Le virus Epstein-Barr (EBV) est un virus herpès oncogène causant la mononucléose infectieuse et est associé à différents types de cancer d'origine épithélial et lymphoïde. Nous avons précédemment démontré que EBV peut infecter et se répliquer dans les monocytes humains. Suite à cette infection, plusieurs fonctions importantes du monocyte s'en trouvent affectées, dont la phagocytose, la biosynthèse de prostaglandine E2 et la production de TNF-oc et d'IFN-a. Il est bien connu que l'IFN de type I (IFN-a/(3) est un puissant médiateur antiviral qui est rapidement sécrété par les cellules infectées lors des stades précoces de l'infection. OBJECTIFS Ce projet vise à caractériser le(s) mécanismes(s) affectant la production d'IFN-a par les monocytes humains infectés par le virus EBV en étudiant notamment la cascade d'activation des protéines JAKs et STATs. MÉTHODES Nous avons évalué l'effet de l'infection des monocytes humains par EBV sur l'activation des facteurs de transcription IRF-3 et IRF-7, sur les protéines de la cascade des JAKs-STATs Qakl, Tyk2, Statl et Stat2) ainsi que sur les protéines SOCS. RÉSULTATS L'activation des protéines IRF-3 et IRF-7 n'est pas affectée, puisqu'il a été possible d'observer une translocation nucléaire de ces facteurs de transcription suite à l'infection par EBV. Cependant, la boucle d'amplification de la voie des JAKs-STATs est supprimée suite à l'inhibition de la phosphorylation de Statl. Le mécanisme d'inhibition de la synthèse d'IFN-a par le virus EBV dans les monocytes humains implique l'activation de la protéine SOCS-3. CONCLUSION Ce projet permet d'approfondir nos connaissances sur les mécanismes immunosuppresseurs mis en œuvre par EBV afin de supprimer les fonctions monocytaires. / Epstein-Barr virus (EBV) is an oncogenic herpesvirus recognized as the causative agent of infectious mononucleosis and is associated with several human malignancies. We have recently provided evidences that human monocytes were permissive to EBV infection and replication. Following infection, EBV can affect various cellular functions of monocytes, such as phagocytosis, biosynthesis of prostaglandins E2 and production of TNF-a and IFN-a. The type I interferon System (IFN-a/p) represents an important elements of host defence against ail kinds of pathogens, mainly viruses. Following infection, virus-infected cells rapidly produce and secrete IFN-a/p. OBJECTIVES The present work aims to determine the mechanism affecting the IFN-a production by the EBV-infected monocytes, in particular by the study of the JAK-STAT pathway. METHODS We examined the effect of EBV infection in monocytes on IRF-3 and IRF-7 nuclear accumulation, on JAKs and STATs proteins activation (Jakl, Tyk2, Statl and Stat2), and on SOCS proteins expression. RESULTS The IRF-3 and IRF-7 activation is not affected, since it was possible to observe a nuclear translocation of these transcription factors following EBV infection. However, the positive-feedback loop of the JAK-STAT pathway was found to be affected by the inhibition of Statl phosphorylation. The mechanism of IFN-a inhibition in EBV-infected monocytes involves the SOCS-3 protein activation. CONCLUSION Further description of EBV inhibitory mechanisms on monocytes immune functions would certainly improve our understanding of the role of these cells in the early stages of EBV pathogenesis.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

鼻咽癌(nasopharyngeal carcinoma, NPC)與艾巴氏病毒(Epstein-Barr virus, EBV)和遺傳及表現遺傳變異有連串關係。儘管鼻咽癌腫瘤的發生機制仍然未知,最近研究顯示微核糖核酸(microRNA, miRNA)通過調節細胞增殖凋亡遷移和侵襲等方式對鼻咽癌的生成起著至關重要的作用。為了確定微核糖核酸與鼻咽癌發生的相關機制及其扮演的角色，我們集中研究微核糖核酸在鼻咽癌腫瘤中所發生的變異，探討這些異常表達的微核糖核酸的功能，並揭開與幹細胞相關的微核糖核酸在鼻咽癌幹細胞樣細胞(cancer stem-like cell, CSC)裏所扮演的角色。 / 通過使用微陣列技術(Agilent Microarray)， 我們運用了 866個人類與 89個病毒微核糖核酸探針,以識別出多個帶有艾巴氏病毒的鼻咽癌腫瘤細胞系裏的微核糖核酸表達圖譜。相比正常的鼻咽上皮細胞系NP69，113個微核糖核酸在鼻咽癌中的差異表達已被鑒定出來。其中58個在鼻咽癌裏下調的微核糖核酸表達，miR-31的轉錄下調現象在鼻咽癌腫瘤細胞系和原發腫瘤中被不斷地發現。在7個帶有艾巴氏病毒的腫瘤細胞系樣本裏, 其中6個(86%)樣本呈miR-31下調跡象。與此同時，以顯微切割技術所得的38個原發腫瘤樣本中全部(100%)都顯示有miR-31下調的跡象。相比之下，所有正常的鼻咽上皮細胞都顯出高表達的miR-31。 / miR-31位於染色體9p21.3上，距離CDKN2A (p16) 0.5Mb處。這是在鼻咽癌細胞裏通常缺失的位置。在X1915和X99186腫瘤細胞系中，已證實在miR-31和CDKN2A位點上都出現了純合性缺失。在四株不具備miR-31缺失的腫瘤細胞系裏，甲基化特異性聚合酶連鎖反應 (methylation-specific PCR, MSP) 和亞硫酸氫鈉測序法(bisulfite sequencing)發現了高甲基化的CpG島。使用5-aza-2’-deoxycytidine (5-Aza-dC) 治療後，鼻咽癌細胞株C666-1被證實恢復了miR-31轉錄。這些結果表明，純合性缺失和啟動子高甲基化是造成miR-31在鼻咽癌裏轉錄失效的主要發生機制。 / 微陣列技術和生物信息學分析找出了一些可能受miR-31影響的基因。其中FIH1和MCM2被確定為在鼻咽癌細胞裏受miR-31影響的基因。我們證實miR-31與FIH1和MCM2 信使核糖核酸的3’UTR處結合會抑制螢光素酶的活性。在鼻咽癌細胞裏miR-31的異位表達也會壓抑FIH1和MCM2蛋白的表達。更重要的是，恢復正常的miR-31表達或敲除FIH1表達能顯著地抑制C666-1細胞的增殖和移動能力。C666-1細胞的克隆形成能力和錨定依賴性生長都顯著地被miR-31的表達所抑制。穩定的miR-31表達亦能抑制鼻咽癌腫瘤在裸鼠體內的生長。