In conclusion, two new methods of cccDNA quantitation were developed and validated. The two assays are complementary to each other and may be used in patients with extreme HBV DNA levels. These cccDNA assays should be further validated in larger studies and may become important tests for diagnostic, prognostic and treatment monitoring purposes. / Over 350 million people worldwide suffer from chronic hepatitis B virus (HBV) infection, which leads to many cases of cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is a critical intracellular replicative intermediate and cannot be eliminated during antiviral therapy. Current methods for cccDNA detection are limited by false positive detection due to the interference by HBV relaxed circular DNA (rcDNA). The tests also have limited sensitivity to detect cccDNA at low concentrations. Hence, we aimed to develop a highly sensitive and highly specific assay for cccDNA detection with wide linear range. / The modified Bowden's assay had the highest intrahepatic cccDNA detection rate (60 positive results out of 61 cases). The detection rate of the modified Bowden's assay is significantly higher than that of the Bowden's assay. On the other hand, the cccDNA detection rate in serum samples was low at 20--27% by all 3 assays. In 5 samples in which cccDNA was undetectable by the Bowden's assay but detectable by the other two assays, a point mutation in the HBV genome was found in the forward primer binding site of the Bowden's assay. This partly explained the false negative results. / The quantification result of cccDNA by the bisulfite conversion assay was significantly lower than that by the Bowden's assay assay (P=0.001) and the modified Bowden's assay (P=0.003). When the total HBV DNA was higher than 107 copies/ml, the serum cccDNA level detected by the bisulfite conversion assay was significantly lower than that detected by the Bowden's assay (P=0.008) and the modified Bowden's assay (P=0.046). When the total HBV DNA is less than 107 copies/ml, there were no significant differences. This suggests that the bisulfite conversion assay was less affected by rcDNA even in samples containing a high viral load. / With this background, two new cccDNA assays were developed and optimized. Bowden's assay was used as a standard to evaluate the performance of new assays. The first new assay (modified Bowden's assay) involved the use of new primers and probes that targeted more conserved regions in the HBV genome. The second assay adopted the bisulfite conversion method, which introduced gene sequence changes into the HBV genome and thereby enhance the specificity of the assay. Capillary sequencing was performed to find mutations in primers and probe range of different assays. / Yu, Ling. / Advisers: Vincent Wai-Sun Wang; Joseph Jao-Yiu Sung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 105-111). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ng Wah. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-113). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF TABLES --- p.X / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Etiological Factors of Hepatocellualr Carcinoma (HCC) --- p.4 / Chapter 1.2.1 --- Dietary Aflatoxins --- p.4 / Chapter 1.2.2 --- Liver Cirrhosis --- p.5 / Chapter 1.2.3 --- Alcohol Abuse --- p.6 / Chapter 1.2.4 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3 --- Literature Review on the Investigations of HBV Integrants in HCC --- p.16 / Chapter 1.3.1 --- Affected Host Junctions --- p.17 / Chapter 1.3.2 --- Viral Junctions --- p.18 / Chapter 1.4 --- Restriction Site Polymerase Chain Reaction (RS-PCR) --- p.19 / Chapter 1.5 --- Aims of Thesis --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Chemicals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Cell Cultures --- p.24 / Chapter 2.1.4 --- Nucleic Acids --- p.24 / Chapter 2.1.5 --- Enzymes --- p.25 / Chapter 2.1.6 --- Equipment --- p.25 / Chapter 2.1.7 --- Software and Web Resources --- p.26 / Chapter 2.2 --- Methods --- p.27 / Chapter 2.2.1 --- DNA Extraction --- p.27 / Chapter 2.2.2 --- RS-PCR --- p.31 / Chapter 2.2.3 --- Sequencing --- p.37 / Chapter 2.2.4 --- Spectral Karyotyping (SKY) --- p.38 / Chapter 2.2.5 --- Fluorescence In situ hybridization --- p.39 / Chapter Chapter 3 --- Investigation of HBV Integration Sites in HCC Cell lines --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Cell Lines --- p.47 / Chapter 3.2.2 --- RS-PCR --- p.47 / Chapter 3.2.3 --- Spectral Karyotyping --- p.48 / Chapter 3.2.4 --- Tyramide Signal Amplification for HBV in FISH Analysis --- p.48 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Identification of HBV Integration Sites in Cell Lines --- p.51 / Chapter 3.3.2 --- Evaluation of RSO Primer Efficiency --- p.52 / Chapter 3.3.3 --- SKY and FISH Analysis --- p.53 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- HBV Insertions in HCC Cell Lines --- p.64 / Chapter 3.4.2 --- Efficacy of RSO Primers --- p.65 / Chapter 3.4.3 --- Investigation of HBV Integration on Chromosomal Rearrangement --- p.65 / Chapter Chapter 4 --- Investigation of Hepatitis B Virus Integration Sites in Primary HCC --- p.67 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Materials and Methods --- p.69 / Chapter 4.2.1 --- Patients --- p.69 / Chapter 4.2.2 --- RS-PCR --- p.70 / Chapter 4.3 --- Results --- p.72 / Chapter 4.3.1 --- HBV Integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.72 / Chapter 4.4 --- Discussion --- p.88 / Chapter 4.4.1 --- HBV integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.88 / Chapter 4.4.2 --- Summary on HBV Integrants Identified --- p.91 / Chapter Chapter 5 --- Proposed Future Studies --- p.98 / Chapter 5.1 --- Correlation of Structural Aberrations with HBV Integrations --- p.99 / Chapter 5.2 --- Transcriptional Expression Study on the Genes Interrupted by or Located near the Virus Host Junctions --- p.100 / Chapter Chapter 6 --- References --- p.101

Lam Kit. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 141-153). / Abstracts in English and Chinese. / Acknowledgment --- p.I / Table of Content --- p.II / List of Tables --- p.VII / List of Figures --- p.IX / Abbreviations --- p.XIV / Abstract --- p.XVI / 論文摘要 --- p.XIX / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatitis B --- p.1 / Chapter 1.1.1 --- Brief Introduction of HBV --- p.1 / Chapter 1.1.2 --- History of Hepatitis B Virus --- p.2 / Chapter 1.1.3 --- Hepatitis B Virus Infection around the World --- p.4 / Chapter 1.1.4 --- Hepatitis B Virus Infection in Hong Kong --- p.5 / Chapter 1.1.5 --- Hepatitis B Virus Infection in China --- p.7 / Chapter 1.1.5.1 --- Update of HBV Infection in China --- p.7 / Chapter 1.1.5.2 --- Problems in China --- p.7 / Chapter 1.2 --- Hepatitis B Virology --- p.8 / Chapter 1.2.1 --- Hepadnaviridae Family --- p.8 / Chapter 1.2.2 --- HBV Particles Types --- p.9 / Chapter 1.2.3 --- The HBV Genome --- p.10 / Chapter 1.2.4 --- The Life Cycle of HBV --- p.12 / Chapter 1.2.5 --- Hepatitis B Surface Antigen (HBsAg) --- p.17 / Chapter 1.3 --- HBV Transmission --- p.19 / Chapter 1.4 --- HBV Therapy --- p.19 / Chapter 1.5 --- Phyllanthus Species --- p.22 / Chapter 1.6 --- Alexander Cells --- p.26 / Chapter 1.7 --- Objectives --- p.29 / Chapter CHAPTER 2 --- COMPARISONS OF AQUEOUS AND ORGANIC EXTRACTS OF THREE PHYLLANTHUS SPECIES OF THEIR IN VITRO ANTI-HBV EFFECTS --- p.30 / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.30 / Chapter 2.2.1 --- Materials --- p.30 / Chapter 2.2.1.1 --- Phyllanthus species --- p.30 / Chapter 2.2.1.2 --- "Chemicals, Antibodies and Instrument" --- p.31 / Chapter 2.2.2 --- Extraction Methods --- p.32 / Chapter 2.2.2.1 --- Aqueous Extraction --- p.33 / Chapter 2.2.2.2 --- Organic Extraction --- p.33 / Chapter 2.2.3 --- Cell line --- p.33 / Chapter 2.2.4 --- Toxicity of Extracts --- p.34 / Chapter 2.2.5 --- IMx Assay --- p.34 / Chapter 2.2.6 --- Semi-quantitative RT-PCR --- p.35 / Chapter 2.2.6.1 --- RNA Extraction --- p.35 / Chapter 2.2.6.2 --- RT-PCR --- p.36 / Chapter 2.2.7 --- Western Blotting --- p.37 / Chapter 2.2.7.1 --- Preparation of Protein Samples --- p.37 / Chapter 2.2.7.