Dissertação (mestrado) - Universidade Federal de Santa Catarina. Programa de Pós-Graduação em Biotecnologia. / Made available in DSpace on 2013-07-16T02:00:14Z (GMT). No. of bitstreams: 0 / O Linfoma de Hodgkin (LH) pode ser definido como uma desordem maligna linfoproliferativa. Embora exista uma importante associação desta doença com o vírus Epstein-Barr (EBV), sua etiologia ainda é desconhecida. A freqüência de casos de LH, positivos para o EBV apresenta variação, não só em populações de diferentes condições sócio-econômicas e geográficas, mas também quando relacionado com o subtipo histológico e a faixa etária de desenvolvimento do LH. A baixa freqüência da associação do EBV ao linfoma de Hodgkin clássico (LHC) no adulto jovem tem sugerido, apesar de poucas evidências, o possível envolvimento de um segundo vírus na patogenia da doença. Por todas estas razões, o presente estudo teve por objetivos avaliar a prevalência do EBV em amostras de biópsias de pacientes com LHC, de Florianópolis, Santa Catarina, além de avaliar a prevalência do vírus TT (TTV) nestas mesmas amostras. Um grupo controle composto por 20 amostras de biópsias de tecido com hiperplasia linfóide inespecífica foi incluído no estudo quando o vírus TT foi pesquisado. A detecção do TTV foi realizada através do ensaio de PCR, e a presença do EBV foi avaliada através da técnica de imunoistoquímica (LMP-1) e hibridização in situ (EBER). Foi observada uma prevalência do EBV de 48% (22/46), com predominância em indivíduos do sexo masculino (61,5%). O subtipo histológico esclerose nodular foi o mais incidente (70%) com uma prevalência de 37% para o EBV. Os casos de LHC dos adultos jovens (15-45 anos) demonstraram uma prevalência menor do EBV quando comparado com os outros grupos etários, sendo observado 21% (4/19) de positividade para o EBV. Nos casos de LHC infantis (<15 anos) foi demonstrada uma positividade para o EBV de 64% (9/14). A positividade para o TTV nos pacientes portadores de LHC quando comparada com o grupo controle, não apresentou diferença significativa. Quando avaliada a co-infecção desses vírus nos diferentes grupos etários, observou-se que o grupo composto por adultos jovens, de menor prevalência para o EBV (21%), mostrou uma maior prevalência do TTV (63%). Não foi possível estabelecer qualquer correlação entre as características clínicas ou o prognóstico da doença com a positividade para o EBV e/ou TTV no presente estudo.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T03:22:40Z (GMT). No. of bitstreams: 1 Pasquotto_Juliana_M.pdf: 2465167 bytes, checksum: dcd2cc122494bab62d8ab495927b1f0e (MD5) Previous issue date: 2008 / Resumo: O Vírus Epstein-Barr (EBV) e o Citomegalovírus (HCMV) são membros da família Herpesvírus. São encontrados em aproximadamente 90% dos indivíduos em idade adulta. A infecção ocorre, geralmente, na infância e é assintomática na maioria dos casos, persistindo de forma latente durante toda a vida do indivíduo. A transmissão destes vírus ocorre principalmente pela saliva, sangue e órgãos transplantados. O EBV está relacionado com a mononucleose infecciosa, doença linfoproliferativa (PTLD), leucemia de células pilosas em pacientes com imunodeficiência congênita ou adquirida e doença de Hodgkin. O risco de um paciente transplantado desenvolver linfoma é 28 a 50 vezes maior do que os indivíduos da população geral. Um dos fatores de risco para o desenvolvimento da PTLD são a variedade e intensidade da imunossupressão utilizada no paciente pós-transplante, idade do receptor e sorologia viral (EBV, CMV). Dependendo da idade do receptor, do tipo de transplante e dos fatores de risco, a prevalência da PTLD pode variar de 0.5% a 22%. Em transplantados pediátricos renais a prevalência chega a atingir 37%. A principal medida terapêutica para o controle da PTLD é a diminuição ou mesmo a retirada total da imunossupressão. Portanto a rejeição do enxerto se torna um problema bastante comum, que compromete a qualidade e/ou expectativa de vida dos pacientes. A introdução de testes laboratoriais rápidos e precoces permite aos clínicos detectar a replicação viral do EBV e diagnosticar, consequentemente, a infecção ativa antes do início da doença. Isso proporciona a oportunidade de iniciar o tratamento específico precocemente. Foram estudadas amostras de sangue e soro de 46 pacientes submetidos a transplantes de células hematopoéticas, acompanhados no Serviço de