Submitted by CLEITON SILVA MARQUES null (biocleitond2@yahoo.com.br) on 2017-06-14T16:42:05Z No. of bitstreams: 1 MS 201513939-9 Dissertação Cleiton Silva Marques.pdf: 1951878 bytes, checksum: 324f65b41f0660609ec022c77b81f85e (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-06-19T13:53:05Z (GMT) No. of bitstreams: 1 marques_cs_me_bot.pdf: 1951878 bytes, checksum: 324f65b41f0660609ec022c77b81f85e (MD5) / Made available in DSpace on 2017-06-19T13:53:05Z (GMT). No. of bitstreams: 1 marques_cs_me_bot.pdf: 1951878 bytes, checksum: 324f65b41f0660609ec022c77b81f85e (MD5) Previous issue date: 2017-05-15 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Presente em aproximadamente 95% da população mundial o Vírus de Esptein-Barr (Epstein-Barr Virus - EBV) está relacionado com 1,5% dos casos de câncer no mundo com destaque para o carcinoma de nasofaringe (Nasopharyngeal Carcinoma – NPC) o qual segundo a Organização Mundial da Saúde, 90% dos carcinomas indiferenciados estão associados ao EBV. Expresso durante o ciclo de latência III e II a proteína latente de membrana 1 (Latent Membrane Protein 1, LMP1), um dos principais produtos oncogênicos do EBV mimetiza o receptor CD40 e mantém-se constitutivamente ativa na célula. Com papel importantíssimo no desenvolvimento do carcinoma de nasofaringe, LMP1 atua na imortalização e proliferação de linfócitos B por meio da deflagração de vias de sinalização intracelular como NFkB e PI3K/AKT. Atualmente são conhecidas sete variantes do gene BNLF1 que codifica LMP1 e apresentam diferenças nas sequências nucleotídicas e aminoacídicas, ambas consideradas oncogênicas por conservarem os domínios CTARs. Essas diferenças encontradas nas variantes de LMP1 podem estar relacionadas à agressividade dos tumores. Entre as variantes de LMP1 foram avaliadas no presente estudo as variantes B95.8 e M81, ambas associadas ao desenvolvimento de NPC. As propriedades transformantes dessas variantes de LMP1 foram analisadas frente ao programa biológico transição epitelial- mesenquimal (Epithelial-Mesenckymal Transition – EMT) que está diretamente relacionado com a progressão dos carcinomas. A avaliação das variantes de LMP1 foi feito por meio de transfecção transiente de células HEK293 e NP69. Células expressando as variantes de LMP1 apresentaram maior capacidade de migração e invasão em relação ao grupo controle. Ainda, células expressando as variantes de LMP1 demonstraram alteração para alguns genes relacionados ao processo de EMT. No entanto, as variantes de LMP1 não apresentam diferenças significativas na regulação do programa de EMT. / FAPESP: 2015/13939-9

Made available in DSpace on 2014-06-12T18:04:23Z (GMT). No. of bitstreams: 2 arquivo6195_1.pdf: 1143920 bytes, checksum: 0d0ca55cd7599cecc0ad08a953f1ec9b (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / O câncer apresenta-se como uma das grandes causa mortis na vida contemporânea, principalmente o de adulto que tem aumentado em função do aumento da expectativa de vida, mudança de hábitos alimentares e modo de vida. O câncer pediátrico está mais claramente relacionado a fatores genéticos herdados ou ligado a embriogênese. Os tipos mais prevalentes são as leucemias, os tumores do sistema nervoso e os linfomas. Os linfomas apresentam-se divididos em: doença de Hodgkin (HD) e linfomas não-Hodgkin (NHL), sendo o segundo responsável por mais da metade dos casos. Os NHL se destacam por várias características morfológicas, fenotípicas e por eventos moleculares que alteram a configuração cromossômica e a expressão de diversos genes, eventos moleculares estes que têm sido relacionados a vírus, porém de uma forma não muito clara e sem um modelo definitivo. No presente trabalho a relação entre a infecção por EBV, a transcrição de RNA do referido vírus e a superexpressão do proto-oncogene c-myc em amostras de linfoma foram estudadas. Analisamos biopsias de tumor de 49 pacientes, dos quais 34 (69,4%) eram do sexo masculino e 15 (31,6%) eram do sexo feminino. Os linfomas não-Hodgkin estudados eram de localização principalmente abdominal e em 63,4% o EBV foi detectado, com transcrição ativa do RNA viral em 29,3%. Já os linfomas de Hodgkin somaram apenas sete e em dois deles foi encontrada infecção por EBV. Nossos estudos indicam forte relação do EBV com o NHL, principalmente de localização abdominal, sendo necessários mais estudos da relação do vírus com a expressão elevada do proto-oncogene c-myc

