Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-06Bitstream added on 2014-06-13T19:44:15Z : No. of bitstreams: 1 leite_frm_dr_arafo.pdf: 1468835 bytes, checksum: e5bbe1112c569efdf39842ed3eb0c03b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A reabsorcao do osso alveolar e uma das principais caracteristicas associadas a progressao da doenca periodontal. Apesar da enorme complexidade da microbiota envolvida, considera-se que bacterias Gram-negativas tenham um papel relevante em sua etiopatogenese. Um dos fatores de virulencia destes microrganismos e representado por um componente de sua parede externa denominado lipopolissacarideo (LPS). A presenca de LPS na proximidade dos tecidos periodontais e capaz de induzir a producao de diversos mediadores inflamatorios que levam a degradacao tanto do tecido conjuntivo quanto osseo. Atualmente acredita-se que a interacao do ligante do receptor-ativador do fator nuclear kappa-B (RANKL) com seu receptor (RANK) presente em precursores hematopoieticos e necessaria e suficiente para a inducao da diferenciacao de osteoclastos. Por outro lado, a ligacao de RANKL com seu falso-receptor, denominado osteoprotegerina (OPG), reduz sua biodisponibilidade e inibe, desta forma, a osteoclastogenese. Assim, a razao da expressao de RANKL e OPG e considerada como o principal determinante do “turnover” do tecido osseo. A producao de RANKL e OPG depende das vias de sinalizacao ativadas, as quais sao influenciadas pela natureza do estimulo extracelular. Atualmente, a familia de receptores NLRs (nod-like receptors) foi identificada como receptor intracelular para componentes bacterianos e agentes moduladores de diferentes vias de sinalizacao. Considerando a relevancia do LPS bacteriano na patogenese da doenca periodontal, o papel do RANKL no processo de reabsorcao ossea e a possivel implicacao das proteinas Nod na transducao de sinais regulando a expressao de RANKL, o objetivo geral deste projeto foi estudar os mecanismos de regulacao da expressao de RANKL induzido por LPS bacteriano em celulas relevantes do periodonto (macrofagos, osteoblastos e fibroblastos). Os objetivos especificos propostos... / Bone resorption is one of the major characteristics of destructive periodontal disease. Despite the great number of different bacterial species in the dental biofilm, Gramnegative microorganisms were demonstrated to have a very important role on periodontal disease pathogenesis. Lipopolysaccharide (LPS) is a bacterial cell wall component, which is acknowledged as one of the main virulence factors of these microorganisms. The mere presence of LPS in proximity with the periodontal tissues initiates the expression and production of inflammatory mediators and other cytokines which can culminate in degradation of both soft and hard tissues. It is currently accepted that the interaction between receptor-activator of nuclear factor kappa-B ligand (RANKL) and its receptor (RANK) is both necessary and sufficient to induce osteoclast differentiation and activation. However, RANKL can interact with its soluble decoy receptor osteoprotegerin (OPG) inhibiting osteoclastogenesis by decreasing the bioavailability of RANKL. Production of RANKL/OPG is the result of the signaling pathways activated by external stimuli. Recently, the NLR (nod-like receptors) family was identified as cytosolic receptors for bacterial components and also, as capable of modulating different signaling pathways. Considering the relevance of LPS and RANKL in bone resorption and the possible implication of Nod proteins in signal transduction regulating RANKL expression, the aim of this study was to evaluate the influence of different intracellular signaling pathways on the regulation of RANKL expression induced by LPS in relevant cells of the periodontium (macrophages, osteoblasts and fibroblasts). The specific objectives proposed were to determine after LPS and interleukin-1 beta stimulation the role of MyD88-dependent and independent signaling pathways, Nod1 and Nod2 on the expression of RANKL, OPG, IL-10 and IFN-beta... (Complete abstract click electronic access below)