Transplante de Medula Óssea (STMO) do Hospital das Clínicas da UNICAMP/HEMOCENTRO. Trabalhamos no estudo para diagnosticar a infecção ativa e quantificar a carga viral do vírus Epstein-Barr (EBV) em pacientes transplantados de células hematopoéticas. Relacionar infecção ativa do vírus Epstein-Barr com o Citomegalovírus (CMV) e verificar a incidência da Doença linfoproliferativa e a Doença do enxerto contra o hospedeiro (GVHD) nos pacientes estudados. Diagnosticamos infecção ativa pelo EBV em 22 (47,8%) pacientes que apresentaram uma carga viral muito baixa (média de 29 cópias/ul). Co-infecção entre EBV e CMV ocorreu em 15/46 pacientes (32,6%). Doença por CMV ocorreu em 7/46 (15,2%) pacientes no trato gastrintestinal. Todos estes doentes apresentaram infecção ativa pelo CMV e 4/7 (57%) apresentaram infecção ativa pelo EBV. Um destes pacientes foi a óbito por doença por CMV. Verificamos que nenhum dos pacientes apresentou doença linfoproliferativa relacionada ao EBV. Os casos de co-infecção ativa EBV+CMV em relação à ocorrência de GVHD foram estatisticamente significantes (p=0.001). Concluímos que a infecção pelo CMV ainda é a maior causa de morbidade e mortalidade nos pacientes após o transplante. A qPCR é uma ferramenta útil para verificar os pacientes que reativaram pelo EBV após o transplante e pode auxiliar na prevenção da doença linfoproliferativa causada pelo EBV juntamente com a identificação dos fatores de risco associados. Verificamos a ocorrência e significância do GVHD e infecção ativa pelo CMV, mas não observamos esses mesmos resultados comparados ao EBV / Abstract: Epstein-Barr Virus (EBV) and Cytomegalovirus (CMV) are members of herpesvirus family. They are found in approximately 90% of the individuals in adult age. The infection generally occurs subclinicaly during the childhood in the major of the cases persisting in latent form during all the life of the individual. The transmission of these viruses occurs mainly for the saliva, blood and transplanted organs. EBV is related with mononucleose infectious, lynfoproliferative disease (PTLD), leukemia of hair cells in patients with congenital or acquired immunodeficiency and Hodgkin¿s disease. The risk of a transplanted patient to develop linfoma is 28 to 50 % more than other individuals of the general population. One of the risk factor for the development of the PTLD is the variety and intensity of the imunossupression used in the patient after-transplant, age of the recipient and viral serology (EBV, CMV). Depending on the age of the recipient, the type of transplant and the risk factor, the prevalence of the PTLD can vary of 0.5% 22%. In renal pediatric transplantation, the prevalence can arrives to reach 37%. The main therapeutical measure for the control of the PTLD is the reduction or total withdrawal of the imunossupression. Therefore the lost of graft is a common problem, that compromises the quality and/or life expectancy of the patients. The introduction of early and rapid laboratorial tests can permit to the physicians to detect the viral response and detect the active EBV infection before the disease. This provides the chance to initiate the specific treatment. We studied samples of blood and serum of 46 patients submitted to hematopoietic stem cell transplantation at the Bone Marrow Transplant unit of the University Hospital of the UNICAMP/HEMOCENTRO. We worked in the study to diagnosis the active infection and quantify the viral load of the Epstein-Barr virus (EBV) in transplanted patients of hematopoetic stem cells, to relate active EBV infection with active CMV infection and to verify the incidence of the lynfoproliferative disease and graft versus host disease (GVHD) in the studied patients. Active EBV infection detected by Real-Time PCR occurred in 22 patients (47,8%). The viral load found was very low (range 29 copies/ul). Co-infection between EBV and CMV occurred in 15/46 patients (32,6%). CMV disease occurred in 7/46 (15,2%) patients in the gastrointestinal tract. All these patients had active CMV infection and 4/7 patients (57%) had active EBV infection. One of these patients died by CMV disease. No patient presented lymphoproliferative disease related to the EBV. The cases of active EBV and CMV (co-infection) infection in relation to the occurrence of GVHD had been statisticaly significant (p=0.001). We conclude that the active CMV infection is already the most cause of morbidity and mortality of the patients after the transplant. The qPCR is a useful tool to verify the patients who had active EBV infection after the transplant and the identification of the risk factors associates prevention the development of the lymfoproliferative disease caused by the EBV. We verify the occurrence and significance of the GVHD and active CMV infection, but we do not observe these same results related to the EBV / Mestrado / Mestre em Farmacologia

