, com 144 homens de 18 e 70 anos de idade, sendo 72 HIV positivos e 72 HIV soronegativos, parceiros de mulheres com patologia associada a infecção pelo HPV. O estudo foi conduzido numa clínica pública de doenças de transmissão sexual em São Paulo (CRT-DST/AIDS), entre fevereiro de 2004 a março de 2005. Os participantes do estudo foram acompanhados por 180 dias para avaliar a persistência, a aquisição e a eliminação do DNA de HPV nos esfregaços penianos por meio da PCR. Este estudo também visou: Correlacionar os aspectos clínicos das lesões genitais com a histologia e a presença de DNA HPV; Comparar a aquisição, persistência, eliminação e ausência da infecção pelo HPV com a carga viral plasmática do HIV, contagem de células T CD4 e uso de terapia anti-retroviral (HAART). RESULTADOS: Não houve associação estatisticamente significativa nos dois grupos em relação a persistência, eliminação, aquisição ou mesmo ausência de HPV durante o seguimento. O grupo HIV positivo apresentou uma maior freqüência dos tipos oncogênicos de HPV em relação ao grupo HIV negativo (P = 0,041), além de uma maior freqüência de múltiplos tipos de HPV durante o seguimento de 180 dias (P = 0,049). As maiores taxas de aquisição e persistência de HPV foram observadas entre portadores de alta carga de HIV, baixo número de células T CD4, e não usuários de HAART. Aqueles em terapia anti-retroviral, com menos cópias de HIV e alto nível de T CD4 apresentaram maiores taxas de eliminação e ausência de HPV. CONCLUSÕES: Homens de ambos grupos podem ser considerados de alto risco, não tendo sido observada diferença na persistência, aquisição e a eliminação de DNA de HPV. Os homens HIV positivos apresentaram uma maior freqüência de infecção múltipla de HPV bem como os tipos mais freqüentes foram os oncogênicos em relação aos HIV negativos durante o seguimento. Os tipos de HPV 16, 6 e 84 foram os mais freqüentes nos homens soropositivos ao HIV, enquanto naqueles HIV negativos, predominaram os tipos de HPV 6, 51 e 84. As lesões clínicas e aceto-brancas observadas nos 2 grupos apresentaram as mesmas características histológicas, sendo coilocitose e papilomatose as mais significativas nas lesões clínicas quando comparada às lesões aceto-brancas. Nas lesões verrucosas, apenas um tipo de HPV foi observado, predominando o tipo 6 ou 11. Em torno de 23% das lesões aceto-brancas eram HPV negativas, sendo que nas positivas predominou o HPV 6. Homens soropositivos ao HIV que estavam usando HAART, com carga viral do HIV alta e contagem de células T CD4 baixa apresentaram maiores taxas de aquisição e persistência da infecção pelo HPV. Entretanto, os que não xvi estavam em terapia anti-retroviral, com carga baixa de HIV e contagem de células T CD4 alta apresentaram maiores taxas de eliminação e ausência de infecção pelo HPV. / BACKGROUND: Co-infection with HPV and HIV modifies its natural history and increases the risk of warts and neoplasia development in the anogenital tract. Cohort studies to address HPV infection in the penis are scarce, mainly in HIV infected individuals. METHODS: A longitudinal study, non-probabilistic, was conducted with 144 men of 18 to 70 years old including 72 HIV-positive and 72 HIV-negative, partners of women with HPV-associated disease. The study was conducted between February 2004 and March 2005 at a large sexually transmitted clinic in São Paulo (CRT-DST/Aids). Men were followed for 180 days to determine persistence, acquisition and clearance of HPV DNA in penile swabs using PCR. In addition, we aimed to correlate the clinical features of genital lesions with histology and the presence of HPV DNA, compare the acquisition, persistence, clearance and absence of HPV infection with plasma HIV viral load, CD4 T-cell count and use of HAART. RESULTS: Both groups showed no significant differences regarding persistence, clearance, acquisition and/or absence of HPV during follow-up. Penile smears of HIV-positive men showed a higher frequency of oncogenic types in relation to the HIV-negative (P = 0.041), as well as a higher frequency of multiple HPV types (P = 0.049). Significantly higher HPV DNA acquisition and persistence rates were observed among HIV-positive men not submitted to HAART, with higher HIV loads and lower CD4+ cells count. Among those men using anti-retroviral therapy, lower viral loads and higher T cell counts, higher rates of clearance and HPV DNA absence were observed. CONCLUSIONS: This male population altogether is considered to be at high risk of HPV DNA infection, which may be the reason why no differences in HPV acquisition, persistence and clearance were observed. HIV-positive men had a higher frequency of multiple HPV infection and the most frequent were oncogenic types. HPV types 16, 6 and 84 were the most frequently found in HIV-positive men, while in HIV-negative men, HPV types 6, 51 and 84 prevailed. Clinical and aceto-white lesions presented the same histological features in both HIV seropositive and -negative men. Koilocytosis and papillomatosis were the most significant histological features found in clinical lesions when compared to the aceto-white lesions. In condylomas, only one type of HPV was present, often HPV 6 or 11. About 23% of aceto-white lesions had no HPV DNA; in HPV-positive lesions, the predominant type was HPV 6. Higher rates of acquisition and persistence of HPV infection occurred in men who were using HAART, with high HIV viral load and low count of CD4. In contrast, those not under anti-retroviral therapy, had low HIV load and high CD4 T cells levels showed higher rates of clearance of HPV infection.

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