Wing Y. Chan. / "March 2000." / Thesis (M.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references. / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Heung May-sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references. / Abstracts in English and Chinese. / Title Page --- p.i / Acknowledgement --- p.ii / Table of Contents --- p.iii / List of Abbreviations --- p.vi / List of Figures --- p.viii / List of Tables --- p.xii / Abstract --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epstein-Barr Virus --- p.1-1 / Chapter 1.1.1 --- Virus structure --- p.1-1 / Chapter 1.1.2 --- Genome structure --- p.1-1 / Chapter 1.1.3 --- Nomenclature for EBV open reading frames --- p.1-2 / Chapter 1.1.4 --- Biology of EBV --- p.1-2 / Chapter 1.1.5 --- EBV latency --- p.1-7 / Chapter 1.1.6 --- EBV latent gene promoters --- p.1-8 / Chapter 1.2 --- EBV Infection and Its Persisence --- p.1-9 / Chapter 1.3 --- DNA Methylation --- p.1-17 / Chapter 1.3.1 --- Aberrant CpG island methylation in cancer --- p.1-18 / Chapter 1.3.2 --- DNA methylation and EBV --- p.1-19 / Chapter 1.4 --- Demethylating Agents --- p.1-21 / Chapter 1.5 --- Aims of the Study --- p.1-23 / Chapter Chapter 2 --- EBV Latency Patterns / Chapter 2.1 --- Introduction --- p.2-1 / Chapter 2.2 --- Materials and Methods --- p.2-1 / Chapter 2.2.1 --- Cell line culture --- p.2-1 / Chapter 2.2.2 --- NPC biopsies culture --- p.2-2 / Chapter 2.2.3 --- RNA extraction --- p.2-3 / Chapter 2.2.4 --- RNA quantification --- p.2-4 / Chapter 2.2.5 --- Deoxyribonuclease I treatment for NPC biopsies --- p.2-5 / Chapter 2.2.6 --- Reverse transcriptase-polymerase chain reaction --- p.2-5 / Chapter 2.2.7 --- Gel Electrophoresis --- p.2-10 / Chapter 2.3 --- Results --- p.2-11 / Chapter 2.3.1 --- Burkitt' s lymphoma and lymphoblastoid cell lines --- p.2-11 / Chapter 2.3.2 --- Nasopharyngeal carcinoma biopsies --- p.2-11 / Chapter 2.4 --- Discussion --- p.2-19 / Chapter Chapter 3 --- Treatment with Demethylating Agents on Rael / Chapter 3.1 --- Introduction --- p.3-1 / Chapter 3.2 --- Materials and Methods --- p.3-2 / Chapter 3.2.1 --- Rael cell line culture --- p.3-2 / Chapter 3.2.2 --- Drug treatment --- p.3-2 / Chapter 3.2.3 --- Viability staining --- p.3-2 / Chapter 3.2.4 --- Statistical analysis --- p.3-3 / Chapter 3.2.5 --- RNA extraction and quantification --- p.3-3 / Chapter 3.2.6 --- RT-PCR and gel electrophoresis --- p.3-3 / Chapter 3.2.7 --- DIG oligonucleotide 3'-end labeling --- p.3-3 / Chapter 3.2.8 --- Southern blot --- p.3-10 / Chapter 3.3 --- Results --- p.3-13 / Chapter 3.3.1 --- 5-azacytidine --- p.3-13 / Chapter 3.3.2 --- 5-aza-2-deoxycytidine --- p.3-26 / Chapter 3.4 --- Discussion --- p.3-39 / Chapter Chapter 4 --- Treatment with Demethylating Agents on NPC Biopsies / Chapter 4.1 --- Introduction --- p.4-1 / Chapter 4.2 --- Materials and Methods --- p.4-2 / Chapter 4.2.1 --- NPC biopsy culture --- p.4-2 / Chapter 4.2.2 --- Drug treatment --- p.4-2 / Chapter 4.2.3 --- RNA extraction and quantification --- p.4-2 / Chapter 4.2.4 --- DNase I treatment for NPC biopsies --- p.4-2 / Chapter 4.2.5 --- RT-PCR and gel electrophoresis --- p.4-2 / Chapter 4.3 --- Results --- p.4-3 / Chapter 4.4 --- Discussion --- p.4-3 / Chapter Chapter 5 --- Conclusion --- p.5-1 / Reference --- p.R-1 / Appendix --- p.A-l

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Cellular immune responses to EBV. Includes bibliographical references (p. 150-178). Also available online through Digital Dissertations.

