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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

子宮頸癌是全球女性中第三常見的癌症。人類乳頭瘤狀病毒（HPV）已被證實為引致子宮頸癌的主要因素。目前已發現了150多種HPV。HPV-52在世界上較為少見，但在亞洲，特別是東亞地區，卻相當流行。 / 本回顧性研究收集了303個HPV-52陽性的子宮頸樣本，其中185個來自香港，118個來自韓國首爾。我們通過對HPV基因組中E6、E7、L1和LCR區域進行擴增和測序，以檢測HPV-52變異株的序列多樣性和致癌風險。 / L1-LCR-E6-E7串聯片段佔據了HPV-52基因組全長的41%。由191條該種序列構建的系統發育樹顯示，HPV-52變異株進化成四個世系。原型系A進化系在香港和首爾都很少見，只占全部樣本的3.7%。B進化系（89.5%）則是最普遍的HPV-52病毒系。E6的最大序列差異為1.6%，L1（2.3%），E7（3.4%）和LCR（4.8%）依次增大。因此，E6作為最保守的基因組區域可作為HPV-52通用引物PCR的靶點，而E7更適宜作為特定變異株的PCR靶點。此外，在短片段序列中發現了可識別HPV-52進化系和進化枝的單核苷酸突變。它們可用於擴增斷裂的DNA片段或大規模實驗中。再者，進化壓力分析顯示E6、E7和L1三個編碼區域都經歷了強烈的淨化選擇作用。 / HPV-52進化系和常見變異株在香港和首爾的分佈情況沒有顯著差異。但E6中的nt 356G>A、nt 378A>C和nt 467C>A (N122K) 核苷酸突變只出現在香港樣本，而L1的nt 6239G>A以及LCR的nt 7395G>A和nt 7911A>C核苷酸突變只在首爾樣本中發現。HPV-52 E6的N122K突變對子宮頸癌有較高的致癌風險（P-value = 0.002）。E6中的nt 378A>C (P-value = 0.014) 同義突變, 以及LCR中的nt 7665G>A (P-value < 0.001)和nt 94G>A (P-value = 0.007)突變，亦與高致癌風險相關。LCR中的nt 7911A>C (P-value = 0.007)和nt 19T>C (P-value = 0.008) 突變則對子宮頸癌的發展有較低風險。HPV-52 E7或L1中的突變與子宮頸癌的發展無明顯關係。上述結果需要通過進一步研究證實。針對HPV-52序列變異的病毒學和作用機理的深入研究是必要的。 / Cervical cancer is the third most common cancer in women worldwide. It has been proven that human papillomavirus (HPV) is the primary causative agent of cervical cancer. To date, more than 150 HPV types have been characterized. HPV-52 is rare around the world but frequently detected in Asia, especially East Asia. / This retrospective study analyzed 303 cervical samples that 185 were collected from Hong Kong, and 118 were collected from Seoul, Korea. All samples were positive for HPV-52. HPV gene regions of E6, E7, L1 and LCR were amplified and sequenced to determine sequence diversity and risk association of HPV-52 variants between the two cities. / The 191 concatenated L1-LCR-E6-E7 sequences that comprised 41% of the whole HPV-52 genome displayed four distinct clusters. The prototype-like lineage A was rare in both cities, only found in 3.7% of all samples. Lineage B (89.5%) was found to be the most prevalent lineage. The maximum sequence divergence of E6 was 1.6%, followed by L1 (2.3%), E7 (3.4%) and LCR (4.8%). E6 being the most conserved region could be a target for HPV-52 consensus PCR, and E7 could be a target for variant-specific PCR. Besides, several single-nucleotide substitutions diagnostic for HPV-52 lineage and clade classification were identified within a few short fragments. They might be useful when handling fragmented DNA and being a more feasible approach in large-scale studies. Moreover, analysis of evolutionary pressure indicated that all the three encoding regions, E6, E7 and L1, underwent strong purifying selection. / No significant difference in the distribution pattern of HPV-52 lineages and common variants between Hong Kong and Seoul was observed. But nucleotide substitutions nt 356G>A, nt 378A>C and nt 467C>A (N122K) were only found in Hong Kong samples; whereas nt 6239G>A, nt 7395G>A and nt 7911A>C were exclusively found in samples from Seoul. A significantly higher risk for cervical cancer was found for the HPV-52 E6 variant N122K (P-value = 0.002). A synonymous substitution of