Cigarette smoke contributes to many diseases. The actions of second and third hand smoke, which have implications for non-smokers and the very young, are just beginning to be appreciated. The overarching hypothesis of this project is that cigarette smoke has different injurious actions on alveolar cells based on chronological age. The purpose here was to learn more about the susceptibility of alveolar cells to cigarette smoke extract (CSE)- induced injury by performing studies on pulmonary alveolar and endothelial cells derived from neonatal, young, and old rats. The aims involved: 1. Developing cell culture models to study age-related effects of cigarette smoke on alveolar type I cells and microvascular endothelial cells from the lung, and 2. Using these models to examine the effects of CSE on markers of oxidative stress, inflammation and aging in alveolar cells harvested from neonatal, young and old rats. Descriptive and experimental studies involved using a variety of cell culture, biochemical and molecular techniques, including gene expression arrays. The most significant findings were that: 1. primary proliferating alveolar type I cells were used to develop novel cell culture model systems, including single culture, co-culture and three-dimensional cultures that were used to study the effects of CSE; 2. Hydrogen peroxide production by endothelial cells was markedly reduced by co-culturing with AT I cells; 3. Gene expression profiling of oxidative stress-specific pathways suggest that genes responsible for both stopping production of H2O2 or mopping-up H2O2 are involved; and 4. Cigarette smoke shortens telomeres of cells from neonates, but unexpectedly preserves telomere length of cells from young and old rats. Data from telomeric pathway-specific gene expression arrays suggest that there are age-related differences in response to gene expression to CSE. The significant conclusions are: 1. Contrary to prior observations, alveolar type I cells demonstrate prolonged proliferative capacity; 2. Alveolar type I cells likely play an important role in ameliorating CSE-induced oxidative stress; and 3. Neonatal alveolar cells may be more susceptible to the deleterious effects of CSE including telomere shortening. These novel model systems and observations provide new ways to study cigarette smoke-associated lung dysfunction.

L’extrémité des chromosomes linéaires est une structure nucléoprotéique très conservée chez les organismes eucaryotes. Elle est constituée du télomère et des régions sous-télomériques répétées (STR) qui sont placées en amont du télomère. Chez la levure bourgeonnante, on trouve deux types de télomère, les télomères XY’ et les télomères X, qui se distinguent par la nature des STR positionnées en amont des répétitions télomériques. Le télomère et les STR sont liés par pas moins de dix protéines qui vont participer au maintien et à la régulation de l’extrémité chromosomique nécessaires à la stabilité du génome. Le télomère protège ainsi le chromosome de dégradations ou encore de fusions avec d’autres chromosomes. Le maintien de la taille du télomère est assuré par la télomérase, une transcriptase inverse, qui permet l’ajout de répétitions pour pallier leur perte lors de la phase de réplication durant le cycle cellulaire. Lorsque la télomérase est absente, deux types particuliers de cellules, les survivants de type I et les survivants de type II, peuvent maintenir leurs télomères grâce aux mécanismes de recombinaison homologue. Chez l’humain, les répétitions télomériques sont également liées par un certain nombre de protéines nécessaires au maintien de la stabilité de l’extrémité chromosomique. L’implication des télomères dans les processus de cancérisation, de vieillissement, mais également dans des maladies congénitales fait de cette structure un pivot dans le domaine de la recherche fondamentale. Dans 10 % des cas de cancers, l’allongement n’est pas dû à une réactivation de la télomérase comme c’est en général le cas, mais est inhérent à des processus de recombinaison homologue, comme chez la levure. Les homologies de séquences, de protéines, mais aussi de mécanismes de régulation des télomères avec les cellules humaines, font de S. cerevisiae un excellent modèle d’étude. Cette thèse se divise en trois chapitres. Les deux premiers traitent de l’interaction du complexe yKu avec les télomères de type XY’ dans le chapitre 1 puis de son interaction avec les télomères de type X dans le chapitre 2. Le chapitre 3 traite du comportement d’un type de survivant chez S. cerevisiae. Le chapitre 1 porte donc sur l’analyse des sites de liaison aux télomères XY’ du complexe yKu par la technique de ChEC in vivo. yKu intervient dans de nombreux processus de régulation des télomères, mais aussi dans un mécanisme de réparation des cassures double-brin de l’ADN (DSBs), la NHEJ (Non homologous end-joining). Les résultats présentés dans cette partie appuient un modèle dans lequel yKu aurait plusieurs sites de liaison aux télomères et dans les répétitions télomériques interstitielles. Nous supposons que la liaison du complexe se ferait lors de la formation d’une cassure de type « one-sided break » générée à la suite du passage de la fourche de réplication à l’intérieur des répétitions télomériques. Le chapitre 2 est également une étude des sites de liaison par la technique de ChEC in vivo du complexe yKu, mais cette fois-ci aux télomères X. Les observations faites dans cette partie viennent corroborer les résultats du chapitre 1 de la liaison de yKu à la jonction entre le télomère et les STRs, de plus elle met en évidence des interactions potentielles du complexe avec les éléments X laissant supposer l’existence d’un potentiel repliement du télomère sur la région sous-télomérique chez la levure. Enfin, le chapitre 3 est axé sur l’étude du comportement des survivants de type I, des cellules post-sénescences qui maintiennent leurs télomères par un processus de recombinaison homologue, le mécanisme de BIR (break-induced replication) en l’absence de télomérase. Les survivants de type I présentent une croissance lente liée à un arrêt du cycle cellulaire en phase G2/M qui dépend de la protéine de contrôle Rad9, dont l’activité est en général induite par des cassures double-brin. Ce chapitre a permis d’apporter des précisions sur la croissance lente probablement inhérente à un berceau télomérique très restreint chez ce type cellulaire.