Heat shock protein (HSP)27 is traditionally described as an intracellular chaperone and signaling molecule, but growing evidence suggests it is released from immune cells where it plays an anti-inflammatory role during atherogenesis. Previously, the O’Brien lab found that overexpression of HSP27 led to augmented HSP27 serum levels in female apolipoprotein E knockout (ApoE-/-) mice, attenuated atherogenesis, and inhibited macrophage foam cell formation via physical binding with scavenger receptor (SR)-A. However, the precise mechanism of atheroprotection remained elusive. This thesis sought to ascertain the mechanism(s) by which HSP27 prevents foam cell formation, and determine if SR-A, a key receptor involved in the uptake of lipid into macrophages, plays an important role in HSP27-mediated atheroprotection. Pre-treatment of human macrophages with recombinant HSP27 (rHSP27) inhibited acytelated low density lipoprotein (acLDL) binding and uptake independent from receptor competition effect. Reduction in uptake was associated with attenuation of expression of SR-A mRNA, total protein, and cell surface expression. To explore the signaling mechanism by which HSP27 modulated SR-A expression it was hypothesized that nuclear factor-kappa B (NF-kB), a major regulator of many atherosclerosis gene programs, is altered by extracellular HSP27. Indeed, rHSP27 markedly activated NF-kB signaling in macrophages. Using an inhibitor of NF-kBsignaling there was an attenuation of rHSP27-induced inhibition of SR-A gene and protein expression, as well as lipid uptake, suggesting that SR-A expression is regulated by NF-kB activation. Lastly, to investigate if SR-A is required for HSP27-mediated atheroprotection in vivo, ApoE-/- and ApoE-/-SR-A-/- mice fed a high fat diet were treated with rHSP25, the mouse orthologue of HSP27, or PBS for 3 weeks. While rHSP25 therapy equally reduced serum cholesterol levels in the mouse cohorts, aortic atherogenesis, assessed using en face and sinus cross-sectional analyses, was attenuated in ApoE-/- mice but not ApoE-/-SR-A-/- mice. In conclusion, rHSP27 inhibits foam cell formation by downregulating SR-A expression. This effect may be associated with NF-kB activation. Reductions in atherosclerotic burden by rHSP27 require SR-A, and are independent of changes in serum cholesterol levels, highlighting the importance of macrophage lipid uptake in atherogenesis. Results presented in this thesis demonstrate that SR-A is a major target for HSP27 atheroprotection in the vessel wall, and provide an impetus for further studies that investigate the potential therapeutic value of HSP27.

Indiana University-Purdue University Indianapolis (IUPUI) / S-nitrosoglutathione reductase (GSNOR) also known as glutathione- dependent formaldehyde dehydrogenase (FDH), is a zinc-dependent dehydrogenase. GSNOR oxidizes long chain alcohols to an aldehyde with the help of a molecule of NAD+. GSNOR was initially identified as FDH because of its role in the formaldehyde detoxification pathway. The only S-nitrosothiol (SNO) substrate recognized by GSNOR is GSNO. A transnitrosation reaction transfers NO from nitrosylated proteins or S-nitrosothiols (RSNO) to glutathione to form S-nitrosoglutathione. This GSNO is finally converted to glutathione disulfide (GSSG) by a two step mechanism. Cellular GSNO is a nitric oxide reservoir that can either transfer to or remove from the proteins a NO group. Reduction of GSNO by GSNOR depletes this reservoir and therefore indirectly regulates protein nitrosylation. GSNOR inhibitors which can increase the basal GSNO levels will be another potential therapy. Several GSNOR inhibitors were identified in our laboratory and the aim of this study was to understand their cellular effects. One of the experiments studied the effect of the compound on protein-SNO. The role of nitric oxide in regulation of NF-κB pathway is reviewed by Bove and van der Vliet. We focused on identification of nitrosylated proteins using protein specific antibodies. We identified nitrosylation of IKKβ. So the question raised was whether nitrosylation of IKKβ affects its activity. IKKβ is responsible for phosphorylation of IκBα and phosphorylation of IκBα results in its degradation and activation of NF-κB pathway. Therefore, we studied the phosphorylation of IκBα in the presence of inhibitor C3. Results showed a dose-dependent decrease of pIκB. So the next question was whether the phosphorylation of IKKβ was affected by nitrosylation. We did not detect any change in pIKKβ with different concentrations of C3. The decreased degradation of IκBα caused by C3 translated into decreased NF-κB activity as seen by a dose-dependent decrease in amounts of ICAM-1 with increasing C3 concentration. This data supports the premise that the activity of transcription factor NF-κB is suppressed by inhibiting GSNOR with compound C3