Orientadores: Manoel Barros Bértolo, Lilian Tereza Lavras Costallat / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T07:51:21Z (GMT). No. of bitstreams: 1 Yazbek_MichelAlexandre_D.pdf: 3193834 bytes, checksum: 4155cea9577385c29405700add4864ef (MD5) Previous issue date: 2014 / Resumo: A etiopatogenia da Artrite Reumatoide (AR) envolve fatores genéticos, imunológicos e ambientais que interagem entre si. Os principais fatores de risco estudados são a presença dos alelos do epítopo compartilhado (shared epitope- SE), dos anticorpos anti-peptídeos cíclicos citrulinados (anti-CCP) e do tabagismo. Há evidências que o Epstein-Barr vírus (EBV), ao interagir com esses fatores de risco, possa causar uma resposta imune anômala em indivíduos susceptíveis. Essas interações também podem contribuir para o desenvolvimento da AR. O objetivo principal desse estudo é estabelecer se há uma associação entre o EBV com os alelos do SE, os anticorpos anti-CCP e o tabagismo em pacientes brasileiros com AR. Os objetivos secundários são: avaliar a correlação entre os alelos do SE, os anticorpos anti-CCP e o tabagismo; detectar a exposição ao EBV e quantificar sua carga viral e estimar o risco de cada fator estudado para o desenvolvimento da AR nessa casuística. Nesse estudo caso-controle, incluímos 140 pacientes brasileiros com AR e 143 controles saudáveis; pareados por idade, sexo e etnia. Foi feita uma caracterização clínico-laboratorial da casuística. Foram realizadas a genotipagem para identificar os alelos do SE, a determinação dos anticorpos anti-CCP pelo método de ELISA e coletada a história de tabagismo de todos os sujeitos da pesquisa. Para avaliar a exposição ao EBV, realizamos a dosagem dos anticorpos anti-Epstein-Barr Nuclear Antigen 1 (anti-EBNA1). Para quantificar a carga viral do EBV, realizamos o método quantitativo da reação em cadeia polimerase em tempo real ou real-time PCR. A análise comparativa entre os grupos mostrou uma positividade significativamente maior para os alelos do SE, anticorpos anti-CCP e tabagismo no grupo de pacientes. A análise dos anticorpos anti-EBNA1 mostrou uma positividade alta, tanto em pacientes como em controles, sem diferença significativa. Entretanto, a quantificação da carga viral pela PCR em tempo real mostrou-se muito maior em pacientes do que em controles (p<0.001). As análises associativas dos anticorpos anti-EBNA1 com os outros fatores estudados não se mostraram significativas; assim como as análises associativas da carga viral do EBV pela PCR em tempo real. A positividade do anti-CCP foi maior em pacientes com os alelos do SE que são tabagistas ou ex-tabagistas (p=0.038). Nas análises de regressão logística, a variável com maior risco para o desenvolvimento da AR foi a positividade dos anticorpos anti-CCP. Apesar dos pacientes com AR apresentarem uma carga viral do EBV aumentada, esse estudo não conseguiu associá-la aos demais fatores de risco estudados. Sugerimos que esses achados possam ocorrer devido a um controle deficitário do EBV em pacientes com AR, mas que não está relacionado aos fatores de risco mais conhecidos da doença. A imunidade celular defeituosa dos pacientes com AR dificulta o controle de uma infecção latente do vírus. Portanto, não é possível estabelecer uma relação causal direta entre o EBV e a AR / Abstract: The pathogenesis of rheumatoid arthritis (RA) involves genetic, immunological and environmental factors. The main risk factors are the presence of the shared epitope alleles (shared epitope- SE), anti-cyclic citrullinated peptide antibodies (Anti-CCP) and smoking. There is evidence that the Epstein-Barr virus (EBV), when interacts with these risk factors, may cause an abnormal immune response in susceptible individuals. These interactions may contribute to the development of RA. The main objective of this study is to establish whether there is an association between EBV and alleles of SE, anti-CCP antibodies and smoking in