Autoimmune diseases are clinical manifestations of a loss-of-tolerance of the immune system against the body’s own substances and healthy tissues. Primary Sjögren’s syndrome (pSS) and systemic lupus erythematosus (SLE) are two chronic inflammatory autoimmune diseases characterized by autoantibody production and an activated type I interferon system. Although the precise mechanisms leading to autoimmune processes are not well defined, recent studies suggest that aberrant DNA methylation and gene expression patterns may play a central role in the pathogenesis of these disorders. The aim of this thesis was to investigate DNA methylation and gene expression in pSS and SLE on a genome-wide scale to advance our understanding of how these factors contribute to the diseases and to identify potential biomarkers and novel treatment targets. In study I, differential DNA methylation was analyzed in multiple tissues from pSS patients and healthy controls. We identified thousands of CpG sites with perturbed methylation; the most prominent finding was a profound hypomethylation at regulatory regions of type I interferon induced genes in pSS. In study II, a cases-case study comparing DNA methylation in pSS patients with high fatigue to patients with low fatigue, we found methylation patterns associated to the degree of fatigue. In study III, RNA-sequencing was applied to investigate the transcriptome of B cells in pSS in comparison to controls. Increased expression of type I and type II interferon regulated genes in pSS was observed, indicating ongoing immune activation in B cells. In study IV, the impact of DNA methylation on disease susceptibility and phenotypic variability in SLE was investigated. We identified DNA methylation patterns associated to disease susceptibility, SLE manifestations and different treatments. In addition, we mapped methylation quantitative trait loci and observed evidence for genetic regulation of DNA methylation in SLE.   In conclusion, the results presented in this thesis provide new insights into the molecular mechanisms underlying autoimmunity in pSS and SLE. The studies confirm the central role of the interferon system in pSS and SLE and further suggest novel genes and mechanisms to be involved in the pathogenesis these autoimmune diseases.