Indiana University-Purdue University Indianapolis (IUPUI) / SIMPL is a transcriptional co-activator that alters the activity of transcription factor, NF-κB. In response to pathogens, cytokines such as Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) signal through the IL-1 and TNF-α receptors, respectively, which are found on various cell types. Activation of these receptors can result in the nuclear localization of NF-κB where it enables the transcription of several different genes key in the innate immune response. Endogenous co-localization of the SIMPL protein with NF-κB in response to these same cytokine signals has yet to be demonstrated. Polyclonal antibody generated against a truncated version of the SIMPL protein was purified from the sera obtained from immunized rabbits using affinity chromatography. The antibody was found to have a high specificity for both the native and denatured form of the protein as demonstrated by the lack of nonspecific bands observed in immunoprecipitations and Western blotting. The antibody was utilized in immunofluorescence experiments on mouse endothelial cells that were either unstimulated or were stimulated (IL-1 or TNF-α). In the absence of cytokine, SIMPL was localized in both the cytoplasm and the nucleus as opposed to NF-κB which was almost exclusively localized in the cytoplasm. In the presence of IL-1, the concentration of SIMPL in the nucleus was increased, and in the presence of TNF-α, the concentration of SIMPL in the nucleus was even greater. Results of this study identified future routes for SIMPL antibody isolation as well as to demonstrate that endogenous SIMPL protein nuclear localization may not be solely dependent upon TNF-α signaling.

No description available.

No description available.

Le neutrophile, le leucocyte le plus abondant de circulation, est un acteur majeur dans la réaction inflammatoire. Bien qu'il soit indispensable pour la défense de l'hôte, le dérèglement de l'activité des neutrophiles a également le potentiel de causer des dommages tissulaires importants. Nous sommes partis de l'hypothèse que l'exposition à long terme à des médiateurs inflammatoires pourrait changer le profil fonctionnel du neutrophile. À cette fin, nous avons incubé des neutrophiles circulants humains avec des cytokines facilitant la survie et trouvé une sous-population de ces cellules pouvant persister pendant plus de 72 heures. Par rapport aux neutrophiles fraîchement isolés, ces neutrophiles de longue durée de vie avaient un phénotype très différent avec des caractéristiques de surface modifiée. Leurs fonctions effectrices notamment le chimiotactisme, la production de superoxyde et de leucotriènes, la dégranulation et la synthèse de cytokines, etc. ont été également modifiées. Comme les médiateurs dérivés de neutrophiles ont été impliqués dans la propagation de l'inflammation dans l'arthrite rhumatoïde, on a essayé ensuite de placer le neutrophile ± reprogrammé ¿ dans ce contexte spécifique. Nous avons trouvé que le liquide synovial et des neutrophiles stimulés par le lipopolysaccharide pouvaient augmenter l'expression de surface du receptor activator of NF-kappa B ligand (RANKL), une molécule essentielle pour l'activation de la résorption osseuse, par le neutrophile. En effet, nous avons montré que le RANKL du neutrophile interagit avec le RANK exprimé par les ostéoclastes, et induit la différenciation des ostéoclastes et la résorption osseuse. En outre, le RANKL associé à la membrane est actif au sein du neutrophile lui-même, induit une phosphorylation du Src homology 2 domain-containing tyrosine phosphatase -1 et inhibe les fonctions des neutrophiles. De plus, nous avons procédé à la co-incubation des neutrophiles avec des synoviocytes d'arthrite rhumatoïde (RA-FLS). Cette co-incubation induit l'expression de RANK dans les neutrophiles. L'activation du RANK par son ligand biologique favorise la survie des neutrophiles, la dégranulation et la synthèse de cytokines. En conclusion, nous présentons des preuves que le neutrophile inflammatoire acquiert de nouvelles fonctions au-delà de celles associées à sa contrepartie classique circulant. Par ailleurs, l'expression de molécules ostéotropiques de l'axe RANKL-RANK-OPG positionne le neutrophile comme un lien cellulaire entre le squelette et le système immunitaire.