E6, nt 378A>C (P-value = 0.014), as well as two nucleotide substitutions of LCR, nt 7665G>A (P-value < 0.001) and nt 94G>A (P-value = 0.007), were also associated with a significant increase in risk for cervical cancer. Two substitutions found to confer a lower risk for cervical cancer were nt 7911A>C (P-value = 0.007) and nt 19T>C (P-value = 0.008), both of which located at LCR. No significant associations between HPV-52 E7 or L1 variants and cervical cancer development were observed. Further studies are needed to confirm these findings, and in-depth investigations into the virological and functional implications of HPV-52 sequence variations are warranted. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Chuqing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-137). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of contents --- p.vii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- 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 --- Genome structure --- p.4 / Chapter 1.2.2 --- Protein function --- p.6 / Chapter 1.2.3 --- Latent and lytic life cycle --- p.9 / Chapter 1.2.4 --- Classification --- p.10 / Chapter 1.3 --- Epidemiology of Human Papillomavirus --- p.14 / Chapter 1.3.1 --- Global burden --- p.14 / Chapter 1.3.2 --- Transmission --- p.18 / Chapter 1.3.3 --- Clinical course --- p.19 / Chapter 1.3.4 --- Prevention --- p.23 / Chapter 1.4 --- Human Papillomavirus Type 52 --- p.25 / Chapter 1.5 --- Objectives --- p.26 / Chapter Chapter Two --- Materials and Methods --- p.27 / Chapter 2.1 --- Study Design --- p.28 / Chapter 2.2 --- Study population --- p.29 / Chapter 2.3 --- DNA extraction --- p.31 / Chapter 2.4 --- Polymerase chain reaction --- p.32 / Chapter 2.4.1 --- Long-fragment PCR approach --- p.33 / Chapter 2.4.2 --- Short-fragment PCR approach --- p.40 / Chapter 2.4.3 --- Purification of PCR products --- p.46 / Chapter 2.5 --- Nucleotide sequencing --- p.47 / Chapter 2.6 --- Data analysis --- p.48 / Chapter 2.6.1 --- Phylogenetic analysis --- p.48 / Chapter 2.6.2 --- Statistical analysis --- p.49 / Chapter Chapter Three --- Results --- p.50 / Chapter 3.1 --- Phylogeny of HPV-52 --- p.53 / Chapter 3.1.1 --- Concatenated sequence of L1-LCR-E6-E7 --- p.53 / Chapter 3.1.2 --- E6 gene --- p.56 / Chapter 3.1.3 --- E7 gene --- p.59 / Chapter 3.1.4 --- L1 gene --- p.62 / Chapter 3.1.5 --- Long control region --- p.67 / Chapter 3.2 --- Nucleotide sequence variation of HPV-52 --- p.70 / Chapter 3.2.1 --- E6 gene --- p.70 / Chapter 3.2.2 --- E7 gene --- p.73 / Chapter 3.2.3 --- L1 gene --- p.75 / Chapter 3.2.4 --- Long control region --- p.81 / Chapter 3.3 --- Geographical distribution of HPV-52 variants --- p.86 / Chapter 3.4 --- Risk association of HPV-52 variants --- p.96 / Chapter Chapter Four --- Discussion --- p.105 / Chapter 4.1 --- Strengths and weaknesses --- p.107 / Chapter 4.2 --- Phylogeny of HPV-52 variants --- p.109 / Chapter 4.2.1 --- Variant lineage classification system of HPV-52 --- p.109 / Chapter 4.2.2 --- Sequence variability of HPV-52 --- p.110 / Chapter 4.2.3 --- Evolutionary pressure on HPV-52 --- p.111 / Chapter 4.3 --- Nucleotide sequence variations of HPV-52 --- p.113 / Chapter 4.3.1 --- E6 gene --- p.113 / Chapter 4.3.2 --- E7 gene --- p.114 / Chapter 4.3.3 --- L1 gene --- p.116 / Chapter 4.3.4 --- Long control region --- p.117 / Chapter 4.4 --- Conclusions --- p.121 / References --- p.124 / Appendices --- p.138

