A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Dentistry Johannesburg, 2015 / Objectives: To analyse the clinico-pathological features of orthokeratinised jaw cysts (OJCs) and to determine whether human papillomavirus (HPV) DNA can be detected in OJCs. Material and methods: The clinical and radiological information of 30 patients diagnosed with OJCs were reviewed and the respective histology samples were studied for light microscopic features characteristic of HPV infection. The 30 OJCs were further evaluated for the presence of HPV by using consensus HPV polymerase chain reaction (PCR). Results: Patients with OJC ranged from 13 to 71-years (mean, 30.9 years; ± 12.9 years). There was a predilection for males (21/30). Most OJCs were found in the mandible (80%) and 44.8% were associated with an impacted tooth. Koilocyte-like characteristics were identified in 70% of cases, while 43.3% of cases showed a verruciform pattern of hyperkeratosis. All 30 OJCs were negative for HPV-DNA. Conclusion: HPV infection does not appear to play a role in the OJC and is not responsible for the wart-like histological changes that may be encountered in OJCs.

Grace Chung Tin Yun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 129-146). / SUMMARY / INTRODUCTION --- p.1 / LITERATURE REVIEW / Chapter 2.1 --- Anatomy and Histology of Uterine Cervix --- p.4 / Chapter 2.2 --- Cervical carcinoma --- p.5 / Chapter A. --- Incidence / Chapter B. --- Pathology and histological classification / Chapter C. --- Etiological factors / Chapter 2.3 --- Human Papillomavirus (HPV) --- p.12 / Chapter 2.4 --- Human papillomavirus and cervical carcinoma --- p.15 / Chapter 2.5 --- Principle of Polymerase Chain Reaction (PCR) --- p.17 / Chapter A. --- DNA structure / Chapter B. --- Repetitive DNA synthesis by PCR / Chapter 2.6 --- Basis of Restriction Fragment Length Polymorphism analysis (RFLP) --- p.18 / Chapter 2.7 --- RFLP analysis and cancer --- p.19 / Chapter 2.8 --- Oncogenes and Suppressor genes --- p.21 / Chapter A. --- Oncogenes / Chapter B. --- Tumour Suppressor Genes / Chapter 2.9 --- Multistep process of carcinogenesis --- p.24 / Chapter 2.10 --- Oncogenes and Suppressor genes in cervical carcinoma --- p.25 / Chapter A. --- Oncogenes / Chapter B. --- Tumour Suppressor Genes / MATERIALS AND METHODS / Chapter 3.1 --- Specimens --- p.41 / Chapter A. --- Cervical scrapes / Chapter B. --- Cervical tissues / Chapter C. --- Blood / Chapter 3.2 --- Extraction of Genomic DNA --- p.42 / Chapter A. --- Cervical scrapes / Chapter B. --- Tissue samples / Chapter C. --- Blood / Chapter 3.3 --- Southern blot --- p.45 / Chapter 3.4 --- Polymerase Chain Reaction (PGR) --- p.47 / Chapter 3.5 --- Nucleic Acid Probes --- p.49 / Chapter A. --- Oligonucleotide probes / Chapter B. --- Human papillomavirus / Chapter C. --- Chromosome-specific RFLP/VNTR probes / Chapter D. --- H-ras and c-myc proto-oncogenes / Chapter 3.6 --- Radioactive Labelling of Probes --- p.51 / Chapter A. --- Oligonucleotide probes / Chapter B. --- Nucleic Acid probes / Chapter C. --- Removal of unincorporated label / Chapter 3.7 --- Nucleic Acid Hybridization --- p.54 / RESULTS / Chapter 4.1 --- Optimize MgCl2 concentration for Polymerase chain reaction --- p.72 / Chapter 4.2 --- Sensitivity of Polymerase chain reaction --- p.72 / Chapter 4.3 --- Detection of HPV in cervical scrapes by PCR --- p.73 / Chapter 4.4 --- Detection of HPV in cervical scrapes using PCR with each primer-set --- p.73 / Chapter 4.5 --- Detection of HPV in cervical tissues using PCR --- p.73 / Chapter 4.6 --- Detection of HPV in cervical