Dissertations / Theses on the topic 'Viral carcinogenesis'

My PhD was focused on two viruses, both responsible of persistent infection: HIV and HBV. The two viruses show high worldwide prevalence and both are associated with high morbidity and mortality. HIV establishes a persistent infection through different mechanisms, first of all with the integration of its DNA into the host genome, thus forming the provirus. However, a consistent part of the viral DNA within the infected cells is unintegrated (circular or not). The project following presented aims to analyse the kinetic of viral replication (measured as p24 production) and HIV DNA integration in primary lymphocytes (CD4+ ad PBMC) and Monocyte-derived Macrophages (MdM) in presence and absence of Integrase inhibitors (INIs). HIV DNA quantification (as proviral, unintegrated and 2-LTR) was assessed at different time points. The presented data reconfirmed, as previously reported in literature, the different kinetic of HIV replication in lymphocytes (CD4+ T cells and PBMCs, much faster) compared to macrophages (more slow), and suggested the presence of a different kinetic of HIV DNA integration between the two cellular systems, too. Both INIs efficiently inhibit the viral replication and the integration of HIV DNA. However, a little but consistent amount of HIV DNA was observed at 30 days post treatment in macrophages, which are the main reservoirs of the infection. By analysing the composition of this little HIV DNA amount, we observed a very little difference between the two drug which could be probably explained by the well-described different kinetic of dissociation of the DTG-enzyme complex. In conclusion these data could give new information about the different kinetic of HIV replication between lymphocytes and macrophages, suggesting also the implication of a different kinetic of HIV DNA integration. Both the integrase inhibitors efficiently inhibit HIV replication and integration. Nonetheless, in presence of INI, although an almost total inhibition of HIV replication was observed, a little but consistent amount of viral DNA was maintained in macrophages. This evidence could have important implication in the study of HIV persistence and viral rebound after treatment interruption. The data above mentioned were presented at the 12th edition of the European congress on HIV e hepatitis, in Barcelona in 2014 (26-28 March), and a manuscript is in preparation. HBV is another important virus able to establish a persistent infection that usually causes cirrhosis and hepatocarcinoma (HCC). HBV could indirectly (through compensative cellular replication) or directly (through event of random integration or due to the viral proteins) promote carcinogenesis. Among the viral proteins the trans-activating HBV protein (HBx) covers a key role in this sense. The project here presented aims to highlight specific mutations in HBX gene associated with HCC in a group of chronically-HBV infected patients. By analysing the sequences of 75 HBV chronically infected patients, we observed the mutation F30V prevalently in the HCC group. This is located within the N-terminal region of HBx which seems to be involved in the negative regulation of the HBx trans-activation function. By analysing the results obtained in vitro, we observed that the mutation would reduce the viral replication, probably due to its localization in a domain within the N-terminal region known to be involve in the dimerization of the protein. HBx protein, both wt and mutated, did not alter the cycle progression of exposed cells. On the contrary, the mutation could to be associated with less cellular susceptibility to apoptotic death related to what observed in presence of the HBx wt. This observed cell survival could probably promote the maintenance of aberrant cellular clones thus favouring the appearance of tumour. Of consequence, considering the obtained results, it is possible to propose that F30V mutation could interfere with HBV replication and can have a role in HBV-driven carcinogenesis by reducing the rate of apoptosis. However, further studies are required in order to understand the potential role of F30V as HCC prognostic factor in HBV chronically infected patients.

