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

Leung, Chung Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 100-113). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / Abbreviations --- p.IX / Table of Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Epidemiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Etiology of heptocellular carcinoma (HCC) --- p.1 / Chapter 1.3 --- HBV genome structure --- p.2 / Chapter 1.4 --- HBV pathogenesis --- p.2 / Chapter 1.5 --- Hepatitis B virus X protein (HBx) --- p.3 / Chapter 1.6 --- Oxidative stress and antioxidant --- p.5 / Chapter 1.6.1 --- Glutathione (GSH) --- p.5 / Chapter 1.6.2 --- Superoxide dismutase (SOD) --- p.7 / Chapter 1.6.3 --- Oxidative stress in HBV-related liver disease and HCC --- p.8 / Chapter 1.7 --- Apoptosis and necrosis --- p.9 / Chapter 1.7.1 --- Apoptotic pathways --- p.9 / Chapter 1.8 --- Role of HBx in apoptosis --- p.10 / Chapter 1.9 --- Transcriptional activity by HBx --- p.12 / Chapter 1.10 --- Chemotherapy drug resistance --- p.13 / Chapter 1.11 --- Objectives of study --- p.14 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Construction of plasmid --- p.23 / Chapter 2.1.1 --- PCR amplification of wild-type and mutant HBx --- p.23 / Chapter 2.1.2 --- Agarose gel extraction --- p.25 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.26 / Chapter 2.1.4 --- Ligation of vectors and gene of interest --- p.26 / Chapter 2.1.5 --- Preparation of competent cells for transformation --- p.27 / Chapter 2.1.6 --- Transformation of plasmid in competent cells --- p.27 / Chapter 2.1.7 --- Plasmid extraction by mini-prep --- p.28 / Chapter 2.1.8 --- DNA sequencing of the inserted genes --- p.29 / Chapter 2.2 --- Transfection --- p.30 / Chapter 2.2.1 --- Cell line --- p.30 / Chapter 2.2.2 --- Lipofectamine transfection --- p.31 / Chapter 2.2.3 --- Construction of stably-transfected cell lines --- p.31 / Chapter 2.3 --- Detection of expression of transfected gene in mRNA level by RT-PCR --- p.32 / Chapter 2.3.1 --- RNA extraction --- p.32 / Chapter 2.3.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.3 --- Agarose gel electrophoresis --- p.36 / Chapter 2.4 --- Detection of expression of transfected gene in protein level by Western blot --- p.36 / Chapter 2.4.1 --- Sample preparation --- p.36 / Chapter 2.4.2 --- Measurement of protein concentration --- p.36 / Chapter 2.4.3 --- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.4.4 --- Transfer of proteins to nitrocellulose membrane --- p.38 / Chapter 2.4.5 --- Immunoblotting of protein --- p.38 / Chapter 2.5 --- Measurement of reduced glutathione (GSH) concentration in cell lines --- p.39 / Chapter 2.5.1 --- Sample preparation --- p.39 / Chapter 2.5.2 --- Measurement of GSH concentration --- p.39 / Chapter 2.6 --- Superoxide dismutase (SOD) activity in cell lines --- p.40 / Chapter 2.6.1 --- Sample preparation --- p.40 / Chapter 2.6.2 --- Measurement of total SOD activity --- p.41 / Chapter 2.6.3 --- Measurement of Cu/ZnSOD and MnSOD by Western blot --- p.42 / Chapter 2.7 --- Cell proliferation assay --- p.43 / Chapter 2.7.1 --- Drugs and concentration --- p.43 / Chapter 2.7.2 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.43 / Chapter 2.7.3 --- Cell proliferation and cytotoxicity of the drugs --- p.44 / Chapter 2.8 --- Detection of apoptosis by flow-cytometry --- p.44 / Chapter 2.8.1 --- Cell culture --- p.44 / Chapter 2.8.2 --- Cell fixation --- p.45 / Chapter 2.8.3 --- Cell staining --- p.45 / Chapter 2.8.4 --- Flow cytometry analysis --- p.46 / Chapter 2.9 --- Detection of protein involved in apoptotic pathway --- p.46 / Chapter 2.9.1 --- Antibodies --- p.46 / Chapter 2.9.2 --- Sample Preparation --- p.47 / Chapter 2.9.3 --- Measurement of protein concentration --- p.48 / Chapter 2.9.4 --- Western blotting --- p.49 / Chapter Chapter 3: --- Establishment of HBx transfected stable cell lines / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Plasmid construction --- p.56 / Chapter 3.2.2 --- Stable transfection of cell lines --- p.57 / Chapter 3.2.3 --- Morphology of wild type and mutant HBx-transfected cell lines --- p.58 / Chapter 3.3 --- Discussion --- p.58 / Chapter Chapter 4: --- Antioxidant level in HBx transfected cell lines / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results --- p.70 / Chapter 4.2.1 --- Glutathione (GSH) concentration in different cell lines --- p.70 / Chapter 4.2.2 --- Superoxide dismutase (SOD) activity in different cell lines --- p.71 / Chapter 4.2.2.1 --- Total SOD activity --- p.71 / Chapter 4.2.2.2 --- Cu/ZnSOD --- p.71 / Chapter 4.2.2.3 --- MnSOD --- p.72 / Chapter 4.3 --- Discussion --- p.72 / Chapter Chapter 5: --- Involvement of HBx in apoptotic pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Results --- p.82 / Chapter 5.2.1 --- Cell proliferation of HBx transfected cells --- p.82 / Chapter 5.2.2 --- Apoptosis of HBx transfected cells --- p.83 / Chapter 5.2.3 --- Cytotoxicity of fluorouracil (5FU) and doxorubicin (DOX) in HBx transfected cells --- p.84 / Chapter 5.2.4 --- Detection of anti-apoptotic proteins cIAP2 and Bcl-2 in HBx-transient and stably transfected cells --- p.84 / Chapter 5.3 --- Discussion --- p.85 / Chapter Chapter 6: --- Concluding remarks and general discussion / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Future work --- p.97 / Chapter 6.3 --- Summary --- p.99 / References --- p.100 / Appendix 1 --- p.114