此外，FIH1的敲除加強了p21和磷酸化p53 (Ser15) 的表達。這些結果暗示了miR-31是一個與鼻咽癌至關重要的微核糖核酸。 它通過了對FIH1的壓制，負面地調節細胞的增殖和移動。 / 使用微核糖核酸微陣列分析後，我們在鼻咽癌細胞中培養的懸浮細胞球裏篩選出差異表達的微核糖核酸。同樣地，實時螢光定量逆轉錄聚合酶鏈反應(qRT-PCR) 亦證實了miR-96和miR-183在C666-1懸浮細胞球裏是被抑制的。此外，miR-96和miR-183的異位表達顯著地降低了C666-1懸浮細胞球形成和克隆形成的能力。這項研究結果暗示, miR-96和miR-183的抑制對鼻咽癌幹細胞樣細胞的形成非常重要。 / 總的來說，某些微核糖核酸已被確定為潛在的鼻咽癌腫瘤抑制基因。 在帶艾巴氏病毒的鼻咽癌裏，miR-31的表達被證實是因純合性缺失和啟動子高甲基化而被下調的。miR-31抑制鼻咽癌細胞的增殖錨定依賴性生長細胞遷移和體內腫瘤的生長。同時，miR-96和miR-183也被發現對維持鼻咽癌的幹細胞樣特性起著一定作用。這些結果表明微核糖核酸對鼻咽癌腫瘤的生成扮演著抑制的角色。對微核糖核酸的機制作進一步全面了解將改進鼻咽癌的治療策略。 / Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) has been reported to be related to a number of genetic and epigenetic changes, however, the molecular mechanism leading to NPC tumorigenesis still remains unclear. Recently, microRNAs (miRNAs) have been demonstrated to play vital roles in NPC development via regulating cell proliferation, apoptosis, and cell migration and invasion. In this study, we aim to elucidate the role of miRNAs in NPC tumorigenesis in this study by identifying the miRNA aberration, investigating the possible functions of these aberrantly expressed miRNAs, and unraveling the role of stemness-related miRNAs in NPC cancer stem-like cells (CSCs). / By using Agilent Microarray with 866 human and 89 viral miRNA probes, miRNA expression profiles of multiple EBV-associated NPC tumor lines were generated. Compared to NP69, a nonmalignant nasopharyngeal epithelial cell line, 113 differentially expressed miRNAs were identified. Among the 58 down-regulated miRNAs in NPC, transcriptional silencing of miR-31 was consistently found in both NPC tumor lines and primary tumors. Down-regulation of miR-31 was detected in 6 of 7 (86%) EBV-positive tumor lines and 38 of 38 (100%) microdissected primary tumors, while all normal nasopharyngeal epithelia showed high expression of miR-31. / miR-31 is located at 0.5 Mb telomeric to CDKN2A (p16) on chromosome 9p21.3, which is commonly deleted in NPC. Homozygous deletion of both miR-31 and CDKN2A loci was confirmed in tumor lines X1915 and X99186. In the four tumor lines with intact miR-31, hypermethylation of 5’ CpG islands was detected by methylation-specific PCR (MSP) and bisulfite sequencing analysis. Restoration of miR-31 transcription was demonstrated in the EBV-positive NPC cell line C666-1 treated with 5-aza-2’-deoxycytidine. These findings suggested that homozygous deletion and promoter hypermethylation are the major mechanisms for transcriptional silencing of miR-31 in NPC. / By microarray and bioinformatic analysis, a number of putative targets of miR-31 were identified. Among these candidates, FIH1 and MCM2 were found to be the targets of miR-31 in NPC. We have shown that binding of miR-31 on FIH1 and MCM2 mRNA 3’UTR suppressed their luciferase activity. Ectopic expression of miR-31 in NPC cells resulted in repression of FIH1 and MCM2 protein expression. Importantly, the restoration of miR-31 or knockdown of FIH1 expression significantly suppressed proliferation as well as migration of C666-1 cells. Clone-forming ability and anchorage-independent growth of C666-1 were significantly inhibited by miR-31 expression. Stably expressed miR-31 was also demonstrated to inhibit NPC tumor growth in nude mice. Furthermore, expression of p21 and phospho-p53 (Ser15) was found to be increased by FIH1 knockdown. These results implied that miR-31 is a critical NPC-associated miRNA which negatively regulates cell proliferation and migration via FIH1 repression. / By miRNA microarray analysis, we have screened for differentially expressed miRNAs in sphere-forming cells of EBV-associated NPC. In concordance with microarray findings, suppression of miR-96 and miR-183 in C666-1 spheroids was confirmed by qRT-PCR. Ectopic expression of miR-96 and miR-183 significantly reduced the sphere-forming and clone-forming ability of C666-1 cells. The findings implied that miR-96 and miR-183 repression is important in the formation of NPC CSCs. / In summary, several miRNAs were identified as potential tumor suppressor genes in NPC. miR-31 was found down-regulated by homozygous deletion or promoter hypermethylation in EBV-associated NPC. It plays roles in NPC pathogenesis by suppressing NPC cell proliferation, clone-forming ability, cell anchorage-independent growth, migration and in vivo tumor growth. Moreover, miR-96 and miR-183 were found to have a role in the maintenance of NPC stem-like properties. These findings suggested important tumor suppressive roles of miRNAs in regulating NPC tumorigenesis, and a better understanding on the miRNA mechanisms may potentiate better therapeutic strategies for NPC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Ching Mei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 177-209). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Thesis / Assessment Committee --- p.vii / Acknowledgements --- p.viii / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xviii / List of publications --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Nasopharyngeal carcinoma (NPC) --- p.1 / Chapter 1.1.1 --- Histopathology and epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.2 / Chapter 1.1.2.1 --- Environmental factors --- p.2 / Chapter 1.1.2.2 --- Genetic factors --- p.3 / Chapter 1.1.2.3 --- Epstein-Barr virus (EBV) infection --- p.3 / Chapter 1.2 --- Molecular pathogenesis of NPC --- p.5 / Chapter 1.2.1 --- Cytogenetic changes --- p.5 / Chapter 1.2.2 --- NPC-associated tumor suppressor genes (TSGs) --- p.6 / Chapter 1.2.3 --- NPC-associated oncogenes --- p.8 / Chapter 1.3 --- MicroRNAs --- p.10 / Chapter 1.3.1 --- Biogenesis of microRNAs --- p.10 / Chapter 1.3.2 --- MicroRNAs and cancers --- p.15 / Chapter 1.3.2.1 --- MicroRNAs - tumor suppressors --- p.15 / Chapter 1.3.2.2 --- MicroRNAs - oncogenes --- p.16 / Chapter 1.4 --- MicroRNAs in nasopharyngeal carcinoma --- p.18 / Chapter 1.4.1 --- MicroRNA profiling in NPC --- p.18 / Chapter 1.4.2 --- OncomiRs in NPC --- p.20 / Chapter 1.4.3 --- Tumor suppressor miRNAs in NPC --- p.22 / Chapter 1.4.4 --- miRNAs and cancer stem-like cells (CSCs) --- p.27 / Chapter 1.4.5 --- Clinical implication of miRNAs in NPC --- p.29 / Chapter 1.5 --- Aims of study --- p.32 / Chapter Chapter 2 --- Materials and methods --- p.34 / Chapter 2.1 --- Patient biopsies --- p.34 / Chapter 2.2 --- NPC cell lines and xenografts --- p.34 / Chapter 2.2.1 --- Cell lines --- p.34 / Chapter 2.2.2 --- Xenografts --- p.36 / Chapter 2.3 --- Total RNA Isolation --- p.39 / Chapter 2.4 --- DNA extraction --- p.39 / Chapter 2.5 --- Protein Extraction --- p.40 / Chapter 2.6 --- Western Blotting --- p.40 / Chapter 2.7 --- Microarray analysis --- p.43 / Chapter 2.7.1 --- MicroRNA microarray --- p.43 / Chapter 2.7.2 --- Gene expression microarray --- p.44 / Chapter 2.8 --- Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) --- p.45 / Chapter 2.8.1 --- Conventional qRT-PCR --- p.45 / Chapter 2.8.2 --- Stem-looped qRT-PCR --- p.46 / Chapter 2.9 --- Preparation of stable clone of miR-31 --- p.51 / Chapter 2.9.1 --- Cloning and plasmid DNA preparation --- p.51 / Chapter 2.9.1.1 --- Bacterial Transformation --- p.51 / Chapter 2.9.1.2 --- Plasmid DNA Extraction --- p.51 / Chapter 2.9.2 --- DNA Sequencing --- p.52 / Chapter 2.9.3 --- Stable transfection --- p.52 / Chapter 2.9.4 --- Clone selection --- p.53 / Chapter 2.10 --- Transient transfection --- p.55 / Chapter 2.11 --- Flow cytometry --- p.55 / Chapter 2.11.1 --- Apoptosis analysis by Annexin V --- p.55 / Chapter 2.11.2 --- Cell cycle analysis by propidium iodide (PI) --- p.56 / Chapter 2.11.3 --- Detection of stem-like cell markers --- p.56 / Chapter 2.12 --- Cell proliferation analysis --- p.56 / Chapter 2.12.1 --- WST-1 assay --- p.56 / Chapter 2.12.2 --- BrdU assay --- p.57 / Chapter 2.13 --- Anchorage-independent growth assay --- p.58 / Chapter 2.14 --- Clone formation assay --- p.58 / Chapter 2.15 --- Cell migration assay --- p.54 / Chapter 2.16 --- In vivo tumorigenicity --- p.59 / Chapter 2.17 --- Dual luciferase reporter assay --- p.60 / Chapter 2.17.1 --- Luciferase reporter vectors --- p.60 / Chapter 2.17.2 --- Luciferase reporter assay --- p.60 / Chapter 2.18 --- Mapping homozygous deletion and genes in chromosome 9p21.3 --- p.64 / Chapter 2.19 --- 5-aza-2’-deoxycytidine (5-Aza-dC) and Trichostatin A (TSA) treatments --- p.64 / Chapter 2.20 --- Methylation specific-PCR (MSP) and bisulfite sequencing analysis --- p.68 / Chapter 2.20.1 --- Bisulfite modification --- p.68 / Chapter 2.20.2 --- Methylation specific-PCR (MSP) --- p.69 / Chapter 2.20.3 --- Bisulfite sequencing analysis --- p.69 / Chapter 2.21 --- Statistical analysis --- p.70 / Chapter 2.22 --- In situ hybridization (ISH) analysis --- p.73 / Chapter Chapter 3 --- Identification of novel deregulated microRNAs in nasopharyngeal carcinoma --- p.74 / Chapter 3.1 --- Introduction --- p.74 / Chapter 3.2 --- Results --- p.80 / Chapter 3.2.1 --- Aberrant expression of microRNAs in NPC --- p.80 / Chapter 3.2.2 --- Homozygous deletion of miR-31 in NPC --- p.90 / Chapter 3.2.3 --- Hypermethylation of 5’ CpG islands of miR-31 in NPC --- p.92 / Chapter 3.2.4 --- Detection of miR-31 expression in normal epithelia and NPC by in situ hybridization --- p.99 / Chapter 3.3 --- Discussion --- p.101 / Chapter Chapter 4 --- Characteristics of miR-31 and its targets in NPC --- p.105 / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Results --- p.107 / Chapter 4.2.1 --- Effects of exogenous miR-31 on NPC cells --- p.107 / Chapter 4.2.1.1 --- miR-31 effect on C666-1 cell proliferation and cell cycle progression --- p.107 / Chapter 4.2.1.2 --- Clone-forming ability and anchorage-independent growth of C666-1 --- p.113 / Chapter 4.2.1.3 --- Migration ability of C666-1 --- p.113 / Chapter 4.2.2 --- Effects of stably expressed miR-31 on NPC cells --- p.117 / Chapter 4.2.2.1 --- Stable clones selection by restoring precursor of miR-31 into C666-1 --- p.117 / Chapter 4.2.2.2 --- Cell proliferation and cell cycle progression in stable clones of miR-31 --- p.117 / Chapter 4.2.2.3 --- Anchorage-independent growth of C666-1 stable clones --- p.117 / Chapter 4.2.2.4 --- Tumorigenicity of C666-1 stable clones expressing miR-31 in vivo --- p.118 / Chapter 4.2.3 --- Identification of miR-31 targets in NPC cells --- p.125 / Chapter 4.2.3.1 --- miR-31 targets FIH1 and MCM2 --- p.125 / Chapter 4.2.3.2 --- Other reported targets of miR-31 in NPC --- p.131 / Chapter 4.2.4 --- Functional analysis of FIH1 in NPC cells --- p.133 / Chapter 4.2.4.1 --- Repression of FIH1 by siRNAs --- p.133 / Chapter 4.2.4.2 --- Proliferation of C666-1 with FIH1 knockdown --- p.133 / Chapter 4.2.4.3 --- Clone-forming and migration ability of C666-1 transfected with siFIH1 --- p.133 / Chapter 4.2.4.4 --- Putative downstream targets of FIH1 --- p.139 / Chapter 4.2.5 --- Identification of novel miR-31 targets by gene expression microarray --- p.139 / Chapter 4.3 --- Discussion --- p.145 / Chapter Chapter 5 --- MicroRNAs regulation on NPC stem-like properties --- p.154 / Chapter 5.1 --- Introduction --- p.154 / Chapter 5.2 --- Results --- p.156 / Chapter 5.2.1 --- MicroRNA expression profiles in NPC sphere-forming cells --- p.155 / Chapter 5.2.2 --- Ectopic expression of miR-183 family and miR-203 in NPC --- p.161 / Chapter 5.2.2.1 --- Sphere-forming ability of NPC cells --- p.161 / Chapter 5.2.2.2 --- Clone-forming ability of C666-1 --- p.161 / Chapter 5.2.3 --- Sphere-forming ability of NPC cells transfected with anti-miR-96 and anti-miR-183 --- p.164 / Chapter 5.2.4 --- Expression of cacner stem cell markers in NPC cells transfected with miR-96 and miR-183 --- p.164 / Chapter 5.3 --- Discussion --- p.167 / Chapter Chapter 6 --- General discussion --- p.170 / Reference --- p.177

The etiology of nasopharyngeal carcinoma (NPC) is based on intricate interactions among environmental factors, genetic susceptibility and Epstein-Barr virus (EBV) infection. Information concerning the role of EBV infection, particularly during the early stage of NPC development is poorly understood. Our laboratory has shown that stable infection of EBV could be achieved in immortalized epithelial cell lines which harbor genetic alterations and altered cell signaling pathway. In this study, these cell models were used to elucidate early events involved in EBV infection in premalignant nasopharyngeal epithelial cell models and their implications on development and progression of nasopharyngeal carcinoma. The response of EBV-infected cells to a stromal inflammatory cytokine, interleukin-6 (IL-6), was examined. EBV infection and long-term propagation of EBV-infected nasopharyngeal epithelial cells confer enhanced sensitivity to STAT3 activation induced by IL-6. IL-6-induced STAT3 activation reinforced their malignant properties in nasopharyngeal epithelial cells and may play a role in the development of nasopharyngeal carcinoma. Furthermore, constitutive STAT3 activation was demonstrated to facilitate malignant transformation of EBV-infected premalignant nasopharyngeal epithelial cells to cancer cells, suggesting that EBV infection and STAT3 activation might synergistically promote the development of NPC. This study also provides support for the existence of a positive feedback loop of IL-6/STAT3/LMP in NP460hTert-EBV cells, which enhanced STAT3 activation in EBV-infected cells. Elevated levels of IL-6Rα expression were observed in EBV-infected NP460hTert cells compared with uninfected cells and were largely responsible for the enhanced sensitivity of IL-6-induced STAT3 activation in these cells. High expression level of IL-6Rα could amplify IL-6 signaling in nasopharyngeal epithelial cells to promote growth proliferation in NP460hTert cells and increase the growth rate of xenografted NPC cells in immune-suppressed animals, suggesting that IL-6Rα overexpression may play a role of contributing to the development of nasopharyngeal carcinoma. The serum concentrations of both IL-6 and sIL-6R were also higher in NPC patients than healthy individuals and may have prognostic values to predict clinical outcome and disease progression in NPC patients. In conclusion, these data support the hypothesis that EBV infection under inflammatory environment may activate aberrant gene expressions and cell signaling to facilitate malignant transformation. The inflammatory cytokine, IL-6, may mediate the role of EBV infection in the development of NPC. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Découvert en 1964, le virus d'Epstein-Barr appartient à la famille des gamma-herpèsvirus. Ce virus à ADN présente une forte prévalence (90% de la population adulte est infectée). Ce fut le premier virus identifié comme associé à des cancers (lymphome de Burkitt et d'Hodgkin, carcinome gastrique et de l'oropharynx). Ce virus a pour spécificité de posséder deux cycles distincts : latent et lytique (production de particules virales). Le facteur de transcription viral EB1 (ou Zebra) est un élément clé lors de l'initiation du cycle lytique et semble une cible importante pour l'élaboration de nouveaux traitements. Une première partie de ce travail concerne la caractérisation d'une protéine cellulaire (l'Ubinucléine) qui interagit et inhibe l'activité de EB1. Cette protéine voyage entre noyaux et jonctions serrées. Elle appartient à la famille des « NACos » (nuclear and adhesion complex components). La fonction de l'Ubinucléine n'est pas connue et sa protéomique quand elle est localisée dans les jonctions serrées, a été réalisée. Des études fonctionnelles montrent que l'Ubinucléine interagit avec plusieurs partenaires cellulaires, emprunte la voie d'endocytose dépendante de la clathrine et que sa localisation cellulaire (nucléaire ou dans les jonctions serrées) est affectée par la PKA. Dans une seconde partie, nous nous sommes intéressés à des molécules inhibitrices du facteur de transcription viral EB1. Après criblage à haut débit de composés chimiques (EMBL-Heidelberg), des tests in-vitro ont permis d'en sélectionner un pour des essais in-vivo. Ce composé chimique inhibe l'activité du facteur de transcription EB1 et bloque précocement la mise en place du cycle lytique dans des cellules de lymphome de Burkitt. Il semble donc intéressant d'améliorer l'efficacité et la spécificité de cette molécule. / Discovered in 1964, Epstein-Barr virus belongs to the Gamma-herpesvirus family. This DNA virus shows an important prevalence (90% of the adult population is infected). It was the first virus identified as associated with cancers (Hodgkin and Burkitt lymphomas, Gastric and oropharyngal carcinomas). It presents two distinct cycles: Latency and Lytic cycle (viral particle production). Viral transcription EB1 (or Zebra) is a key element for lytic cycle initiation and seems to be an important target for future treatment development. A first part of this work concerned the characterization of the cellular protein Ubinuclein that inhibits EB1 activity. This protein travels between nucleus and tight junctions. It belongs to the “NACos” (nuclear and adhesion complex components) protein family. Ubinuclein function is not known and its proteomic was performed when it was localized at tight junction. Next, functional studies showed that Ubinuclein interacts with various cellular partners and goes though the clathrin dependent endocytosis pathway. It's localisation (nuclear or at tight junction) changes with PKA activity. In the second part of this work, we focus on viral transcription factor EB1 inhibitors. After high-throughput screening of compounds (EMBL-Heidelberg), in-vitro assays allowed to select one molecule for in-vivo experiments. This compound inhibits the activity of the transcription factor EB1 and stops early lytic cycle establishment in Burkitt lymphoma cells. Further work needs to be done to increase efficacy and specificity of this molecule.

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-25T12:06:32Z No. of bitstreams: 1 Arquivototal.pdf: 2117961 bytes, checksum: 458d516added97ab516b1c8ae4ec5ddd (MD5) / Made available in DSpace on 2018-04-25T12:06:32Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 2117961 bytes, checksum: 458d516added97ab516b1c8ae4ec5ddd (MD5) Previous issue date: 2017-10-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The microRNAs are of great interest in studies on viral infections. In the context of the Epstein-Barr virus (EBV), they act as important regulators of the expression of viral and cellular genes, facilitating viral persistence and oncogenesis. The present work aims to identify viral microRNAs, their target mRNAs and the essential target proteins specifically for Human gammaherpesvirus 