Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, Dec. 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D. in Toxicology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 121-141). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "May 1998." Typescript. Vita. Includes bibliographical references (leaves 172-189). Also available on the Internet.

Thesis (Ph.D.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on Oct. 6, 2008). Includes bibliographical references (p. 213-240). Available online via the OhioLINK ETD Center. Also available in print.

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Dec. 2004. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 84-104).

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.

A caracterização de vias de sinalização alteradas em células tumorais, e a validação de fármacos inibidores de transdução de sinal que interajam com as mesmas de modo a atuar como terapia principal ou adjuvante no tratamento de neoplasias sólidas e hematopoiéticas, é um campo de grande interesse em oncologia. A necessidade da caracterização de novos alvos moleculares advém da incidência considerável de recidivas, quadros de quimiorresistência e efeitos adversos associados ao tratamento com os agentes antitumorais clássicos. Nesta tese, desenvolvemos a hipótese de que o fator de transcrição NFκB poderia estar envolvido com processos de proliferação, antiapoptose e quimiorresistência em células neoplásicas, caracterizando-se como um potencial alvo para interferência farmacológica. Para isso, avaliamos: i) o papel de NFκB na resposta celular a ERO e no controle da decisão entre morte e proliferação celular; ii) o estado de ativação de NFκB em modelos tumorais in vitro e in vivo; iii) o potencial citotóxico e seletividade dos inibidores de NFκB, seus mecanismos de ação, atividade em células neoplásicas e em linhagens quimiorresistentes. Para isso, foram utilizadas abordagens in vitro (cultivos celulares), in vivo (modelo animal de implante tumoral), e análises de bancos de expressão gênica através de ferramentas de biologia de sistemas. Os resultados demonstraram que NFκB está superestimulado em linhagens de leucemia e de glioblastoma quando comparado com leucócitos e astrócitos não-transformados. O tratamento com inibidores farmacológicos de NFκB causou morte celular programada em um mecanismo seletivo para células tumorais, potenciando os efeitos de antitumorais clássicos tanto em linhagens selvagens quanto em células quimiorresistentes. Além disso, os inibidores BAY117082 e curcumina apresentaram atividade in vivo em modelo animal de glioma sem evidência de citotoxicidade aguda. Em suma, os dados aqui apresentados sugerem que o fator de NFκB constitui-se um potencial alvo para inibição farmacológica no tratamento de neoplasias. Neste contexto, a diversidade da expressão de NFκB em diferentes tipos tumorais e a segurança associada ao uso clínico de seus inibidores permanece por ser avaliada. / Characterization of new molecular targets is one of the most important challenges in oncology. In this context, there is a growing interest in characterization of deregulated cell signaling pathways in solid and hematopoietic cancer cells in order to leverage the development of specific cell signaling inhibitors to act as principal or adjuvant therapy, and to circumvent the frequently observed chemoresistance and relapse in cancer patients. In this study, we hypothesized that the transcription factor NFκB could be involved on proliferation, antiapoptosis response and chemoresistance in cancer cells thus being a potential target for pharmacological interference. Attempting to test this, we evaluated: i) the role of NFκB in cell response against reactive oxygen species and in control of cell death and proliferation signaling; ii) NFκB activation status in cancer and noncancerous cells in vitro and in vivo; iii) cytotoxicity, selectivity and mechanisms of action of NFκB inhibitors in glioma and leukemia cells besides its biological activity against chemotherapy-resistant cell lines. Experiments were performed based on in vitro (cancer cell lines and primary cultures) and in vivo (tumor implants) models of cell growth. Moreover, in some experiments, bench-directed analysis of public gene expression databases followed by bioinformatic approach was used to ensure the significance and reliability of experimental data. Our findings showed an overstimulation of NFκB in leukemic and glioblastoma cell lines compared to healthy leucocytes and non-transformed astrocytes, respectively. Treatment with pharmacological inhibitors of NFκB induced programmed cell death in a cancer cells selective manner and potentiated the effects of classical anticancer drugs in both wild-type and chemoresistant cell lines. Indeed, the pharmacological NFκB inhibitors BAY117082 and curcumin showed significant anticancer efficacy in a model of brain-implanted gliomas without evidence of acute toxicity. Overall, the herein presented data suggest that NFκB is a potential target for cell death induction in leukemia and glioblastomas. Evaluation of its expression profile in different type of cancers beyond testing efficacy and safety of NFκB pharmacological inhibitors for clinical usefulness remains to be investigated.