INTRODUÇÃO: Onicomicose, infecção das unhas por fungo é a mais freqüente das doenças ungueais, constituindo aproximadamente metade de todas as alterações ungueais. Pode ser causada por dermatófitos, leveduras ou fungos filamentosos não dermatófitos. OBJETIVO: Caracterizar as onicomicoses causadas por fungos filamentosos não dermatófitos. (1) Verificar, dentre as suspeitas clínicas de onicomicose, qual a freqüência da recuperação de fungos,(2) Verificar, dentre as suspeitas clínicas de onicomicose, quais as espécies de fungos recuperadas, (3) Verificar, dentre o total das espécies identificadas, qual a freqüência das espécies de fungos filamentosos não dermatófitos. MÉTODOS: Duzentos e cinco indivíduos com suspeita clínica de onicomicose foram estudados no período de dezembro de 2003 a novembro de 2004, por meio de exames micológicos diretos e cultura para fungos. RESULTADOS: O diagnóstico de onicomicose foi estabelecido, pelo exame micológico direto, em 170 indivíduos. O diagnóstico etiológico foi estabelecido pela cultura para fungos. Dentre os 107 agentes identificados, os dermatófitos foram identificados em 78,52% (N=84), as leveduras em 13,08% (N=14) e os FFND em 8,40% (N=9) das vezes. CONCLUSÃO: É necessário que se estabeleça o diagnóstico etiológico dos casos de onicomicoses, já que os fungos filamentosos não dermatófitos ocorrem com freqüência considerável e são indistinguíveis daquelas por dermatófitos. / INTRODUCTION: Onychomycosis, a nail fungus infection is the most frequent nail disease, constituting about half of all nail disorder. It can be caused by dermatophytes, yeasts and non- dermatophytes filamentous fungi. OBJECTIVE: Characterize the onychomycosis caused by filamentous fungi non- dermatophytes. (1) Verify after a clinical suspicion of onychomycosis, which are the frequency of fungi recovery, (2) examine, after a clinical suspicion of onychomycosis, which species of fungi are recovered, (3) Checking, among the total of identified species , which are the frequency of the species of filamentous fungi not dermatophytes. METHODS: Two hundred and five patients with clinical suspicion of onychomycosis were studied in the period from December 2003 to November 2004, through direct mycological examination and culture for fungus. RESULTS: The diagnosis of onychomycosis has been established by direct mycological examination, in 170 individuals. The etiological diagnosis was established by the culture for fungus. Among the 107 persons identified, the dermatophytes were recovered in 78.52% (N = 84), the yeast in 13.08% (N = 14) and filamentous fungi non- dermatophytes in 8,40% (N = 9). CONCLUSION: It is necessary to establish the etiological diagnosis of the cases of onychomycosis, as filamentous fungi non- dermatophytes occur often than ever considered and are its clinic features are indistinguishable from those of the dermatophytes.