Thesis (MScMedSc))--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Despite the availability of Hepatitis B virus (HBV) vaccination for over three decades, this infection remains a major public health problem. Whilst the WHO recommends giving a birth dose of the vaccine, in South Africa, routine infant HBV vaccination commences at six weeks of age. This schedule is based on data from the pre-HIV era which showed transmission occurred via the horizontal, rather than the vertical route. In the era of HIV however, maternal HIV co-infection may release HBV from immune control, resulting in higher HBV loads and increasing the risk of vertical transmission. The aim of this study was to determine the prevalence and character of HBV infection in HIV-exposed infected and uninfected infants. Residual plasma samples from routine HIV nucleic acid testing of 1000 HIV-exposed infants aged between 0 and 18 months from the Western Cape were tested. Samples were tested for HBsAg by ELISA (Murex HBsAg Version 3) and confirmed by neutralisation. HBV DNA was quantified using an in-house real-time PCR assay. Infants with HBsAg positive samples were followed up and a blood sample was collected from mother and child. Those HBsAg positive samples were tested for HBeAg/antiHBe (Diasorin) and HBsAg negative samples were tested for antiHBs. HBV DNA was quantified. The surface gene was sequenced and the HBV genotype determined by phylogenetic analysis using HepSEQ (www.hepseq.org.uk). Whole genome sequencing was also performed. Of 1000 samples tested, four samples were positive for HBsAg and/or HBV DNA, indicating a prevalence of HBV transmission of 0.4%. At follow-up, two of three infected infants were positive for HBsAg, with HBV viral loads of greater than 108 IU/ml. The third infant was found to have cleared his infection and the fourth child was lost to follow up. These infected infants had all received HBV vaccination. All four mothers were HBeAg positive. Sequencing analysis showed the HBV strains from the two infants and four mothers belonged to subgenotype A1. The two mother-child paired sequences were identical. The data from this study shows that vertical transmission of HBV infection in HIV-exposed infants from the Western Cape is occurring, despite vaccination. Data from the Western Cape, showing an HBV prevalence of 3.4% in HIV-infected pregnant women, and those presented here suggest a vertical transmission rate of HBV of 12%. This is despite the widespread use of tenofovir and lamivudine in HIV-infected women with low CD4 counts. This study provides data supporting calls to bring HBV vaccination closer to the time of birth. Further work is urgently needed to confirm these findings and to determine the rates of transmission in HIV-unexposed infants. / AFRIKAANSE OPSOMMING: Ten spyte van die beskikbaarheid van die Hepatitis B virus (HBV) inenting vir meer as drie dekades, hierdie infeksie bly 'n groot openbare gesondheid probleem. Terwyl die WGO aan beveel dat'n geboorte dosis van die entstof, in Suid-Afrika, roetine baba HBV inenting op die ouderdom van ses weke gegee word. Hierdie skedule is gebaseer op data van die pre-MIV era wat getoon het dat die oordrag plaasgevind het via die horisontale, eerder as die vertikale roete. In die era van MIV egter, moeder MIV ko-infeksie kan HBV vrylaat van immuun beheer, wat lei in hoër HBV vlakke en die verhoging van die risiko van vertikale oordrag. Die doel van hierdie studie was om die voorkoms en karakter van die HBV infeksie in MIV-besmette en onbesmette babas te bepaal. Residuele plasma monsters van roetine-MIV-nukleïensuur toetse van 'n 1000 MIV-blootgestelde babas tussen die ouderdomme van 0 en 18 maande van die Wes-Kaap was getoets. Monsters was getoets vir HBsAg deur ELISA (Murex HBsAg Version 3) en bevestig deur neutralisering. HBV DNA is gekwantifiseer deur gebruik te maak van 'n in-huis real-time PCR assay. Babas met HBsAg positiewe monsters was opgevolg en 'n bloedmonster is versamel van moeder en kind. Die HBsAg positiewe monsters was getoets vir HBeAg/antiHBe (Diasorin) en