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.2.7.3 --- Protein Transfer --- p.38 / Chapter 2.2.7.4 --- Immumnoblotting --- p.39 / Chapter 2.2.7.5 --- Protein Assay --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Toxicity of the Extracts --- p.40 / Chapter 2.3.2 --- Effects on HBsAg Secretion and Viral Gene Expression --- p.45 / Chapter 2.3.2 --- Analysis of Intracellular Viral Proteins --- p.58 / Chapter 2.4 --- Discussion --- p.63 / Chapter CHAPTER 3 --- ISOLATION AND CHARACTERIZATION OF ACTIVE COMPONIENT FROM AN ORGANIC EXTRACT OF PHYLLANTHUS URINARIA (GUANGDONG) --- p.68 / Chapter 3.1 --- Introduction --- p.68 / Chapter 3.2 --- Materials and Methods --- p.69 / Chapter 3.2.1 --- Materials --- p.69 / Chapter 3.2.2 --- Methods --- p.70 / Chapter 3.2.2.1 --- Ethanol Extraction --- p.70 / Chapter 3.2.2.2 --- Partitions --- p.70 / Chapter 3.2.2.3 --- Column Purification --- p.71 / Chapter 3.2.2.4 --- Analytical Thin Layer Chromatographic (TLC) --- p.71 / Chapter 3.2.2.5 --- Crystallization --- p.71 / Chapter 3.3 --- Results --- p.72 / Chapter 3.3.1 --- Analysis of Four Fractions after Partitions --- p.72 / Chapter 3.3.2 --- Screening of the Active Fraction after Column Chromatography of Fraction B --- p.76 / Chapter 3.3.3 --- Screening of the Active Fraction after Column Chromatography of Fraction6 --- p.79 / Chapter 3.3.4 --- Crystallization and Identification of the Isolated component --- p.82 / Chapter 3.3.5 --- Study Anti-HBV effects of pheophorbide a --- p.91 / Chapter 3.4 --- Discussion --- p.97 / Chapter CHAPTER 4 --- STUDY OF PRE S I PROMOTER ACTIVITY OF HBV --- p.103 / Chapter 4.1 --- Introduction --- p.103 / Chapter 4.2 --- Materials and Methods --- p.108 / Chapter 4.2.1 --- Materials --- p.108 / Chapter 4.2.2 --- Methods --- p.109 / Chapter 4.2.2.1 --- Cell line --- p.109 / Chapter 4.2.2.2 --- Clonning of Pre SI Promoter from HBV Genome --- p.109 / Chapter 4.2.2.3 --- Gene Clean --- p.110 / Chapter 4.2.2.4 --- Restriction Enzyme Digestion --- p.111 / Chapter 4.2.2.5 --- Synthesis of T-Overhang EcoR V Cut pBluescript® II KS (-) --- p.111 / Chapter 4.2.2.6 --- Ligation --- p.112 / Chapter 4.2.2.7 --- DH5α Competent Cells Preparation --- p.112 / Chapter 4.2.2.8 --- Transformation --- p.113 / Chapter 4.2.2.9 --- Plasmid Purification --- p.113 / Chapter 4.2.2.10 --- Transfection --- p.114 / Chapter 4.2.2.11 --- Luciferase Assay --- p.115 / Chapter 4.3 --- Results --- p.116 / Chapter 4.3.1 --- Cloning of the Pre S I Promoter --- p.116 / Chapter 4.3.2 --- Sequences of the Pre S I Promoter --- p.121 / Chapter 4.3.3 --- Pre S I Promoter Activities in Hep 3B Cell Line --- p.123 / Chapter 4.3.4 --- Effects of Herbal Extracts on Pre S I Promoter --- p.126 / Chapter 4.4 --- Discussion --- p.130 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.134 / REFERENCES --- p.141

Hung Wah Johnson. / Thesis submitted in: August 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 116-121). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.iv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Motivation --- p.1 / Chapter 1.2 --- Problem Statement --- p.3 / Chapter 1.3 --- Outline of the thesis --- p.6 / Chapter 2 --- Background --- p.8 / Chapter 2.1 --- Biological Background --- p.8 / Chapter 2.2 --- Sequence Alignments --- p.9 / Chapter 2.2.1 --- Pairwise Sequences Alignment --- p.11 / Chapter 2.2.2 --- Multiple Sequences Alignment --- p.15 / Chapter 2.3 --- Neighbor Joining Tree --- p.16 / Chapter 2.4 --- Marker Extractions --- p.18 / Chapter 2.5 --- Neural Network --- p.19 / Chapter 2.6 --- Conclusion --- p.22 / Chapter 3 --- Related Work --- p.23 / Chapter 3.1 --- FASTA --- p.23 / Chapter 3.2 --- Suffix Tree --- p.25 / Chapter 4 --- Sub-Sequence Segmentation Tree --- p.28 / Chapter 4.1 --- Introduction --- p.28 / Chapter 4.2 --- Problem Statement --- p.29 / Chapter 4.3 --- Design --- p.33 / Chapter 4.4 --- Time and space complexity analysis --- p.38 / Chapter 4.4.1 --- Performance Evaluation --- p.40 / Chapter 4.5 --- Summary --- p.48 / Chapter 5 --- Applications: Global Sequences Alignment --- p.51 / Chapter 5.1 --- Introduction --- p.51 / Chapter 5.2 --- Problem Statement --- p.53 / Chapter 5.3 --- Pairwise Alignment --- p.53 / Chapter 5.3.1 --- Algorithm --- p.53 / Chapter 5.3.2 --- Time and Space Complexity Analysis --- p.64 / Chapter 5.4 --- Multiple Sequences Alignment --- p.67 / Chapter 5.4.1 --- The Clustalw Algorithm --- p.68 / Chapter 5.4.2 --- MSA Using SSST --- p.70 / Chapter 5.4.3 --- Time and Space Complexity Analysis --- p.70 / Chapter 5.5 --- Experiments --- p.71 / Chapter 5.5.1 --- Experiment Setting --- p.72 / Chapter 5.5.2 --- Experimental Results --- p.72 / Chapter 5.6 --- Summary --- p.80 / Chapter 6 --- Applications: Marker Extractions --- p.81 / Chapter 6.1 --- Introduction --- p.81 / Chapter 6.2 --- Problem Statement --- p.82 / Chapter 6.3 --- The Multiple Sequence Alignment Approach --- p.85 / Chapter 6.3.1 --- Design --- p.85 / Chapter 6.4 --- Reference Sequence Alignment Approach --- p.88 / Chapter 6.4.1 --- Design --- p.90 / Chapter 6.5 --- Time and Space Complexity Analysis --- p.95 / Chapter 6.6 --- Experiments --- p.95 / Chapter 6.7 --- Summary --- p.99 / Chapter 7 --- HBV Application Framework --- p.101 / Chapter 7.1 --- Motivations --- p.101 / Chapter 7.2 --- The Procedure Flow of the Application --- p.102 / Chapter 7.2.1 --- Markers Extractions --- p.103 / Chapter 7.2.2 --- Rules Training and Prediction --- p.103 / Chapter 7.3 --- Results --- p.105 / Chapter 7.3.1 --- Clustering --- p.106 / Chapter 7.3.2 --- Classification --- p.107 / Chapter 7.4 --- Summary --- p.110 / Chapter 8 --- Conclusions --- p.112 / Chapter 8.1 --- Contributions --- p.112 / Chapter 8.2 --- Future Works --- p.114 / Chapter 8.2.1 --- HMM Learning --- p.114 / Chapter 8.2.2 --- Splice Sites Learning --- p.114 / Chapter 8.2.3 --- Faster Algorithm for Multiple Sequences Alignment --- p.115 / Bibliography --- p.121

A number of recent research studies have been done on different species of the plants of the genus Phyllanthus. The plants are widely distributed in most tropical and subtropical countries and have long been used for the treatment of liver diseases in China and India. / Hepatitis B virus (HBV) is a major pathogen of human viral hepatitis. It has been estimated that 350 million people are chronic carriers of HBV throughout the world. Increasing evidence indicates that persistent viral infection of the liver is associated with cirrhosis and hepatocellular carcinoma. Hepatitis B virus belongs to a family of DNA viruses called hepadnaviruses. The current treatments of HBV infection with interferon or lamivudine have several disadvantages, and there appears to be much room for improvement in terms of medical treatment. / My project research focuses on two poorly characterized Indian Phyllanthus species called Phyllanthus nanus ("PN") and Phyllanthus niruri ("PI"). In my studies, random amplified polymorphic DNA ("RAPD") technique and high performance liquid chromatography ("HPLC") fingerprinting were used to authenticate different species of Phyllanthus. Both aqueous and ethanolic extracts of PN and PI were prepared to study their cytotoxicity in hepatoma cell lines. The effect of these extracts on hepatitis B virus was also examined in the HBV-genome integrated cell lines - PLC/PRF/5 (Alexander) and HepG2 2.2.15. Microparticle enzyme immunoassay ("MEIA") and enzyme-linked immunosorbent assay were used to measure the amount of hepatitis B surface antigen ("HBsAg") and hepatitis B e antigen ("HBeAg") secretion from the cell lines. RT-PCR was used to detect the change in HBsAg mRNA's expression level in the drug-treated cell lines. Real-time PCR was also employed to examine the effect of drug treatment on the level of HBV DNA replication and the amount of virions secreted into the medium. The experimental results showed that both the aqueous and ethanolic extracts of PN and PI exerted suppressive effect on HBsAg secretion and HBsAg mRNA level. The PN and PI ethanolic extracts also showed mild suppression of viral replication in vitro. The ethanolic extract of PN seemed to be more potent in suppressing HBV than the other extracts tested. (Abstract shortened by UMI.) / Lam Wai Yip. / "June 2005." / Advisers: Mary Waye; Vincent Ooi. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 217-234). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that is prevalent in Southeast Asia and China, where hepatitis B viral (HBV) infection is the main etiologic factor. Despite a high incidence of HCC developing in patients with HBV-induced liver cirrhosis, the molecular events underlying the malignant liver progression remain largely unclear. In an effort to characterize the genetic abnormalities involved in the HBV-related liver carcinogenesis, genome-wide exploration by metaphase comparative genomic hybridization (CGH) was performed on 100 cirrhotic HCC tumors that were derived from chronic hepatitis B carriers. CGH analysis indicated chromosomal instability in both early and advanced stage tumors where common genomic copy gains on 1q, 8q and 17q, and deletions on 4q, 8p, 13q, 16q and 17p found in both groups are suggestive of early events in hepatocarcinogenesis. Nevertheless, a combined univariate and multivariate statistical analyses highlighted for the first time preferential regional 3q26-q28, 7q21-q22 and 7q34-q36 gains in association with advanced stage HCC. The novel aberrant gains identified here thus formed basis for further mapping analysis for causative genes related to HCC progression in this thesis. / Near 50% of the advanced stage HCC manifested copy gains of chr 7q21-q22. High resolution mapping analysis by cDNA microarray-based CGH nominated 13 amplified candidates within the region 7q21-q22 Analysis on the mRNA expresson levels of these genes in a cohort of primary HCC compared to paired adjacent non-tumorous liver tissues by quantitative RT-PCR (qRT-PCR) indicated the up-regulation of the PFTK1 (PFTAIRE protein kinase 1) gene as the only candidate that demonstrated a close association with advanced metastatic tumors. The effects of PFTK1 on cell proliferation, migration and invasive phenotypes were further studied to substantiate its role in HCC progression. Upon gene suppression of PFTK1 in vitro by RNA interference (RNAi), a significant reduction in chemotactic migration, cellular invasion and an inhibition on cell motility were indicated, albeit cell proliferation remained unaffected. / Sub-cellular localization study of translated PFTK1 protein indicated protein localization in both the nucleus and cytoplasm. This has led to the further investigations of potential PFTK1 function at both the transcriptional and protein levels. (Abstract shortened by UMI.) / Sy Ming Hui. / "July 2005." / Advisers: Winnie Yeo; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3571. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 124-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

HBV DNA replicates through a genomic RNA intermediate. The HBV reverse transcriptase lacks proof-reading activity, resulting in a much higher mutation rate for the HBV genome compared with other DNA viruses. HBV DNA thus is often present in quasi-species in an individual. One or more species may be favorably selected by factors like host immune clearance and use of antiviral drugs. / Hepatitis B virus (HBV) infected millions of people worldwide. Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). / In summary, this study developed and validated two platforms for (1) HBV mutation discovery; and (2) HBV mutation detection in viral quasi-species. These tools may be useful for research on HBV drug resistant mutations, clinical instructing and monitoring of antiviral treatment. / In this study, I have developed high-throughput methods for (1) discovery of novel HBV mutations; and (2) highly multiplexed detection of known HBV mutations, both in the background of HBV quasi-species. Patients undergoing long-term lamivudine treatment were used for mutation discovery. For mutation discovery in quasi-species, the MassCLEAVE(TM) technology, a method based on base-specific RNA cleavage and automated Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS), was used. I found that MassCLEAVE(TM) can be used to discover mutations present as minorities. Additionally, a synergistic effect was found between direct sequencing and MassCLEAVE(TM) in identifying minority mutations. Multi-PLEX, a method based on single nucleotide extension and automated MALDI-TOF MS, was used to develop a highly