Le suppresseur de tumeur DOK1 (downstream of tyrosine kinases1) est une protéine régulatrice de voies de signalisation impliquées dans des processus cellulaires tel que la prolifération, la migration et l'apoptose. Le rôle suppresseur de tumeur de DOK1 a été démontré dans des modèles animaux. Les souris knock-out pour DOK1 présentent une forte susceptibilité de développer des leucémies, des tumeurs malignes hématologiques, des adénocarcinomes pulmonaires, ainsi que des sarcomes histiocytaires agressifs. En outre, nous avons rapporté précédemment que le gène DOK1 peut être muté et son expression réprimée dans différentes tumeurs malignes humaines, telles que les lignées cellulaires de lymphome de Burkitt (BL) et la leucémie lymphoïde chronique (LLC). Cependant, les mécanismes de dérégulation de DOK1 restent inconnus, notamment dans les processus de carcinogenèse induite ou non par des oncovirus. Dans ce projet de thèse, nous avons d'abord caractérisé le promoteur de DOK1 et le rôle du facteur de transcription E2F1 comme le principal régulateur de l'expression de DOK1. Nous avons démontré pour la première fois la contribution de DOK1 dans la réponse cellulaire au stress par son rôle suppresseur de prolifération cellulaire et promoteur d'apoptose. Nous avons trouvé que l'expression du gène DOK1 est réprimée dans une variété de cancers humains, y compris le cancer de la tête et du cou, les lymphomes de Burkitt et les cancers du poumon. Cette répression est due à l'hyperméthylation aberrante de DOK1. Nous avons donc étudié les événements épigénétiques, qui sont souvent altérés dans les cancers, et leurs implications dans la répression de DOK1 dans les lignes cellulaire cancéreuses de la tête et du cou. Nous nous sommes par la suite intéressés aux mécanismes de dérégulation de DOK1 par le virus d'Epstein Barr dans le cadre de sa propriété oncogénique dans les lymphocytes B humains ainsi que dans les lignes cancéreuses du lymphome de Burkitt. Nos résultats apportent de nouvelles informations sur les mécanismes de régulation de l'expression de DOK1 dans la carcinogenèse induite ou non par des oncovirus, ce qui pourrait le définir comme un biomarqueur potentiel de cancer et comme une cible intéressante pour des thérapies épigénétiques
The newly identified tumor suppressor DOK1 (downstream of tyrosine kinases1) inhibits cell proliferation, negatively regulates MAP kinase activity, opposes leukemogenesis, and promotes cell spreading, motility, and apoptosis. DOK1 also plays a role in the regulation of immune cell activation, including B cells. The tumor suppressor role of DOK1 was demonstrated in animal models. DOK1 knockout mice show a high susceptibility to develop leukemia, hematological malignancies as well as lung adenocarcinomas and aggressive histiocytic sarcoma. In addition, we previously reported that the DOK1 gene can be mutated and its expression is down-regulated in human malignancies such as Burkitt’s lymphoma cell lines (BL) and chronic lymphocytic leukemia (CLL). However, very little is known about the mechanisms underlying DOK1 gene regulation and silencing in viral- and non viral-related tumorigenesis. In the present project, we first characterized the DOK1 promoter. We have shown the role of E2F1 transcription factor as the major regulator of DOK1 expression and how DOK1 plays a role in DNA stress response though opposing cell proliferation and promoting apoptosis. We demonstrated that DOK1 gene expression is repressed in a variety of human cancers, including head and neck, Burkitt’s lymphoma and lung cancers, as a result of aberrant hypermethylation. We investigated the link between the epigenetic events and DOK1 silencing in non viral head and neck cancer cell lines, and by Epstein Barr virus in relation to its oncogenic activity in human B cells and neoplasia such as Burkitt’s lymphoma. These data provide novel insights into the regulation of DOK1 in viral and non viral-related carcinogenesis, and could define it as a potential cancer biomarker and an attractive target for epigeneticbased therapy

MicroRNAs (miRNAs) function as key regulators in immune responses and cancer development. In the contexts of infection with oncogenic viruses, miRNAs are engaged in viral persistence, latency establishment and maintenance, and oncogenesis. In this review, we summarize the potential roles and mechanisms of viral and cellular miRNAs in the host-pathogen interactions during infection with selected tumor viruses and HIV, which include (i) repressing viral replication and facilitating latency establishment by targeting viral transcripts, (ii) evading innate and adaptive immune responses via toll-like receptors, RIG-I-like receptors, T-cell receptor, and B-cell receptor pathways by targeting signaling molecules such as TRAF6, IRAK1, IKKε, and MyD88, as well as downstream targets including regulatory cytokines such as tumor necrosis factor α, interferon γ, interleukin 10, and transforming growth factor β, (iii) antagonizing intrinsic and extrinsic apoptosis pathways by targeting pro-apoptotic or anti-apoptotic gene transcripts such as the Bcl-2 family and caspase-3, (iv) modulating cell proliferation and survival through regulation of the Wnt, PI3K/Akt, Erk/MAPK, and Jak/STAT signaling pathways, as well as the signaling pathways triggered by viral oncoproteins such as Epstein-Barr Virus LMP1, by targeting Wnt-inhibiting factor 1, SHIP, pTEN, and SOCSs, and (v) regulating cell cycle progression by targeting cell cycle inhibitors such as p21/WAF1 and p27/KIP1. Further elucidation of the interaction between miRNAs and these key biological events will facilitate our understanding of the pathogenesis of viral latency and oncogenesis and may lead to the identification of miRNAs as novel targets for developing new therapeutic or preventive interventions.

