Gut-associated lymphoid tissues such as Peyer’s patches (PP) are inductive sites for immune response in the intestine. Unlike other peripheral lymphoid tissues, gut-associated lymphoid tissues lack afferent lymphatics and can directly sample mucosal antigens by specialized epithelial cells in the follicular associated epithelia (FAE), known as M cells. M cells derive from Lgr5+ intestinal stem cells in intestinal crypts, where the daughter cells of Lgr5+ cells differentiate into M cells after stimulation from the cytokine receptor activator of nuclear factor-κB ligand (RANKL). RANKL is produced by stromal cells within the sub-epithelial dome (SED) residing below the FAE. The transcytosis of antigens across the FAE by M cells is an important initial step in the induction of efficient mucosal immune responses against certain pathogenic bacteria as well as the commensal bacterial flora. However some pathogens, for example orally-acquired prions, may also exploit M cells to infect the host. M cells have been implicated in the uptake of orally acquired prions from the gut lumen. After oral exposure, the accumulation of prions in PP is important for their efficient spread to the nervous system. Previous studies have also shown that pathogen-induced inflammation increases M cell density and this effect can be mimicked by exogenous administration of RANKL. This has led to the hypothesis tested in this thesis that inflammation-related enhancement of M cell differentiation aids the delivery of prions into the lamina propria of villi. The administration of RANKL resulted in increased M cell density in the gut epithelium of mice. Consequently, RANKL treatment enhanced the accumulation of orally-administered prions in PP, decreased disease incubation time and increased prion disease susceptibility. These data indicate the importance of M cells in prion disease pathogenesis and highlight the potential of M cells as vaccine targets against prion disease. The fate and terminal differentiation of distinct intestinal epithelial cell lineages from their uncommitted precursors is dependent on their intrinsic expression of one or more specific transcription factors during their development. Alongside inducing M cell differentiation, RANKL stimulation can also induce the nuclear translocation of the NF-κB transcription factor subunit c-Rel. A comparison of the genes encoding the individual NF-κB subunits c-Rel, Rel-A and Rel-B revealed that they were expressed at the mRNA level in the FAE and by M cells. A c-Rel-deficiency in mice did not influence the expression of RANKL or RANK in PP. The subsequent induction of M cell maturation in the FAE was also unaffected in, indicating that c-Rel is dispensable for the RANKL-mediated differentiation and functional maturation of M cells. The factors implicated in Lgr5+ intestinal stem cell proliferation and their differentiation into M cells are poorly understood. Some reports have indicated that crypt-associated macrophages may provide extrinsic factors that assist Lgr5+ intestinal stem cell proliferation. In this thesis, the ablation of macrophages in the gut resulted in dysregulation of crypt microarchitecture, depleting Paneth cells and the Lgr5+ stem cells. This adversely affected the subsequent differentiation of intestinal epithelial cell lineages and impeded the functional development of M cells. These data reveal a previously unknown role for macrophages in the maintenance of intestinal crypts and intestinal stem cell proliferation and differentiation.