The high incidence rate of Nasopharyngeal Carcinoma (NPC) in southern China, including Hong Kong, has attracted worldwide attention. According to the Center for Health Protection in Hong Kong, there were 841 new cases of NPC, with 655 cases of males and 186 cases of females in 2013. The development of NPC is highly associated with the infection of one human herpes virus, the Epstein-Barr virus (EBV). Given that the homodimerization of one of the EBV endogenous protein-Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for both viral genome maintenance and infected-cell survival, thus the interference of EBNA1 homodimerization would be a novel strategy for the inhibition of EBV-positive tumours. In this thesis we devote to conjugate several kinds of organic fluorophores with various EBV-specific peptides in order to achieve the highly responsive and selective imaging, as well as the effective inhibition of EBV-positive tumours in vitro and in vivo. The first research focused on the conjugation of a styrene pyridine fluorophore with two EBNA1-specific peptides, aiming to develop a dual-probe for the imaging and inhibition of EBV-positive tumour cells. Then we tried to introduce a Nuclear Localization Sequence into the EBNA1-specific peptide, and used an Intra-molecular Charge Transfer characterized fluorophore for the following second research, it showed an impressively responsive signal when the probe binds with EBNA1 both in vitro and in vivo, more importantly, only 4 μg probe can inhibit 92.8% of growth inhibition of an EBV-positive tumour. Along this line, our last research centred on the further improvement of the imaging by taking advantage of lanthanide.

A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in the branch of Anatomical Pathology. 21 July 2017. / Aims and objectives The study design aimed to assess and validate various laboratory techniques in the detection of EBV in HIV positive patients with diffuse large B cell lymphoma. The sensitivity and specificity of each technique was determined, as was the presence of an asymptomatic (latent) or lytic phase infection and the viral strain. DLBL samples occurring in HIV seropositive patients were used as a vehicle for these laboratory procedures which included chromogenic in situ hybridisation (EBER), immunohistochemistry (EBNA 2, LMP 1), real time PCR, (EBNA 1, LMP 2 and BZLF 1) and nested PCR (EBNA 2). Materials and Methods 46 cases of previously diagnosed DLBL from HIV positive individuals were identified and retrieved from the archives of the Department of Anatomical Pathology of the University of Witwatersrand and NHLS. All in-situ hybridisation, immunohistochemical and PCR laboratory procedures were carried out in accordance with the Standard Operating Procedures of the Anatomical Pathology Molecular Laboratory, using appropriate negative and positive controls throughout. Ethical clearance was obtained (M140273). Results/Conclusion A 20% frequency of EBV in HIV positive DLBL cases was established. All EBV infections were found to be in the lytic phase, with an almost equal distribution of latency patterns II and III and an equal distribution of EBV strains 1 and 2. EBER in situ hybridisation was confirmed to be the most sensitive and reliable method of viral detection, and the presence of the BZLF 1 gene determined by real time PCR was found to be a reliable indicator of a lytic infection. / LG2018

