Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 129-144).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

近年来肠病毒71亚型（EV71）的大规模流行已成为全世界特别是亚太地区的一个严重的公共卫生问题。EV71感染可以引起腹泻，皮疹，手足口病等等一些自愈性疾病。然而在部分儿童患者中，EV71可能导致严重的神经性疾病。目前，关于EV71感染后宿主细胞的反应机制的报导比较少。在本次研究中，我们运用蛋白组学方法对EV71感染后的人横纹肌瘤细胞的蛋白表达情况进行了分析，最终发现了42个差异表达的蛋白（>2倍的变化，P <0.05），其中21个下调， 21个上调。进一步分析表明，这些蛋白主要参与了细胞内代谢，生物学调控，细胞构建，信息传递和细胞死亡的调控。 接下来我们选择了其中一个变化比较大的蛋白：HSP27，对其功能进行了深入分析。我们的研究结果显示：EV71感染的早期阶段，HSP27在转录和翻译水平上都有明显上调。降低HSP27表达可以减少EV71的复制，过表达HSP27则可以提高病毒复制。通过使用特异的磷酸化蛋白抗体，我们发现HSP27第15位以及78位的丝氨酸有明显的磷酸化修饰，而82位的丝氨酸则没有发生改变。使用p38激酶抑制剂预先处理细胞可以降低HSP27的磷酸化修饰，从而抑制EV71的复制。进一步分析表明，HSP27可以帮助EV71蛋白酶2A对真核翻译起始因子eIF4G的剪切，从而加强病毒自身蛋白的翻译，最终促进了病毒的感染。这项研究结果阐明了宿主细胞EV71的反应机制，有利于我们对病毒致病机制的研究，并为EV71的抗病毒研究提供了一个新的药物靶标。 / The outbreaks of enterovirus 71 (EV71) infections have become a major public health issue worldwide, especially in the Asia-Pacific region. EV71 infection can be asymptomatic or cause diarrhea, rashes, and hand, foot, and mouth disease (HFMD). However, EV71 can also cause severe neurological disease even death. To date, little is known about the molecular mechanisms of the host response to EV71 infection. In this study, the expression patterns of host genes in EV71 infected human rhabdomyosarcoma cells were analyzed by using two-dimensional proteomics assays. In total, 42 protein spots were found to be differentially expressed (>2 fold changes, p<0.05) in three pairs of gels, of which 21 proteins were found to be down-regulated while 21 were up-regulated. Data analysis suggested that proteins associated with metabolic process, biological regulation, cellular component organization, cell communication and death were most modified. HSP27, one of the most altered proteins during EV71 infection, was selected to determine its fundamental roles upon EV71 infection. We show that HSP27 is rapidly up-regulated both at the transcriptional and the translational levels at the early stage of EV71 infection. Depleting cellular HSP27 expression reduced EV71 replication, while over-expression of HSP27 greatly enhanced viral infection. By using the phosphorylated specific antibodies, serine residues 15, 78, but not the 82 were found to be phosphorylated during EV71 infection. The phosphorylation depended on the activation of the mitogen-activated protein kinase p38 signaling pathway. After treating with p38 kinase inhibitors, EV71 replication was coordinately decreased. Further analysis showed that HSP27 affected the protease 2A mediated eIF4G cleavage and assisted the IRES driven translation, thus facilitated the EV71 replication. The findings in this work not only provided a global view of the host responses to EV71 infection, but demonstrated HSP27 to be a valid target for anti-EV71 drug development. / Detailed summary in vernacular field only. / Yi, Lina. / "September 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 94-103). / 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.iii / Acknowledgement --- p.iv / Publications --- p.v / Table of Contents --- p.vii / List of Tables and Figures --- p.x / List of Abbreviation --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Enterovirus 71 --- p.2 / Chapter 1.1.1 --- Clinical features --- p.2 / Chapter 1.1.2 --- Molecular epidemiology of EV71 --- p.5 / Chapter 1.1.3 --- The virology of EV71 --- p.8 / Chapter 1.1.4 --- Pathogenesis --- p.18 / Chapter 1.1.5 --- Treatment of EV71 infection --- p.20 / Chapter 1.2 --- The heat shock protein 27 --- p.23 / Chapter 1.2.1 --- Properties of HSP27 --- p.23 / Chapter 1.2.2 --- Functions of Hsp27 --- p.26 / Chapter 1.2.5 --- Phosphorylation of Hsp27 --- p.28 / Chapter 1.2.6 --- Hsp27 and Viral infection --- p.31 / Chapter 1.3 --- Thesis hypothesis and objective --- p.33 / Chapter Chapter 2 --- Materials and Methods --- p.34 / Chapter 2.1 --- Cells and Virus propagation --- p.35 / Chapter 2.2 --- Viral infection --- p.35 / Chapter 2.3 --- 2-DE and image analysis --- p.36 / Chapter 2.4 --- MALDI-TOF-MS --- p.37 / Chapter 2.5 --- Database analysis --- p.38 / Chapter 2.6 --- Bioinformatic analysis --- p.38 / Chapter 2.7 --- Plasmids --- p.39 / Chapter 2.8 --- siRNA synthesis --- p.41 / Chapter 2.9 --- Transfection and cell treatment --- p.41 / Chapter 2.10 --- RNA extraction and cDNA synthesis --- p.41 / Chapter 2.11 --- Real-Time Quantitative PCR --- p.42 / Chapter 2.12 --- Western Blotting analysis --- p.44 / Chapter 2.13 --- Luciferase assays --- p.44 / Chapter 2.14 --- Statistical Analysis --- p.45 / Chapter Chapter 3 --- Proteomic analysis of cellular