Die Entstehung verschiedener Tumorentitäten kann häufig mit einer vorliegenden Virusinfektion korreliert werden. So lässt sich beispielsweise ein Zusammenhang zwischen der Infektion mit den zur Gruppe der Herpesviren gehörenden Epstein-Barr-Viren (EBV) und der Entwicklung von Burkitt-Lymphomen, Hodgkin-Lymphomen sowie Nasopharynx-Karzinomen herstellen. Für die Transformation einer mit EBV infizierten humanen B-Zelle ist die Expression des Latenten Membranproteins 1 (LMP1) des Epstein-Barr-Virus essentiell. LMP1 ist ein Membranprotein mit einem funktionellen C-terminus, das Homologien zu den Tumornekrosefaktoren (TNF)- und Toll-like-Rezeptoren aufweist. Im Zusammenspiel mit anderen viralen Faktoren trägt LMP1 durch die von ihm aktivierten Signalwege (z.B. NF-κB, JNK, p38) zur Immortalisierung und malignen Entartung von EBV-infizierten Zellen bei. Einer dieser Signalwege ist der Jak/STAT-Signalweg. Da dessen EBV-abhängige Aktivierung bislang nur unzureichend geklärt ist, ist es Zielsetzung dieser Arbeit, die durch das Epstein-Barr-Virus vermittelte Aktivierung des Jak/STAT-Signalweges in Burkitt-Lymphomzellen näher zu untersuchen. Als Negativregulatoren des Jak/STAT-Signalweges sind hierbei die SOCS-Moleküle (Suppressor of Cytokine Signaling) von Bedeutung. Von besonderem Interesse sind dabei die Mechanismen der Aktivierung von SOCS3 durch LMP1 und dem damit verbundenen möglichen Einfluss auf den transformierten Zustand einer Zelle. In dieser Arbeit konnte gezeigt werden, dass in den untersuchten Burkitt-Lymphomzellen das exprimierte virale Onkoprotein LMP1 des Epstein-Barr-Virus ausreichend ist, den Jak/STAT-Signalweg zu aktivieren und SOCS3 zu induzieren. Die Aktivierung des Jak/STAT-Signalweges wird indirekt, das heißt über EBV-abhängig aktivierte Zytokinsignalwege, reguliert. Eine zusätzliche direkte Aktivierung durch eine Interaktion von Komponenten des Jak/STAT-Signalweges mit LMP1 kann derzeit jedoch nicht völlig ausgeschlossen werden.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Cutaneous lupus erythematosus (CLE) is an autoimmune disease that resolves with or without permanent scars depending on the subtype. Interferons (IFNs), including the skin specific IFNλ mainly activate STAT1, which results in inflammation in CLE and may play a significant role in scar formation in chronic discoid CLE. IL-22 activates STAT3 and it is emerging as a mediator with significant impact on normal wound repair, epidermal hyperproliferation and prevention of fibrosis. This work focussed on understanding the regulation and functional impact of IL-22 and IFNλ on skin cells. The counter-regulatory effect of IL-22 on the activities of IFNλ was assessed through downstream interferon stimulated genes (ISGs) expression in healthy and CLE keratinocytes. Cell proliferation and gap closure were investigated in skin resident cells using cell trace dye and scratch assay. Dermal fibroblasts were assessed for the presence of IFNλR1 and IL-22R1, downstream activities of the receptors. Results showed that IL-22 accelerated “scratch” closure in keratinocytes while IFNλ caused a delay in closure. IL-22 significantly downregulated IFNλ-induced chemokines expression in healthy, but not CLE keratinocytes. Reduced IL-22R1 expression and “STAT3 signature genes” was observed in CLE keratinocytes. A key finding of this project is that dermal fibroblasts respond to both IFNλ and IL-22. This work shows that IL-22 can reduce the damaging effect of IFNs in inflamed skin and also identifies dermal fibroblasts as important cells in skin immune responses. In conclusion, IL-10 family members can have both beneficial and destructive effects on the skin organ depending on the micro milieu and cell-type involved. Manipulating the balance of IL-10 family members in the skin may offer new therapeutic approach for both psoriasis and CLE.

Made available in DSpace on 2015-07-22T20:49:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O melanoma é um tumor de origem neuroectodérmica resultante da proliferação e transformação maligna dos melanócitos, os quais se originam de precursores da crista neural que então migram para a pele e folículos capilares durante o processo de embriogênese, e se apresentam como células especializadas que produzem melanina, encontradas na camada basal da epiderme e nos folículos do cabelo onde sua homeostase é regulada pelos queratinócitos da epiderme. A última década assistiu a grandes avanços em aspectos celulares e moleculares do melanoma. As implicações terapêuticas contribuíram para o entendimento das vias de regulação nas células tumorais que resultam de complexas alterações em múltiplas cascatas de sinalização, controlando a mobilidade, controle do crescimento, invasão, metabolismo, liberação de citocinas e capacidade de escapar da resposta imune. No presente trabalho, o silenciamento com shRNAi de uma proteína reguladora da via de sinalização JAK / STAT denominada SOCS- 1 foi capaz de reverter o fenótipo tumorigênico das linhagens de melanoma murino B16F10-Nex2 inibindo a progressão e desenvolvimento tumoral e assim, um novo marcador crítico do melanoma metastático pode ser estudado de maneira isolada. Nos ensaios in vitro o silenciamento de SOCS-1 inibiu o crescimento e regulação do ciclo celular, motilidade e a invasão de Matrigel pelas células de melanoma. O ensaio clonogênico demonstrou a participação de SOCS-1 como um modulador de resistência ao anoikis. Além disso, o silenciamento de SOCS-1 culminou na redução da expressão de receptores tais como receptor do fator de crescimento endotelial (EGF), receptor de insulina e fator de crescimento fibroblástico (FGF). Nos ensaios in vivo o silenciamento de SOCS-1 inibiu o crescimento tumoral e metastático. Coletivamente, estes dados sugerem que o silenciamento da expressão da proteína SOCS-1 em células de melanoma é um evento crítico, levando a um perfil não tumorigênico. O silenciamento da expressão de SOCS-1 é uma abordagem nova e promissora para aumentar os alvos terapêuticos e assim prevenir o desenvolvimento e a progressão do melanoma. / Melanoma is the most aggressive form of skin cancer and its incidence has increased dramatically over the years. The origin of melanoma is neuroectodermal and it results from the proliferation and malignant transformation of melanocytes that originate from the neural crest and migrate to the skin and hair follicles during embryogenesis. The last decade has seen major advances in both cellular and molecular aspects of melanoma biology and therapeutic implications. Knowledge on the regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now recognized that melanoma development is regulated by multiple cascade signaling pathways that affect, motility, growth control, invasion, metabolism, cytokine release and the ability to escape immune response. Here, we demonstrate a novel signaling mechanism whereby silencing of SOCS-1 protein, a negative regulator of Jak/Stat pathway, leads to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS-1 silencing with small hairpin RNA inhibits in vitro growth by cell cycle regulation and S phase arrest, motility inhibition and decreased melanoma cell invasion through Matrigel. Clonogenic assay showed that SOCS-1 acted as a modulator of anoikis resistance. Additionally, downregulation of SOCS-1 expression decreased the expression of epidermal growth factor (EGF), insulin receptor and fibroblast growth factor receptor (FGFR). We further demonstrate by in vivo assay that SOCS-1 silencing inhibits tumor growth and metastatic spread in the lungs. Collectively, these data show that silencing of SOCS-1 protein expression in melanoma cells is a critical event, leading to nontumorigenesis and incapacity to metastasize. / TEDE / BV UNIFESP: Teses e dissertações

HCV infection is associated with immune dysregulation and B cell Non-Hodgkins lymphoma (HCV-NHL). We have previously shown in vitro that HCV core protein differentially regulates T and B cell functions through two negative signaling pathways, programmed death-1 (PD-1) and suppressor of cytokine signaling-1 (SOCS-1). In this report, we performed a detailed immunologic analysis of T and B cell functions in the setting of HCV-NHL. We observed that T cells isolated from patients with HCV-NHL exhibited an exhausted phenotype including decreased expression of viral-specific and non-specific activation markers; whereas B cells exhibited an activated phenotype including over-expression of cell activation markers and immunoglobulins compared to healthy subjects. Individuals with HCV alone or NHL alone exhibited abnormal T and B cell phenotypes, but to a lesser extent compared to HCV-NHL. This differential activation of T and B lymphocytes was inversely associated with the expression of PD-1 and SOCS-1. Interestingly, blocking PD-1 during TCR activation inhibited SOCS-1 gene expression, suggesting that these regulatory pathways are linked in T cells. Importantly, blocking PD-1 also restored the impaired T cell functions observed in the setting of HCV-NHL. These results support a coordinated mechanism by which HCV might cause immune dysregulation that is associated to HCV-NHL.