tissues by Southern blot hybridization --- p.74 / Chapter 4.7 --- Analysis of cervical tissues with chromosome 3-specific probes --- p.75 / Chapter 4.8 --- Analysis of cervical tissues with proto-oncogene probes H-ras and c-myc --- p.77 / Chapter 4.9 --- Analysis of cervical tissues with chromosome 17-specific probes mapped to 17pl3 --- p.77 / DISCUSSION / Chapter 5.1 --- Evaluation of methods --- p.111 / Chapter 5.2 --- Analysis of HPV using PCR --- p.114 / Chapter 5.3 --- PCR vs Southern blot hybridization in HPV detection --- p.117 / Chapter 5.4 --- HPV DNA status in cervical neoplasia and cervical cancer --- p.118 / Chapter 5.5 --- Genetic lesions in cervical neoplasia and cervical cancer --- p.119 / Chapter 5.6 --- HPV infection and genetic lesions in cervical cancer --- p.125 / CONCLUSION --- p.127 / REFERENCE --- p.129

Thèse doctorat : Sciences de la Vie : Strasbourg 1 : 2005. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 18 p.

Orientador: Christiane Pienna Soares / Banca: Luis Octávio Regasini / Banca: Denise Crispim Tavares / Resumo: Atualmente, o câncer cervical é considerado a segunda causa mais comum entre as mulheres e encontra-se associado ao Papilomavírus Humano (HPV), que infecta células epiteliais escamosas, dando origem a grandes lesões. A nitensidina A é um alcalóide guanidínico isolado de Pterogyne nitens Tul. (Fabaceae), que possui atividade anti-inflamatória e anti-oxidante, além de um amplo aspecto de atividades biológicas que está sendo estudado. Foi testada nas concentrações de 20,0 a 0,25 µg/mL (diluição seriada 1:3) e nos tempos de 24 h e 48 h nas linhagens imortalizadas com HPV-16 (SiHa) e não imortalizadas (C33A). Para avaliar a citotoxicidade da nitensidina A o teste MTT foi realizado sendo possível observar um efeito concentração-resposta em duas linhagens testadas (SiHa e C33A, CI50 = 10,31 µg/mL e 11,91 µg/mL, respectivamente) por 24 h. Com o intuito de avaliar o potencial efeito indutor de apoptose, foi realizado o ensaio de anexina V por citometria de fluxo. Em ambas as linhagens e tempos de tratamento notaram-se uma morte mais relevante por apoptose precoce, sendo que há diferença apenas na maior concentração (20,0 g/mL) na linhagem SiHa, que também induz morte por apoptose tardia/necrose. Para completar esse ensaio foi realizado o teste do Hoechst-iodeto de Propídeo, diferenciando assim apoptose precoce, tardia e necrose. Na linhagem SiHa tratada por 24 h nas menores concentrações houve morte celular por apoptose precoce, e na maior concentração por apoptose tardia (81,4±1,4 %). Por 48 h, em todas as concentrações a morte celular foi por apoptose tardia. Na linhagem C33A em 24 h não foi observado morte celular significativa. Em 48 h, a maior concentração induziu apoptose tardia (57,7±20,6 %). O modelo de estudo de implante tumoral xenográfico de células SiHa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently, cervical cancer has been considered the second most common among women and is associated with human papillomavirus (HPV) which infects squamous epithelial cells, resulting in major injuries. Nitensidine A is a guanidine alkaloid isolated from Pterogyne nitens Tul. (Fabaceae), which has anti-inflammatory and antioxidant, as well as a broad spectrum of biological activities being studied. It was tested at concentrations from 20.0 to 0.25 mg/mL (serial dilution 1:3) and for 24 h and 48 h in immortalized with HPV-16 (SiHa) and non-immortalized (C33A) cell lines. To evaluate the cytotoxicity of nitensidine A MTT test was performed and it was possible to observe a concentration-response effect