HBsAg negatiewe monsters was getoets vir antiHBs. HBV DNA was gekwantifiseer. Die oppervlak gene volgorde en genotipes was bepaal deur filogenetiese analise met behulp van HepSEQ (www.hepseq.org.uk). Die hele genoom-volgordebepaling was ook uitgevoer. Van die 1000 monsters wat getoets was, was vier monsters positief vir HBsAg en of HBV DNA, dit dui op 'n voorkoms van HBV oordrag van 0.4%. By op volg, twee van die drie besmette babas was positief vir HBsAg, met HBV virale vlakke van groter as 108 IE/ml. Die derde baba was gevind dat sy infeksie opgeklaar het en die vierde kind was verlore as gevolg van op volg. Hierdie besmette babas het almal HBV inenting ontvang. Al vier moeders was HBeAg positief. Volgordebepaling analise het getoon die HBV stamme van die twee babas en vier moeders behoort aan subgenotype A1. Die twee moeder-kind gepaarde rye was homoloë. Die data van hierdie studie toon dat die vertikale oordrag van HBV infeksie in MIV-blootgestelde babas van die Wes-Kaap vind plaas, ten spyte van inenting. Data van die Wes-Kaap, wat 'n HBV voorkoms van 3.4% in MIV-besmette swanger vroue, en dié wat hier aangebied is dui op 'n vertikale oordrag koers van 12% van die HBV. Dit is ten spyte van die wydverspreide gebruik van tenofovir en lamivudine in MIV-geïnfekteerde vroue met 'n lae CD4-telling. Hierdie studie bied data wat ondersteunende oproepe van HBV inenting nader aan die tyd van die geboorte bring. Verdere werk is dringend nodig om die bevindinge te bevestig en die pryse van die oordrag in MIV-blootgestelde babas te bepaal. / National Health Laboratory Service Research Trust / Poliomyelitis Research Foundation (PRF) / Harry Crossley Foundation / Stellenbosch University

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Deng Xue-Long. / "January 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 196-230). / 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.

Thesis (PhD (Med))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background and Objective: The most common cause of severe proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease (MCD). This is also the pattern observed in white and Indian children in South Africa (SA). By contrast, black and mixed race/coloured children of Southern Africa in the 1960s to 1990s were shown to have a different pattern of NS. One of the main differences was the frequency of hepatitis B virus (HBV) associated glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of this project was a clinicopathological study of this subgroup of nephrotic children to document the disease further and in particular to seek correlations between pathological and clinical features including prognosis. A central focus was to document the detailed ultrastructural examination of the renal biopsies of these children and to correlate the spectrum of pathological features with demographic, clinical, laboratory and prognostic features. The hypothesis was that the clinicopathological features of HBV MGN in children differed substantially from idiopathic MGN in general (children and adults) and also from HBV MGN in adults and that HBV MGN in children should be viewed as a distinct disease. Patients and methods: The childhood (12 years and younger) patient cohort was 309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from Namibia. The study group was 71 children with HBV MGN who were followed up to 2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN in childhood as this centre only had 2 such patients during the study period.) Demographic, clinical, laboratory and renal pathology data were collected, compared and correlated. Results: HBV associated MGN was the most frequent cause of NS in the Namibian subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood nephrotic cohort, by far the dominant subgroup. The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years) at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen (HBeAg) was identified in the serum of 87% of children tested. Laboratory features different from idiopathic MGN included more prominent haematuria, mildly raised serum transaminases and more frequently lowered serum C3 and C4 levels. Light microscopic examination of renal biopsies showed mesangial proliferation in all patients but with minimal glomerular sclerosis and interstitial disease. On ultrastructural examination mesangial and subendothelial deposits were common and prominent as was mesangial interposition. The MGN of HBV in children therefore frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies displayed severe mesangial interposition in addition to the subepithelial deposits of MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like bodies and tubuloreticular inclusion bodies were both found in more than 80% of biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits in the glomeruli. This was the first time this feature was demonstrated in Africa. The 46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort developed chronic renal failure (CRF). Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset of CRF was more than 19 years with the longest being 23 years. The 358 cases of childhood HBV MGN from Southern Africa constitute 37% of the reported childhood patients. Comparative data A comparison was made between the 71 children with HBV MGN, 12 adults with HBV MGN and 33 adults with idiopathic MGN. The main differences were that both HBV MGN groups included only coloured and black patients and were more predominantly male while the idiopathic MGN group included all races. In the HBV patients, haematuria was more frequent and severe, liver enzymes were frequently raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of adult MGN patients had normal C4 levels while the childhood HBV MGN group had reduced C4 levels. The immune complex pattern in both of the HBV MGN adult and childhood groups on biopsy was similar with more mesangial and subendothelial deposits as well as mesangial interposition than the idiopathic group. Despite this similarity between the two HBV groups, both adult groups showed more glomerular sclerosis and interstitial disease than the childhood group. The clinical outcome of the children’s cohort was better than the other 2 groups with remission (52%) more frequent at 4 years (p< 0.01) and better renal and patient survival. Including the 83 cases from this series, at least 1243 renal biopsy proven cases of HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar (children 79%; adults 84%) and significantly greater than for idiopathic MGN. Conclusions: The findings confirm that HBV MGN in children is a distinct form of GN which broadens the classical morphologic description of MGN by often including a number of mesangiocapillary GN features. The subgroup of renal biopsies with the most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary GN group. The MGN spectrum as a whole comprised 86% of the HBV positive childhood group. HBV MGN was the most frequent association with NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group (19%) in the SA children. It showed a relatively high spontaneous remission rate but at least 10% of the children developed renal failure. Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. Extended follow up (more than 15 years) was required to demonstrate renal failure in some patients in the poor outcome group. Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this disease in SA. HBV MGN has been a valuable and possibly unique model of human GN and MGN in particular in that the HBeAg has been identified in both the serum and glomeruli enabling confirmation of the aetiological role of HBeAg. / AFRIKAANSE OPSOMMING: Agtergrond en Doelwit: Die algemeenste oorsaak van erge proteïenurie/nefrotiese sindroom (NS) in kinders wêreldwyd is minimale veranderingsiekte. Hierdie patroon kom ook voor in blanke- en Indiër kinders in Suid-Afrika. In teenstelling hiermee is aangetoon dat swart en kleurling/gemengde ras kinders in Suider Afrika tussen die jare 1960s tot 1990s ’n ander patroon van nefrotiese sindroom gehad het. Een van die hoof verskille was die algemene voorkoms van hepatitis B virus (HBV) geassosieerde glomerulonefritis, gewoonlik membraneuse glomerulonefritis (MGN). Die doelwit van hierdie projek was ’n klinies-patologiese studie van hierdie subgroep van nefrotiese kinders ten einde die siekte verder te beskryf en veral om korrelasies te tref tussen patologiese en kliniese kenmerke insluitende prognose. Die gedetaileerde ultrastrukturele ondersoek van die kinders se nierbiopsies en die korrelasie van die spektrum patologiese kenmerke met demografiese, kliniese, laboratorium en prognostiese kenmerke was ‘n sentrale fokusarea. Die hipotese was dat die klinies-patologiese kenmerke van HBV MGN in kinders wesenlik van idiopatiese MGN in die algemeen verskil (in kinders en volwassenes) en ook van HBV MGN in volwassenes, en dat die beeld in kinders as ’n afsonderlike siekte beskou behoort te word. Pasiënte en metodes: Die kinder kohort (12 jaar en jonger) was 309 kinders met erge proteïenurie/nefrotiese sindroom wie in Tygerberg Hospitaal (TBH) behandel was oor ‘n 21 jarige periode vanaf 1974 tot 1995, insluitende 67 kinders van Namibië. Die studiegroep was 71 kinders met HBV MGN wie waar moontlik tot 2005 opgevolg was. Die vergelykende volwasse groep was 45 volwassenes met MGN van wie 12 HBV MGN gehad het en 33 idiopatiese MGN. (’n Vergelyking met idiopatiese MGN in kinders kon nie gedoen word nie omdat hierdie