4 tipo 2. First, we used the StructRNAfinder and IntaRNA programs to perform the identification tasks of miRNAs and target mRNAs, respectively. Subsequently, we created a network of proteins from the data obtained from the tool online virusmentha and visualized in the program cytoscape. In addition, we performed a topological analysis of the protein interaction network with the cytoscape tool. We found 55 viral miRNAs and 46 mRNAs and identified 10 proteins that were prominent in the protein interaction network. The BTRF1 and BSFR1 proteins exert a greater control in the protein network, although they are proteins of the integument with unknown functions. Thus, we suggest that the BTRF1 and BSRF1 proteins are potential targets for understanding and controlling EBV infections. / Os microRNAs são de grande interesse em estudos sobre infecções virais. No contexto do vírus Epstein-Barr (EBV), eles atuam como importantes reguladores da expressão de genes virais e celulares, facilitando a persistência viral e a oncogênese. O presente trabalho objetiva identificar microRNAs virais, seus mRNAs alvos e as proteínas alvos essenciais específicas para o Human gammaherpesvirus 4 tipo 2. Primeiramente, utilizamos os programas StructRNAfinder e o IntaRNA para realizar as tarefas de idenficação de miRNAs e mRNAs alvos, respectivamente. Posteriormente, criamos uma rede de proteínas a partir dos dados obtidos da ferramenta online virusmentha e visualizamos no programa cytoscape. Além disso, realizamos uma análise topológica da rede de interação de proteínas com a ferramenta cytoscape. Encontramos 55 miRNAs virais e 46 mRNAs e identificamos 10 proteínas que se destacavam na rede de interação de proteínas. As proteínas BTRF1 e BSFR1 exercem um maior controle na rede de proteínas, embora sejam proteínas do tegumento com funções desconhecidas. Assim, sugerimos que as proteínas BTRF1 e BSRF1 sejam alvos potenciais para compreender e controlar as infecções por EBV.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The oral hairy leukoplakia (OHL) is a non-malignant epithelial lesions that usually occurs along the side edges of the tongue caused by the Epstein -Barr virus (EBV). This disease is almost exclusively seen in immunocompromised patients, particularly in individuals infected with human immunodeficiency virus (HIV). However, the OHL is related to immunosuppression in general, been described in patients receiving immunosuppressive therapy. The diagnosis of OHL in its clinical or subclinical form, can be done through the detection of EBV cytopathic effects in epithelial cells. Thus, the proposition of this study was to evaluate the oral hairy leukoplakia as a possible marker of immune impairment, through a cytological study in patients undergoing immunosuppressive therapy. 40 patients with autoimmune diseases were evaluated under immunosuppressive treatment, recruited from spontaneous demand of a population of patients regularly treated at the Department of Rheumatology, University Hospital, Federal University of Sergipe. Intraoral history and physical examination were performed, and soon after, two sweeps lateral border of the tongue, one for each side, then the construct two smears on conventional glass slides to be stained by the Papanicolaou technique were performed. Subsequently consulted medical records in order to collect data regarding the diagnosis of the underlying disease, time since diagnosis, type and duration of immunosuppressive therapy and WBC count. Stained by Papanicolaou smears were evaluated for representative nuclear changes cytopathic effect of EBV, which represent the criterion of cytological diagnosis of oral hairy leukoplakia. Most of the examined patients were females (70%) and the overall mean age was 40.5±16.1 years. Subclinical LPO was observed in 52.5% of patients. Thus, the use of shaved bilateral language for cytological diagnosis of subclinical LPO as clinical and laboratory monitoring tool immunosuppressed patients, in order, ultimately, minimize the occurrence of opportunistic diseases through modulation of therapy used is suggested. / A leucoplasia pilosa oral (LPO) é uma lesão epitelial não maligna que ocorre normalmente nas bordas laterais da língua, causada pelo vírus Epstein-Barr (EBV). Esta patologia é quase que exclusivamente vista em pacientes imunocomprometidos, particularmente em indivíduos infectados pelo vírus da imunodeficiência humana (HIV). Contudo, a LPO está relacionada com imunossupressão em geral, sendo descrita em pacientes que receberam terapia imunossupressora. O diagnóstico da LPO, na sua forma clínica ou subclínica, pode ser feito