INTRODUÇÃO: Onicomicose, infecção das unhas por fungo é a mais freqüente das doenças ungueais, constituindo aproximadamente metade de todas as alterações ungueais. Pode ser causada por dermatófitos, leveduras ou fungos filamentosos não dermatófitos. OBJETIVO: Caracterizar as onicomicoses causadas por fungos filamentosos não dermatófitos. (1) Verificar, dentre as suspeitas clínicas de onicomicose, qual a freqüência da recuperação de fungos,(2) Verificar, dentre as suspeitas clínicas de onicomicose, quais as espécies de fungos recuperadas, (3) Verificar, dentre o total das espécies identificadas, qual a freqüência das espécies de fungos filamentosos não dermatófitos. MÉTODOS: Duzentos e cinco indivíduos com suspeita clínica de onicomicose foram estudados no período de dezembro de 2003 a novembro de 2004, por meio de exames micológicos diretos e cultura para fungos. RESULTADOS: O diagnóstico de onicomicose foi estabelecido, pelo exame micológico direto, em 170 indivíduos. O diagnóstico etiológico foi estabelecido pela cultura para fungos. Dentre os 107 agentes identificados, os dermatófitos foram identificados em 78,52% (N=84), as leveduras em 13,08% (N=14) e os FFND em 8,40% (N=9) das vezes. CONCLUSÃO: É necessário que se estabeleça o diagnóstico etiológico dos casos de onicomicoses, já que os fungos filamentosos não dermatófitos ocorrem com freqüência considerável e são indistinguíveis daquelas por dermatófitos. / INTRODUCTION: Onychomycosis, a nail fungus infection is the most frequent nail disease, constituting about half of all nail disorder. It can be caused by dermatophytes, yeasts and non- dermatophytes filamentous fungi. OBJECTIVE: Characterize the onychomycosis caused by filamentous fungi non- dermatophytes. (1) Verify after a clinical suspicion of onychomycosis, which are the frequency of fungi recovery, (2) examine, after a clinical suspicion of onychomycosis, which species of fungi are recovered, (3) Checking, among the total of identified species , which are the frequency of the species of filamentous fungi not dermatophytes. METHODS: Two hundred and five patients with clinical suspicion of onychomycosis were studied in the period from December 2003 to November 2004, through direct mycological examination and culture for fungus. RESULTS: The diagnosis of onychomycosis has been established by direct mycological examination, in 170 individuals. The etiological diagnosis was established by the culture for fungus. Among the 107 persons identified, the dermatophytes were recovered in 78.52% (N = 84), the yeast in 13.08% (N = 14) and filamentous fungi non- dermatophytes in 8,40% (N = 9). CONCLUSION: It is necessary to establish the etiological diagnosis of the cases of onychomycosis, as filamentous fungi non- dermatophytes occur often than ever considered and are its clinic features are indistinguishable from those of the dermatophytes.

Xanthomonas oryzae pv. oryae (Xoo) est l'agent causal de la bactériose vasculaire du riz (BLB), maladie entraînant des pertes de rendement importantes dans la plupart des régions rizicoles, notamment en Afrique. La virulence des souches Asiatiques de Xoo dépend des effecteurs de la famille AvrBs3/PthA ou TAL (pour Transcription Activator-Like). Des études appro fondies sur le mode d'action des effecteurs TAL ont montré que l'activité de virulence ou d'avirulence que les TAL confèrent à la bactérie dépend essentiellement de leur interaction avec les gènes de sensibilité et/ou de résistance correspondants chez le riz. Les souches de Xoo originaires d'Afrique isolées récemment ne présentent que huit effecteurs de type TAL dans leur génome et leur rôle dans la virulence est jusqu'à présent inconnu. Les travaux de cette thèse ont porté sur la caractérisation des effecteurs de type TAL dans la souche africaine de Xoo BAI3. Une mutagenèse systématique par recombinaison homologue sur l'ensemble des huit gènes tal de la souche BAI3 a été effectuée et a conduit à l'identification et à la caractérisation du gène talC. TalC se caractérise par 21.5 répétitions et est phylogénétiquement proche des TAL de Xanthomonas oryzae pv. oryzicola (Xoc). Le mutant BAI3ΔtalC est incapable de développer les symptômes de la maladie sur plante sensible. Toutefois, les bactéries se multiplient très bien au niveau de l'apex foliaire proche du site d'inoculation, laissant supposer que TalC est requis pour la colonisation du système vasculaire. Parmi les cibles directes potentielles identifiées via l'analyse du transcriptome de feuilles de riz sensible (BAI3 versus BAI3ΔtalC), Os11N3 code une protéine de la famille des noduline-3 MtN3 et est fortement induit durant l'infection par la souche sauvage BAI3. Nous avons identifié dans la région promotrice de Os11N3 une séquence