Master of Science / Department of Statistics / Christopher Vahl / In animal health research, it is quite common for a clinical trial to be designed to demonstrate the efficacy of a new drug where a binary response variable is measured on an individual experimental animal (i.e., the observational unit). However, the investigational treatments are applied to groups of animals instead of an individual animal. This means the experimental unit is the group of animals and the response variable could be modeled with the binomial distribution. Also, the responses of animals within the same experimental unit may then be statistically dependent on each other. The usual logit model for a binary response assumes that all observations are independent. In this report, a logit model with a random error term representing the group of animals is considered. This is model belongs to a class of models referred to as generalized linear mixed models and is commonly fit using the SAS System procedure PROC GLIMMIX. Furthermore, practitioners often adjust the denominator degrees of freedom of the test statistic produced by PROC GLIMMIX using one of several different methods. In this report, a simulation study was performed over a variety of different parameter settings to compare the effects on the type I error rate and power of two methods for adjusting the denominator degrees of freedom, namely “DDFM = KENWARDROGER” and “DDFM = NONE”. Despite its reputation for fine performance in linear mixed models with normally distributed errors, the “DDFM = KENWARDROGER” option tended to perform poorly more often than the “DDFM = NONE” option in the logistic regression model with one random effect.

<p>It is estimated that 1 in 500 Americans are inflicted with type I diabetes (T1D) with approximately 18,000 children and adolescents diagnosed each year. Islet/β cell replacement with long-lasting glucose-sensing and insulin-releasing functions has the potential to eliminate the need for insulin injections and minimize complications for individuals with T1D. However, limitations remain precluding it from widespread clinical use, including i) limited donor supply, ii) significant loss of functional islet mass upon transplantation, iv) limited functional longevity, and v) need for life-long systemic immunosuppression. To restore glucose-responsive insulin-release back to the patient’s body without the need for systemic immunosuppression, our approach involves a subcutaneous injection using a novel fibril-forming biologic, type I oligomeric collagen (Oligomer). Oligomer protects and in situ encapsulates replacement cells beneath the skin by transitioning from a liquid to a stable collagen-fibril scaffold, within seconds, just like those found in the body’s tissues. Preclinical validation studies in streptozotocin-induced diabetic mice show that replacement of islets at a dose of 500 or 800, results in a rapid (within 24 hours) reversal of hyperglycemia. All animals receiving syngeneic islets maintained euglycemia for beyond 90 days, while >80% of animals receiving allogeneic or xenogeneic (rat) islets remained euglycemia for at least 50 days. Histopathological analysis of Oligomer-islet implants showed normal morphology with no apparent evidence of a foreign body response and immune cell infiltrate. To our knowledge, this is the first report of an injectable subQ islet transplant strategy that yields rapid lowering and extended glycemic control without systemic immunosuppression.</p>

Osteoporose é a doença osteometabólica mais comum na população idosa. É definida como redução difusa da qualidade óssea que surge por um desbalanço entre a reabsorção e a formação óssea. Vários fatores etiológicos acontecem juntos levando à redução das proteínas ósseas, entre eles: deficiência hormonal, senilidade, subnutrição e imobilização. Um fator etiológico que, além dos processos de mutação, leva a alteração da estrutura do colágeno no processo de envelhecimento é o ageing, que se caracterizado por mudanças estruturais na matriz extracelular e virtualmente atinge todos os tecidos e sistemas orgânicos. Mudanças nas ligações cruzadas têm sido considerada como o papel principal do ageing e essas ocorrem através de 2 principais maneiras: a primeira é um processo enzimático pela ação da lisil oxidase e a segunda é um processo não-enzimático e recebe o nome de glicação. Essa última envolve reação da glicose com arginina (Arg) e lisina (Lys) sendo a principal causa de disfunção dos tecidos colágeno na idade avançada. No ageing há hidroxilação da lisina (Lys) levando modificação nas ligações cruzadas, o que gera uma estrutura de colágeno alterada. Além disso, a alteração em especial da arginina (Arg) afeta a crucial interação célula-matriz envolvendo motifs como o RGD (Arg-Gly-Asp), o que altera a mineralização e remodelagem óssea. Essas modificações envolvendo o intervalo 70-110 do período D do colágeno tipo I, que corresponde a interface overlap:gap tem uma séria repercussão, pois segundo o modelo de mineralização, qualquer variação de carga nesse local afeta a interação com a fosfoforina, uma proteína ácida que parece ser o gatilho para o processo de mineralização da dentina, e que também, de alguma forma, deve ocorrer no tecido ósseo. Assim, em um colágeno bioquimicamente modificado pelas reações do ageing leva, entre outras coisas, a uma alteração na mineralização, e essa alteração seria mais um fator a ser considerado no estudo da osteoporose. / Osteoporosis is the most commom osteometabolic disease in the elderly. It´s definied like difuse reduction of bone density that arouses from a delicate debalance between bone reabsorption and formation. Several etiologic factors happens together and they result in the reduction of bone proteins, like hormonal deficiency, senility, bad nutrition and imobilization. Another etiologic factor that causes the collagen struture modification in old age it\'s ageing, characterized for structure modification on extracelular matrix and virtually affect all tissue and organic system. Modification on cross-link have been considered like main reason of ageing and these modifications occur through two main ways: the first one is an enzimatic process for the lysil oxidase action and the second is a non-enzimatic process denominated glycation. The last process involve the glucose reaction with arginine (Arg) and lysine (Lys) being the main cause of the collagen tissue disfunction in advanced age. In ageing process there is a lysine (Lys) hidroxilation modification the cross-link what results a fragilized collagen structure. Besides, the special modification of arginine (Arg) affects the crucial interaction of the cell matrix involving motifs like RGD (Arg-Gly-Asp), around the interval 70-110 of period collagen type I, that corresponds to the interface overlap:gap has a serious repercussion, because according to the mineralization standard, which charge alteration in this place affects the interaction with phosphoforin an acid protein that seems to be the responsible of mineralization process. In this manners, in a biochemically modified collagen causes, among other things, to a mineralization alteration, and it has its consequences on osteoporosis.

A imunoterapia baseada em MoDC constitui uma estratégia para tratamento de indivíduos HIV+. Protocolos para obtenção de MoDC em geral utilizam IL-4 e GM-CSF (IL4-DC). Alguns estudos utilizam as IFN-DC (IFN-&alpha; + GM-CSF), que exibem um fenótipo combinado de DC mielóide, DC plasmocitóide (pDC) e célula NK. Esse perfil misto pode aperfeiçoar a imunoterapia para pacientes HIV+. Para tanto, monócitos de pacientes HIV+ foram cultivados com GM-CSF e IL-4 ou IFN-&alpha; por 5 dias e estimuladas por 48 horas com pulso de HIV inativado por AT-2 e/ou coquetel de citocinas pró-inflamatórias. Avaliamos a expressão de moléculas de superfície de IFN-DC e ativação de linfócitos T por citometria de fluxo; produção de citocinas IL-12 e IL-10 por ELISA. IFN-DC apresentaram morfologia e fenótipo basais ativados e características de pDC e célula NK, diferente das IL4-DC. As IFN-DC foram capazes de produzir IL-12, estimular a proliferação e produção de IFN-&gamma; de linfócitos TCD4 e CD8, porém similares às IL4-DCs. IFN-DC são capazes de estimular resposta de linfócitos T tanto quanto IL4-DC. / Immunotherapy based on MDDCs is a strategy for treating HIV-infected patients. Alternatively to the conventional protocol for DC differentiation based on IL-4 and GM-CSF (IL4-DC) some studies suggest the use of IFN-DC (IFN-&alpha; + GM-CSF). These cells exhibit a combined phenotype of myeloid DC, plasmacytoid DC (pDC) and NK. Considering the mixed profile of IFN-DCs alternative protocols can bring novel elements for immunotherapy. Monocytes isolated from HIV-infected patients were cultured in the presence of GM-CSF and IL-4 or IFN-&alpha;. On day 5 DCs were pulsed with AT-2-inactivated HIV and stimulated for 48 hours with a cocktail of proinflammatory cytokines. We assessed IFN-DC surface markers expression and T cell activation by flow cytometry; IL-10 and IL-12 production by ELISA. IFN-DC showed activated morphological and phenotypic features during basal state of maturity and exhibited features of pDC and NK different from IL4-DC. The IFN-DC like IL4-DC were able to produce IL-12 and stimulated T cells. So, the IFN-DC were able to stimulate the T cells as well as IL4-DCs.