Made available in DSpace on 2016-03-10T13:16:17Z (GMT). No. of bitstreams: 2 diana_viveiros_ioc_dout_2014.pdf: 2553058 bytes, checksum: cd6b615f6279917df0cb1eea71997b6f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14 / Doenças pulmonares crônicas, como a asma e a silicose, são caracterizadas por inflamação das vias aéreas e fibrose que levam à marcada deterioração da função pulmonar. O objetivo da presente tese é investigar o impacto das propriedades anti-inflamatórias e antifibróticas do composto LASSBio-897 na asma e silicose experimental. Utilizaram-se metodologias diversas, incluindo a quantificação da reatividade de vias aéreas por pletismografia barométrica, além da quantificação do teor tecidual de mediadores inflamatórios por ELISA e do infiltrado celular pulmonar com técnicas de histologia e imunohistoquímica. Foram ainda quantificados a produção de citocinas por fibroblastos e macrófagos \201Cin vitro\201D. O tratamento oral profilático com LASSBio-897 (2 e 5 mg/kg) preveniu a instalação do quadro de hiper-reatividade brônquica, o infiltrado celular e a geração de citocinas pró-inflamatórias em modelos de asma aguda em camundongos. Por outro lado, o tratamento mostrou-se inativo quando feito após a instalação da resposta asmática. Resultados mais promissores foram obtidos na condição da silicose experimental, onde o tratamento oral terapêutico com LASSBio-897 (2 e 5 mg/kg) claramente inibiu o comprometimento da função pulmonar, o processo inflamatório e a fibrose observados induzidos pela exposição à sílica. O composto não interferiu com a atividade de mastócitos e células epiteliais estimuladas com sílica \201Cin vitro\201D No entanto, a administração de LASSBio-897 reduziu a expressão de F4/80 e \03B1-SMA no tecido pulmonar de animais silicóticos, evidenciando assim sua atividade sobre macrófagos e miofibroblastos, respectivamente. Ensaios \201Cin vitro\201D confirmaram esse efeito, uma vez que o composto reduziu a liberação de TNF-\03B1 por macrófagos alveolares estimulados com sílica e inibiu a proliferação e produção de colágeno induzida por IL-13 em fibroblastos pulmonares oriundos de animais silicóticos. Por fim, a administração do composto resultou na redução da expressão tecidual de NF-\03BAB após o desafio com sílica. Conclui-se que o composto LASSBio-897 pode prevenir a instalação do quadro de asma aguda, sem contudo alterar a resposta asmática já instalada. A capacidade do LASSBio-897 de reverter a resposta inflamatória e fibrótica pulmonar induzida por partículas de sílica sugere que uma aplicação na silicose, mais do que na asma, seja uma perspectiva verdadeiramente promissora / Chronic lung diseases such as asthma and silicosis are characterized by airway inflammation and fibrosis leading to marked deterioration of lung function. The aim of this thesis is to investigate the effect of compound LASSBio - 897 on experimental asthma and silicosis. Various pathol ogical parameters were assessed, including airway hyper - reactivity by barometric plethysmography, lung tissue content of inflammatory mediators by ELISA and cellular infiltration using histology and immunohistochemistry techniques. Cytokine generation from fibroblasts and macrophages in vitro was also quantified. Oral prophylactic treatment with LASSBio - 897 (2 and 5 mg/kg) prevented airway bronchial hyper - reactivity, leukocyte recruitment and cytokine generation in a model of acute asthma in mice. However, this treatment was inactive when performed after installation of the asthmatic response. More promising results were obtained in the experimental silicosis condition, where the LASSBio - 897 therapeutic treatment (2 and 5 mg/kg, oral) clearly inhib ited airway hyper - reactivity, as well as inflammation and peribronchial fibrosis induced by silica particles. Furthermore, LASSBio - 897 did not interfere with mast cells and epithelial cells activity after silica stimulation in vitro . However, LASSBio - 897 c learly reduced F4/80 and α - SMA expression in the lung of silicotic mice, showing its inhibitory effect on macrophages and myofibroblasts, respectively. In vitro assays confirmed this effect, since the compound reduced TNF - α release by an alveolar macrophag e cell lineage after silica exposure, and inhibited proliferation and collagen production induced by IL - 13 in lung fibroblasts recovered from silicotic mice. Finally, LASSBio - 897 reduced sil ica - induced upregulation of NF - κ B expression in the lung tissue. I t is concluded that LASSBio - 897 can prophylactically prevent the development of acute asthma, without altering the already installed asthmatic response. The capacity of LASSBio - 897 to reverse silica but not allergen - induced lung inflammation and fibrosis s uggests the application in silicosis, rather than in asthma, as a truly promising perspective

