Ng Dean Yew, Dennis. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 85-98). / Abstract --- p.i / Acknowledgments --- p.iii / Table of contents --- p.iv / List of figures --- p.vii / List of tables --- p.ix / List of abbreviation --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- History --- p.1 / Chapter 1.2. --- Classification and structure of Epstein-Barr Virus --- p.2 / Chapter 1.3. --- Genomic organization of EBV --- p.3 / Chapter 1.4. --- Replication cycle of EBV --- p.5 / Chapter 1.5. --- EBV latent and lytic cycle proteins --- p.6 / Chapter 1.6. --- Clinical diseases associated with EBV Infection --- p.11 / Chapter 1.7. --- Association of EBV and NPC --- p.13 / Chapter 1.8. --- EBV serological markers in the diagnosis of NPC --- p.13 / Chapter 1.9. --- Sources of EBV-specific DNase --- p.15 / Chapter 1.10. --- Characteristics of Epstein-Barr virus alkaline DNase --- p.15 / Chapter 1.11. --- Aim of the project --- p.18 / Chapter Chapter 2 --- Materials & Methods / Chapter 2.1. --- Molecular cloning --- p.19 / Chapter 2.1.1. --- Cell culture --- p.19 / Chapter 2.1.2. --- mRNA purification --- p.19 / Chapter 2.1.3. --- First strand cDNA synthesis --- p.21 / Chapter 2.1.4. --- Polymerase chain reaction (PCR) of cDNA --- p.21 / Chapter 2.1.5. --- Purification of PCR product after gel electrophoresis --- p.22 / Chapter 2.1.6. --- Ligation of PCR amplified DNase gene into pUC18 Sma/BAP vector --- p.23 / Chapter 2.1.7. --- Transformation by electroporation --- p.24 / Chapter 2.1.7.1. --- Cell preparation --- p.24 / Chapter 2.1.7.2. --- Electroporation procedure --- p.25 / Chapter 2.2. --- Extraction ofplasmid DNA --- p.28 / Chapter 2.2.1. --- Boiling preparation --- p.28 / Chapter 2.2.2. --- Plasmid digestion --- p.29 / Chapter 2.3. --- Large-scale purification ofplasmid --- p.29 / Chapter 2.4. --- Small-scale purification ofplasmid --- p.32 / Chapter 2.5. --- DNA sequencing --- p.33 / Chapter 2.5.1. --- Annealing of primer to template DNA --- p.33 / Chapter 2.5.2. --- Labelling reaction --- p.34 / Chapter 2.5.3. --- Sequencing termination reaction --- p.35 / Chapter 2.5.4. --- Prepartion of sequencing gel --- p.36 / Chapter 2.5.5. --- Autoradiography of sequencing gel --- p.38 / Chapter 2.6. --- Epitope mapping --- p.39 / Chapter 2.6.1. --- Processing of EBV- specific DNase peptides --- p.39 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Molecular cloning --- p.41 / Chapter 3.1.1. --- Cell culture --- p.41 / Chapter 3.1.2. --- mRNA purification --- p.42 / Chapter 3.1.3. --- PCR amplification --- p.42 / Chapter 3. 1.4 --- DNA purification of PCR product --- p.42 / Chapter 3.1.5. --- Molecular cloning of PCR amplified DNase gene into pUC18 SmaI/BAP vector --- p.44 / Chapter 3.1.6. --- Transformation by electroporation --- p.46 / Chapter 3.1.7. --- Extraction of plasmid DNA --- p.48 / Chapter 3.1.7.1. --- Boiling preparation --- p.48 / Chapter 3.1.8. --- Plasmid digestion --- p.51 / Chapter 3.2. --- DNA sequencing --- p.51 / Chapter 3.2.1. --- Comparison of B95-8 EBV-speicific DNase gene with gene sequence of EBV in GeneBank --- p.50 / Chapter 3.2.2. --- Comparison of 5' end of Raji & B95-8 EBV derived EBV-specific DNase gene --- p.57 / Chapter 3.2.3. --- Comparison of the 3'end of the Raji and B95-8 denved EBV-specific DNase gene --- p.63 / Chapter 3.2.4. --- Amino acid sequence homology between B95-8 & Raji EBV-specific DNase --- p.64 / Chapter 3.2.5. --- Amino acid sequence comparison between the 3' end of the B95-8 EBV DNase protein with that of the Raji EBV DNase protein --- p.62 / Chapter 