multiplexed assay for simultaneous detection of 60 HBV mutations including all functionally known HBV mutations and other frequently observed mutations during antiviral treatment with unknown functions. This multiplex assay was tested on a large cohort of single and multiple drug-resistant patients and was shown to be highly accurate in detecting HBV viral mutations in quasi-species. / Nucleotide and nucleoside analogues (NAs) are widely used for antiviral therapy by effectively suppressing viral DNA replication. However, long-term administration may select for drug-resistant mutant strains, leading to treatment failure and liver disease progression. A number of HBV mutations such as rtM204V/I, rtN236T and rtL180M within the HBV reverse transcriptase are known to confer drug resistance. Detection of these known mutations is useful genotypic markers for monitoring antiviral treatment. In addition, novel drug resistant mutations continue to be discovered. / by Luan, Ju. / Adviser: Chunming Ding. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

After alignment of 300 HBV sequences randomly downloaded from GenBank, we found that the frequency of A1762T and G1764A mutations in genotype C was found as high as 64%, while 34% was found for other genotypes (A, B, D to H). Besides, recent clinical studies have also shown that A1762T/G1764A mutations occur frequently in HCC patients with genotype B infection (81%, 30 of 37 patients), but were relatively lower in asymptomatic carriers (43%, 22 of 51 patients). These indicate that the contribution of A1762T/G1764A mutations to liver cancer might not be limited to genotype C. As the double mutations are present within the region of HBV Enhancer II/Basal core promoter (BCP) and cause residue substitution of HBx (Lys130Met and Val131Ile); therefore, their effects on the promoter and HBx activities were examined. / Chronic infection of hepatitis B virus (HBV) increases the risk of hepatocellular carcinoma (HCC) by more than 100-fold. However, the underlying molecular mechanism of this process is not fully understood. Several recent studies have shown that A1762T and G1764A mutations of HBV were associated with the aggressiveness of liver disease, in which inactive carriers would develop active hepatitis, and eventually liver cirrhosis and HCC. In Asia, genotypes B and C are the predominant genotypes of HBV infections. Our longitudinal five-year follow-up study of 426 chronic hepatitis B patients in Hong Kong found that the genotype C HBV (normally with A1762T/G1764A mutations) was closely associated with higher risk of HCC than genotype B HBV (non-frequent mutations with A1762T/G1764A). / In this study, systemic site-directed mutagenesis studies, promoter assays, replication capacity assays and overexpression of HBx assays were carried out to demonstrate the molecular mechanisms of these mutations for the increases risk of HCC. Three conclusions were drawn from this study. (1) A1762T and/or G1764A mutations of HBV could reduce BCP activities in a synergistic manner with 1764A contributing more. Reversed T1762A and/or A1764G mutations increase the BCP activities also in a synergistic manner with 1764G contributing more; (2) HBx could increase HBV BCP activity, HBV replication and HBsAg expression. The Lys130Met and Val131Ile mutations of HBx could further increase the above abilities while the A1762T/G1764A double mutations in the BCP region could not affect the interaction of HBx and HBV BCP; (3) The G1677T/A1679C and T1706C mutations could increase the BCP activity; The ectopic expression of HBx could further increase the BCP activity while the mutated HBx (130Met and 131Ile) has less effect on these mutated promoters. / Dong, Qingming. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A case-control study was previously carried out to identify HCC-associated genomic markers on HBV. Some of them are clustered at the preS1 and X promoter regions of HBV genotype B and core promoter of HBV subgenotype Cs. The functional significance of these markers to the virus was investigated in our study. Our result showed that one of those markers, the G1613A mutation on core promoter, can significantly increase the promoter activity in a genotype-dependent manner and the effect is reversible by the A-to-G back mutation. We have established an in vitro full-length HBV genome transfection system and the result suggested that the G1613A mutation suppressed the e antigen (HBeAg) secretion and enhanced virus DNA production by downregulating the precore (preC) mRNA transcription. In consistence to the clinical study, the mutation was associated to serum HBV DNA level higher than 6 log copies/1M in female HBV carriers in a univariate analysis. In addition, we demonstrated that the G1613A mutation is a hot spot mutation situated on the negative regulatory element (NRE) on the core promoter in an alignment analysis. To further investigate the molecular mechanism of the mutation, two unknown protein complexes had been shown to bind on the NRE. They showed different binding affinity to the G1613-wild-type and A1613-mutant NRE sequence. Moreover, we showed that in vitro synthesized RFX1 protein could bind to the mutated NRE probe at a higher affinity than that to wild-type NRE probe. Overall, our result suggests that the G1613A mutation exerts its effect by differential binding to some proteins via the NRE region. Studying the mechanism of the mutations may provide insights to the viral pathogenesis and HBV-associated HCC, which has long been a health burden in Asia-Pacific countries. / Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Approximately 60-80% of world's HCC is related to HBV, and it is the third most common cause of cancer death in Asia-Pacific region. Almost 400 million people are chronically infected with HBV and one-third was likely to die of complications of cirrhosis, including liver failure and HCC. As there is a shortage of effective curative treatments, detection and prognosis of the risk of cancer development will be essential to improve survival of patients with chronic HBV infection. / Li, Man Shan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 198-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

<p>Hepatitis E virus (HEV) je uzročnik akutne hepatis E infekcije kod ljudi. HEV se prenosi putem zagaĎene vode i odgovoran je za nastanak mnogobrojnih epidemija velikih razmera u zemljama u razvoju Azije i Afrike. Hepatitis E je prvi put kod svinja izolovan 1997. godine, a kasnije je dokazan i kod ostalih ţivotinjskih vrsta, kao &scaron;to su: divlje svinje, jelen, zečevi, pacovi, ptice i ostalo.<br />Prva istraţivanja prisustva HEV infekcije kod domaćih i divljih svinja u Srbiji sprovedena su 2008. godine. HEV RNK je dokazana u 30% uzoraka fecesa i 45% uzoraka organa (Petrovic et al., 2008). Analizom uzoraka krvnih seruma svinja u individualnim gazdinstvima ustanovljena je seroprevalencija od 34,6% (Lupulovic et al, 2010). Cilj ovog istraţivanja je ispitivanje ra&scaron;irenosti HEV infekcije kod svinja na farmama na teritoriji Vojvodine, kao i ispitivanje HEV seroprevalencije kod ljudi u Vojvodini.<br />Od metoda za istraţivanje kori&scaron;ćene su: nekomercijalni ELISA test (in-house ELISA), komercijalni ELISA test, Western-blot metod, real-time RT -PCR i imunohistohemijska metoda za detekciju HEV antigena.<br />Materijal za ispitivanje su bili uzorci krvi svinja (300) sa 3 farme na teritoriji Juţne Bačke i Srema i uzorci krvi ljudi (294), kao i uzorci fecesa, ţuči, jetre i mesa sakupljeni u klanicama od 95 tovljenika i 50 prasadi.<br />Prisustvo specifičnih antitela IgG klase protiv hepatitis E virusa dokazano je kod svinja na sve tri ispitujuće farme. Primenom in house ELISA testa ustanovljena je seroprevalencija od 37% na farmi A, 31% na farmi B i 54% na farmi C, dok je primenom komercijalnog ELISA testa utvrĎeno 40% seropozitivnih svinja na farmi A, 41% na fami B i 65% na farmi C. Uporednom analizom rezultata dobijenih sa oba ELISA testa, ustanovljena je prosečna seroprevalencija od 40,66% in house ELISA testom, odnosno 48,66% komercijalnim ELISA testom.