The NF-κB transcription factor p65/RelA controls hundreds of genes involved in inflammation, immunity, cell proliferation and survival. Cells treated with the inflammatory cytokine TNF-α show repeated nuclear-cytoplasmic translocations of p65 maintained by multiple negative feedback loops. Application of pulses of TNF-α at various set intervals can entrain the translocation frequency and cause different patterns of NF-κB-dependent gene transcription. Translocation frequency can also be altered by biological and environmental factors such as temperature and diclofenac treatment. These observations have led to the hypothesis that the dynamics of NF-κB play a role in the transcriptional responses dependent on particular cellular states. NF-κB can physically interact with members of other molecular networks including glucocorticoid signalling, the circadian clock and the cell cycle. In a previous study, two tumour cell lines showed differential p65 dynamics when treated with TNF-α at different cell cycle stages which concomitantly also altered cell cycle length. Evidence suggested that physical interactions between p65 and the cell cycle regulators E2F1 and E2F4 could, at least in part, be responsible for these effects. This thesis describes studies designed to investigate if similar responses exist in more normal cells. Although transcript levels of NF-κB target genes were suppressed in MEF cells treated with TNF-α during S phase relative to G1/S, NF-κB translocations appeared unaffected. Knowledge of the interactions with other molecular networks is central to understanding the context-dependent role of NF-κB, and the complexity of these dynamic interactions requires a Systems Biology approach. Within this study, a model of the interaction between NF-κB and the cell cycle was tested for its ability to recapitulate biological data from HeLa cells. Also, the effect of cell cycle timing of TNF-α treatment on NF-κB dynamics in HeLa cells was examined using ImageStream flow cytometry. It is suggested that a Systems Biology approach could be used to extend a study, initial results of which are presented here, of the interaction between NF-κB and glucocorticoid signalling. Understanding the interactions between NF-κB signalling and other networks should have clinical benefits, for example for cancer treatment dependent on circadian timing.

Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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) / Orientador: Carlos Rossa Junior / Coorientador: Joni Augusto Cirelli / Banca: José Augusto Cezar Sampaio / Banca: Luis Carlos Spolidório / Banca: Luiz Carlos de Mattos / Banca: Cleni Mara Marzocchi Machado / Doutor

Abstract Neutrophils are the major cellular component of acute inflammatory response. The mechanism by which fMLP or PAF activates neutrophils is not fully elucidated. Stimulation of MAPKs and activation of NF-kappa B in neutrophils regulate various cell functions, including superoxide production. Neutrophils isolated from blood taken from healthy donors, were incubated with specific inhibitors, GF109203X (PKC inhibitor), calphostin C (PKC-gamma isoform inhibitor), wortmannin (PI3K inhibitor), U73122 (PLC inhibitor), aristolochic acid (PLA2 inhibitor), SKF96365 (SOC channel inhibitor), EGTA (extracellular calcium chelator), SB203580 (p38 MAPK inhibitor), and PD98059 (MEK inhibitor), followed by fMLP or PAF treatment. MAPK activation by fMLP or PAF is based on immunoblot analysis. NF-kappa B activation is detected by EMSA, and superoxide production is measured by flow cytometry. The data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK MAPK phosphorylation was involved PI3K, PKC, PLA2, PLC, and extracellular calcium, wheres fMLP-induced phosphorylation doesn¡¦t involve PKC

Dissertation (Ph.D.)--University of Toledo, 2009. / "Signaling" misspelled as "singaling" on title page of document. "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Non-Latin script record Bibliography: p. 94-108.

Dissertation (Ph.D.)--University of Toledo, 2009. / Typescript. "Submitted as a partial fulfillment of the requirements for the Doctor of Philosophy degree in Biology." Bibliography: leaves 135-146.

Zugl.: Berlin, Freie Univ., Diss., 2008 / Includes bibliographic references.

Thesis (Ph. D.)--University of Kentucky, 2002. / Title from document title page. Document formatted into pages; contains vii, 158 p. : ill. Includes abstract. Includes bibliographical references (p. 128-155).

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 92 - 100.