Human papillomavirus (HPV) infections play a crucial role in human carcinogenesis. Greater than 96% of all cervical carcinomas are positive for high-risk HPV infections; especially types 16 and 18. High-risk HPV onco-proteins, E6 and E7, are consistently expressed in such cancers and function by inactivating p53 and pRb tumor suppressors, respectively. The presence of high-risk HPVs is also correlated with anogenital cancers. In this study, we examined the effect of high-risk HPV type 16 E6 and E7 oncoproteins in two normal human colorectal epithelial cell lines, NCE1 and NCE5. We report that the expression of E6/E7 proteins, alone, induced cellular transformation of both cell lines; consequently, NCE1-E6/E7 and NCE5-E6/E7 form colonies in soft agar with respect to their wild type cells. This is accompanied by cell cycle deregulation, as is demonstrated by the over-expression of cyclin dependant kinases (cdks) and their respective cyclins. Furthermore, we demonstrate that E6/E7 oncoprotein transduction induces migration of colorectal epithelial cells. More still, well analyzed Id gene expression, a family member of the helix-loop-helix (HLH) transcription factors involved in the regulation of cell invasion and metastasis of human cancer cells. In parallel, using tissue microarray analysis we found that the four members of the Id protein family are correlated with the presence of HPV type 16 and 18 in human colon cancer tissues. Our data suggests that high-risk HPV infections are sufficient to induce cellular transformation of normal human colorectal cells, in vitro. Furthermore, the correlation with the Id family of proteins may present a novel set of markers associated with HPV induced colorectal carcinogenesis. Our results may suggest a new approach to detect and prevent colorectal cancer.

It is known that many types of leukemias and lymphomas are of viral origin. A new strain of immunologically deficient mice, the BALB/c x C57B1/6 beige nude mice, has been observed to develop spontaneous lymphomas of unknown origin at a high frequency. It is possible the tumors originate from a retroviral infection, which we attempted to show by detection of viral reverse transcriptase (RT) activity. We measured the (RT) activity in the supernatants of cocultures from the spleen and lymph node tissues of the beige nude animals by two methods, tritiated thymidine triphosphate incorporation in a standard RT assay, and the commercially available RT-DetectTM (DuPont) method. Of all supernatants tested, none showed a significant amount of RT activity compared with a cell line that was known to be actively producing the retrovirus MuLV. Upon electron microscopic analysis of the tumor-like cells grown in coculture, no viral particles were observed. Flow cytometric analysis of the tumor-like cells showed two general phenotypes; one predominately of a helper T cell type, and the other of a less differentiated immature thymocyte type.
Department of Biology

This thesis examines cancer and mortality rates among people diagnosed with hepatitis B (HBV) and C (HCV) infection in New South Wales (NSW) from 1990 through 2002, by linking hepatitis notifications with the NSW Central Cancer Registry (CCR) and National Death Index. Of the 39101 HBV, 75834 HCV and 2604 HBV/HCV co-infection notifications included 1052, 1761 and 85 were linked to cancer notifications and 1233, 4008 and 186 were linked to death notifications respectively. Of 2072 hepatocellular carcinoma (HCC) notifications to the CCR 323, 267 and 85 were linked to HBV, HCV and HBV/HCV co-infection notifications. Incidence of HCC was 6.5, 4.0 and 5.9 per 1000 person years for HBV, HCV and HBV/HCV co-infected groups. Risk of HCC in those diagnosed with hepatitis was 20 to 30 times greater than the standard population. There was a marginally statistically significant increased risk of immunoproliferative malignancies associated with HCV infection (SIR=5.6 95% CI 1.8 ???17.5). Risk of death for those with hepatitis was significantly greater, 1.5 to 5 fold, than the general population with the greatest risk among those with HBV/HCV co-infection. The primary cause of HBV deaths was liver related, particularly HCC, whereas in the HCV groups drug related deaths were most frequent. Among people with HCV, risk of dying from drug related causes was significantly greater than from liver related causes (p=0.012), with the greatest increased risk in females age 15- 24 years (SMR 56.9, 95%CI 39.2???79.9). Median age at diagnosis of HCC varied markedly by country of birth and hepatitis group: HBV 66, 63 and 57years ; HCV 51, 68 and 71 years; unlinked 69, 70 and 64 years for Australian, European, and Asian-born groups, respectively (P<0.0001 for all groups). While the risk of cancer, particularly HCC, is elevated among people with HBV and HCV infection, the absolute risk remains low. Young people with HCV face a higher mortality risk from continued drug use than from liver damage related to their HCV infection. The influence of IDU in the epidemiology of HCC in New South Wales was possibly reflected in the varying distributions of age and country of birth.