in two tested lines (SiHa and C33A, IC50 = 10.31 mg/mL and 11.91 mg/mL, respectively) for 24 h. In order to assess the potential effect of inducing apoptosis, we performed the annexin V assay by flow cytometry. In both strains and treatment times were noted a death most significant by early apoptosis, and a difference only at highest concentration (20.0 mg/mL) in SiHa, which also induces death by late apoptosis / necrosis. To complete this test, it was performed the Hoechst-propidium iodide assay, thereby differentiating early, late apoptosis and necrosis. In SiHa treated for 24 h at lower concentrations there was cell death by early apoptosis, and late apoptosis at highest concentration (81.4 ± 1.4%). For 48 h at all concentrations cell death was by late apoptosis. In C33A at 24 h was not observed significant cell death. At 48 h, the highest concentration induced late apoptosis (57.7 ± 20.6 %). The study model of xenographic tumor implant of SiHa cells in nude mice BALB/c treated with nitensidine... (Complete abstract click electronic access below) / Mestre

Orientador: Sophie Françoise Mauricette Derchain / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T02:01:22Z (GMT). No. of bitstreams: 1 Figueiredo_PriscilaGarcia_D.pdf: 2027663 bytes, checksum: 8870b21112f34b9523a25e0440c2ea6f (MD5) Previous issue date: 2006 / Resumo: Introdução: a vaginose bacteriana (VB) representa uma condição clínica caracterizada pela substituição da flora vaginal normal por bactérias predominantemente anaeróbias ou facultativas. Paralelamente, estudos têm demonstrado que a expressão da ciclo-oxigenase-2 (COX-2), enzima marcadora de inflamação, encontra-se alterada em processos neoplásicos. Objetivo: avaliar a relação entre a expressão da COX-2 e a presença de VB em mulheres portadoras de lesões escamosas intra-epiteliais cervicais. Sujeitos e métodos: para este estudo de corte transversal, foram selecionadas 228 mulheres portadoras de anormalidades citológicas compatíveis com lesões induzidas pelo Papilomavirus humano (HPV), no período de Fevereiro de 2001 a Abril de 2004. A VB foi diagnosticada através da presença de pelo menos 20% de clue cells no esfregaço de Papanicolaou. A detecção do HPV de alto risco oncogênico, foi realizada através do exame de Captura Híbrida II (CH II), sendo estas amostras colhidas antes da realização da biópsia ou da conização. O diagnóstico histológico foi compatível com colo normal ou cervicite em 11 casos (5%), neoplasia intra-epitelial cervical (NIC) grau 1 (NIC 1) em 35 (15 %), NIC 2 em 31 (14%) e NIC 3 em 151 casos (66%). A expressão citoplasmática da COX-2 foi determinada por imuno-histoquímica e avaliada no espécime de tecido que apresentava a lesão mais grave. Resultados: a VB foi diagnosticada em 38 (17%) das 228 mulheres. O HPV de alto risco oncogênico foi detectado em 192 (84%) mulheres. O diagnóstico de VB foi significativamente maior em mulheres com infecção pelo HPV (p=0,05). A distribuição da VB foi similar entre os diferentes graus histológicos da NIC (p=0,42). A presença de VB foi significativamente menor em mulheres com expressão moderada ou forte da COX-2 (p=0,02; OR=0,4 IC 95% 0,2 a 0,9). Quando se relacionou a detecção do HPV com a expressão da COX-2, observou-se uma proporção significativamente maior de mulheres infectadas pelo HPV com expressão moderada ou forte da COX-2 (p=0.04). A expressão da COX-2 não esteve significativamente associada à gravidade da NIC (p=0.24). Conclusões: a VB esteve associada com a presença de infecção pelo HPV e com a menor expressão da COX-2. A expressão da COX-2 foi maior em