The NF-kB signalling pathway is a critical mediator of the cellular responses to inflammatory cytokines. The IκB kinase (IKK) complex, which is composed of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ/NEMO) acts as the master regulator of NF-κB transcription factor activity. Seminal genetic studies in knockout (KO) mouse embryonic fibroblasts (MEFs) have defined two pathways of NF-κB activation; a canonical pathway, activated in response to cytokines such as TNFα/IL-1β, that requires NEMO and predominantly IKKβ catalytic activity; and a non-canonical pathway, activated in response to a subset of TNF-family cytokines, which requires IKKα and NIK kinase. We have generated and validated CRISPR-Cas9 IKKα, IKKβ and IKKα/β DKO HCT116 colorectal cancer cell lines to interrogate novel functions of the I kappa B kinases in colorectal cancer, including the relative contributions of these kinases to the activation of NF-κB signalling pathways downstream of TNFα induction. Contrary to the seminal studies in KO MEFs, IKKα appeared to make a more significant contribution to canonical NF-κB induction in these cells than IKKβ. Western blot studies demonstrated that both IKKs contributed to the phosphorylation and degradation of IκB and the phosphorylation of the NF-κB subunit, p65 at Serine 536. However, high-content immunofluorescence studies demonstrated that IKKα KO cells were defective in TNFα-induced nuclear translocation of p65 compared to WT and IKKβ KO cells. Additionally, NF-κB-driven luciferase reporter assays showed that IKKα, but not IKKβ, KO cells exhibited significantly reduced NF-κB-dependent gene expression following TNFα stimulation. We also have evidence to suggest that the phosphorylation site at Serine 468 on p65, previously defined as an IKKβ-dependent site, is in-fact an IKKα-dependent site in these cells. Furthermore, IKKα knockout revealed a potentially important role for IKKα activity in preventing the stabilisation of NIK protein following prolonged TNFα stimulation. RNA sequencing analysis of wild-type, IKKα KO, IKKβ KO and IKKα/β DKO cells stimulated with TNFα was performed to identify genes whose expression were differentially deregulated by IKK KO. These analyses confirmed the importance of IKKα for canonical NF-κB gene expression. Furthermore, IKKβ knockout had unexpected effects on the expression of a broad range of genes involved in chromatin organisation, cytoskeletal organisation, mitotic cell cycle control and the DNA damage response. During the characterisation of IKK KO cells it was discovered that the expression of NEMO was downregulated at the protein, but not mRNA level by approximately 50% in IKKα KO cells and 90% in IKKα/β DKO cells. IKKβ KO cells, meanwhile, exhibited wild-type NEMO expression. Emetine-chase and radioactive pulse chase labelling experiments demonstrated that the half-life of NEMO in IKKα and IKKα/β DKO cells was significantly shortened due to enhanced proteasomal turnover. Bioinformatics analyses predicted significant regions of intrinsic structural disorder within NEMO, particularly at the N- and C-termini, the former of which overlapped with the IKK binding domain. On this basis, the susceptibility of NEMO to in vitro degradation by the 20S proteasome was examined, with NEMO proving be a highly effective substrate of the 20S proteasome. Importantly, IKKα and IKKβ were both shown to protect NEMO from proteasomal degradation, leading us to propose a model whereby interaction with IKK kinase subunits sequesters/masks intrinsically disordered regions in NEMO that would otherwise make NEMO a highly effective substrate for ubiquitin-dependent and/or ubiquitin-independent proteasomal degradation. BMS-345541 is a commercially available allosteric inhibitor of IKKβ that has been used extensively in numerous studies, including a report that proposed novel functions for IKKβ in mitotic cell cycle progression (Blazkova et al., 2007). Similar antiproliferative effects to those reported by Blazkova et al., were observed during the characterisation of a novel ATP-competitive inhibitor of IKKβ, AZD2230. In depth characterisation of the selectivity of AZD2230 and BMS-345541, however, revealed that the antiproliferative effects of AZD2230 and BMS-345541 are, in fact, due to off-target inhibition, potentially at the level of RNA Polymerase II C-terminal domain phosphorylation, and hence general transcription. Collectively, these studies reveal novel functions of the IKK kinases in NF-κB signalling and inform therapeutic strategies for targeting chronic canonical NF-κB activation in colorectal cancer.

No description available.

Thesis (M.S.)--Indiana University, 2009. / Title from screen (viewed on October 1, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Sonal P. Sanghani, Paresh C. Sanghani, William F. Bosron. Includes vita. Non-Latin script record Includes bibliographical references (leaves 53-56).