sentrum net twee sulke pasiënte tydens die studietyd behandel het.) Demografiese, kliniese, laboratorium en nierpatologie inligting is versamel, vergelyk en gekorreleer. Resultate: HBV geassosieerde MGN was die algemeenste oorsaak van NS in die Namibiese subgroep, 25/67 (37%) en die derde mees algemeen, 71/309 (23%) in die kinder kohort as geheel. Die MGN groep was 86% (71/83) van die totale HBV kinder nefrotiese kohort en verreweg die oorheersende subgroep. Die gemiddelde ouderdom van die 71 kinders met HBV MGN by presentering was 6.0 jaar (reikwydte 2-12 jaar) en seuns het 80% van die groep behels. Hepatitis B omhullingsantigeen (envelope antigen- HBeAg) is aangetoon in die serum van 87% van die kinders wie daarvoor getoets is. Laboratoriumkenmerke wat van idiopatiese MGN verskil het, het ingesluit meer prominente hematurie, gering verhoogde serum transaminases en meer dikwels verlaagde serum C3 en C4 vlakke. Ligmikroskopiese ondersoek van die nierbiopsies het mesangiale proliferasie in elke pasiënt getoon, maar met minimale glomerulêre sklerose en interstisiële siekte. Met ultrastrukturele ondersoek was mesangiale en subendoteliële neerslae asook mesangiale interposisie algemeen. Die MGN van HBV in kinders het dus dikwels kenmerke van mesangiokapillêre glomerulonefritis getoon bo en behalwe die subepiteliële neerslae van MGN. Die ondergroep van 23 van wie die nierbiopsies erge mesangiale interposisie aangetoon het asook die subepiteliale neerslae van MGN is die gemengde HBV MGN-mesangiokapillêre GN groep genoem. Virustipe liggaampies en tubuloretikulêre insluitingsliggaampies is in meer as 80% van die biopsies bevestig. HBeAg was in die subepiteliële neerslae identifiseer. Dit was die eerste keer dat hierdie kenmerk in Afrika identifiseer is. Die 46 Suid-Afrikaanse kinders het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking (KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer. Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder pasiënte. Vergelykende data ’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir idiopatiese MGN. Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre GN groep geklassifiseer. Die MGN spektrum in geheel het 86% van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer. Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van die swak uitkomsgroep aan te toon. Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol van HBeAg te bevestig.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The HBV RNAse H has been cloned into the PET43a vector, which contains the NusA protein which works as a solubilizing fusion protein. The fusion NUS-RNAse H protein was cleaved by enterokinase; the cleaved RNAse H is about 17 Kda which remains soluble and active. A fluorescence assay utilizing a quenching mechanism was used to characterize the activity of NUS-RNAse H and cleaved RNAse H proteins. The beacon is a RNA:DNA hybrid oligonucleotide labeled with a 5'DABCYL and a 3'fluorescein, when RNAse H digests the RNA, DABCYL is released resulting in high fluorescence. The digestion of the RNA was also confirmed by gel analysis. The protein was identified by N-terminal amino acid sequence analysis of the fusion protein, SDS-PAGE, western blot utilizing HBV positive sera for primary antibodies, and enzyme immunoassay by peroxidase labeling of HBV RNAse H. Structural analysis of the protein was done by circular dichroism, tryptophan fluorescence, the generation of a model from HIV RNAse H and initial crystals which unfortunately did not diffract. The ability to produce good amounts soluble RNAse H, the development of a sensitive assay to test for activity and the solution of the crystal structure will help develop new anti-viral inhibitors.

The Hepatitis B core antigen (HBcAg) has been proposed to be an ideal candidate for use as an adjuvant due to its immunogenicity, and tolerance to manipulations such as insertions of epitopes or covalent attachment of ligands. HBcAg is a complex macromolecule containing protein and nucleic acid. We investigated the effect of the removal and reconstitution of nucleic acids upon its structure. It’s been shown that the RNA content of hepadnavirus core antigens can be reduced significantly, but not be completely removed. Following removal of some of the RNA, antigens retain the ability to bind added nucleic acids, in particular, "immune-enhancing" synthetic oligonucleotides without affecting the structure of antigen or disrupting its ability to spontaneously self-assemble into core particles. The removal and addition of nucleic acids was successfully applied to an altered woodchuck core antigen, with a nucleic acid-based malaria epitope addition, giving rise to a potential vaccine adjuvant platform for malaria.