3.3. --- Epitope mapping --- p.67 / Chapter 3.3.1. --- Amino acid key --- p.67 / Chapter 3.3.2. --- Amino acid sequence of peptides --- p.73 / Chapter 3.3.2. --- O.D. readings at 492nm of five histologically proven NPC sera --- p.74 / Chapter Chapter 4 --- Discussions / Chapter 4.1. --- Overall strategy --- p.75 / Chapter 4 2 --- Significance of EBV-specific DNase as marker for NPC --- p.76 / Chapter 4.3. --- Characterization of EBV-specific DNase --- p.76 / Chapter 4.4. --- Molecular cloning of PCR amplified gene into PUC18 SmaI/BAP vector --- p.77 / Chapter 4.4.1. --- Cell culture --- p.77 / Chapter 4.4.2. --- PCR amplification --- p.73 / Chapter 4.4.3. --- "Blunting,kinasing and ligation of EBV-specific DNase cDNA into pUC18 vector" --- p.78 / Chapter 4.4 .4 --- .Transformation by electroporation --- p.80 / Chapter 4.4.5. --- Restriction enzyme digestion of pUC18/EBV-DNase plasmid … --- p.81 / Chapter 4.5. --- DNA sequencing --- p.81 / Chapter 4.6. --- Epitope mapping --- p.83 / Reference --- p.85

by Liu Chun Ki, Kevin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 126-142). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.ii / Table of contents --- p.iii / List of figures --- p.viii / List of tables --- p.x / List of abbreviations --- p.xi / Chapter Chapter 1 --- Introduction Epstein-Barr Virus / Chapter 1.1 --- History --- p.1 / Chapter 1.2 --- Classification --- p.2 / Chapter 1.3 --- Virus and genome structure --- p.3 / Chapter 1.4 --- Epidemiology --- p.6 / Chapter 1.4.1 --- Prevalence of infection --- p.6 / Chapter 1.4.2 --- Modes of transmission --- p.7 / Chapter 1.5 --- Pathogenesis of EBV --- p.7 / Chapter 1.5.1 --- "Adsorption, penetration and dissemination" --- p.7 / Chapter 1.5.2 --- Lytic infection cycle --- p.8 / Chapter 1.5.3 --- Latent infection cycle --- p.9 / Chapter 1.5.4 --- Functions of the EBV-specific proteins associated with latent infection cycle proteins --- p.10 / Chapter 1.5.4.1 --- EBNA1 --- p.10 / Chapter 1.5.4.2 --- EBNA2 --- p.11 / Chapter 1.5.4.3 --- "EBNA 3A, 3B and 3C" --- p.11 / Chapter 1.5.4.4 --- EBNA LP --- p.12 / Chapter 1.5.4.5 --- LMP1 --- p.13 / Chapter 1.5.4.6 --- Characteristics of EBV LMP 2 gene --- p.14 / Chapter 1.5.4.7 --- Functions of LMP 2A --- p.15 / Chapter 1.5.4.8 --- Functions of LMP 2B --- p.18 / Chapter 1.6 --- Clinical significance of EBV --- p.20 / Chapter 1.6.1 --- Infectious mononucleosis (IM) --- p.20 / Chapter 1.6.2 --- Burkitt's lymphoma (BL) --- p.20 / Chapter 1.6.3 --- Nasopharyngeal carcinoma (NPC) --- p.21 / Chapter 1.6.4 --- Hodgkin's lymphoma (HL) --- p.21 / Chapter 1.7 --- Immune response to EBV infection --- p.22 / Chapter 1.7.1 --- Humoral immune response --- p.22 / Chapter 1.7.2 --- Cellular immune response --- p.22 / Chapter 1.8 --- Diagnosis of EBV infection --- p.26 / Chapter 1.9 --- Treatment and prevention --- p.27 / Chapter 1.10 --- Nasopharygneal Carcinoma (NPC) --- p.28 / Chapter 1.10.1 --- Epidemiology --- p.28 / Chapter 1.10.2 --- Etiology --- p.28 / Chapter 1.10.2.1 --- Environmental factor associated with NPC --- p.30 / Chapter 1.10.2.2 --- Genetic factors associated with NPC --- p.31 / Chapter 1.10.2.3 --- Association of NPC and EBV --- p.31 / Chapter 1.10.3 --- Diagnosis ofNPC --- p.32 / Chapter 1.10.4 --- Treatment --- p.33 / Chapter 1.11 --- Objective of the project --- p.34 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- EBV-containing cell cultures --- p.35 / Chapter 2.2 --- Extraction of total RNA --- p.36 / Chapter 2.2.1 --- Cell lysis --- p.36 / Chapter 2.2.2 --- Protein digestion --- p.36 / Chapter 2.2.3 --- DNA