Os linfócitos T regulatórios (Tregs) desempenham um papel essencial no controle da tolerância periférica, regulando a homeostase do sistema imune, e, por este motivo, são consideradas a população celular - com fenótipo regulatório - mais importante do sistema imune. Sob estímulos específicos, as Tregs podem ser geradas naturalmente no timo (nTregs), ou serem induzidas na periferia (pTreg) a partir de células T naive. É possível gerar Tregs in vitro (iTreg) a partir de células T CD4+ naive isoladas de sangue de cordão umbilical, suplementando a cultura com TGF-?, atRA e IL-2. Haja vista o grande potencial terapêutico das iTregs, a comunidade científica apresenta grande interesse no entendimento dos mecanismos envolvidos em sua geração. Os mais recentes trabalhos indicam um importante papel da via NF-?B na geração destas células, especialmente no que tange o componente c-Rel. Entretanto, na literatura, não existem dados suficientes sobre a avaliação dos genes potencialmente orquestrados por este fator. Permeado por todo este questionamento, o presente trabalho avalia a interação de c-Rel com as regiões promotoras do genoma, em células T naive e nas células iTregs geradas in vitro. Em adição, também avalia o efeito do silenciamento da proteína c-Rel no perfil de geração das iTregs. Para isso, foi utilizada a metodologia de imunoprecipitação de cromatina atrelada à técnica de PCR em tempo real com primers, que cobrem as regiões promotoras de alguns genes com papel importante na biologia de células T naive e de iTreg. Também foi avaliado a eficiência de transfecção de siRNA contra c-Rel e também do siRNA marcado com molécula fluorescente FITC em conjunto com diferentes concentrações e diferentes reagentes de transfecção, utilizando células Jurkat e T naive. Nossos resultados mostraram uma melhor eficiência de transfecção do siRNA FITC, considerando as células viáveis transfectadas, com os agentes DharmaFECT®1 (22,14%), DharmaFECT®4 (43,14%) e DMRIE-C (27,59%) em células Jurkat e DharmaFECT®1 (16,35%) e DMRIE-C (25,94%) em células T naive, nas maiores concentrações, entretanto não é muito eficiente para a avaliação do silenciamento proteico por qPCR. Além disso demonstramos que em uma pureza média de 80,46% de células Tnaive isoladas obtivemos a geração das iTregs (CD4+CD25+CD127-FOXP3+) com 98,42%, em média, na expressão do marcador FOXP3, e essas células iTregs apresentam maior ligação de cRel aos promotores de genes como IL2RA (CD25) (média de 8,26 vezes), CD69 (3,71 vezes), regiões do gene FOXP3 (região promotora (20,15 vezes), região CNS - kb1 (16,9 vezes), kb2 (17,2 vezes) e kb3 (23,29 vezes)) e do próprio Rel (8,12 vezes). Estes resultados evidenciam os potenciais mecanismos de regulação exercidos por c-Rel, durante o processo de geração das iTregs. / Regulatory T lymphocytes (Tregs) play an essential role in the control of peripheral tolerance, regulating the homeostasis of the immune system, and are considered the cellular population - with regulatory phenotype - most important of the immune system. Tregs can be generated naturally in the thymus (nTregs), or be induced at the periphery (pTreg) from naive T cells, in dependence of specifcs stimuli. In cell culture, supplemented with factors such as IL-2, TGF-? and atRA, it is possible to generate in vitro Tregs (iTreg) from naive CD4 + T cells isolated from umbilical cord blood. Knowing the great therapeutic potential of iTregs, the scientific community has shown great interest in understanding the mechanisms involved in this process. Recent works indicate an important role of the NF-?B pathway in the generation of these cells, especially relating the involvement of the c-Rel componente, however, in the literature, there are not enough data on the evaluation of genes potentially orchestrated by this factor. With all this questioning, this thesis aimed to evaluate the interaction of c-Rel with the some promoter regions of the genome, naive T cells and in vitro generated iTregs cells, in addition, we have also attempted to evaluate the effect of c-Rel protein silencing on the generation profile of iTregs. For this, the methodology of chromatin immunoprecipitation coupled to the real-time PCR technique with primers was used, which cover the promoter regions of some genes with important role in the biology of naive and iTreg T cells, The efficiency of siRNA transfection against c-Rel and also the siRNA labeled with FITC fluorescence molecule in conjunction with different concentrations and different transfection reagentes, using Jurkat and naive T cells, was also evaluated. Our results showed a better transfection efficiency of the FITC siRNA, considering the viable cells transfected with the highest concentrations of the reagents DharmaFECT®1 (22,14%), DharmaFECT®4 (43,14%) e DMRIE-C (27,59%) with Jurkat cells and DharmaFECT®1 (16,35%) e DMRIE-C (25,94%) with naive T cells, however, was not very efficient for the evaluation by qPCR of silencing, that is, the reduction of c-Rel mRNA and RNA of other genes. In addition, we demonstrated that in an average purity of 80.46% of isolated naive T cells we obtained the generation of iTregs (CD4 + CD25 + CD127-FOXP3 +) with 98.42%, on average, in the expression of the FOXP3 marker, and in these iTregs cells, when compared to naive T cells, c-Rel subunit has bound to the promoters of the genes as IL2RA (CD25) (average de 8,26 folds), CD69 (3,71 folds), regions of the FOXP3 gene (promoter region (20,15 folds), CNS regions - kb1 (16,9 folds), kb2 (17,2 folds) and kb3 (23,29 folds)) and c-Rel promoter - Rel (8,12 folds). Our results demonstrate the possible regulatory mechanisms, such as some of the main roles of the c-Rel subunit during the generation process of iTregs.

No description available.