<br />Sprovedena su i istraţivanja prisustva specifičnih antitela IgG klase protiv HEV u krvnim serumima dobrovoljnih davalaca krvi i kod pacijenata. Primenom in house ELISA testa utvrĎena je<br />seroprevalencija od 15% kod dobrovoljnih davalaca krvi, dok su uzorci krvi pacijenata bili seronegativni. Testiranjem komercijalnim ELISA testom kod dobrovoljnih davalaca krvi pozitivan serolo&scaron;ki nalaz je ustanovljen kod 17,86% dobrovoljnih davalaca krvi (pregledani su serumi koji su u in house testu dali pozitivan ili sumnjiv nalaz, kao i odreĎen broj seronegativnih uzoraka), a kod pacijenata 2,12%.<br />Kao tzv. &bdquo;zlatni standard&ldquo; za definisanje rezultata sa sumnjivim serolo&scaron;kim nalazom čije su ekstinkcije bile blizu cut off vrednosti u in house ELISA testu, kori&scaron;ćen je Western blot metod. Pozitivan rezultat je potvrĎen kod 6 od ukupno pregledanih 11 uzoraka krvi svinja, odnosno od ukupno pregledanih 11 uzoraka seruma ljudi, pozitivan nalaz je ustanovljen kod 7 uzoraka.<br />Uzorci sa klanica pregledani su real-time RT- PCR metodom, a HEV RNK je dokazana u u fecesu (54%), ţuči (26%), jetri (16%) i mesu (10%) prasadi. Kod tovljenika prisustvo HEV RNK je potvrĎeno samo u fecesu (7,27%), dok su svi uzorci tkiva bili negativni.<br />Patahistolo&scaron;kim pregledom dokazane su mikroskopske lezije II stepena kod 3 uzorka (11,53%) jetri prasadi od ukupno pregledanih 26 uzoraka sa pozitivnim RT-PCR. Imunohistohemijskim pregledom uzoraka jetre prasadi nije dokazano prisustvo antigena hepatitis E virusa.<br />Definisani su protokoli laboratorijskog ispitivanja hepatitis E infekcije kod svinja i ljudi, kao i u uzorcima mesa i jetri svinja u klanicama</p> / <p>Hepatitis E virus (HEV) is the causative agent of acute hepatitis E infection in humans. HEV is transmitted through contaminated water and is responsible for the outbreaks of many large-scale epidemics in the developing countries of Asia and Africa. Swine HEV was first isolated in 1997, and was later detected in other animal species, such are: wild boar, deer, rabbits, rats, birds and more.<br />The first investigations of HEV infection in domestic and wild pigs in Serbia were carried out in 2008. HEV RNA was detected in 30% of faecal samples and 45% of the tissue samples (Petrovic et al., 2008). Analysing the blood samples of beckyard pigs, the seroprevalence of 34,6% was determined (Lupulovic et al, 2010). The aim of this study was to investigate the prevalence of HEV infection in pigs on farms in Vojvodina, as well as testing the HEV seroprevalence in humans.<br />The methods used for this study were: non-commercial ELISA (in house ELISA), the commercial ELISA, Western blot method, real-time RT-PCR and immunohistochemical method for the detection of HEV antigen.<br />Material for the study were: blood samples of pigs (300) from 3 farms on the territory of South Backa and Srem and blood samples of people (294), as well as faeces, bile, liver and meat collected in slaughterhouses from 95 fatteners and 50 piglets.<br />The presence of specific IgG antibodies against the hepatitis E virus in pigs has been detected on all three examinated farms. Upon the application of in house ELISA, the seroprevalence of 37% was establised on farm A, 31% in farm B and 54% on farm C, while using a commercial ELISA , 40% of seropositive pigs were detected on farm A, 41% of fami B and 65% Farm C. The comparative analysis of the results obtained with both ELISA, determined the average seroprevalence of 40,66% by in house ELISA and 48,66% by commercial ELISA.<br />The research of the presence of specific IgG antibodies against HEV in the serum of blood donors and patients were also conducted. Upon the application of in house ELISA, the seroprevalence of 15% were recorded in blood donors, while blood samples of patients were seronegative. Testing by commercial ELISA, positiv serological findings were diagnosed in 17,86% of blood donors (serums with positive or suspicious results in in house ELISA and a number of seronegative samples were tested), and in patients 2, 12%.<br />As so-called &quot;gold standard&quot; for defining the serological results with suspiciousserological findings, which extinctions were close to cut off values in in house ELISA, we used the Western</p>