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

Differential Display technique was used for gene profiling in trnasformed human umbilical vein endothelial cell line (ECV 304) and primary human umbilical vein endothelial cells (HUVECs) to study the cellular response to viral infection. After screening the mRNA from uninfected and infected HUVECs and ECV 304 cells with 16 different random primers we identified 8 gene targets. These genes included the human inhibitor of apoptosis-1 (h-IAP1), 2'-5' oligoadenylate synthetase (2'-5' OAS), 2'-5' oligoadenylate synthetase-like (2'-5' OAS-like), Galectin-9 (Gal-9), MxA, Mx1, Regulator of g-protein signaling (RGS2) and endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). We found that HUVECs were a better model to study gene expression dureing dengue 2 virus infection but not the transformed cell line, ECV 304. Of the 41 primer combinations utilized in ECV 304 cells detected only one upregulated gene, h-IAP1 and 8 out of the 16 primer combinations tried for HUVECs. We hypothesize the activation of two novel signaling pathways (Tumor necrosis factor- alpha (TNF-alpha), Interleukin1-beta (IL1-beta) in endothelial cells during D2V infection. ALso, our data detected genes that are activated in the Type-I IFN (IFN alpha/beta) signaling pathway during dengue 2 virus infection in HUVEC.

Type I diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreas. Destruction of the body's own proteins, cells, and tissues is precipitated by the dysfunction of cytokine production, protein modification, and signaling pathways in immune cell subtypes. Tumor Necrosis Factor α (TNFα) and its receptors Tumor Necrosis Factor 1 (TNFR1) also known as p55 and TNFRSF1A, and Tumor Necrosis Factor 2 (TNFR2) also known as P75 and TNFRSF1B play a crucial role in this autoimmune process. TNFα has been shown to stimulate cell death through TNFR1 signaling by the caspase system, while promoting cell survival through TNFR2 signaling using the Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells (NF-𝜅B) pathway. Recent findings show a defect in immuno-proteasomes found in autoreactive T cells in people with T1D. This defect causes improper signaling transduction when TNFα binds to TNFR2. The inability to save the cell by activating the NF-𝜅B pathway eventually leads instead to apoptosis using the caspase system. A decrease in TNFα or increase in soluble TNFα receptors might be an explanation for these autoreactive T cells to evade the host immune system, and allow them to cause destruction of the pancreas. We hypothesize that patients with T1D will show abnormal distribution of TNFα and its receptors at basal levels, as well as when stimulated with interleukins, cytokines, and bacteria such as interleukin-2 (IL-2), lipotechoic acid (LTA), granulocyte macrophage-colony stimulating factor (GM-CSF), and Bacillus Calmette-Guérin (BCG). To test this hypothesis, we obtained peripheral blood from T1D patients (n=102) and controls (n=89) and performed in vitro stimulation assays. After a 48-hour incubation, tissue culture supernatants were collected and analyzed for TNF and its receptors production by ELISA, as well as densities of cell membrane receptors by flow cytometry. The data from this study showed significant differences in basal levels of TNFα, TNFR1, and TNFR2 on both the membrane and in the serum between patients and controls. Patients contained a greater percentage of CD4, 8, and 14 - TNFR2 and not TNFR1 double positive cells than their healthy control counterparts. Patient's sera also contained higher levels of all three markers, sTNFα, sTNFR1, and sTNFR2 than the controls. However, no significant differences were found between patient and controls when stimulated with the various compounds listed above.