mulheres infectadas pelo HPV, embora não tenha havido associação entre a expressão da COX-2 e a gravidade da NIC / Abstract: Background: bacterial vaginosis (BV) is a syndrome characterized by a reduction in the normal Lactobacillus microflora and an excessive growth of microorganisms such as anaerobes and facultative. In parallel, studies suggest that COX-2 expression, an enzyme related to inflammatory response, is altered due to neoplastic processes. Objectives: to assess the relation between COX-2 expression and BV in women with cervical squamous intraepithelial lesions. Subjects and methods: a cross-sectional study enrolled 228 women due to cytological abnormalities in Papanicolaou smears, through February 2001 to April 2004. The finding of 20% or more clue cells on Papanicolaou smears was considered positive for the presence of BV. High-risk HPV detection was assessed through Hybrid Capture II (HCII). Collection of samples for Papanicolaou smears and for HCII was performed immediately before biopsy or conization. Pathological diagnoses were classified as normal/cervicitis in 11 cases (5%), cervical intraepithelial neoplasia (CIN) grade 1 (CIN 1) in 35 (15 %), CIN 2 in 31 (14%) and CIN 3 in 151 (66%). Cytoplasmic expression of COX-2, evaluated from the tissue block that harbored the most significant lesion, was ascertained through immunohistochemistry. Results: BV was rendered as positive in 38 (17%) of 228 women. High-risk HPV detection was present in 192 (84%) women. The diagnosis of BV was significantly higher in women with HPV infection (p=0.05). The prevalence of BV was similar across different grade of CIN (p=0.42). Presence of BV was significantly lower among women with moderate and strong expression of COX-2 (p=0.02; OR=0.4 IC95% 0.2 to 0.9). When analyzing HPV detection according to the COX-2 expression, a significantly higher proportion of women infected by high-risk HPV showed a moderate and strong expression of COX-2 (p=0.04). COX-2 expression was not associated with the severity of CIN (p=0.24). Conclusions: the presence of BV was related to HPV infection and negative or weak COX-2 expression. COX-2 expression was higher in HPV infected women, although no difference was observed in relation to the severity of CIN / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Obstetrics & Gynaecology. Johannesburg, 2017. / Background and objectives Evidence supporting an association between HPV infection and pre-eclampsia has recently been published. Pre-eclampsia is a common, serious complication of pregnancy of complex aetiology that to date has not been fully described. Human papillomavirus (HPV) is a ubiquitous, DNA-virus with tropism for human mucosal commonly found in the female genital tract. Association between placental HPV infection and preterm labour and pregnancy loss has previously been described. This study tested the hypothesis that an association exists between HPV in the placenta or the cervix and clinical pre-eclampsia, or levels of its associated biomarkers, soluble fms-like tyrosine kinase (sFLT1) and placental growth factor (PlGF). Methods Women with pre-eclampsia were matched to healthy controls. All subjects were delivered by caesarean section, and cervical and placental samples were collected at the time of delivery. These samples were tested for HPV using a polymerase chain reaction (PCR) assay. Serum levels of soluble fms-like tyrosine kinase (sFLT1) and placental growth factor (PlGF) at the time of delivery were tested. Placental and cervical HPV was compared to the outcomes of clinical pre-eclampsia and serum