Immune evasion is an emerging hallmark of cancer. Dysregulation of the major histocompatibility complex class I (MHC-I) is a frequent mechanism of immune evasion utilized by tumor cells and is particularly relevant to the pathogenesis of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). A better understanding of MHC-I dysregulation in B-cell lymphomas is necessary to identify factors related to the risk, development, and progression of these tumors. In this thesis, we investigate the role of MHC-I dysregulation in DLBCL and cHL through the application of computational approaches to study genomic data. First, we introduce some background information about the normal function of MHC-I in the immune response to cancer and viral infection as well as the phenomenon of MHC-I dysregulation in the context of cancer. We provide an overview of how factors such as germline zygosity of HLA class I (HLA-I) genes and somatic alterations in the genes B2M and HLA-I that encode the protein subunits of MHC-I contribute to the development of DLBCL and cHL. Second, we present a study of the effects of HLA-I allele zygosity on survival in a cohort of 519 DLBCL patients treated with R-CHOP immunochemotherapy stratified by molecular subtype. Homozygosity in HLA-I was associated with a worse overall survival in patients whose tumors were classified in the “EZB” subtype, associated with somatic mutation in the epigenetic regulator EZH2. We find an association between the zygosity of the genes HLA-B and -C specifically and overall survival in EZB-DLBCL. These findings indicate that HLA-I zygosity may be a risk factor for worse clinical prognosis in patients with the EZB subtype of DLBCL. Third, we present a study of the genetic landscape of cHL tumors that are associated with infection with Epstein-Barr virus (EBV). We analyze inherited HLA-I allele types, somatic mutations, copy number changes, and mutational signatures in a cohort of 57 cHL patients (15 EBV-positive). We find that EBV-positive cHL is genetically distinct from EBV-negative cHL and is characterized by lower somatic mutation load and different activities of mutation signatures. Further, we find that cHL tumors are characterized by different patterns of MHC-I dysregulation depending on the EBV infection status. Germline homozygosity in HLA-I was associated with the EBV-positive subtype of cHL, while somatic alterations in HLA-I were associated with the EBV-negative subtype of cHL. These results suggest that inherited HLA-I homozygosity may be a risk factor for the EBV-positive subtype of cHL. Fourth, we expand our study of HLA-I in virus-associated cHL to perform a comparative analysis of virus-positive and virus-negative tumors in nine cancers linked to five viruses. We find that virus-positive tumors occur more frequently in males and show geographical disparities in incidence. Genomic analysis of 1,658 tumors reveals virus-positive tumors exhibit distinct mutation signatures, recurrent mutations in the RNA helicases DDX3X and EIF4A1, and a lower somatic mutation burden compared to virus-negative tumors of the same cancer type. We find that germline homozygosity in HLA-I is a potential risk factor for the development of EBV-positive cHL, but not other common virus-associated solid or hematological malignancies. Finally, we present in the Appendix a study of the genomic characterization of plasmablastic lymphoma (PBL), a rare EBV-associated B-cell lymphoma that occurs in the context of immunodeficiency caused by human immunodeficiency virus (HIV) infection. We find that PBL is characterized by mutations leading to constitutive activation of the JAK-STAT pathway. We additionally identify recurrent mutations in immune-related genes, such as B2M. These findings indicate a potential role for MHC-I and immune dysregulation in the pathogenesis of other B-cell lymphomas.

No description available.

No description available.

Le virus Epstein-Barr (EBV) est un virus herpès oncogène qui a développé au cours de son évolution un arsenal de contre-mesures lui permettant de déjouer les différentes facettes de la réponse immunitaire de l'hôte. Une partie importante de cette réponse est assurée par les monocytes, qui constituent une composante majeure de la première ligne de défense contre les infections virales. Cependant, le rôle des monocytes dans la pathogenèse du virus EBV ainsi que les tactiques utilisées par celui-ci pour déjouer cet aspect de la réponse immunitaire restent encore largement méconnus. La présente étude démontre que l'EBV peut infecter et se répliquer dans les monocytes humains. En effet, la pénétration et la translocation des particules virales aux noyaux ont pu être mises en évidence par microscopie électronique. De plus, l'expression de gènes viraux associés au cycle lytique, ainsi que la libération de particules virales infectieuses par les monocytes infectés confirment la capacité du virus à se répliquer dans ces cellules. Nos résultats ont également démontré que d'importantes fonctions du monocyte sont affectées suite à l'infection. C'est notamment le cas de la phagocytose, dont l'activité est réduite de plus de 50% dans les cellules infectées. De plus, EBV inhibe également la génération de prostaglandine (PG) E2 par les monocytes en interférant avec l'expression de la COX-2, principale enzyme impliquée dans la biosynthèse des PGs. Des études plus approfondies ont démontré que la protéine virale ZEBRA, laquelle est rapidement exprimée dans les monocytes infectés, inhibe la transactivation du promoteur de la COX-2 par les facteurs de transcription NF-KB et CREB. Cette inhibition est causée par l'interaction physique de ZEBRA avec la TATA-binding protein (TBP), une composante clé du complexe d'initiation de transcription. En résumé, nos résultats suggèrent que l'infection des monocytes par EBV et l'altération de leurs fonctions biologiques pourraient constituer un nouveau mécanisme visant à affaiblir la réponse immunitaire et ainsi favoriser la propagation virale dans les premiers stades de l'infection.

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.