A person is exposed to many pathogens throughout their lifetime, and with the resolution of each infection, there remains a pool of pathogen-specific immune cells that protect that person from re-infection with the same pathogen. However, there is a great deal of evidence to suggest that the pool of pathogen-specific memory cells can also participate in the immune response to future infections with unrelated pathogens. Many believe T cells to be cross-reactive in nature because of their interaction with self antigens during development in the thymus and their interaction with foreign antigens once in the periphery. There are many features of the interaction between a T cell and its ligand that facilitate this cross-reactive nature. Based on solved crystal structures, relatively few contacts are required for a stable interaction, and that interaction is often mediated by the flexible CDR3 loops of the T cell receptor that accommodate ligands of various structure. There is also evidence in the murine and human systems that subsets of virus-specific memory CD8 T cells take on an activated phenotype upon infection with an unrelated virus. In murine models, these memory T cell subsets could kill target cells, secrete several cytokines, and proliferate in response to a cross-reactive stimulation, suggesting that a cross-reactive T cell response could impact the outcome of a viral infection. In fact, upon heterologous infection, mice immune to a previous virus were often protected, having lower titers of the second unrelated virus, their epitope-specific and T cell receptor repertoires were often skewed, and they were more prone to immune-mediated pathologies. All of these observations coincided with the presence of cross-reactive T cell responses. Thus, we define heterologous immunity as changes in viral replication and the disease pathology associated with that viral infection as a result of the host's history of infection, and this can be mediated, in part, by cross-reactive CD8 T cell responses. Since many human viral infections are associated with a wide range of disease states, we questioned whether cross-reactive CD8 T cell responses occurred as commonly as they appeared to occur in the murine models and whether they influenced the outcome of such infections. Epstein-Barr virus (EBV) infects over 90% of the U. S. population and has a large genome with the capacity to encode a multitude of T cell epitopes. The first part of this thesis research focuses on the identification of cross-reactive CD8 T cell responses with specificity for known epitopes derived from EBV, a common human virus. We directed our study to HLA-A2-restricted responses because of the common expression of this MHC Class I allele in the U. S. population. This study resulted in the detection of cross-reactive responses with five different specificities that involved either the immunodominant lytic EBV-BMLF1280 epitope or the latent EBNA 3A596epitope. Three of the cross-reactive responses had specificity for epitopes derived from another unrelated, but common, human virus, influenza A virus (IV). Each of these cross- reactive responses had the potential to participate in the collective immune response to acute EBV infection. EBV is also well-suited as a model system to study heterologous immunity in humans, as infection at an early age is frequently asymptomatic, while the same infection during adolescence often results in an immune-mediated syndrome, infectious mononucleosis (IM). Since older individuals have presumably been exposed to more pathogens in their lifetime and, therefore, would have memory CD8 T cell pools with more extensive specificities, we hypothesized that acute EBV infection activated cross-reactive memory CD8 T cell responses that promoted the development of IM. In order to determine if the cross-reactive responses we identified above contributed to the immune response to acute EBV infection, we first screened the blood of IM patients for cross-reactive T cells with specificity for EBV-BMLFl280 and IV-M158. The total number of M1-specific T cells of 5 of 8 patients was increased at presentation with IM, which was suggestive of their specific activation during the EBV infection since a bystander mechanism would have resulted in 8 out of 8 patients having increased numbers of M1-specific T cells. Our hypothesis was further supported by the fact that we clearly detected cross-reactive T cells capable of recognizing both BMLF1 and M1 epitopes in the blood of 2 of the 5 IM patients with an augmented M1-specific T cell