sFLT1 and PlGF levels. Results and conclusion While clinically apparent disease was associated with increased levels of sFLT1 and decreased levels of PlGF, HPV was not detected in any of the placental specimens using the PCR assay. As a result, no association was found between placental HPV detection and clinically apparent pre-eclampsia or deranged serum levels of sFLT1 or PlGF. HPV was very common in cervical samples and showed a non-significant trend towards negative association with clinical pre-eclampsia and sFLT1, and a positive association with PlGF. This may be an effect of cervical HPV infection on the vascular endothelial growth factor (VEGF) signalling system that may explain its association with miscarriage. / LG2018

Resumo: O papilomavírus humano (HPV) está associado à um amplo espectro de lesões em humanos e tem sido associado à carcinogênese oral. Uma das metodologias mais sensíveis e especifica para sua detecção é a reação em cadeia de polimerase (PCR). O objetivo deste estudo foi investigar a presença do DNA do HPV em carcinoma epidermóide de lábio e correlacioná-la com fatores de risco, características clínicas, anatomopatológicas e sobrevida dos pacientes estudados. A presença do DNA do HPV foi investigada através da nested PCR em 30 amostras parafinadas de carcinoma epidermóide de lábio. O DNA viral foi detectado em 43,33% (13/30) das amostras. A presença do DNA do vírus foi relacionada com: idade, sexo, etnia, graduação histológica, etilismo, tabagismo, exposição freqüente à radiação solar, estadiamento clínico e sobrevida livre de doença e nenhuma correlação entre a presença do DNA viral e os parâmetros avaliados foi observada. Estes resultados sugerem que HPV não teve participação no processo de carcinogênese dos carcinomas epidermóides de lábio estudados. / Abstract: Human papillomavirus (HPV) is associated with a wide spectrum of lesions in humans and has been related to the oral carcinogenesis. The most sensitive and specific methodology for its detection is the polymerase chain reaction (PCR). The aim of this study was to investigate HPV DNA presence in lip squamous cell carcinoma and to correlate it with demographic, clinicalpathologic characteristics and survival data. HPV DNA was investigated by nested PCR in 30 paraffin-embedded tissues of lip squamous cell carcinomas. HPV DNA was detected in 43,33% (13/30) of samples. Presence of viral DNA was not associated with the following factors: age, sex, race, histologic grade, etilism, tabagism, history of solar radiation exposition, clinical staging and survival of the patients. This results suggest that HPV is not related to the lip squamous cell carcinomas carcinogenesis. / Orientador: Glauco Issamu Miyahara / Coorientador: José Fernando Garcia / Banca: Cáris Maroni Nunes / Banca: Ricardo Della Coletta / Mestre

Orientador: Glauco Issamu Miyahara / Coorientador: Cáris Maroni Nunes / Banca: Sandra Helena Penha de Oliveira / Banca: Izabel Regina Fischer Rubira-Bullen / Resumo: A leucoplasia bucal é considerada uma lesão cancerizável para o desenvolvimento do carcinoma espinocelular (CEC), e vários fatores de risco podem estar relacionados a essa carcinogênese, incluindo o papiloma vírus humano (HPV). Na mucosa bucal tem sido feita a associação da leucoplasia bucal com o HPV. O objetivo desse estudo foi detectar a presença do DNA do HPV em amostras de tecidos fresco, saliva, plasma e células exfoliadas orais, extraídas de pacientes com e sem leucoplasia bucal, analisadas através da técnica de nested PCR (nPCR). Para este estudo foram avaliados 32 pacientes portadores