Brazilian patients with RA. Secondary objectives are the assessment of the correlation between alleles of SE, anti-CCP antibodies and smoking; the detection of EBV; the quantification of EBV viral load and the estimation of the likelihood of each analyzed factor for the development of RA in this sample. In this case-control study, we included 140 Brazilian patients with RA and 143 healthy controls; matched for age, sex and ethnicity. We performed a clinical and laboratory characterization of the sample. Genotyping was performed to identify SE alleles, anti-CCP antibodies were examined by ELISA and smoking information was collected from all subjects. To assess the exposure to EBV, we examined anti-Epstein-Barr Nuclear Antigen 1 (anti-EBNA1) antibodies. To quantify the viral load of EBV, we performed the quantitative method of polymerase chain reaction in real time or real-time PCR. The comparative analysis between groups showed a significantly higher positivity for the alleles of SE, anti-CCP antibodies and smoking in patients. The analysis of anti-EBNA1 antibodies showed a high positivity, both in patients and in controls, with no significant difference. However, the quantification of viral load by real-time PCR proved to be much higher in patients than in controls (p <0.001). Associative analysis of anti-EBNA1 antibodies with other factors studied were not significant; as well as the association analyzes of the EBV viral load by PCR in real time. The positivity of anti-CCP antibodies was higher in patients with SE alleles which are smoker or ex-smoker (p = 0.038). In logistic regression analyzes, the variable with higher risk for RA was the positivity of anti-CCP antibodies. Although patients with RA present an increased EBV viral load, this study did not link EBV to the other risk factors studied. We suggest that these findings may be due to a deficient control of EBV in RA patients, which is unrelated to the better-known disease risk factors. Defective cellular immunity of patients with RA complicates the control of latent virus infection. Therefore it is not possible to establish a direct causal relationship between EBV and RA / Doutorado / Clinica Medica / Doutor em Clínica Médica

Epstein-Barr virus (EBV) induced infectious mononucleosis (IM) is characterized by the activation and expansion of T lymphocytes and the induction of cytotoxic responses able to mediate the lysis of EBV-uninfected, allogeneic MHC mismatched and EBV-infected autologous target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) were used to examine the nature of these cellular immune responses. Activated lymphocytes, as identified by HLA-DR expression, associated with EBV induced IM were shown to be a heterogeneous population containing significantly elevated cytotoxic/suppressor (CD8+) T cells, helper/inducer (CD4+) T cells and natural killer (NK, CD16+) cells. CD8+ T cells were the primary activated population, representing 24% of the total lymphocyte population and 60-70% of the CD8+ T cell population. The activated CD4+ T cells and natural killer (NK) cells accounted for 7% and 4% of the total lymphocyte population, respectively. Analysis of serum soluble interleukin 2 receptors (IL-2R) and CD8 molecules demonstrated significantly (p<0.001) elevated levels in the sera of IM patients compared with normal controls. These elevated levels of serum IL-2R am CD8 molecules correlated, (r=0. 67 and r=0.82, respectively) with increased percentages of CD8/HLA-DR positive T cells (i.e., activated CD8 T cells). Increased levels of soluble cell surface molecules peaked during the acute phase and normalized as the patients progressed toward convalescence. Individual patients demonstrated strong correlations between the percentage of CD8/HLA-DR positive cells and soluble CD8 levels. The functional significance of the serum IL-2R and CD8 molecules is presently unknown. However, the strong correlative data between serum CD8, and to a lesser extent IL-2R, and CD8 T cell activation suggests that serum CD8 levels may provide a sensitive measure of CD8 T cell activation in systemic infections. The ability of freshly isolated acute IM PBMC to lyse allogeneic, EBV-infected lymphoblastoid cell lines (LCL), demonstrated the ability of acute IM effector cells to lyse MHC mismatched target cells. Effector cells from acute IM patients lysed allogeneic DM-LCL and AF-LCL target cells by 