The prevalence of Hepatitis C Virus (HCV) in the US is estimated at 3.5 million with 18,153 deaths in 2016. It is the most common bloodborne infection, with a higher age-adjusted mortality rate than Hepatitis B Virus or Human Immunodeficiency Virus. Without treatment, nearly 1.1 million people will die from HCV by 2060. About 41,200 new cases of HCV were reported in 41 states in the US in 2016. The reported cases of acute HCV in 2016 is 2.3 per 100,000 in Tennessee, which is more than twice the national goal set by Healthy People 2020. This is a descriptive study to ascertain the HCV prevalence and usefulness of screening in medical outreach settings (MO) compared to indigent healthcare clinics (IHC) in northeast Tennessee. This study period was from April 2017 – February 2019. Participants (n=250), were adults, who engaged in routine, opt-out HCV testing at 4 IHC and 3 MO sites in the Tri-Cities, TN region. During the screening, demographic information- age, gender, race- were collected and the de-identified data were analyzed using Statistical Analysis System (SAS 9.3) to perform a descriptive analysis. Also, several discrete Chi-Square tests of independence between the demographic variables, screening locations, and HCV antibody prevalence was conducted. A total of 250 clients were screened for HCV. The majority of clients screened were non-Hispanic whites 228 (91.20%); females 136 (54.40%); young adults 131 (52.40%) and at IHC clinics 187 (74.80%). Screening showed HCV antibody prevalence of 14.8%. The majority of positive cases were non-Hispanic whites 36 (97.30%; P=0.1561); females 19 (51.35%; P=0.6867) and young adults 23 (62.16%; P=0.286). The prevalence at the IHC clinics and MO settings were 36 (97.30%; P=0.0006) and 1(2.70%) respectively. This analysis shows the higher yield of targeted HCV screening at IHC clinics. Focused HCV screening is critical in the era of opioid epidemic, particularly when direct-acting antiviral agents (DAAs) which offer a Sustained Virologic Response (SVR) rate of more than 90% are available. The use of case control or cohort study designs to establish causality is recommended for improving focused HCV screening.

Tong Hung Man Joanna.
"June 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 137-149).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

博士
臺北醫學大學
醫學科學研究所
96
Accumulating evidence shows that tumor DNA can be found in the circulation of patients with cervical cancer. The presence of such tumor DNA in the blood may be of diagnostic and prognostic value. HPV DNA has been found in serum or plasma samples from cervical cancer patients with detection rates varying from 7% to 45%. The discrepancy may be due to different target materials (serum or plasma), method of extracting DNA, tools of analysis (conventional PCR, real-time PCR, or PCR-enzyme immunoassay), and differing primers used (L1, E6, E7). Therefore, information regarding the comparison of detection rates of HPV DNA in circulating blood is limited. The first part of this study provides a prospective study of HPV DNA detection at a single diagnostic time point. Real-time PCR is used to detect the low viral loads of HPV DNA in blood. The results show that more than one-fourth (27%) of patients with invasive cervical cancer had HPV DNA detected in their blood samples. Approximately 50% of patients with confirmed HPV 16, 18 or 52 positive cervical cancers had HPV DNA detected in their blood. This study also used serial follow-up data on HPV DNA viral load among cervical cancer patients after treatment to understand its clinical significance. Six cervical cancer patients with HPV DNA viral loads undetectable in their blood after treatment showed no recurrence during follow-up. In longitudinal follow-up, eight out of ten cervical cancer patients with viral loads of HPV DNA detectable in the blood at 3 months after treatment were associated with recurrence. Among these, seven of eight patients had distant metastases. Although the study was limited to a small number of patients and a short period of follow-up, it is worth pointing out that detection of circulating HPV DNA after treatment could predict recurrence. It is postulated that blood HPV DNA might be a useful marker to select subsets of patients who need more aggressive treatment. The presence and quantity of HPV DNA in blood are likely to be a reflection of metastasis and may be of prognostic value. The second part of this study focuses on the role of integration of HPV type 52 and 58 in cervical cancer patients. The integration of HPV DNA into the host genome is thought to occur early in cancer development and to be an important event in malignant transformation of cervical cancer. However, most studies on the integration of HPV DNA focus on type 16 and a few on type 18. While HPV type 52 and 58 are oncogenic types with relatively low prevalence in cervical cancer in the Americas, Europe, Africa and Southeast Asia, they are as prevalent as the known high-risk (for cervical cancer) HPV types 16 and 18 in Taiwan and other Asian countries. To analyze whether integration or high viral loads of human papillomavirus (HPV) are essential for malignant transformation of HPV type 52 and 58 as well as type 16 and 18, cervical swabs from 178 consecutive patients, including 81 with invasive cervical cancers and 97 with cervical