frequency. Furthermore, the M1-specific TCR repertoires of those two patients were dramatically skewed, which was an indication of cross-reactive M1-specific T cell expansions and, therefore, participation in the lymphoproliferation characteristic of IM. In addition, T cell lines derived from 3 out of 8 healthy donors with previous exposure to both viruses contained a subset of T cells that responded to both BMLF1 and M1 epitopes, suggesting that these cross-reactive cells are often maintained in memory. These cross-reactive T cells were cytotoxic and produced MIP-1β, IFNγ, and TNFα, functions which could potentially promote the symptoms of IM and, indeed, may have been contributed to the severe case of IM noted in one patient. The final part of this thesis research focused on defining the structure of the cross-reactive TCR that recognized both BMLF1 and M1 epitopes, which have only 33% sequence similarity. In addition, we examined the cross-reactive TCR repertoire organization of multiple individuals to determine the breath and, therefore, the likelihood that this cross-reactive T cell response will occur. These studies revealed that a wide range of Vα and Vβ families can mediate interaction with both epitopes and that the cross-reactive TCR repertoire was unique to each individual, relying heavily on the T cell clones present in that individual's private BMLF1- and M1-specific repertoires. We also observed an increased frequency of TCRs with longer CDR3 regions within the cross-reactive repertoire, which were often extended by non-bulky amino acid residues that could provide these TCRs with more flexibility in order. to accommodate the two different epitope structures. Given that we detected a cross-reactive T cell response with specificity for two immunodominant epitopes derived from two of the most common human viruses among people that share one of the most common MHC Class I alleles in the U. S. population, we predict that cross-reactive T cells are common components of human immune responses. The variability in the magnitude and specificity of each cross-reactive T cell response is dependent on each individual's unique history of infection and th,eir unique TCR repertoire, and such responses likely represent one of many factors that could explain the individual variability in disease severity associated with EBV and many other human viral infections.

This thesis is the construct of a computational system for studying the nasopharyngeal carcinoma (NPC) using high-throughput sequencing data. The system involves several components, including discovery of gene fusion in NPC cell line, construction of Esptein-Barr virus (EBV) genome, and evaluation on contaminated sequencing data alignment approaches. We successfully discovered a gene fusion (UBR5-ZNF423) in a NPC cell line (C666-1) which was verified by lab experiments and found in 8.3% of primary tumors. It was discovered the regulation of this gene affect the growth of cancer cell. We constructed the EBV genome in C666-1. It serves as an important reference for studying this important NPC cell line, which was the only NPC cell line in the world for a long time. We also evaluated three mapping approaches. Two of them are designed to filter out potential mouse contamination reads on human sequencing data, which can originate from NPC human-in-mouse xenografts. We found that special care should always be applied to contaminated data. Although direct mapping can give acceptable results if in most cases, the combined-based approached is suggested. It can effectively reduce false positive variants and maintain good enough numbers of true positive variants. Filtering approach is an alternative to the combined-based approach that can also effectively reduce contamination when memory is not sufficient. / 本論文利用電腦有系統地研究鼻咽癌，當中的數據利用了高通量測序技術來定序。其中章節包括在鼻咽癌胞系中尋找融合基因、組建潛藏於人體可引致鼻咽癌的EB病毒基因組、還有評價幾種可處理受污染序列的序列排列方法。我們成功地在鼻咽癌胞系（C666-1）中發現出一個融合基因（UBR5-ZNF423），並在實驗中確定此成果，其中發現在原發腫瘤中有8.3%的樣本中找出此融合基因。此外，也發現這融合基因調控會影響到癌細胞的生長。C666-1鼻咽癌胞系在過往有一段很長的時間裡，都是全世界唯一的鼻咽癌胞系，因此它有非常重要的參考價值，在此研究，我們組建了在C666-1裡的EB病毒基因組，使它作為研究C666-1的參考樣本。另外，我們評價了三種處理排列的方法，其中兩種的設計能過濾部分人類序列數據當中老鼠基因組的污染，老鼠基因組的污染可以來自於異種移植，即把人類癌細腫瘤移植於老鼠身上種植，我們建議在情況許可下都使用特殊的處理方法而不是直接作序列排列。直接作序列排列數據雖然已有合理的表現，但相比之下組合基因組式序列排列方法能有效減少錯誤肯定的遺傳變異，並同時保留足夠多正確肯定的遺傳變異，所以組合基因組式序列排列方法應在情況許可下都使用它。過濾式序列排列方法也是一種特殊的處理方法，它也能有效減少錯誤肯定的遺傳變異，它對記憶體的需求比組合基因組式序列排列方法少，可在電腦的記憶體不足時使用它。 / Tso, Kai Yuen. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 112-120). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only.

Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.