34% (n=7) and 23% (n=6), respectively, compared with 4% (n=5) and 0% (n=5), respectively, for normal controls. MAb-dependent complement depletion of CD3+ or CD8+ cells with anti-CD3 and anti-CD8 mAb decreased the non-MHC restricted cytolysis of LCL by 96% and 89%, respectively. In contrast, complement depletion with NK-cell specific mAbs Leu 11b and NKH-1, resulted in only a slight decrease (<35%) in the lysis of these LCL (46%). Depletion with anti-HLA-DR also significantly (p<0.001) decreased the lysis of LCL. Depletions with anti-CD4 demonstrated no decrease in LCL-lysis. MAbs OKT3 and OKT8 inhibited the non-MHC restricted cytolysis by 87% and 82%, respectively. We interpret these results as evidence that, 1) lysis of allogeneic cells is mediated primarily by CD3+, CD8+, HLA-DR+, cytotoxic T lymphocytes (CTL); and 2) these acute IM cytotoxic T cells utilize the T cell receptor and the CD8 antigen as an accessory molecule. An active role for target cell MHC class I molecules in the recognition and subsequent lysis of target cells is supported by a number of observations: 1) the MHC class I reactive mAbs W6/32 and BBM.1 significantly (p<0.05) inhibited the lysis of 63463-LCL by 65% and 57%, respectively; 2) acute IM effector T cells did not lyse the MHC class I negative Daudi cell line; 3) allogeneic MHC class I matched LCL mediated strong competitive inhibition (72% at 10:1 competitor to target cell ratio) vs 29% competitive inhibition for an allogeneic MHC class I mismatched LCL; and 4) NK-cell depleted effector cells from one patient mediated preferential lysis of the K562 cell line expressing MHC class I. HLA-A2 molecules. We interpret these results as evidence that target cell MHC class I molecules (or associated determinants) are the target antigen(s) for the allogeneic MHC cytotoxic response. The role of EBV in this acute allogeneic response was examined using target cell lines devoid of EBV genome. Acute IM CTL mediated lysis of the allogeneic HSB-2 T cell line (45%), and allogeneic HTLV-I transformed T cell lines (16%). The lysis of the HSB-2 T cell line was inhibited by anti-OKT3 (58% inhibition), W6/32 (53%) and BBM.1 (42%). Similarily, lysis of HTLV-I T cell lines was inhibited by W6/32 (69% inhibition), BBM.1 (69%) and OKT3 (38%). These data demonstrate that EBV antigenic expression is not required for allogeneic recognition and subsequent lysis of these allogeneic target cells. Effector cells from acute IM patients (n=5) were able to lyse their autologous EBV-infected LCL (mean lysis=21%), but were unable to lyse the EBV-uninfected autologous HTLV-I T cell line. These same effectors, however, were able to mediate lysis of both allogeneic B cell lines (21% lysis) and allogeneic T cell lines (8% lysis). These data are consistent with the observations by Strang et al. (1987a), who recently cloned virus specific/MHC-restricted CTL cloned from acute IM PBMC. These virus specific/MHC-restricted T cells presumably mediate the lysis of the autologous EBV-transformed B cell lines but not the autologous EBV-uninfected T cell lines. Whether the CTL which lyse the autologous EBV-transformed LCL are also responsible for the observed allogeneic reactivity was examined with cold target competition using autologous and allogeneic LCL. Lysis of autologous LCL was inhibited only by autologous competitor cells (64% inhibition compared with 24% for allogeneic LCL). Likewise, lysis of the allogeneic LCL was inhibited only by the allogeneic competitor cells (85% inhibition compared with 30% for autologous LCL). These data demonstrated no competition between allogeneic and autologous LCL and therefore support the concept that lysis of autologous LCL and allogeneic target cells is mediated by distinct effector populations. These data help us to understand the unusual immune response observed during acute IM. The strong allogeneic cytotoxic response is thought to represent polyclonal CD8 T cell activities induced by EBV-infected and transformed B cells which circulate in vivo. In addition, a population of CD8 CTL exist which mediate the lysis of autologous EBV-transformed B cells. These CTL likely represent virus-specific/MHC-restricted CTL and presumably play a major role in the control of EBV infections. The role, if any, of the markedly expanded alloreactive CTL population in the elimination of EBV infected and transformed B cells remains to be clarified.