através da detecção dos efeitos citopáticos do EBV nas células epiteliais. Assim, a proposição deste estudo foi avaliar a leucoplasia pilosa oral como um possível marcador de comprometimento imune, através de um estudo citopatológico em pacientes submetidos à terapia imunossupressora. Foram avaliados 40 pacientes portadores de doenças autoimunes, sob tratamento imunossupressor, recrutados a partir de demanda espontânea de uma população de pacientes regularmente atendidos no Serviço de Reumatologia do Hospital Universitário da Universidade Federal de Sergipe. Foram realizados anamnese e exame físico intraoral e, logo após, foram realizadas duas raspagens de borda lateral da língua, uma para cada lado, seguida da confecção de dois esfregaços em lâminas de vidro convencionais para serem corados pela técnica de Papanicolaou. Posteriormente, consultado prontuário médico a fim de colher dados referentes ao diagnóstico da doença de base, tempo de diagnóstico, tipo e tempo de terapia imunossupressora e contagem leucocitária. Os esfregaços corados por Papanicolaou foram avaliados quanto às alterações nucleares representativas do efeito citopático do EBV, que representam o critério de diagnóstico citopatológico da leucoplasia pilosa oral. A maioria dos pacientes examinados pertencia ao gênero feminino (70%) e a média geral de idade encontrada foi de 40,5±16,1 anos. A LPO subclínica foi observada em 52,5% dos pacientes. Assim, sugere-se a utilização do raspado bilateral da língua para diagnóstico citopatológico de LPO subclínica como ferramenta de acompanhamento clínico-laboratorial de pacientes imunossuprimidos, a fim de, em última instância, minimizar a ocorrência de doenças oportunistas através da modulação da terapia empregada.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The Epstein-Barr virus (EBV) has been related to the tumorigenesis of the gastric carcinomas, varying from 1.3-19.3% according to the studied population. Several studies have demonstrated strong evidences of its relation in this process, such as the monoclonality of the viral genome and its the presence in almost all tumor cells. However, most of the mechanisms used by the virus to control this process are still unknown. In this context, the present study aimed to investigate the frequency of the EBV and the association with the BCL-2, BAX and c-MYC proteins. Therefore, 100 cases of gastric carcinoma (67 males and 33 females), obtained from two hospitals in Fortaleza, were assessed to detect the EBV by PCR and in situ hybridization (aimed to the EBER1 transcript) using the standard method and GenPointÂ. Immunohistochemistry technique was done to evaluate the expression of the referred cellular proteins, by streptavidin-biotin-peroxidase method. The distribution by sex, age, tumor anatomic site and the histopathologic analysis, in general, reproduced the pattern of the world scientific bibliographies. Regarding virus detection by in situ hybridization, 8 (8%) cases were positive, 6 of these had shown diffuse pattern of staining, and 2 had demonstrated focal pattern. From 100 cases, only 2 presented infected lymphocytes. In general, the EBV demonstrated higher association with: males (87.5%[p=0.265]), tumors situated in the cardia (37.5% [p=0.549]), advanced stage (IIIB and IV), intestinal type (87.5%[p=0.136]), and moderately differentiated (75%).There were no EBV-positive cases which exhibited BCL-2 staining. Although the BAX and the c-MYC (nuclear) proteins have demonstrated significant positivity index and scores averages in the EBV-positive group, these were lower than the values of the EBV-negative group, notably the c-MYC nuclear protein (Mann-Withney test LI p=0.039 and HS p=0.045). The cytoplasmic staining of the c-MYC protein revealed slightly higher staining values in the EBV-positive group. The balance between the BCL-2 and BAX proteins demonstrated that the majority of the evaluated cases had exhibited apoptosis-orientation, however 62.3% of the EBV-positive cases exhibited equilibrium between these proteins. Twenty-nine cases (28 negative and 1 positive) were submitted to the biotinyl tyramide system (in situ hybridization method - GenPointÂ), demonstrating the same results obtained by the standard technique. From the 61 cases assessed by PCR, 35 (57.4%) were positive, being verified a low concordance index (kappa = -0.026 [Â0.069]) with the standard in situ hybridization technique. The 30bp deletion of LMP1 gene was investigated in 24 out of 35 positive cases, being verified in 37.5% of these. The results obtained in the present study, concerning the EBV frequency and the correlation with