nucléotidique ciblée par TalC et démontré qu'elle était fonctionnelle via des expériences d'expression en trans dans N. benthamiana. Les travaux de cette thèse montrent pour la première fois que les effecteurs de type TAL contribuent de manière importante à la virulence de la souche africaine Xoo BAI3. Ces travaux contribueront à l'amélioration génétique des lignées de riz pour la résistance à la bactériose vasculaire du riz en Afrique. / Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of Bacterial Leaf Blight (BLB) on rice, a serious disease causing important yield losses in the main rice growing regions including Africa. The virulence of Asian Xoo strains mainly depends on the type III effectors of avrBs3/pthA gene family, namely TAL (for Transcription Activator Like) effectors. In depth studies on the function of TAL effectors revealed that the virulence and/or the avirulence activities conferred by these effectors requires the binding and the induction of the corresponding S and/or R genes. African Xoo strains was shown to harbor 8 TAL effectors in their genomes. However, the contribution of these TAL effectors to Xoo virulence is still unknown. This work reports on the identification and characterization of TAL effectors in the African Xoo strain BAI3R. A random mutagenesis based on homologous recombination in the genes encoding TAL effector was conducted in Xoo str ain BAI3R and led to the identification of talC. TalC harbors 21.5 repeats in its central domain and is phylogenetically more related to TAL effectors of Xanthomonas oryzae pv. oryzicola (Xoc). The BAI3RΔtalC mutant is seriously impaired in its virulence on susceptible rice varieties. Interestingly, bacteria are still able to grow at wild-type levels in the apex of the leaf, suggesting a requirement of talc for vascular colonization. Potential direct host targets were identified by conducting a transcriptomic analysis of rice leaves challenged with Xoo strain BAI3R vs. BAI3RΔtalC. Among the identified targets, the rice gene Os11N3 was found to be highly induced upon infection by the wild type strain but not the mutant one. A DNA target box for TalC was located in the Os11N3 upstream region and proved to be functional using GUS assays. We also show that the Os11N3 341-bp upstream region is transcriptionnally activated by TalC. Our results demonstrated for the first time that TAL effectors play an important role in the virulence of Xoo strain BAI3R. Our work will contribute to better improve rice for resistance to bacterial leaf blight.

L’objectif de cette thèse a été d’enrichir nos connaissances sur les bactériesintracellulaires strictes et spécialement Coxiella burnetii, agent responsable de la fièvre Q.Pour ce faire, nous avons d’une part amélioré les techniques de préparation de l’ARN pourles études transcriptionnelles et d’autre part utilisé la technologie des puces à ADN pouranalyser le transcriptome ainsi que la diversité génomique de C. burnetii.L’utilisation d’échantillons cliniques dans les études transcriptionnelles est limitéepar la quantité de matériel disponible qui ne permet pas d’analyser simultanément les profilsdu pathogène et de son hôte au cours de l’infection. De ce fait, nous avons développé, sur unmodèle d’escarres obtenus à partir de malades de fièvre boutonneuse méditerranéenne, unestratégie basée sur l’hybridation soustractive pour séparer les ARN eucaryotes etprocaryotes dans le but d’entreprendre des hybridations de puces à ADN.C. burnetii est une bactérie hautement résistante aux stress environnementaux commele changement de pH, la dessiccation, mais aussi le changement de température. Nous avonsparticulièrement étudié la réponse précoce de C. burnetii lors d’une exposition à une hauteet faible température. L’analyse globale du profil transcriptionnel a montré que la réponsede C. burnetii était limitée et similaire pour les différents stress appliqués. Cependant,malgré cette faible réponse, il apparait clairement qu’une accumulation de ppGpp, un arrêtde la croissance et des modifications de la membrane et de la paroi cellulaire permettraient àC. burnetii de résister à ces stress. Toutes ces régulations géniques convergent vers unchangement d’état de la bactérie vers une forme pseudo-sporulée. De plus, nous avonsobservé une organisation spatiale des gènes différentiellement exprimés. Nos analyses bioinformatiquesont montré que ces clusters de régulation ne répondaient ni au paradigmepromoteur - facteur de transcription – opéron, ni à des réseaux biologiques. Ayant retrouvéce phénomène dans plusieurs autres études transcriptionnelles chez d’autres bactériesintracellulaires, nous spéculons que ces clusters de régulations pourraient être dus à unerégulation épigénétique qu’il reste à caractériser.Différentes