Orientador: Patrícia de Oliveira Prada / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T05:33:45Z (GMT). No. of bitstreams: 1 Weissmann_Lais_M.pdf: 2304567 bytes, checksum: b275887306c298a5ec1fd2910b146e00 (MD5) Previous issue date: 2013 / Resumo: Recentemente, demonstrou-se que IkB quinase e (IKK?) aumenta a resistência à insulina (RI) induzida por dieta hiperlipídica em tecido adiposo e hepático. Assim, os objetivos do presente estudo foram investigar: 1) a expressão e ativação de IKK? no hipotálamo de camundongos DIO e db/db; 2) o papel da IKK? hipotalâmica no metabolismo energético e da glicose, e na ação e sinalização da insulina e leptina. Observamos maior expressão e ativação de IKK? no hipotálamo de camundongos db/db e DIO quando comparados aos controles. Cinco dias de injeção intracerebroventricular (ICV) de CAY (inibidor farmacológico da IKK?) ou de IKK? siRNA, via mini-bomba ICV, reduziu 80% da atividade de IKK?, a fosforilação do IRS1Ser307 e não alterou a fosforilação de IKK?/? em hipotálamo. A inibição IKK? (ICV): reduziu a adiposidade e ingestão alimentar, aumentou o consumo de O2 e expressão de UCP-1 no adiposo marrom, melhorou os efeitos anorexigênicos da insulina e de leptina, aumentou a fosforilação do receptor de insulina, da AKT, JAK2 e STAT3 em hipotálamo de camundongos DIO e db/db. Esses resultados persistiram com pair-feeding e pair-weight. A inibição com CAY (ICV) reduziu: a glicemia de jejum, produção hepática de glicose e expressão da PEPCK em fígado de animais DIO e db/db. Em resumo, os dados sugerem que IKK? hipotalâmica está ativada em modelos de obesidade e participa no desenvolvimento da resistência à insulina e leptina. Sua inibição reduz a adiposidade, ingestão alimentar e aumenta o gasto energético. A IKK? além de influenciar o metabolismo energético, também tem um papel no metabolismo da glicose de animais obesos. Assim, pode-se sugerir que a IKK? representa uma ligação entre a obesidade e resistência à insulina e leptina no hipotálamo e pode ser um alvo terapêutico para a resistência à insulina, obesidade e diabetes / Abstract: It was demonstrated that IkB kinase epsilon (IKK?) increased insulin resistance (IR) in liver and adipose tissue of diet-induced obesity (DIO) mice. The aims of this study are to investigate: 1) the expression and activation of IKK? in the hypothalamus from DIO mice and db/db, 2) the role of hypothalamic IKK? in the energy and glucose metabolism, and in insulin and leptin action/signaling. We observed increased expression and activation of IKK? in the hypothalamus from db/db and DIO mice compared with controls. Five days of intracerebroventricular (ICV) injections of CAY (pharmacological inhibitor of IKK?) or siRNA, via ICV mini-pump, reduced 80% of IKK? activity as well asthe phosphorylation of IRS1Ser307 and did not alter the phosphorylation of IKK?/? in hypothalamus from obese mice. Inhibition of IKK? (ICV) reduced fat mass, food intake and increased O2 consumption and expression of UCP-1 in the brown adipose tissue, improved anorexigenic effects of leptin and insulin, increased insulin receptor, AKT, JAK2 and STAT3 phosphorylation in the hypothalamus of DIO and db/db mice. These results persisted even if in pair-fed and pair-weight mice. Inhibition of IKK? with ICV CAY reduced: fasting glycemia, hepatic glucose production and the expression of PEPCK in liver of DIO and db/db animals. In summary, the data suggest that hypothalamic IKK? is activated in models of obesity and participates in the development of insulin and leptin resistance. Its inhibition reduces adiposity, food intake and increases energy expenditure. Hypothalamic IKK? influences the energy and glucose metabolism in obese mice. Thus, it can be suggested that the IKK? represents a link between obesity and insulin resistance in the hypothalamus and may be a new therapeutic target to treat insulin resistance, obesity and diabetes / Mestrado / Clinica Medica / Mestra em Clínica Médica

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

Dissertation (Ph.D.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Table of contents (p. iii) incorrect starting page number for third bibliography (says p. 123, actually is p. 121) and incorrect starting page number for abstract (says p. 156, actually is p. 154). Bibliography: p. 76-83, 108-111, 121-153.

Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 105-116).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 102-123).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 98-113).

Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 131-146). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;