de leucoplasia bucal e 24 pacientes selecionados de forma caso-controle. Foi realizada a extração de DNA das amostras, e em seguida a sua amplificação através da PCR. A nPCR para detecção do HPV revelou a presença do vírus em 68,75% das amostras de tecido fresco, 50% no plasma, 28,1% no citobrush, 62,5% na saliva no grupo de pacientes com leucoplasia em comparação com 45,8%, 54,2%, 45,8%, 50% nas amostras de tecido fresco, plasma, citobrush e saliva do grupo controle respectivamente. Baseado no presente estudo o HPV poderia ser um co-fator etiológico na patogênese da leucoplasia oral. / Abstract: The oral leukoplakia is considered as a pre-malignant lesion for the development of the oral squamous cell carcinoma, and several risk factors can be related to this carcinogenesis, including the human papillomavirus (HPV). HPV is the main cause of cervix cancer, and your role in the oral carcinogenesis is still controversial. The aims of this study was to detect the presence of HPV DNA in fresh tissue samples, plasma and oral exfoliated cells extracted from patients 32 with oral leukoplakia, and 24 controls analyzed through the technique of nPCR and make a comparison among these biological materials sources. It was performed the extraction of DNA from 37 patients with oral leukoplakia, and amplification of the human β-globin gene was carried out in all samples to confirm the presence and integrity of DNA. Nested PCR assays revealed the presence of HPV DNA in 68.75% of fresh tissue, 50.0% of plasma, 62.5% of saliva, and in 28.1% of oral exfoliated cells extracted from patients. Based on the current experiment, HPV could potentially be an etiologic co-factor in the pathogenesis of oral leukoplakia. / Mestre

人類乳頭瘤病毒 (HPV) 是引起宮頸癌的必要條件。在高危型HPV中，以HPV16在癌症樣本中最為常見，其全球盛行率達50%以上。近年來，用以辨認HPV16變異子譜系的序列特徵已經建立。雖然這個系統建基於全球的HPV16變異株，但是它只包含了四個亞洲地區。為了改善這個系統於亞洲樣本的準確性，是次研究收集了更多亞洲地區的序列。 / 是次研究提供了在香港和韓國收集的HPV16樣本的系統發生史及序列變異 (LCR、E6 和 E7)。此外，是次研究也檢測了HPV16變異株的在兩地的分佈和致癌風險。 / 是次研究從香港和韓國收集了329個HPV16呈陽性的宮頸樣本。利用LCR、E6、E7 和整合的LCR-E6基因序列以極大似然法來構建HPV16變異株的系統發生樹。序列變異會按照系統發生樹之拓撲結構來分類並詳細描述。卡方檢驗或費雪精確性檢定用於分析HPV16變異株在兩地的分佈和致癌風險。 / 是次研究結果顯示用以辨認HPV16變異子譜系的序列特徵需加以改善。我們建議採用A7287C/T作為亞洲子譜系的序列特徵，以替代原有的A7287C。有關HPV16變異株的地理分佈，亞洲和歐洲的變異株在香港 (亞洲變異株: 70%，歐洲變異株: 25.3%) 和韓國 (亞洲變異株: 61.2%，歐洲變異株: 20.2%) 均十分普遍。另外，1和2型亞美變異株在香港和韓國的分佈有著明顯差別 (1型亞美變異株: 2% 與12.4%，P < 0.001; 2型亞美變異株: 0% 與2.8%，P = 0.04)。 / 另外，是次研究發現亞洲子譜系於韓國民族中呈較高致癌風險 [比值比 (95% 置信區間) 2.02 (1.03-3.99)]。在進化支中，E6的第五進化支[2.44 (1.27-4.74)]和E7的第三進化支[2.02 (1.03-3.99)]也於韓國民族中呈較高致癌風險。在SNP中，E6 T178G [2.17 (1.11-4.23)]、兩個E7的SNPs (A647G [1.73 (0.88-3.42)]、T846C [2.27 (1.16-4.49)]) 和9個LCR SNPs (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] 和 C24T [2.36 (1.20-4.66)])於韓國民族中也呈較高致癌風險。這些進化支和SNPs都與亞洲子譜系有關聯。在香港方面，兩個LCR SNPs (A7289C [1.89 (0.92-3.87)] 和 T7781C [2.07 (0.92-4.71)])呈較高致癌風險。 / 是次研究發現的高危SNPs和進化支需要進一步的大型流行病學研究和生物化學實驗來核實。這些序列特徵可作為生物標誌物以檢測出與HPV有關的早期宮頸病變。 / Human papillomavirus (HPV) is necessary for the development of cervical cancer. Of those high-risk HPV types, HPV16 is the most common type detected in cervical cancer and accounts for a prevalence of greater than 50% worldwide. Recently, a sequence signature-based system for identifying the sub-lineages of HPV16 variants has been established. Although this system was developed from HPV16 variants collected worldwide, only four Asian regions were included. To improve the accuracy of this sub-lineage classification system for Asian samples, more sequence data from Asian regions were included in the current study. / The current study provided data on the phylogeny and the sequence variation of Long