intraepithelial neoplasias (CIN) II-III, were collected and examined to determine the prevalence, physical status and viral load of HPV type 16, 18, 52 and 58 DNA using genechip and real-time PCR (polymerase chain reaction) analysis. The infrequent integration of HPV 52 and 58 DNA in cervical cancer suggests that it is not a prerequisite for progression to cervical cancer. By contrast, integration appears to be a critical step for carcinogenesis of HPV 16 and 18 DNA. High viral loads (E6) of HPV 16, 18 and 52 DNA may be predictive of the transition of CIN II-III to cervical cancer. The results indicate that both viral DNA physical status and viral loads of HPV are important factors in the carcinogenesis of different HPV types. This study successfully used the median log of viral loads of HPV 16, 18 and 52 DNA to predict the presence of cervical cancer. The selected cut-off values of the median log of viral loads in HPV 16, 18 and 52 DNA achieved 62.5-83.3% sensitivity and a 0-25% false positive rate in predicting the presence of cervical cancer. The ROC curve analyses indicated that the model could accurately predict the diagnostic group of CIN II-III or cervical cancer in 73.8%, 92.9%, and 88.5% of patients with positive HPV 16, 18 and 52, respectively. The third part of this study focuses on low-grade squamous intraepithelial lesions (LSILs). Approximately 50% of atypical squamous cells of undetermined significance (ASCUS) and 80% of LSILs are infected by oncogenic types of HPV. HPV DNA testing for patients with ASCUS provides useful information and allows referral of patients for immediate colposcopy to detect high-grade squamous intraepithelial lesions (HSILs) and cancer. By contrast, oncogenic HPV DNA testing is not informative for triage of patients with LSILs because a high percentage of LSIL patients are HPV positive. A repeat Pap smear in 3 to 6 months or direct biopsy under colposcopy is generally used in clinical practice. Development of alternative triage strategies for women with LSILs would be valuable in distinguishing women with LSILs that have high probabilities of progression to HSILs from women with LSILs that have spontaneously regressed. The 2-year cumulative risks were evaluated for HSIL attributable to HPV 16, 18, 52, and 58, the most common oncogenic types in pre-invasive cervical lesions including LSILs and HSILs in Asia, and questioned as to whether the integration of HPV oncogenes into the host genome contributed to the risk of LSILs progressing to HSILs. In addition, it was determined whether or not E6 viral load and its change contributed to the risk of LSILs progressing to HSILs during the interval between baseline diagnosis of LSIL by Pap smear and a 6-month follow-up visit by repeat Pap smear. It was found that women with LSILs whose viral loads increased between baseline and 6 month follow-up had a 45% risk of developing HSIL, which was seven-fold greater than those without increased viral loads (OR = 7.6, 95% CI = 1.9 to 29.4, p < 0.01), as evaluated by real-time PCR. The risk was calculated at 44%, a six-fold greater risk than those without increased viral loads (OR = 6.1, 95% CI = 1.6 to 22.7, p < 0.01), as evaluated by HC2. The two viral load measures correlated well (Person’s coefficient, r = 0.687, p < 0.001). The results indicate that evaluation of viral load changes (increased or not increased) through repeat HPV DNA testing could predict progression of disease in LSIL cases of HPV types 16, 18, 52, and 58, which correlates to clinical implications. In summary, this research strives to understand the role of HPV DNA viral loads and integration in the carcinogenesis of cervical cancer by searching for a useful marker applicable in clinical practice to predict disease progression in pre-invasive and invasive cervical cancer.

The usual approach to the study of signaling pathways in biological systems is to assume that high numbers of cells and of perfectly mixed molecules within cells are involved. To study the temporal evolution of the system averaged over the cell population, ordinary differential equations are usually used. However, this approach has been shown to be inadequate if few copies of molecules and/or cells are present. In such situation, a stochastic or a hybrid stochastic/deterministic approach needs to be used. Moreover, considering a perfectly mixed system in cases where spatial effects are present can be an over-simplifying assumption. This can be corrected by adding diffusion terms to the ordinary differential equations describing chemical reactions and proliferation kinetics. However, there exist cases in which both stochastic and spatial effects have to be considered. We study the relevance of differential equations, stochastic Gillespie algorithm, and deterministic and stochastic reaction-diffusion models for the study of important biological processes, such as viral infection and early carcinogenesis. To that end we have developed two optimized libraries of C functions for R (r-project.org) to simulate biological systems using Petri Nets, in a pure deterministic, pure stochastic, or hybrid deterministic/stochastic fashion, with and without spatial effects. We discuss our findings in the terms of specific biological systems including signaling in innate immune response, early carcinogenesis and spatial spread of viral infection.