Xu, Meng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 112-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lou, Pak Kin. / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 136-170). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / List of Publications --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Aims of Study --- p.1 / Chapter 1.2. --- Literature Review --- p.2 / Chapter 1.2.1. --- Nasopharyngeal Carcinoma --- p.2 / Chapter 1.2.1.1. --- Overview --- p.2 / Chapter 1.2.1.2. --- Histopathology --- p.2 / Chapter 1.2.1.3. --- Epidemiology --- p.3 / Chapter 1.2.1.4. --- Etiology --- p.5 / Chapter 1.2.1.4.1. --- Epstein-Barr Virus (EBV) Latent Infection --- p.5 / Chapter 1.2.1.4.2. --- Environmental Factors --- p.5 / Chapter 1.2.1.4.3. --- Genetic Factors --- p.6 / Chapter 1.2.1.5. --- Molecular Pathogenesis --- p.7 / Chapter 1.2.1.5.1. --- Chromosomal Alterations --- p.7 / Chapter 1.2.1.5.2. --- NPC-associated Tumour Suppressor Genes --- p.7 / Chapter 1.2.1.5.3. --- NPC-associated Oncogenes --- p.8 / Chapter 1.2.2. --- Epstein-Barr Virus --- p.9 / Chapter 1.2.2.1. --- Overview --- p.9 / Chapter 1.2.2.2. --- Lytic and Latent Infection of EBV --- p.9 / Chapter 1.2.2.3. --- EBV Latency Programs and Associated --- p.10 / Malignancies --- p.11 / Chapter 1.2.2.4. --- The Role of EBV in NPC --- p.12 / Chapter 1.2.3. --- NF-kB Signalling Pathways --- p.12 / Chapter 1.2.3.1. --- Overview --- p.12 / Chapter 1.2.3.2. --- Pathway Components --- p.12 / Chapter 1.2.3.2.1. --- NF-kB Subunits --- p.16 / Chapter 1.2.3.2.2. --- Inhibitors of kB (IkBs) --- p.16 / Chapter 1.2.3.2.3. --- IkB Kinases (IKKs) --- p.17 / Chapter 1.2.3.3. --- NF-kB Activation and Signalling --- p.17 / Chapter 1.2.3.3.1. --- The Canonical Pathway --- p.18 / Chapter 1.2.3.3.2. --- The Non-canonical Pathway --- p.18 / Chapter 1.2.3.3.3. --- Physiological Functions of NF-kB --- p.19 / Chapter 1.2.3.4. --- NF-kB Signalling and Tumourigenesis --- p.20 / Chapter 1.2.3.4.1. --- Oncogenic Activation of NF-kB in Hematological Malignancies --- p.20 / Chapter 1.2.3.4.2. --- Oncogenic Activation of NF-kB in Solid and Epithelial Tumours --- p.22 / Chapter Chapter 2 --- Material and Methods --- p.22 / Chapter 2.1. --- Tumour Specimens --- p.24 / Chapter 2.2. --- NPC Tumour Lines and Immortalized NP Cell Lines --- p.24 / Chapter 2.2.1. --- Cell Lines --- p.24 / Chapter 2.2.2. --- Xenografts --- p.27 / Chapter 2.3. --- DNA Sequence Analysis --- p.27 / Chapter 2.3.1. --- Genomic DNA Extraction --- p.27 / Chapter 2.3.2. --- Polymerase Chain Reaction (PCR) --- p.28 / Chapter 2.3.3. --- DNA Sequencing --- p.32 / Chapter 2.4. --- RNA Expression Analysis --- p.32 / Chapter 2.4.1. --- Total RNA Extraction and Reverse Transcription --- p.33 / Chapter 2.4.2. --- Quantitative Real-time Polymerase Chain Reaction (QRT-PCR) --- p.35 / Chapter 2.5. --- Protein Expression Analysis --- p.35 / Chapter 2.5.1. --- Total Protein Extraction --- p.35 / Chapter 2.5.2. --- Nuclear and Cytoplasmic Protein Isolation --- p.36 / Chapter 2.5.3. --- Western Blotting --- p.39 / Chapter 2.6. --- Immunohistochemical Staining --- p.41 / Chapter 2.7. --- Statistical Analysis --- p.41 / Chapter 2.8. --- Immunoprecipitation --- p.43 / Chapter 2.9. --- Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay --- p.44 / Chapter 2.10. --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.45 / Chapter 2.11. --- Plasmid Preparation --- p.45 / Chapter 2.11.1. --- Plasmids --- p.45 / Chapter 2.11.2. --- Bacterial Transformation and Plasmid DNA Extraction --- p.46 / Chapter 2.12. --- Transfections --- p.46 / Chapter 2.12.1. --- Transient Transfection --- p.46 / Chapter 2.12.2. --- Stable Transfection --- p.47 / Chapter 2.13. --- Immunofluorescence --- p.47 / Chapter 2.14. --- Cell Proliferation and Viability Analysis --- p.47 / Chapter 2.15. --- Small Interfering RNA (siRNA) Knockdown --- p.49 / Chapter 2.16. --- Expression Microarray --- p.49 / Chapter 2.16.1. --- Agilent Oligonucleotide Microarray --- p.50 / Chapter 2.16.2. --- Data Analysis --- p.51 / Chapter Chapter 3 --- Activation of NF-kB Signals in NPC --- p.51 / Chapter 3.1. --- Introduction --- p.52 / Chapter 3.2. --- Results --- p.52 / Chapter 3.2.1. --- Expression Pattern of NF-kB Subunits in NPC Tumour Lines --- p.55 / Chapter 3.2.2. --- Distinct NF-kB Complexes in NPC Tumour Lines --- p.60 / Chapter 3.2.3. --- Expression of NF-kB Subunits in NPC Primary Tumours --- p.67 / Chapter 3.3. --- Discussion / Chapter Chapter 4 --- Alterations of NF-kB Components in NPC --- p.71 / Chapter 4.1. --- Introduction --- p.72 / Chapter 4.2. --- Results --- p.72 / Chapter 4.2.1. --- Homozygous Deletion of IicBa and TRAF3 in NPC Tumour Lines --- p.76 / Chapter 4.2.2. --- Mutation of TRAF2 and A20 in NPC Tumour Lines / Chapter 4.2.3. --- Aberrant Expression of Multiple NF-kB Signalling Components in NPC Tumour Lines --- p.80 / Chapter 4.2.4. --- Expression of NF-kB Signalling Components in NPC --- p.85 / Primary Tumour --- p.92 / Chapter 4.3. --- Discussion --- p.99 / Chapter Chapter 5 --- Identification of Downstream Targets for NPC-associated NF-kB Signalling --- p.99 / Chapter 0.1. --- Introduction --- p.99 / Chapter 0.2. --- Results --- p.100 / Chapter 0.2.1. --- Target Genes Modulated by p50 --- p.100 / Chapter 0.2.2. --- Functional Annotation of p50 Target Genes --- p.105 / Chapter 0.2.3. --- Target Genes Modulated by RelB --- p.105 / Chapter 0.2.4. --- Functional Annotation of RelB Target Genes --- p.105 / Chapter 0.2.5. --- Functional Annotation of Genes Modulated by both p50 and RelB --- p.111 / Chapter 0.3. --- Discussion --- p.118 / Chapter Chapter 6 --- Functional Role of TRAF3 Inactivation in NPC --- p.118 / Chapter 0.1. --- Introduction --- p.118 / Chapter 0.2. --- Results --- p.118 / Chapter 0.2.1. --- Effect of TRAF3 Restoration on NF-kB Activity --- p.119 / Chapter 0.2.2. --- Effect of TRAF3 Expression on Cell Proliferation --- p.123 / Chapter 0.2.3. --- TRAF3 Expression Modulates Interferon Transcription in NPC Cells --- p.128 / Chapter 0.3. --- Discussion / Chapter Chapter 7 --- General Discussion --- p.132 / Chapter Chapter 8 --- Conclusion / Chapter Chapter 9 --- References / Appendix --- p.136

Tong Hung Man Joanna. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 137-149). / 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.

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.

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