méthodes de typage ont déjà été mises au point pour classer les isolats deC. burnetii dans le but d'explorer son pouvoir pathogène. Ici, nous présentons une méthodede génomotypage basée sur la présence ou l'absence de gènes à l'aide de puces à ADN.Nous avons testé notre stratégie de génomotypage sur 52 isolats provenant de différenteszones géographiques, de différents hôtes et de patients présentant différentes manifestationscliniques. L'analyse a révélé la présence de 10 génomotypes organisés en 3 groupes avecune topologie congruente à celle observée avec le Multi Spacer Typing. Nous avons aussidécouvert 4 génomotypes particulièrement associés à la fièvre Q aiguë, alors que tous lesgénomotypes étaient associés à la forme chronique. De plus, le génomotypage a révélé queles isolats retrouvés dans les tiques dures, y compris la souche de référence Nine Mileappartiennent au même genomotype.Globalement, les données que nous avons obtenues confirment le fait que les puces àADN sont un outil adapté pour l’analyse de la pathogénicité de C .burnetti mais aussi desautres bactéries intracellulaires strictes. Cependant, de nouvelles technologies plusrésolutives comme le DNA ou RNAseq semblent être plus prometteuses mais restent encoreà optimiser / The objective of this thesis was to increase knowledge of obligate intracellular bacteriaand specifically, the causative agent of Q fever C. burnetii. In this regard, we have improvedstrategies to purify RNA for the transcriptional studies. We also used the technologymicroarrays to analyze the transcriptome and genomic diversity of C. burnetii.The use of clinical samples in the transcriptional studies is limited by the amount ofmaterial available and thus the transcriptional profiles of the pathogen and its host duringinfection can not be simultaneously analyze. We developed, with a model of eschars obtainedfrom Mediterranean spotted fever patients, a strategy based on subtractive hybridization toseparate RNA eukaryotic and prokaryotic cells in order to perform microarray experiments.Analysis of the survival strategies used by this bacterium to adapt to new environmentalconditions is critical for our understanding of C. burnetii pathogenicity. Here, we report theearly transcriptional response of C. burnetii under temperature stresses. Our data show thatC. burnetii exhibited minor changes in gene regulation under short exposure to heat or coldshock. While small differences were observed, C. burnetii seemed to respond similarly to coldand heat shock. The expression profiles obtained using microarrays produced in-house wereconfirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentiallyexpressed in at least one condition, with a fold change of up to 4. Globally, the differentiallyexpressed genes in C. burnetii were associated with bacterial division, (p)ppGpp synthesis,wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycansynthesis. These findings could be associated with growth arrest and witnessed transformationof the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes weredifferentially expressed. These clusters do not belong to operons or genetic networks; theyhave no evident associated functions and are not under the control of the same promoters. Wealso found undescribed but comparable clusters of regulation in previously reportedtranscriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeriamonocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stressespermits the recognition of unpredicted clusters of regulation for which the trigger mechanismremains unidentified but which may be the result of a new mechanism of epigenetic regulation.Different typing methods have been previously developed to classify C. burnetii isolatesin order to explore its pathogenicity. Here, we report a comprehensive genomotyping methodbased on presence or absence of genes using microarray. The genomotyping method was thentested on 52 isolates obtained from different geographic areas, different hosts and isolated frompatient with different clinical manifestations. The analysis reveals the presence of10 genomotypes organized in 3 groups with a topology congruent with that of Multi SpacerTyping. We also found out 4 genomotypes especially associated with acute Q fever whereas allthe genomotypes could be associated to chronic human infection. Serendipity, genomotypingreveals that hard ticks isolates including Nine Mile belong to the same genomotype.Overall, the data we obtained confirm that DNA microarrays are a suitable tool forexploring pathogenicity of C. Burnetti and other obligate intracellular bacteria. However newtechnologies such as DNAseq or RNAseq seem more promising but still need to optimize andalso are still expensive compared to microarray.