Li, Sai Kam.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 162-179).
Abstracts in English and Chinese.
Abstract --- p.i
Abstract in Chinese (摘要） --- p.ii
Acknowledgements --- p.iii
Table of Content --- p.iv
Abbreviations --- p.xi
List of Figures --- p.xiv
List of Tables --- p.xvii
Chapter CHAPTER 1 --- INTRODUCTION
Chapter 1.1 --- Hepatitis B Virus
Chapter 1.1.1 --- General information --- p.1
Chapter 1.1.2 --- Classification --- p.2
Chapter 1.1.3 --- Virus life cycle and genome --- p.3
Chapter 1.1.4 --- Hepatitis B virus X protein (HBx) --- p.7
Chapter 1.2 --- Enigmatic functions of HB --- p.x
Chapter 1.2.1 --- HBx as a transactivator --- p.10
Chapter 1.2.2 --- HBx as a cell cycle regulator --- p.12
Chapter 1.2.3 --- HBx as an apoptosis modulator --- p.13
Chapter 1.3 --- Etiology of HBV-mediated hepatocarcinogenesis --- p.14
Chapter 1.4 --- Clinical mutants of HBV --- p.16
Chapter 1.5 --- Hypothesis and aims of the research --- p.16
Chapter 1.6 --- Basis of Tet-On system --- p.18
Chapter CHPATER 2 --- EXPERIMENT MATERIALS
Chapter 2.1 --- Cell culture
Chapter 2.1.1 --- Cell-lines --- p.21
Chapter 2.1.2 --- Culture medium --- p.22
Chapter 2.1.3 --- Culture medium supplements --- p.23
Chapter 2.2 --- Reagents for subcloning
Chapter 2.2.1 --- Reagents for polymerase chain reaction (PCR) --- p.24
Chapter 2.2.2 --- Reagents for restriction enzyme digestion --- p.24
Chapter 2.2.3 --- Reagents for ligation --- p.25
Chapter 2.2.4 --- Reagents for electrophoresis --- p.25
Chapter 2.2.5 --- Reagents for E. coli DH5a preparation --- p.25
Chapter 2.2.6 --- Materials for bacterial culture work --- p.27
Chapter 2.3 --- Reagents for subcellular localization study
Chapter 2.3.1 --- Reagents for cell staining --- p.28
Chapter 2.3.2 --- Reagents for mounting slides --- p.29
Chapter 2.3.3 --- Materials for site-directed mutagenesis --- p.29
Chapter 2.4 --- Reagents for cell cycle analysis and cellular proliferation
Chapter 2.4.1 --- Reagents for cell cycle analysis --- p.29
Chapter 2.4.2 --- Reagents for cellular proliferation study --- p.30
Chapter 2.5 --- Reagents for protein expression study
Chapter 2.5.1 --- Cell lysis buffer --- p.30
Chapter 2.5.2 --- Reagents for SDS-PAGE --- p.30
Chapter 2.5.3 --- Reagents for Western blot --- p.33
Chapter 2.5.4 --- Antibodies --- p.34
Chapter 2.6 --- Reagents for gene expression study
Chapter 2.6.1 --- Reagents for RNA extraction --- p.36
Chapter 2.6.2 --- Reagents for first strand cDNA synthesis --- p.37
Chapter 2.6.3 --- Reagents for real-time PCR --- p.37
Chapter 2.7 --- Reagents for establishment of Tet-On inducible stable cell-lines
Chapter 2.7.1 --- Reagents for MTT assay --- p.38
Chapter 2.7.2 --- Reagents for selection of stable clones --- p.38
Chapter 2.8 --- Vectors used in the project
Chapter 2.8.1 --- Vectors for subcellular localization study --- p.39
Chapter 2.8.2 --- Vectors for establishment of Tet-on inducible cell-lines --- p.39
Chapter 2.9 --- Primers used in the project
Chapter 2.9.1 --- Primers used for subcloning --- p.42
Chapter 2.9.2 --- Primers used for site-directed mutagenesis --- p.43
Chapter 2.9.3 --- Primers used in real-time chain polymerase reaction --- p.43
Chapter CHAPTER 3 --- RESEARCH METHODS
Chapter 3.1 --- Subcloning of HBx and mutant genes into a green fluorescence protein (GFP) expression vector
Chapter 3.1.1 --- Amplification of HBxWt，HBxΔC44 and HBxAN60 genes --- p.45