Les protéines receptor-interacting protein kinase 1 (RIP1) et RIP3 ont été identifiées comme intervenant dans la régulation de la mort cellulaire apoptotique ou nécroptotique mais également dans la survie cellulaire. Ces deux protéines possèdent un domaine sérine/thréonine kinase, un domaine d'interaction spécifique RHIM (RIP homotypic interacting motif) et diffèrent dans leur domaine C-terminal car seule RIP1 possède un domaine de mort. Ces protéines font partie d'un ensemble de protéines régulatrices nommé ripoptosome. Des études ont montré une altération du ripoptosome dans les leucémies lymphoïdes chroniques (LLC) et les leucémies aigües lymphoïdes (LAL). Nous nous sommes intéressés aux leucémies aigües myéloïdes (LAM). L'analyse de l'expression des protéines RIP1 et RIP3 a été réalisée dans des blastes triées CD34+ de patients atteints de LAM ou dans des cellules CD34+ de donneurs sains en Q-RT-PCR. Les premières analyses montrent que RIP3 est significativement sous-exprimée chez les patients atteints de LAM en comparaison avec les cellules CD34+ issues de donneurs sains. Aucune différence n'a été mise en évidence pour l'expression de RIP1 dans les deux types de cellules CD34+. Afin de comprendre l'implication de l'extinction de RIP3 dans les LAM, nous avons étudié sa réexpression dans une lignée cellulaire leucémique murine (DA1-3b) où RIP3 n'est pas exprimée par métylation de son promoteur, au moyen d'un système d'expression conditionnelle (LacSwith II, IPTG). Après 10h d'induction de l'expression, on constate que la protéine RIP3 sauvage (RIP3-WT) induit une apoptose dans les cellules DA1-3b. Afin de déterminer l'implication des domaines de RIP3, nous avons utilisé une protéine mutante kinase Dead (RIP3-KD, activité kinase abolie) et une protéine mutante dans la séquence d'interaction spécifique avec RIP1 (RIP3-RHIM). L'analyse de la mortalité cellulaire en cytométrie en flux et en microscopie électronique montre que les protéines RIP3-WT et -KD induisent toutes les deux la mort apoptotique des cellules DA1-3b respectivement de 15% et de 50% après 10h d'expression. On constate donc que la protéine RIP3-KD induit une mort plus importante et plus précoce que la protéine sauvage. La protéine RIP3 mutée dans son domaine RHIM ne peut plus induire de mort cellulaire. Il semble donc que le domaine kinase de RIP3 jouerait un rôle régulateur dans la mort cellulaire induite par RIP3. L'utilisation du modèle de leucémie murine DA1-3b a permis de réaliser un étude in vivo de l'expression conditionnelle de RIP3-WT et -KD. Seule l'expression de RIP3-KD est capable de prolonger significativement la survie des souris.De plus, il a été démontré que RIP3 pouvait également induire la nécroptose dans les cellules lorsque l'apoptose ne peut aboutir, notament lorsque les caspases sont inhibées à l'aide d'un inhibiteur de pan-caspases le Z-VAD-FMK. Le traitement des cellules exprimant RIP3-WT par 50µM de Z-VAD-FMK induit une plus forte mortalité (45%) des cellules tandis que dans les cellules exprimant RIP3-KD, l'inhibiteur des caspases inhibe complètement le processus apoptotique et permet la survie des cellules (10%). Une étude en microscopie électronique a permis de déterminer que la présence de Z-VAD-FMK induit un switch de l'apoptose vers la nécroptose. Il semble donc que le domaine de kinase possède un rôle important dans la signalisation de la nécroptose car la protéine RIP3-KD n'est plus capable d'initier le switch entre l'apoptose et la nécroptose. Quelques données préliminaires semblent indiquer que les calpaïnes ainsi que la caspase 12 pourraient également être impliquées dans la balance apoptose/nécroptose. [...]

Ip Wai-Ki. / "July 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 261-290). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Abstract not available