Der Transkriptionsfaktor NF-κB ist von großer Bedeutung, da er verschiedene zelluläre Prozesse wie Proliferation, Apoptose, Invasion oder Inflammation beeinflusst. Im Gegensatz zu den meisten anderen Zelltypen ist in der humanen Epidermis ein wachstumsinhibierender Effekt mit der Aktivierung von NF-ĸB assoziiert. Die epidermale Homöostase dient der Aufrechterhaltung der intakten Hautbarriere und beschreibt das Gleichgewicht zwischen proliferierenden und differenzierenden epidermalen Keratinozyten. Schädliche äußere Einflüsse wie übermäßige Sonnenlichtexposition können die epidermale Homöostase stören, was zur Entstehung epidermaler Neoplasien wie aktinischer Keratosen oder Plattenepithelkarzinomen beiträgt. Das Ziel dieser Arbeit war die Expressions- und Funktionsanalyse der einzelnen NF-κB Proteinuntereinheiten in humanen Keratinozyten in vitro. Die siRNA-vermittelte transiente Reduktion von c-Rel beeinflusste deutlich das Zellschicksal von Keratinozyten. Obwohl vorangegangene Experimente durch eine stärkere Zellproliferation nach Inhibierung der NF-ĸB Proteine p50 und p65 das Gegenteil suggerierten, konnte für die Reduktion von c Rel ein inhibierender Effekt auf das Zellwachstum festgestellt werden. Außerdem zeigten sich eine veränderte Zellzyklusphasenverteilung sowie eine Akkumulation mitotischer Zellen mit aberranter, hauptsächlich monopolarer Spindelformation. Die zusätzlich detektierte Apoptose-Induktion könnte aus dem verlängerten mitotischen Arrest der c-Rel Knockdown Keratinozyten resultieren. Insgesamt lässt sich ein regulatorischer Effekt von c-Rel beim Eintritt in die Mitose oder der mitotischen Progression vermuten, wobei die beteiligten Zielgene noch zu identifizieren sind. Des Weiteren bewirkte der c-Rel Knockdown phänotypische Veränderung von HaCaT Keratinozyten mit tendenziell spindelzellartiger Elongation und einem insgesamt veränderten Wachstumsmuster. Die Adhäsion und besonders die Wundheilung von c-Rel reduzierten HaCaT Zellen war vermindert, möglicherweise bedingt durch ein reduziertes Stressfaservorkommen. Dieser Effekt zeigte sich allerdings nicht in c Rel herunter regulierten primären Keratinozyten, was auf Mutationen der spontan immortalisierten HaCaT Keratinozytenzelllinie zurückzuführen sein könnte. Zusammenfassend konnte in dieser Arbeit ein neuer Aspekt der einzelnen NF-ĸB Proteine aufgezeigt werden, besonders in Bezug auf die Proteinuntereinheit c-Rel. Hieraus resultiert ein besseres Verständnis der vielfältigen und komplexen Regulation von NF-κB abhängigen Funktionen und deren Effekte auf die epidermale Homöostase.

Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines occurs at the transcriptional level. In Hep3B cells, the transcription factors C/EBPβ, STAT3, and Rel p50 have been shown to participate in this process. A C/EBP binding site centered at -53 and an overlapping nonconsensus κB site on the promoter are critical for CRP expression. We have previously found that an oligonucleotide containing a κB site diminished binding of C/EBPβ to the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B nuclei facilitate the formation of C/EBPβ-complexes. The current studies were undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB, play such a role. Mutation of the nonconsensus κB site did not abolish binding of C/EBPβ to its binding site, indicating that this site was not necessary for the formation of C/EBPβ-complexes. Depletion of Rel proteins from Hep3B nuclei led to decreased formation of C/EBPβ-complexes on a CRP promoter-derived oligonucleotide that contained only the intact C/EBP binding site but not the nonconsensus κB site. This finding indicates that Rel proteins are involved in the binding of C/EBPβ to its binding site by a κB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs) revealed that it was c-Rel that facilitated formation of C/EBPβ-complexes and that c-Rel bound directly to C/EBPβ-complexes formed on the C/EBP site. Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase activity and enhanced endogenous CRP expression in cells transfected with C/EBPβ. We conclude that c-Rel regulates CRP expression without the requirement of binding to a κB site, and binds directly to C/EBPβ to facilitate the binding of C/EBPβ to the CRP promoter.

No description available.

No description available.

No description available.

Type 1 diabetes is an autoimmune disease resulting in the selective destruction of the insulin producing β-cells in the pancreas. Pro-inflammatory cytokines and the free radical nitric oxide (NO) have been implicated in mediating the destruction of β-cells, possibly through activation of the mitogen activated protein kinases (MAPKs) JNK, ERK and p38. In addition to MAPKs, cytokine signaling also results in activation of the transcription factor nuclear factor-kappaB (NF-κB). The upstream signaling events leading to MAPK and NF-κB activation in β-cells are not well known. The work presented in this thesis therefore aims at characterizing the regulation of MAPKs and NF-κB in human islets, with emphasis on the role of the MAPK activator MAP/ERK kinase kinase-1 (MEKK-1) in islet cell death. It was found that MEKK-1 was phosphorylated in response to the nitric oxide donor DETA/NONOate (DETA/NO), the β-cell toxin streptozotocin (STZ) and pro-inflammatory cytokines and that MEKK-1 downstream signaling in response to the same treatments involved activation of JNK but not ERK and p38. MEKK-1 was also found to be essential for cytokine-induced NF-κB activation. MEKK-1 downregulation protected human islet cells from DETA/NO-, STZ, and cytokine-induced cell death. Furthermore, overexpression of the NF-κB subunit c-Rel protected human islet cells from STZ and hydrogen peroxide-induced cell death indicating that NF-κB activity protects against cell death in human islets. In summary, these results support an essential role for MEKK-1 in the activation of JNK and NF-κB, with important consequences for human islet cell death and that strategies preventing human islets death by inhibition of the JNK pathway instead of NF-κB might be suitable.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.