Chapter 3.1.2 --- Purification of PCR products --- p.46
Chapter 3.1.3 --- Restriction enzyme digestion --- p.47
Chapter 3.1.4 --- Ligation of gene products with pEGFP-C 1 vector --- p.47
Chapter 3.1.5 --- Preparation of chemically competent bacterial cells E. coli strain DH5α --- p.47
Chapter 3.1.6 --- Transformation of the ligation product into competent cells --- p.48
Chapter 3.1.7 --- PCR confirmation of successful ligation --- p.48
Chapter 3.1.8 --- Small scale preparation of bacterial plasmid DNA --- p.49
Chapter 3.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.50
Chapter 3.1.10 --- Large scale preparation of target recombinant plasmid DNA --- p.50
Chapter 3.2 --- Subcellular localization pattern study
Chapter 3.2.1 --- Cell transfection --- p.51
Chapter 3.2.2 --- Mitochondria and nucleus staining --- p.52
Chapter 3.2.3 --- Epi-fluorescence microscopy --- p.53
Chapter 3.2.4 --- Analysis of fluorescence images --- p.53
Chapter 3.2.5 --- In vitro site-directed mutagenesis --- p.53
Chapter 3.3 --- Cell cycle phase analysis with flow cytometry
Chapter 3.3.1 --- Cell transfection --- p.55
Chapter 3.3.2 --- Cell staining --- p.55
Chapter 3.3.3 --- Flow cytometry --- p.55
Chapter 3.4 --- Cellular proliferation quantification by BrdU proliferation assay
Chapter 3.4.1 --- Cell transfection --- p.57
Chapter 3.4.2 --- BrdU ELISA measurement --- p.57
Chapter 3.5 --- Protein expression
Chapter 3.5.1 --- Cell lysate collection --- p.58
Chapter 3.5.2 --- Quantification of protein samples --- p.59
Chapter 3.5.3 --- SDS-PAGE --- p.59
Chapter 3.5.4 --- Western blot --- p.60
Chapter 3.5.5 --- Western blot luminal detection --- p.60
Chapter 3.6 --- Gene expression
Chapter 3.6.1 --- Primer design --- p.61
Chapter 3.6.2 --- Cell transfection --- p.61
Chapter 3.6.3 --- RNA extraction --- p.61
Chapter 3.6.4 --- Reverse transcription for first strand complementary DNA (cDNA) --- p.63
Chapter 3.6.5 --- Quantitative real-time PCR --- p.63
Chapter 3.7 --- Establishment of Tet-On inducible stable cell-lines
Chapter 3.7.1 --- Subcloning of HBx gene into pTRE2 vector --- p.64
Chapter 3.7.2 --- Construction of WRL68/Tet-On stable cell-lines --- p.64
Chapter 3.7.3 --- Construction of WRL68/Tet-On HBx and mutants expression cell-lines --- p.68
Chapter 3.7.4 --- Characterization of Tet-On gene expression monoclones --- p.69
Chapter 3.8 --- Statistical analyses --- p.70
Chapter CHPATER 4 --- STUDY ON MITOCHONDRIA TARGETING
Chapter 4.1 --- Establishment of pEGFP-Cl-HBx and mutants constructs --- p.71
Chapter 4.2 --- Transactivation C-terminus domain is essential for granular localization --- p.73
Chapter 4.3 --- Wild-type HBx localizes in mitochondria --- p.76
Chapter 4.4 --- C-terminal transactivation domain is sufficient for mitochondria targeting --- p.79
Chapter 4.5 --- Mapping of the HBx region crucial for mitochondria targeting --- p.81
Chapter 4.6 --- The 111-117 amino acids in HBx do not work as a signal peptide --- p.83
Chapter 4.7 --- Site-directed mutagenesis identifies the key amino acid at 115 in HBx for mitochondrial targeting --- p.85
Chapter CHAPTER 5 --- CELL PROLIFERATION AND REGULATION
Chapter 5.1 --- Alteration of S-phase distribution in cell cycle --- p.88
Chapter 5.2 --- Analysis of DNA synthesis using BrdU proliferation ELISA --- p.92
Chapter 5.3 --- Differential molecular regulation of cell cycle --- p.94