Cutaneous lupus erythematosus (CLE) is an autoimmune disease that resolves with or without permanent scars depending on the subtype. Interferons (IFNs), including the skin specific IFNλ mainly activate STAT1, which results in inflammation in CLE and may play a significant role in scar formation in chronic discoid CLE. IL-22 activates STAT3 and it is emerging as a mediator with significant impact on normal wound repair, epidermal hyperproliferation and prevention of fibrosis. This work focussed on understanding the regulation and functional impact of IL-22 and IFNλ on skin cells. The counter-regulatory effect of IL-22 on the activities of IFNλ was assessed through downstream interferon stimulated genes (ISGs) expression in healthy and CLE keratinocytes. Cell proliferation and gap closure were investigated in skin resident cells using cell trace dye and scratch assay. Dermal fibroblasts were assessed for the presence of IFNλR1 and IL-22R1, downstream activities of the receptors. Results showed that IL-22 accelerated “scratch” closure in keratinocytes while IFNλ caused a delay in closure. IL-22 significantly downregulated IFNλ-induced chemokines expression in healthy, but not CLE keratinocytes. Reduced IL-22R1 expression and “STAT3 signature genes” was observed in CLE keratinocytes. A key finding of this project is that dermal fibroblasts respond to both IFNλ and IL-22. This work shows that IL-22 can reduce the damaging effect of IFNs in inflamed skin and also identifies dermal fibroblasts as important cells in skin immune responses. In conclusion, IL-10 family members can have both beneficial and destructive effects on the skin organ depending on the micro milieu and cell-type involved. Manipulating the balance of IL-10 family members in the skin may offer new therapeutic approach for both psoriasis and CLE. / University of Bradford and Centre for Skin Sciences

No description available.

No description available.

Les virus influenza aviaires faiblement pathogènes (IAFP) ciblent principalement les épithéliums des voies respiratoires et intestinales chez les poulets (Gallus gallus) infectés. Cependant, les virus influenza aviaires hautement pathogènes (IAHP) mènent à une maladie systémique fatale avec une localisation particulière aux endothéliums. L’objectif de cette thèse a été d’explorer les relations entre la réplication des virus influenza aviaires (IA) et la réponse antivirale de l’hôte dans deux modèles cellulaires originaux obtenus chez le poulet : des cellules épithéliales pulmonaires (CLEC213) et des cellules endothéliales d’aortes (chAEC). Les résultats clés sont les suivants : (i) la réplication productive des virus IA dans les chAEC dépend du clivage de l’hémagglutinine et de l’échappement viral à la réponse immunitaire innée ; (ii) les CLEC213 sont très permissives aux virus IA et présentent une faible réponse antivirale médiée par la signalisation TLR3 et MDA5 ; (iii) les fonctions régulatrices de SOCS1 et SOCS3, sur le signal des interférons et des cytokines, sont conservées chez le poulet. Nous proposons que certains virus IA peuvent exploiter les fonctions pro-virales de SOCS1 et SOCS3 à leur avantage de manière spécifique au type cellulaire. / Low pathogenic avian influenza (LPAI) viruses essentially target the epithelia of the respiratory and intestinal tract in the infected chicken host (Gallus gallus). However, highly pathogenic avian influenza (HPAI) viruses induce a peracute fatal systemic disease and exhibit a striking endothelial cell tropism. The objective of the present thesis was to explore the interdependencies of AI virus replication and the antiviral host response in two novel avian cell culture models: chicken lung epithelial cells (CLEC213) and chicken aortic endothelial cells (chAEC). The salient findings from this study are that (i) productive AI virus replication in chAEC is dependent on hemagglutinin cleavability and appears to be related to innate immune escape; (ii) CLEC213 are highly permissive to AI virus infection, due to a cell type-specific diminished TLR3- and/or MDA5-mediated antiviral signaling response; (iii) the interferon and cytokine regulatory functions of SOCS1 and SOCS3 are conserved in the chicken. Based on our data, we propose a model that predicts that certain AI viruses may exploit the proviral functions of SOCS1 and SOCS3 in a cell type-specific manner.