MAGALHÃES, P. A. F. Acidose metabólica agrava a lesão renal aguda isquêmica em ratos. 2016. 110 f. Tese (Doutorado em Ciências Farmacêuticas) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-03-29T13:57:35Z No. of bitstreams: 1 2016_tese_pafmagalhaes.pdf: 3724835 bytes, checksum: 2211058119db6e128e61846550417799 (MD5) / Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2016-03-29T13:57:48Z (GMT) No. of bitstreams: 1 2016_tese_pafmagalhaes.pdf: 3724835 bytes, checksum: 2211058119db6e128e61846550417799 (MD5) / Made available in DSpace on 2016-03-29T13:57:48Z (GMT). No. of bitstreams: 1 2016_tese_pafmagalhaes.pdf: 3724835 bytes, checksum: 2211058119db6e128e61846550417799 (MD5) Previous issue date: 2016-02-18 / Lesão renal por isquemia/reperfusão (I/R) e acidose metabólica (MA) são duas condições críticas que podem ocorrer simultaneamente na prática clínica. O resultado dessa combinação pode ser prejudicial para os rins, mas esta questão não tem sido exaustivamente estudada até hoje. O presente estudo avaliou em ratos a influência do baixo pH sistêmico em vários parâmetros da função renal mediante lesão renal por I/R. A acidose metabólica foi induzida em ratos Wistar machos através da ingestão de cloreto de amônio (NH4CI) dissolvido em água da torneira, começando 2 dias antes da agressão isquêmica e mantida durante todo o estudo. Isquemia/reperfusão renal foi induzida por clampeamento de ambas as artérias renais durante 45 min, seguido por 48 h de reperfusão. Foram estudados quatro grupos de animais: controle (submetido à cirurgia sham, n = 8), I/R (n = 8), acidose metabólica (AM; solução de NH4CI 0,28 M e cirurgia sham, n = 6), e AM+I/R (solução de NH4CI 0,28 M + I/R, n = 9). Em comparação com grupo I/R, ratos AM+I/R exibiram redução significativa de pH sanguíneo, bicarbonato plasmático (pBic), e excesso de base (SBE), com declínio no ritmo de filtração glomerular e função tubular. Foram detectados sinais de lesão tubular microscópica. Imunofluorescência mostrou que a combinação entre acidose metabólica e isquemia/reperpusão renal aumentou nitidamente a expressão do fator nuclear kappa B (NF-B) e da heme oxigenase-1 (HO-1), mas não interferiu na diminuição da expressão da óxido nítrico sintase endotelial (eNOS) causada por I/R. Os resultados sugerem que a lesão renal induzida por isquemia/reperfusão é agravada pela acidose.