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.

Dans les pays occidentaux, les lymphomes T périphériques et NK représentent environ 10% des lymphomes non-Hodgkiniens. Le lymphome NK/T de type nasal est l'une des entités de présentation extra-ganglionnaire les plus fréquentes, en Asie, et en Amérique Centrale et du Sud. Il survient classiquement dans la sphère nasopharyngée avec une prédilection pour les adultes jeunes. Morphologiquement, la tumeur est souvent angiocentrique avec une invasion de la paroi des vaisseaux par les cellules tumorales d'aspect variable. Ces lymphomes ont le plus souvent une origine NK avec un phénotype CD3+ (cytoplasmique), CD5-, CD56+, CD4-/CD8-, expression des molécules cytotoxiques et absence de réarrangement des gènes des récepteurs T. Le virus d'Epstein-Barr est présent dans la quasi-totalité des cellules tumorales dans sa forme clonage épisomale, avec une latence de type II, suggérant son rôle dans l'oncogenèse. A côté des mutations fréquentes des gènes FAS et TP53 (p53) et des méthylations de TP73 et CDKN2A (p16), des délétions du bras long du chromosome 6q sont fréquemment observées. Très récemment, des méthylations et des mutations des gènes suppresseurs de tumeur PRDM1, ATG5, et AIM1 localisés en 6q21 ont été retrouvées dans les lignées de lymphome NK/T de type nasal. Nous avons réalisé une analyse combinée du profil d'expression génique et du profil génomique par hybridation comparative sur puces, d'échantillons tumoraux de lymphome NK/T de type nasal (n=9) et de lignées, comparés à celle de lymphocytes NK normaux et de lymphomes T périphériques, sans autre spécificité (PTCL, NOS). Nous avons identifié la signature moléculaire particulière du lymphome NK/T de type nasal caractérisée par un haut niveau des trascrits de marqueurs de cellules NK et de molécules cytotoxiques, notamment de granzyme H dans les lymphomes NK/T de type nasal comparé aux PTCL, NOS. Par immunohistochimie, nous avons validé l'expression "spécifique" de granzyme H par les cellules tumorales du lymphome NK/T de type nasal, qui pourrait constituer un nouveau marqueur de ces lymphomes. Comparé aux cellules NK normales, le lymphome NK/T de type nasal a une signature plus proche des cellules NK activées que des NK au repos et sur-expriment des gènes associés à la biologie vasculaire, des gènes induits par l'EBV, et PDGFRA. Nous avons confirmé l'expression protéique de PDGFRAa et de sa forme phosphorylée, et montré in vitro la sensibilité de la lignée tumorale MEC04 à l'imatinib mesytale. La dérégulation des voies de signalisation AKT, JAK-STAT et NF-kB, suggérée par les analyses bioinformatiques, a été corroborée par la mise en évidence d'une expression nucléaire des formes phosphorylées d'AKT, de STAT3 et de RelA dans les lymphomes NK/T de type nasal. De plus, plusieurs gènes dérégulés dans ces voies moléculaires sont localisés dans des régions altérées de manière récurrente par des gains ou des pertes (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). En plus de l'activation constitutive de STAT3 confirmée par l'expression nucléaire de phospho-STAT3, l'inhibition de croissance et l'augmentation de la mort cellulaire des cellules de la lignée MEC04 résultant de l'inhibition de STAT3 conforte le rôle de STAT3 dan la lymphomagenèse du lymphome NK/T nasal. L'analyse intégrée a également mis en évidence la dérégulation du gène suppresseur de tumeur HACE1 en 6q21, confirmée par RT-PCR quantitative. Bien que les mécanismes exacts conduisant à l'activation de plusieurs voies moléculaires, de même qu'à la dérégultaion de HACE1 ne soient pas déterminés, nos résultats identifient plusieurs voies oncogéniques impliquées dans le lymphome NK/T de type nasal ainsi que de nouveaux biomarqueurs diagnostiques - comme granzyme H - et des cicles thérapeutiques d'intérêt. L'étude en cours du profil d'expression des microARNs pourrait apporter un éclairage sur les mécanismes impliqués dans certaines voies identifiées / In Western