digestion --- p.37 / Chapter 2.2.4 --- Elution of total RNA --- p.37 / Chapter 2.2.5 --- Purity and electrophoresis analysis of total RNA --- p.38 / Chapter 2.3 --- First strand cDNA synthesis --- p.38 / Chapter 2.4 --- PCR amplification of LMP 2 cDNA --- p.39 / Chapter 2.5 --- Isolation of the PCR amplified LMP 2 cDNA --- p.40 / Chapter 2.6 --- Purification of the PCR amplified LMP 2 cDNA --- p.41 / Chapter 2.7 --- Confirmation of the PCR amplified cDNA --- p.42 / Chapter 2.7.1 --- Nested PCR --- p.42 / Chapter 2.7.2 --- Restriction enzyme digestion --- p.44 / Chapter 2.8 --- Ligation of insert LMP 2 cDNA with vector --- p.45 / Chapter 2.9 --- Transformation of competent cells JM109 --- p.45 / Chapter 2.10 --- Screening of the recombinant clones --- p.47 / Chapter 2.11 --- Small scale purification of plasmid DNA --- p.47 / Chapter 2.12 --- Determination of the size of the insert DNA --- p.48 / Chapter 2.13 --- DNA sequencing --- p.48 / Chapter 2.13.1 --- The cycle sequencing reaction --- p.48 / Chapter 2.13.2 --- Preparation of the acrylamide gel and TBE buffer --- p.51 / Chapter 2.13.3 --- Running conditions of the electrophoresis --- p.52 / Chapter 2.13.4 --- "Processing, editing and exporting the sequences" --- p.52 / Chapter 2.14 --- Data analysis --- p.53 / Chapter 2.14.1 --- Sequence analysis --- p.53 / Chapter 2.14.2 --- Amino acid analysis --- p.53 / Chapter 2.14.3 --- Protein secondary structure analysis --- p.53 / Chapter 2.14.4 --- Hydrophobicity analysis --- p.54 / Chapter 2.14.5 --- Isoelectric point analysis --- p.54 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Cell Cultures --- p.55 / Chapter 3.2 --- Extraction of total RNA --- p.56 / Chapter 3.3 --- PCR amplification --- p.61 / Chapter 3.4 --- Isolation of PCR amplified LMP 2 cDNA --- p.62 / Chapter 3.5 --- Confirmation of the PCR amplified cDNA --- p.66 / Chapter 3.5.1 --- Nested PCR --- p.66 / Chapter 3.5.2 --- Restriction enzyme digestion --- p.71 / Chapter 3.6 --- Transformation and screening --- p.77 / Chapter 3.7 --- Extraction of plasmid DNA and its digestion with restriction enzyme --- p.78 / Chapter 3.8 --- DNA sequencing --- p.83 / Chapter 3.8.1 --- DNA sequence comparison --- p.84 / Chapter 3.9 --- Amino acid sequence homology --- p.89 / Chapter 3.9.1 --- Amino acid sequence comparison --- p.90 / Chapter 3.10 --- Hydrophobicity analysis --- p.92 / Chapter 3.10.1 --- Comparison of hydrophobicity of B95-8 derived LMP2 with GeneBank --- p.93 / Chapter 3.10.2 --- Comparison of hydrophobicity of CB 14022-derived LMP2 with GeneBank --- p.95 / Chapter 3.10.3 --- Comparison of hydrophobicity of Raji-derived LMP2 with GeneBank --- p.97 / Chapter 3.11 --- Protein secondary structure analysis --- p.100 / Chapter 3.11.1 --- Comparison of secondary structure of B95-8-derived LMP2 with GeneBank --- p.100 / Chapter 3.11.2 --- Comparison of secondary structure of CB 14022-derived LMP2 with GeneBank --- p.100 / Chapter 3.11.3 --- Comparison of secondary structure of Raji-derived LMP2 with GeneBank --- p.101 / Chapter 3.12 --- Isoelectric point analysis --- p.103 / Chapter Chapter 4 --- Discussions / Chapter 4.1 --- Overall strategy for the cloning and sequencing of EBV LMP 2 gene --- p.106 / Chapter 4.2 --- Implications of the results obtained in sequencing --- p.107 / Chapter 4.3 --- Results interpretation --- p.108 / Chapter 4.3.1 --- Cell culture --- p.108 / Chapter 4.3.2 --- Extraction of total RNA --- p.108 / Chapter 4.3.3 --- PCR amplification --- p.109 / Chapter 4.3.4 --- Confirmation of the PCR amplified cDNAs using nested PCR --- p.109 / Chapter 4.3.5 --- Confirmation of the PCR amplified cDNAs using restriction enzyme digestion --- p.110 / Chapter 4.3.6 --- Ligation of EBV LMP 2 cDNA to pGEM-T Easy Vector --- p.111 / Chapter 4.3.7 --- Transformation and screening --- p.114 / Chapter 4.3.8 --- Extraction of plasmid DNA and digestion with restriction enzyme --- p.115 / Chapter 4.4 --- DNA sequencing and sequence homology --- p.116 / Chapter 4.5 --- Amino acid sequence homology --- p.117 / Chapter 4.6 --- Hydrophobicity analysis --- p.119 / Chapter 4.7 --- Protein secondary structure analysis --- p.120 / Chapter 4.8 --- Isoelectric point analysis --- p.122 / Chapter 4.9 --- Summary of results --- p.122 / Chapter 4.10 --- Conclusions --- p.124 / References --- p.126

by Cheung Siu Tim. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 155-160). / 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.

Epstein-Barr Virus (EBV) has two states of infection, latent and lytic. During the latent state the viral genome remains stable in cells as episomes and replicates with cellular DNA. During the lytic cycle the viral DNA becomes amplified and packaged in newly formed virions. An unsolved problem is whether newly replicated EBV DNA produced upon lytic cycle activation is associated with histones, and if so, whether these histones are acetylated. This question has biological significance as knowing the chromatin structure of genes is important in determining their function and expression profile. Our hypothesis is that newly synthesized EBV lytic DNA is associated with histones and the histone tails are selectively acetylated. To investigate our hypothesis we performed chromatin immunoprecipitation (ChIP) in HH514-16 cells, a Burkitts Lymphoma cell line, during latent and lytic replication. We used quantitative PCR (qPCR) to detect the relative concentration of DNA among the different samples. We tested three different variables: type of inducing agent, duration of treatment, and different regulatory regions in the genome of Epstein-Barr Virus. We found that in cells induced into the lytic cycle with Trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), association of newly replicated EBV DNA with acetylated histone 3 (H3) increased ~ 6-10 fold. This increase in association was greatest 72 hrs after treatment. Furthermore, activation of lytic viral replication in HH514-16 cells using a different inducing agent, Azacytidine (AZC), which is known to function as a DNA methyltransferase inhibitor, increased binding of H3 with viral DNA ~8 fold. However, unlike TSA, AZC increased the acetylation state of histones bound to newly synthesized viral DNA only ~ 2 fold. Changing the regulatory region of the EBV genome analyzed in qPCR did not affect our results. Our results suggest that newly replicated viral DNA is associated with histones, a fraction of which are acetylated. The degree of acetylation likely depends on the agent used to induce the lytic cycle. H3 is highly acetylated when an HDACi is used and less acetylated when AZC is used. Our study provides new insight on the epigenetic profile of newly replicated viral DNA during the lytic cycle. It remains to be determined whether histones are packaged together with viral genomes into virions and whether the chromatin state of virion DNA affects gene expression after the virus enters uninfected cells.

Zugl.: Berlin, Freie Universiẗat, Diss., 2009.

Universiẗat, Diss., 2004--München.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Lic.-avh. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 3 uppsatser.