Xanthomonas axonopodis pv. manihotis (Xam) est une bactérie à gram négatif causant le Cassava Bacterial Blight (CBB) sur Manihot esculenta Crantz. Le manioc représente une des sources les plus importantes de carbohydrates pour près d'un milliard de personnes sur terre et une source importante d'énergie du fait de sa forte concentration en amidon. Le CBB constitue une limitation importante à la production massive de manioc et nos connaissances sur cette maladie sont encore insuffisantes. La pathogénie de nombreuses phytobactéries dépend de l'injection d'effecteurs de type III via un système de sécrétion de type III dans la cellule eucaryote hôte Parmi tous les effecteurs référencés aujourd'hui, les effecteurs de type TAL pour Transcription Activator-Like sont particulièrement intéressant. Une fois injectés dans la cellule végétale, les effecteurs TAL sont importés au noyau et y modulent l'expression de gènes cibles au bénéfice de la bactérie. Chez Xam, TALE1Xam est le seul gène de cette famille qui a été étudié au niveau fonctionnel. Cette étude a pour objectif majeur d'identifier les gènes de manioc dont l'expression est modifiée en présence de TALE1Xam. Le transcriptome de plantes de manioc inoculées avec XamΔTALE1Xam vs. XamΔTALE1Xam (TALE1Xam) a été analysé par RNAseq. Les données obtenues confrontées à la recherche bioinformatique de promoteurs de gènes potentiellement directement activés par TALE1Xam ont permis d'établir une liste de gènes ciblés par TALE1Xam candidats. Un candidat majeur ressort de cette analyse comme étant particulièrement intéressant, il s'agit d'un gène codant un facteur de transcription de type B3 régulant l'activité de protéines de type "Heat Shock". L'analyse fonctionnelle de ce candidat permettra de valider sa fonction en tant que gène de sensibilité du manioc à Xam. / Xanthomonas axonopodis pv. manihotis (Xam) is a gram negative bacteria causing the Cassava Bacterial Blight (CBB) in Manihot esculenta Crantz . Cassava represents one of the most important sources of carbohydrates for around one billion people around the world as well as a source of energy due to its high starch levels content. The CBB disease represents an important limitation for cassava massive production and little is known about this pathosystem. Bacterial pathogenicity often relies on the injection in eucaryotic host cells of effector proteins via a type III secretion system (TTSS). Between all the type III effectors described up to now, Transcription Activator-Like Type III effectors (TALE) appear as particularly interesting. Once injected into the plant cell, TAL effectors go into the nucleus cell and modulate the expression of target host genes to the benefit of the invading bacteria by interacting directly with plant DNA. In Xam, only one gene belonging to this family has been functionally studied so far. It consists on TALE1xam. This work aim to identify cassava genes whose expression will be modified upon the presence of TALE1xam. By means of cassava plants challenged with Xam Δ TALE1xam vs. Xam + TALE1xam together with the TAL effectors code, statistical analyses between RNAseq experiments and a microarray containing 5700 cassava genes, we seek out direct TALE1xam target genes. Hence, through transcriptomic, functional qRT validation and specific artificial TALEs design we proposed that TALE1xam is potentially interacting with a Heat Shock Transcription Factor B3. Moreover we argue that this gene is responsible of the susceptibility during Xam infection. Furthermore this work represents the first complete transcriptomic approach done in the cassava/Xam interaction and open enormous possibilities to understand and study CBB.