countries, mature natural killer (NK)- and T-cell lymphomas account for 15% to 20% of aggressive lymphomas and around 10 % of all non-Hodgkin lymphomas. This number is higher in Asia, with 25% in Japan and 39% in Taiwan. Among those T- and NK-cell lymphomas with primary extranodal presentation, extranodal NK/T-cell lymphoma of nasal type (nasal NKTCL) is one of the most common entities in Asian, Central and South American populations. It classically arises in the nasal region showing a predilection for young adults with male predominance. This tumor morphologically exhibits an angiocentric and angio destructive growth pattern, admixed with polymorphous non-neoplastic infiltrates. Most tumor cells have a cytoplasmic CD3+, CD5-, CD56+, CD4-/CD8- phenotype with expression of cytotoxic granule-associated proteins and without rearrangement of T-cell receptors genes. Killer immunoglobulin-like receptors have been reproted to be expressed in a subset of this lymphoma and its expression might be associated with prognosis. Epstein-Barr virus is present in virtually all neoplastic cells in its clonal episomal form with type II latency program, implying a role in oncogenesis. Although the results were variable between different studies, methylations of TP73 (p73) and CDKN2A (p16) and mutations of FAS and TP53 (p53) were frequently found in nasal NKTCL. Genomic alterations have also been reported in nasal NKTCL with frequent deletion in chromosome 6q. A very recent study also identified both methylations and mutations of three putative tumor suppressor genes PRDM, ATG5, and AIM1 mapping to del6q21 in nasal NKTCL cell times. We performed integrative gene expression profiling and array-based comparative genomic hybridization analyses of nasal NKTCL tumors as well as tumour-derived cell lines, compared to that of normal NK cells and peripheral T-cell lymphomas, not otherwise specified (PTCL, NOS). We identified the distinctive molecular signature of nasal NKTCL with high transcript levels for NK-cell markers ans cytotoxic molecules, especially granzyme H in nasal NKTCL compared to PTCL, NOS. By immunohistochemistry, we validated expression of grnzyme H which appears a novel sensitive biomarker of nasal NKTCL. Compared to normal NK cells, nasal NKTCL tumors were closer to activated than resting cells and overexpressed several genes related to vascular biology, EBV-induced genes and PDGFRA. Notably, we confirmed the expression of PDGFRa and its phosphorylated form at the protein level, and in vitro the MEC04, nasal NKTCL-cell line, was sensitive to imatinib mesylate. Deregulation of the AKT, JAK-STAT and NF-kB pathways suggested by bioinformatical analysis, was corroborated by nuclear expression of phosphorylated AKT, STAT3 and RelA in nasal NKTCL, and several deregulated genes in these pathways mapped to regions of recurrent copy number aberrations (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). In addition to constitutive activation of STAT3 as confirmed by the demonstration of phosphorylated STAT3 in the nuclei of neoplastic nasal NKTCL cells, growth inhibition and cell death of nasal NKTCL cells induced by STAT3 inhibition implied the role of STAT3 in the nasal NK/T-cell lymphomagenesis. Integrative analysis and qRT-PCR analysis also evidenced deregulation of another tumor suppressor HACE1 in the frequently deleed 6q21 region. Although the exact mechanism of activation of several pathways as well as that of HACE1 deregulation remains to be determined, our studies highlight emerging oncogenic pathways in nasal NKTCL and identify novel diagnostic and therapeutic targets. The ongoing investigation of microRNA expression profiling might shed light in a better understanding of the pathogenesis of nasal NKTCL and especially of the activation of oncogenic pathways. Connectivity map analysis may also help to depict other targeted therapies useful to improve the prognosis of this agressive lymphoma

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy