Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-14T12:30:34Z No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:05:05Z (GMT) No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Made available in DSpace on 2016-10-22T13:05:05Z (GMT). No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) Previous issue date: 2008-02-15 / Nos países tropicais, as perdas causadas pela infestação de carrapatos em bovinos acarretam um grande impacto no sistema de produção animal. Recentes estudos têm mostrado a importância de fatores genéticos ligados a resistência a carrapato em Bos taurus indicus e Bos taurus taurus e que as citocinas têm um papel crítico na prevenção ou progressão de doenças. O objetivo desse trabalho foi avaliar os níveis de expressão dos genes IL-10 e IL-4 relacionados ao perfil imunológico Th2 associado à susceptibilidade ao carrapato e os genes IL-2 e IFN- relacionados ao perfil imunológico Th1 associado à resistência ao parasito. Além destes genes, analisou-se o perfil de expressão do gene TLR-2, importante no processo de reconhecimento de patógenos e os genes IL-8 e TNF-α importantes no processo inflamatório inicial. Seis animais mais resistentes e seis animais mais susceptíveis de uma população F2 de 332 animais, originária do cruzamento de animais F1(½ Holandês: ½ Gir), foram selecionados baseado na contagem de carrapatos e valor genético. Amostras de tecido foram coletadas de pele no 5° e 12° dias após a infestação para extração de RNA total. As PCRs em tempo real foram realizadas usando o gene GAPDH como controle endógeno. Os animais resistentes e susceptíveis apresentaram aumento de expressão do gene IL-10 no 5° (p<0,01) e 12 ° dias após a infestação (p<0,05). O gene IL-2, nos animais resistentes e susceptíveis, no 5° dia após a infestação não apresentou alteração da expressão sendo que 12° dia, em ambos os grupos de animais, este gene passou a ser mais expresso em relação ao animal controle sugerindo um perfil de resposta imunológica do tipo de Th2 nos animais resistentes e susceptíveis nos primeiros dias após a infestação. O gene IL-4 apresentou uma tendência ao aumento de expressão nos animais resistentes e susceptíveis em relação ao controle, sendo o perfil Th2 sugerido atribuído a IL-10 produzida por linfócitos T regulatórios (p>0,05). O gene TNF- apresentou aumento de expressão nos animais susceptíveis no 5° dia após a infestação com posterior diminuição no 12° dia após a infestação (p<0,05). Nos animais resistentes não foi observada alteração da expressão deste gene, isto sugere que ele possa estar mais atuante no início do processo inflamatório, logo após a fixação do carrapato. A mesma observação estende-se para o gene IL-8, em que não foi verificada alteração de expressão nos animais resistentes, embora nos animais susceptíveis este gene apresentou diminuição da expressão no 12° dia após a infestação (p<0,05). Quanto ao gene IFN-, não houve diferença de expressão entre os animais resistentes e susceptíveis, sendo que este gene parece não estar relacionado ao mecanismo de resistência. O gene TLR-2 apresentou diminuição da expressão em ambos os grupos de animais. Estes resultados sugerem que a resposta imune adquirida avaliada neste trabalho não apresenta papel preponderante no mecanismo de resistência e que resposta imune inata poderia está envolvida no mecanismo de resistência ao carrapato. Portanto, avaliação da resposta imunológica horas após a fixação do carrapato poderia nos fornecer resultados mais conclusivos. / In tropical countries losses caused by tick infestation in cattle lead to a major impact on animal production systems. Recent studies have shown the importance of genetic factors linked to tick resistance in Bos indicus and Bos taurus as well as the critical role in the prevention or progression of diseases mediated by cytokines. The aim of this work was to evaluate gene expression of IL-10 and IL-4 in relation to tick susceptibility associated with the Th2 profile and gene expression of IL-2 and IFN- in relation to tick resistance associated with the Th1 profile. In addition, the expression of TLR-2, important in the process the recognition of pathogens, and TNF-α and IL-8 genes, important in the initial inflammatory process, were evaluated. Six tick-resistant and six tick-susceptible animals from a F2 population of 332 animals, originated from the cross of F1 animals (½ Holstein: ½ Gir), were selected based on tick count and breeding value for tick resistance. Skin biopsies were collected in the 5th and 12th days after tick infestation. The GAPDH was used as endogenous control to normalize the amount of starting cDNA target in the real-time PCR assay. Both resistant and susceptible animals showed increased gene expression of IL-10 in the 5th and 12th days after infestation in relation to control animal (p<0.05). The IL-2 gene showed no change of expression in the 5th day after infestation for the resistant and susceptible animals. In the 12th post infestation, both resistant and susceptible animals showed increased gene expression in relation to control animal. These results suggest an enhancement of Th2 profile through the increase of IL-10 mRNA levels and a possible inhibition of the Th1 pattern in both groups (resistant and susceptible) starting 5 days after infestation and return to normal by day 12. Despite our results suggest the occurrence of the Th2 profile, the susceptible and resistant animals did not show variation on gene expression for IL-4 in relation to control animal. The susceptible animals showed increased expression of TNF-α in the 5th day after infestation. However, in the 12th day post infestation it was noted a decrease in the gene expression level. The resistant animals showed no change in the expression of this gene in relation to control animals suggesting that TNF-α could be more actively expressed in the early steps of the inflammatory process. Similarly, the resistant animals showed no variation in the expression of IL-8 while the susceptible animals showed increased expression in the 12th day post infestation. There were no differences of expression between resistant and susceptible animals in relation to IFN-γ what suggests that this gene might not be involved in the resistance mechanism. The TLR-2 gene showed decreased expression in both resistant and susceptible animals (p<0.05). Finally, there was no difference in expression between susceptible and resistant animals in relation to all selected genes in the 5th and 12th days after infestation. These results suggest that the acquired immunity evaluated in this work might not have preponderant role in the resistance mechanism. The innate immunity might be playing a major role in the bovine tick resistance/susceptibility mechanism in early hours after infestation.

Background &amp; Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system. Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats. Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet. / Doctoral thesis

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Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the immune-mediated destruction of insulin-producing beta-cells of pancreatic islets, culminating in critical insulin deficiency. Both genetic and environmental factors likely orchestrate an immune-mediated functional loss of beta cell mass, leading to the clinical manifestation of disease and lifelong dependence on insulin therapy. Additional evidence suggests the role of innate and adaptive immune mechanisms leading to inflammation in beta cells mediated by proinflammatory cytokines and chemokines, activation of beta-cell-reactive T cells,and failure of immune tolerance. Viral infections have been proposed as causal determinants or initiating triggers for T1D but remain unproven. Understanding the relationship between viral infections and the development of T1D is essential for T1D prevention. Importantly, virus-induced innate immune responses, particularly type I interferon (IFN-I, IFN-a/b), have been implicated in the initiation of islet autoimmunity and development of T1D. The goal of my thesis project is to investigate how the IFN-I signaling pathway affects the development of T1D using the LEW.1WR1 rat model of autoimmune diabetes. My hypothesis is that disrupting IFN-Isignaling via functional deficiency of the IFN-I interferon receptor (IFNAR) prevents or delays the development of virus-induced diabetes.For this purpose, I generated IFNAR subunit 1(IFNAR1)-deficient LEW.1WR1 rats using CRISPR-Cas9 genome editing and confirmed the functional disruption of IFNAR1. The absence of IFNAR1 results in a significant delay in onset and frequency of diabetes following poly I:C challenge and reduces the incidence of insulitis after poly I:C treatment. The frequency of diabetes induced by Kilham rat virus (KRV) is also reduced in IFNAR1-deficient LEW.1WR1 rats. Furthermore, I observe a decrease in CD8+T cells in spleens from KRV-infected IFNAR1-deficient rats relative to that in KRV-infected wild-type rats. While splenic regulatory T cells are depleted in WT rats during KRV-infection, no such decrease is observed in KRV-infected IFNAR1-deficient rats. A comprehensive bulk RNA-seq analysis reveals a decrease of interferon-stimulated genes and inflammatory gene expression in IFNAR1-deficient rats relative to wild-type rats following KRV challenge. Collectively, the results from these studies provided mechanistic insights into the essential role of virus-induced, IFN-I-initiated innate immune responses in the early phase of autoimmune diabetes pathogenesis.

To understand molecular mechanisms of individual steps of virus infection is a prerequisite for successful design of specific and effective antiviral drugs. Polyomaviruses, replicating in the cell nucleus, travel from plasma membrane to the endoplasmic reticulum (ER) in endosomes. However, it is not clear how they deliver their DNA genomes from ER to the nucleus. In this thesis, we found that partially disassembled virions of the Murine polyomavirus (MPyV) interact with importin β1 at around 6 hours post infection. Mutational disruption of the nuclear localization signal (NLS) of the major capsid protein, VP1, and/or common NLS sequence of the minor capsid proteins VP2 and VP3 did not affect the structure and composition of virions, but it resulted in decreased viral infectivity (up to 80%). Virions are thus released from ER to cytosol and translocate to the nucleus via nucleopores. Mutation analyses of NLSs of individual capsid proteins showed that MPyV virions can utilize VP1 and VP2/VP3 NLSs in concert. However, one functional NLS, either that of VP1 or VP2/3 seems to be sufficient for the delivery of VP1-VP2/3 complexes into the nucleus, although none of these proteins is delivered into the nucleus separately. Thus, the conformation of NLS regions given by the presence of all three capsid...