protein alterations in response to EV71 infection --- p.46 / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Results --- p.48 / Chapter 3.2.1 --- EV71 infection of the RD cells --- p.48 / Chapter 3.2.2 --- 2-DE profiling of EV71 infected and non-infected RD cells --- p.49 / Chapter 3.2.3 --- Identification of differentially expressed proteins --- p.50 / Chapter 3.2.4 --- Functional classification --- p.52 / Chapter 3.2.5 --- GO enrichment analysis --- p.54 / Chapter 3.2.6 --- Protein validation by Western blot --- p.56 / Chapter 3.3 --- Discussion --- p.57 / Chapter Chapter 4 --- HSP27 effects on EV71 infection --- p.62 / Chapter 4.1 --- Introduction --- p.63 / Chapter 4.2 --- Results --- p.64 / Chapter 4.2.1 --- Increased Hsp27 expression in EV71 infected cells --- p.64 / Chapter 4.2.2 --- Suppression of Hsp27 inhibits EV71 replication --- p.65 / Chapter 4.2.3 --- Over-expression of Hsp27 increases EV71 replication --- p.66 / Chapter 4.2.4 --- Hsp27 is rapidly phosphorylated during EV71 infection --- p.67 / Chapter 4.2.5 --- Pathways involved in Hsp27 phosphorylation --- p.68 / Chapter 4.2.6 --- Role of Hsp27 phosphorylation during EV71 infection --- p.68 / Chapter 4.3 --- Discussion --- p.70 / Chapter Chapter 5 --- HSP27 facilitate EV71 IRES driven translation --- p.75 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- Results --- p.79 / Chapter 5.2.1 --- Hsp27 increase viral IRES activity --- p.79 / Chapter 5.2.2 --- Hsp27 affects EV71 2A mediated eIF4G cleavage --- p.80 / Chapter 5.3 --- Discussion --- p.82 / Chapter Chapter 6 --- Summary and Perspectives --- p.87 / Chapter 6.1 --- Summary --- p.88 / Chapter 6.2 --- Perspectives --- p.89 / Reference --- p.93

Lu, Jing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 119-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19625号 / 医博第4132号 / 新制||医||1015(附属図書館) / 32661 / 京都大学大学院医学研究科医学専攻 / (主査)教授 高田 穣, 教授 松田 文彦, 教授 小泉 昭夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introdução: a síndrome antifosfolípide (SAF) primária é uma vasculopatia autoimune mediada por autoanticorpos com trombose como sua principal manifestação clínica. A presença de anticorpos antifosfolípides (aPL), embora relevante para confirmar o diagnóstico, não parece ser suficiente para explicar completamente a fisiopatologia da doença e um segundo gatilho é usualmente necessário. Além das hipóteses de infecções virais e insulto inflamatório como possíveis desencadeantes, parece que os receptores toll like (TLR) e o Interferon (IFN) tipo I são possíveis protagonistas nesse processo, contribuindo para o início da trombose. Recentemente, dois pequenos estudos demonstraram que uma porcentagem relevante de pacientes com SAF primária tem uma regulação positiva de genes IFN em células mononucleares do sangue periférico (CMSP). Entretanto, 20% e 28% dos pacientes nessas duas coortes tiveram anticorpos anti-dsDNA positivos, um autoanticorpo altamente específico do lúpus eritematoso sistêmico (LES). Objetivo: avaliar se os pacientes com SAF bem caracterizados apresentam assinatura para interferon nas células mononucleares periféricas. Secundariamente foram avaliadas possíveis associações clínico laboratoriais com a assinatura de IFN. Métodos: foram selecionados 53 pacientes do sexo feminino com diagnóstico de SAF primária de acordo com os critérios de Sidney, com idade igual ou maior a 18 anos, selecionados no Ambulatório de SAF da Disciplina de Reumatologia do HCFMUSP, pareados por sexo e idade com 50 controles saudáveis. Um terceiro grupo com 29 paciente com antecedente de trombofilias não imunomediadas também foi incluido. Após a coleta de sangue as CMSPs foram purificadas por metodologia de Ficoll. A expressão gênica das CMSPs foi realizada através do TaqMan® RNA Assay em placas TLDA. Foram pesquisados 41 genes induzidos por IFN (GIIs). Uma análise de componente principal (ACP) foi realizada para determinar quais genes deveriam compor a assinatura de IFN. O teste de z-score foi utilizado para normalizar e calcular a assinatura de IFN para cada paciente. O cutoff da assinatura de IFN foi definido por uma curva ROC, e foi escolhido o ponto que maximizava a sensibilidade e especificidade. Características demográficas, clínicas e laboratoriais foram analisadas buscando por associações com a assinatura de IFN. Resultados: 11 genes estavam superexpressos nos pacientes com SAF em comparação aos controles. Após a análise de ACP foram escolhidos 6 genes que representavam mais de 95% do comportamento da amostra para compor a assinatura de IFN: DNAJA1, IFI27, IFI6, IFIT5, MX1 e TYK2. O cutoff encontrado pela curva ROC foi de 3,9 folds (AUC = 0,706, S = 0,49, E = 0,86, VPP = 0,79, VPN = 0,61). A assinatura de IFN estava presente em 49% dos pacientes com SAF primário vs. 14% dos controles saudáveis e 17% dos controles positivos (p < 0,001). Foi encontrada associação entre a assinatura de IFN e uma ocorrência mais precoce do primeiro evento clínico (p = 0,023), e com ocorrência de eventos obstétricos (em especial pré-eclâmpsia, p = 0,032). Não foi econtrada nenhuma associação entre a assinatura de IFN e número de eventos trombóticos, exames laboratoriais, comorbidades, antecedentes familiares de doenças autoimunes, e escores de risco de retrombose. De todos os tratamentos em uso a única associação encontrada foi entre uma menor assinatura de IFN e o uso de estatinas (p = 0,026). Conclusão: esse estudo indica que pacientes com SAF primária bem caracterizados apresentam uma assinatura de IFN tipo I, não observada em outras trombofilias não imunidade-mediadas ou em controles saudáveis. Também demonstrou-se que essa superexpressão de genes regulados por IFN tipo I está associada a um início mais precoce dos eventos e pré-eclâmpsia. Mais estudos são necessários para determinar se este subgrupo de pacientes se beneficiará de intervenções terapêuticas direcionadas à via de sinalização IFN tipo I / Introduction: primary antiphospholipid syndrome (PAPS) is an autoimmune vasculopathy mediated by autoantibodies with thrombosis as its main clinical manifestation. The presence of antiphospholipid antibodies, while relevant to confirm the diagnosis, does not seem to be sufficient to fully explain the pathophysiology and a second trigger is usually needed. Besides the hypotheses of viral infections and inflammatory insult as possible triggers, type I Interferon (IFN) has been pointed as a possible protagonist. Recently, two studies have demonstrated that a relevant percentage of PAPS patients have an up-regulation of IFN genes in peripheral blood mononuclear cells (PBMC). However, 20% and 28% of patients in these 2 cohorts, had antidsDNA positive antibodies, a highly specific Systemic Lupus Erythematosus (SLE) autoantibody. Objective: The aim of this study is to determine the prevalence of type I IFN signature in PBMC of patients with PAPS without specific SLE autoantibodies and search for it with clinical and laboratorial associations. Methods: 53 PAPS patients (according to Sydney´s criteria) were consecutively selected and age-matched with 50 healthy controls. A third group, with non-immune-mediated thrombophilia patients, was also included. The expression of 41 IFN induced genes was analysed using real time quantitative PCR (TaqMan Low Density Array). A principal component analysis (PCA) was used to determine which genes should compose the IFN signature and z-score was calculated. The IFN signature score cut-off was defined with a ROC curve, as the point that maximized both the specificity and sensitivity. Clinical and laboratorial features were analysed searching for associations with IFN signature. Results: 11 IFN genes were highly expressed in primary APS patients. After PCA, 6 genes remained in the IFN signature: DNAJA1, IFIT5, IFI27, MX1, IFI6, TYK2. The ROC cutoff was 3,9 folds (AUC = 0.706, S = 0.49, E = 0.86, VPP = 0.79, VPN = 0.61). The type I IFN signature was present in 49% of patients with primary APS compared to 14.0% of healthy controls and 17% of non-immune-mediated thrombophilia patients (p < 0.0001). The mean IFN score was significantly higher in PAPS patients (4.0 fold higher, p < 0.0001) than in controls. A higher IFN signature was associated with a younger age at the first APS event (p = 0.023) and with the presence of obstetric events, especially with preeclampsia (p = 0.032). There was no association between IFN signature and number of thrombotic events, laboratory exams, comorbidities, family history of autoimmune diseases, and thrombosis risk scores. Treatment with statins was associated with lower levels of IFN scores (p = 0.026). Conclusion: our result indicates that PAPS patients, without lupus specific antibodies, have an enhanced type I IFN gene signature, not observed in non-immune mediated thrombophilia. We also provide novel data demonstrating that this overexpression of type I IFN-regulated genes is associated with an earlier onset of APS events and preeclampsia. Further studies are necessary to determine if this subgroup of patients will benefit of interventions targeting the type I IFN signalling pathway