control region (LCR), E6 and E7 open reading frames (ORFs) of HPV16 isolates collected in Hong Kong and Korea. The distribution of HPV16 variants between two regions and the risk association of HPV16 variants with cervical cancer development were also examined. / A total of 329 HPV16-positive cervical samples were collected from Hong Kong and Korea. The phylogenetic trees were constructed for the LCR, E6, E7 and concatenated LCR-E6 sequences using the maximum likelihood method. The sequence variation of each region was delineated and grouped according to the tree topology. The distribution and risk association of HPV16 variants were examined using the chi-square test or Fisher’s exact test as appropriate. / The results showed that the previously described sequence signatures for classifying sub-lineages of HPV16 variants required further improvement, especially for the Asian sub-lineage. We proposed A7287C/T as a signature SNP of the Asian sub-lineage rather than A7287C as suggested by Cornet et al. In regard to the distribution of HPV16 variants, the Asian (As) and European (Eur) variants were commonly found in Hong Kong (As: 70%, Eur: 25.3%) and Korea (As: 61.2%, Eur: 20.2%). Furthermore, Asian American-1 and 2 (AA1 and AA2) variants were found to distribute significantly different between Hong Kong and Korea (AA1: 2% versus 12.4%, P < 0.001; AA2: 0% versus 2.8%, P = 0.04). / A key finding was that variants of the Asian sub-lineage carried a higher oncogenicity among Korean population [odds ratio (95% confidence interval) = 2.02 (1.03-3.99)]. In clade level, E6 clade 5 [2.44 (1.27-4.74)] and E7 clade 3 [2.02 (1.03-3.99)] were found to carry a higher oncogenicity among Korean population. In SNP level, E6 T178G [2.17 (1.11-4.23)], two SNPs of E7 ORF (A647G [1.73 (0.88-3.42)] and T846C [2.27 (1.16-4.49)]) and nine SNPs of LCR (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] and C24T [2.36 (1.20-4.66)]) were also found to carry a higher oncogenicity among Korean population. Those clades and SNPs were linked to the Asian sub-lineage. In contrast, only two SNPs of LCR (A7289C [1.89 (0.92-3.87)] and T7781C [2.07 (0.92-4.71)]) were found to associate with a higher oncogenicity among Hong Kong population. / The risk associations of SNPs, clades of the HPV16 Asian sub-lineage revealed by the current study should be verified by large-scale epidemiological studies and biochemical experiments. These signatures may serve as biomarkers for early detection of HPV-related cervical neoplasia. / 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. / Detailed summary in vernacular field only. / Ma, Tsz Ue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-156). / Abstracts also in Chinese. / Abstract of Thesis --- p.I / 論文摘要 --- p.V / Acknowledgements --- p.VIII / Contents --- p.X / Figures --- p.XIII / Tables --- p.XIV / Abbreviations --- p.XVI / Chapter Chapter 1 --- 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 Organization and Protein Functions --- p.4 / Chapter 1.2.1.1 --- E5 Protein --- p.7 / Chapter 1.2.1.2 --- E6 Protein --- p.8 / Chapter 1.2.1.3 --- E7 Protein --- p.9 / Chapter 1.2.2 --- Life Cycle of Human Papillomavirus --- p.10 / Chapter 1.2.3 --- Taxonomy of Human Papillomavirus --- p.12 / Chapter 1.3 --- Cervical Cancer --- p.16 / Chapter 1.3.1 --- Natural History --- p.16 / Chapter 1.3.2 --- Risk Factors --- p.17 / Chapter 1.4 --- Epidemiology of Cervical Cancer --- p.19 / Chapter 1.4.1 --- Global Disease Burden --- p.19 / Chapter 1.4.2 --- Disease Burden in Hong Kong --- p.21 / Chapter 1.4.3 --- Disease Burden in South Korea --- p.22 / Chapter 1.5 --- Human Papillomavirus Type 16 --- p.23 / Chapter 1.6 --- Background and Objectives --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Study Design --- p.31 / Chapter 2.2 --- Study Samples --- p.35 / Chapter 2.2.1 --- HPV16-Positive Samples --- p.35 / Chapter 2.2.2 --- Samples with Unknown HPV Status --- p.36 / Chapter 2.3 --- Laboratory Methods --- p.39 / Chapter 2.3.1 --- DNA Extraction --- p.39 / Chapter 2.3.2 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.2.1 --- PGMY09/11 PCR --- p.40 / Chapter 2.3.2.2 --- HPV16-Specific PCR --- p.42 / Chapter 2.3.3 --- Genotyping of HPV --- p.48 / Chapter 2.3.4 --- Purification of PCR Products --- p.51 / Chapter 2.3.5 --- Sequencing Reaction --- p.52 / Chapter 2.4 --- Data Analysis --- p.54 / Chapter 2.4.1 --- Sequence Edit and Alignment --- p.54 / Chapter 2.4.2 --- Sequence Variation of HPV16 Variants --- p.56 / Chapter 2.4.3 --- Construction of Phylogenetic Tree --- p.56 / Chapter 2.4.4 --- Distribution and Comparison of HPV16 Variants in Hong Kong and Korea --- p.57 / Chapter 2.4.5 --- Distribution of HPV16 Variants in Normal and Cancer Samples and Risk Association Study --- p.58 / Chapter Chapter 3 --- Results --- p.59 / Chapter 3.1 --- Study Samples --- p.60 / Chapter 3.1.1 --- HPV16-Positive Samples --- p.60 / Chapter 3.1.2 --- Samples with Unknown HPV Status --- p.61 / Chapter 3.2 --- Sub-lineage Identification of HPV16 Variants --- p.63 / Chapter 3.2.1 --- Based on the Phylogenetic Analysis in the Current Study --- p.63 / Chapter 3.2.1.1 --- Concatenated LCR-E6 Phylogenetic Tree --- p.63 / Chapter 3.2.1.2 --- LCR Phylogenetic Tree --- p.66 / Chapter 3.2.1.3 --- E6 Phylogenetic Tree --- p.69 / Chapter 3.2.2 --- Based on the Single Nucleotide Polymorphisms Proposed by Cornet et al. --- p.74 / Chapter 3.2.2.1 --- Single Nucleotide Polymorphisms of LCR Sequence --- p.74 / Chapter 3.2.2.2 --- Single Nucleotide Polymorphisms of E6 Open Reading Frame --- p.78 / Chapter 3.3 --- Sequence Variation of HPV16 Variants --- p.82 / Chapter 3.3.1 --- LCR Sequence --- p.82 / Chapter 3.3.2 --- E6 Open Reading Frame --- p.91 / Chapter 3.3.3 --- E7 Open Reading Frame --- p.95 / Chapter 3.4 --- Distribution of HPV16 Variants in Hong Kong and Korea --- p.100 / Chapter 3.4.1 --- Sub-lineage Level --- p.100 / Chapter 3.4.2 --- Clade Level of E6 Open Reading Frame --- p.101 / Chapter 3.4.3 --- Clade Level of E7 Open Reading Frame --- p.102 / Chapter 3.4.4 --- Single Nucleotide Polymorphisms Level --- p.105 / Chapter 3.4.4.1 --- LCR Sequence --- p.105 / Chapter 3.4.4.2 --- E6 Open Reading Frame --- p.107 / Chapter 3.4.4.3 --- E7 Open Reading Frame --- p.108 / Chapter 3.5 --- Risk Association and distribution of HPV16 Variants in normal and Cancer samples --- p.112 / Chapter 3.5.1 --- Sub-lineage Level --- p.112 / Chapter 3.5.2 --- Clade Level of E6 Open Reading Frame --- p.114 / Chapter 3.5.3 --- Clade Level of E7 Open Reading Frame --- p.115 / Chapter 3.5.4 --- Single Nucleotide Polymorphisms Level --- p.122 / Chapter 3.5.4.1 --- LCR Sequence --- p.122 / Chapter 3.5.4.2 --- E6 Open Reading Frame --- p.125 / Chapter 3.5.4.3 --- E7 Open Reading Frame --- p.126 / Chapter Chapter 4 --- Discussion --- p.132 / Chapter 4.1 --- HPV16 Variant Sub-lineages --- p.133 / Chapter 4.2 --- Comparison of HPV16 variants between Hong Kong and Korea --- p.137 / Chapter 4.3 --- Risk Association of HPV16 Variants --- p.138 / Chapter 4.4 --- Strength and Weakness --- p.144 / Chapter 4.5 --- Implications for Future Work --- p.146 / References --- p.148

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