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Das human pathogene Varicella-Zoster Virus (VZV) gehört zur Familie der Herpesviren und ist weltweit verbreitet. Die Primärinfektion verursacht Varicellen, welche durch einen bläschenartigen Hautausschlag charakterisiert ist. Im Anschluss daran etabliert VZV eine lebenslange Latenz und verursacht nach Reaktivierung Herpes Zoster. Seit 2004 ist der Lebendimpfstoff aus attenuierten Virionen des VZV-Stammes V-Oka in Deutschland empfohlen. Im Gegensatz zur Infektion mit zirkulierenden virulenten VZV Stämmen tritt nach Verimpfung des Vakzin-Stammes V-Oka kein Exanthem auf. Die Haut ist der Hauptreplikationsort von VZV und immunologische Unterschiede zwischen virulentem VZV und dem Vakzin-Stamm treten hier am deutlichsten auf. In der vorliegenden Arbeit konnte eine neue Immunevasionsstrategie virulenter VZV Stämme aufgedeckt werden, welche erklären könnte, wie virulente VZV Stämme frühe antivirale Immunantworten umgehen. In Hautläsionen von Herpes Zoster Patienten konnte eine massive Infiltration von myeloiden inflammatorischen Dendritischen Zellen beobachtet werden. In vitro Studien mit Monozyten abgeleiteten Dendritischen Zellen (DC), welche inflammatorische DC repräsentieren, zeigten, eine signifikant erhöhte Expression von CD1c Molekülen nach Infektion mit dem Vakzin-Stamm, sowie virulentem VZV. Funktionelle Untersuchungen mit intraepithelialen CD1c-restringierten gamma delta T Zellen zeigten, dass DC nach Infektion mit dem Vakzin-Stamm phänotypisch und funktionell reiften und somit die T Zellen zur IFN-gamma Sekretion stimulierten. Im Gegensatz dazu wurde die funktionelle Reifung von DC, die mit virulentem VZV infiziert waren, geblockt. Folglich wurde kein bioaktives IL-12 sezerniert, welches als entscheidendes Cytokin zum Aufbau einer antiviralen T-Helfer 1 Immunantwort beiträgt. Darüber hinaus konnte gezeigt werden, dass virulentes VZV die Signalkaskade des Toll-like Rezeptors 2 (TLR2) in DC inhibiert und somit die IL-12 Produktion verhindert. / Varicella-zoster virus (VZV) which belongs to the family of herpesviruses is restricted to humans and distributed worldwide. Primary infection of VZV causes chickenpox characterized by a disseminated rash. Thereafter, VZV establishes a lifelong latency and can be reactivated to cause herpes zoster. Since 2004 the attenuated strain V-Oka of VZV was licensed for Germany to immunize children against VZV infection. In contrast to infection by circulating virulent VZV strains, vaccination with V-Oka remains asymptomatic. The skin is the major replication site of VZV and immunological differences between virulent VZV and the vaccine should become most apparent within this immune organ. In summary, this study discovered a new immune evasion strategy of virulent VZV strains which might explain how virulent VZV strains overcome innate antiviral responses. A strong infiltration of myeloid-derived inflammatory DCs has been detected in skin lesions of herpes zoster patients. In vitro studies with monocyte-derived dendritic cells (DCs), reflecting inflammatory DCs, showed that they were efficiently infected by both, the vaccine and a virulent VZV strain. Intriguingly, a significant upregulation of CD1c molecules on VZV-infected DCs was observed. Functional investigations using intraepithelial CD1c-restricted gamma delta T cells revealed that DCs infected with the vaccine virus were fully instructed to mature, thereby promoting IFN-gamma secretion of gamma-delta T cells. In striking contrast, DCs infected with virulent VZV strains were efficiently blocked to mature functionally. In detail, they did not secrete bioactive IL-12 which is an instrumental cytokine for generation of antiviral T helper 1 responses. Moreover, virulent VZV blocked Toll-like receptor 2 (TLR2) signaling in DCs thereby preventing production of bioactive IL-12 which in turn inhibited IFN-gamma secretion by gamma-delta T cells.

Albert Font Haro ABSTRACT Modulation of plasmacytoid dendritic cell function: role of immunoreceptors TIM-3 and BDCA-2 Plasmacytoid dendritic cells (pDCs) are key players in the antiviral response as well as in linking innate and adaptive immune response. They express endosomal toll-like receptors 7 and 9, which can detect ssRNA and unmethylated CpG DNA, respectively. Due to the constitutive expression of the transcription factor IRF7, pDCs are able to rapidly produce massive quantities of type I (α, β, ω) and type III (1, 2, 3, 4) interferons (IFN-I and IFN-III) as well as pro- inflammatory cytokines such as IL-1, IL-6 and TNF-α. After maturation, they also function as antigen-presenting cells. Despite intense research, the mechanisms of IFN and pro-inflammatory cytokines production and regulation are still poorly understood. Using the pDC cell line GEN2.2 and also primary human pDCs, we shed light on the role of kinases MEK and SYK in IFN-I production and regulation. We found that SYK is not only involved in the regulatory receptor (RR)-mediated BCR-like pathway that represents the negative regulation of IFN-I and IFN-III secretion but also in the positive TLR7/9-mediated signal transduction pathway that leads to IFN-I production, representing the immunogenic function. We also found that MEK plays a...