Chapter 5.4 --- Regulation of the mRNA expression levels of cyclin-dependent kinases inhibitors p2raf/cipl and p27kipl --- p.98
Chapter CHAPTER 6 --- TRANSACTIVATION AND RAS/RAF/MAPK PHOSPHORYLATION
Chapter 6.1 --- Determination of p53-dependency of p21、vaf/cipl expression --- p.101
Chapter 6.2 --- Ras/Raf/MAPK pathway activation by HBx variants
Chapter 6.2.1 --- ERK1/2 phophorylation by HBx variants --- p.104
Chapter 6.2.2 --- ERK inhibition blocks the regulation effect on p53Wt and p21waf/cipl --- p.107
Chapter 6.3 --- Transactivation activity on oncogenes/ proto-oncogenes
Chapter 6.3.1 --- Effect on c-myc (NM´ؤ002467) mRNA expression --- p.109
Chapter 6.3.2 --- Effect on RhoC (NM_017744) and Rabl4 (NM´ؤ016322) mRNA expression --- p.112
Chapter CHAPTER 7 --- CONSTRUCTION OF TET-ON INDUCIBLE CELL-LINES
Chapter 7.1 --- Establishment of WRL/Tet-On monoclonal cell-lines Page
Chapter 7.1.1 --- Determination of geneticin selection dosage --- p.116
Chapter 7.1.2 --- Selection of the best WRL/TOn clone using luciferase assay --- p.118
Chapter 7.2 --- Establishment of inducible WRL/TOn/Gene monoclonal cell-lines
Chapter 7.2.1 --- Determination of hygromycin selection dosage --- p.120
Chapter 7.2.2 --- Selection of positive WRL/TOn/Gene clones with viral genes --- p.122
Chapter 7.3 --- Characterization of TOXDC1 cell-line
Chapter 7.3.1 --- Cell morphology --- p.125
Chapter 7.3.2 --- Growth pattern of TOXDC1 --- p.126
Chapter 7.3.3 --- HBxAC44 induced p21waf/cipl mRNA expression --- p.127
Chapter 7.3.4 --- Doxycycline concentration dependent HBxAC44 expression in TOXDC1 --- p.129
Chapter CHAPTER 8 --- DISCUSSION
Chapter 8.1 --- Selection of cell model
Chapter 8.1.1 --- Selection of cell models --- p.130
Chapter 8.1.2 --- Selection of truncation mutant --- p.131
Chapter 8.2 --- Differential sub-cellular localization of HBx and its variants
Chapter 8.2.1 --- Mechanisms of mitochondria targeting --- p.132
Chapter 8.2.2 --- Mitochondria as site of HBx-induced apoptosis --- p.134
Chapter 8.2.3 --- Stimulation of calcium release from mitochondria by wild-type HBx --- p.135
Chapter 8.3 --- Cell cycle distribution profiling and its regulations
Chapter 8.3.1 --- Cell cycle pattern and cell proliferation --- p.136
Chapter 8.3.2 --- Differential cell cycle molecular pathway activation --- p.138
Chapter 8.4 --- Ras/Raf/MAPK mediated transactivation by HBxWt and its mutants
Chapter 8.4.1 --- p53-mediated p21waf/cipl expression --- p.142
Chapter 8.4.2 --- ERK-mediated p21waf/cipl and wild-type p53 mRNA expression --- p.143
Chapter 8.4.3 --- Regulation of oncogenes/ proto-oncogenes expression --- p.147
Chapter 8.5 --- General discussions on differential effects of HBxWt and HBxAC44 --- p.149
Chapter 8.6 --- Establishment of Tet-On/HBxAC44 cell-line TOXDC1 --- p.153
Chapter 8.7 --- Conclusions --- p.154
Chapter 8.8 --- Future Prospects
Chapter 8.8.1 --- From mitochondria targeting to calcium signaling --- p.157
Chapter 8.8.2 --- Construction of a complete cell cycle regulation pathway --- p.158
Chapter 8.8.3 --- Elucidation of the transcriptional transactivation regulation --- p.159
Chapter 8.8.4 --- To make the best use of the Tet-on stable cell-line TOXDC1 --- p.159
Chapter 8.8.5 --- Study with other carboxy-terminal truncation mutants --- p.160
Chapter 8.8.6 --- In vivo study --- p.160
REFERENCES --- p.162

Xu, Meng.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 112-129).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.