clinic-histopathologic data, reproduced findings of researches done in several world regions. The correlation with the proteins suggests that in vivo the virus is not related to the overexpression of BCL-2 and c-MYC (nuclear) that could act in synergism to promote the tumor development. The suppression of the BAX expression might represent a viral mechanism for apoptosis inhibition. The results of the cytoplasmic c-MYC point to a possible involvement of the EBV with transport mechanisms of the nuclear membrane, resulting in its accumulation in the cytoplasm. The low frequency of infected lymphocytes indicates that they are not the main responsible of the high number of positivity in the PCR technique. It could be, at least in part, due to the infected normal and/or pre-neoplastic epithelium, suggesting a new latency pattern which not express the EBER1. / O vÃrus Epstein-Barr (EBV) tem sido associado com a tumorigÃnese dos adenocarcinomas gÃstricos, variando entre 1,3-19,3% de acordo com a populaÃÃo estudada. Diversos estudos tÃm demonstrado importantes evidÃncias do envolvimento do EBV nesse processo, tais como a monoclonalidade do genoma viral e a presenÃa do vÃrus em quase todas as cÃlulas tumorais do sitio primÃrio e em cÃlulas metastÃticas. No entanto, os mecanismos utilizados pelo vÃrus para orquestrar a transformaÃÃo tumoral, ainda nÃo foram totalmente elucidados. Neste contexto, o presente estudo objetivou investigar a freqÃÃncia do EBV e a associaÃÃo com as proteÃnas BCL-2, BAX e c-MYC. Para tanto, 100 casos de adenocarcinomas gÃstricos (67 homens e 33 mulheres), obtidos de dois hospitais de Fortaleza, foram analisados quanto à presenÃa do EBV, detectado atravÃs das tÃcnicas de PCR e de hibridaÃÃo in situ (direcionada ao transcrito viral EBER1) pelo mÃtodo usual e GenPointÂ. Procedeu-se tambÃm, estudo imuno-histoquÃmico das referidas proteÃnas celulares, atravÃs do mÃtodo da estreptoavidina-biotina-peroxidase. A distribuiÃÃo por sexo, idade, sÃtio anatÃmico do tumor e as anÃlises histopatolÃgicas, de modo geral, reproduziram as tendÃncias da literatura mundial. Pela tÃcnica de hibridaÃÃo in situ, 8 (8%) casos foram positivos, 6 destes apresentaram marcaÃÃo difusa e 2 apresentaram marcaÃÃo focal. Apenas 2 apresentaram linfÃcitos infectados. De modo geral, o EBV apresentou maior associaÃÃo com o sexo masculino (87,5% [p=0,265]), com tumores situados na cÃrdia (37,5% [p=0,549]), de estadiamento avanÃado (IIIB e IV), do tipo intestinal (87,5% [p=0,136]) e moderadamente diferenciados (75%). Nenhum dos casos EBV-positivos exibiram marcaÃÃo para BCL-2. Embora as proteÃnas BAX e c-MYC (nuclear) apresentaram Ãndices de positividade e mÃdias de escores significativos no grupo EBV-positivo, estes foram inferiores aos valores do grupo EBV-negativo, sobretudo a proteÃna c-MYC nuclear (Teste de Mann-Withney LI p=0,039 e HS p=0,045). A marcaÃÃo citoplasmÃtica da proteÃna c-MYC revelou valores de marcaÃÃo discretamente superiores no grupo EBV-positivo. O balanÃo entre as proteÃnas BCL-2 e BAX demonstrou que a maioria dos casos estudados apresentavam tendÃncia à apoptose, mas 62,5% dos casos EBV-positivos exibiram um equilÃbrio. Vinte e nove casos (28 negativos e 1 positivo) foram submetidos a outro mÃtodo de hibridaÃÃo in situ que emprega o sistema da biotinil-tiramida (GenPointÂ),demonstrando resultados idÃnticos aos obtidos pela tÃcnica convencional. De 61 casos analisados atravÃs da tÃcnica de PCR, 35 (57,4%) foram positivos, sendo constatado um baixÃssimo Ãndice de concordÃncia (kappa = -0,026 [Â0,069]) com a tÃcnica de hibridaÃÃo in situ. Em 24/35 casos positivos, a deleÃÃo de 30pb do gene LMP1 foi investigada, sendo constatada em 37,5% destes. Os resultados obtidos no presente estudo quanto à freqÃÃncia do EBV e a correlaÃÃo com critÃrios clÃnico-histopatolÃgicos, reproduziram os achados de estudos realizados em diversas partes do mundo. A correlaÃÃo com as proteÃnas sugere que in vivo, o vÃrus nÃo esteja relacionado com a expressÃo de BCL-2 e de c-MYC (nuclear), que poderiam atuar em sinergismo favorecendo o desenvolvimento tumoral. A supressÃo da expressÃo de BAX, pode representar um mecanismo viral para inibiÃÃo da apoptose. Os resultados da c-MYC citoplasmÃtica apontam para um possÃvel envolvimento do EBV com mecanismos de transporte da membrana nuclear, determinando o acÃmulo da proteÃna no citoplasma. A baixa freqÃÃncia de linfÃcitos infectados indica que os mesmos nÃo sÃo os principais responsÃveis pela elevada positividade da tÃcnica de PCR, devendo ser ao menos em parte, decorrente de epitÃlio normal e/ou prÃ-neoplÃsico infectado sugerindo um padrÃo de latÃncia que nÃo expresse EBER1.