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.

Lo, Chun Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 71-80). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.iv / List of figures --- p.vi / Abbreviations --- p.vii / Contents --- p.viii / Chapter Chapter One --- General Introduction --- p.Page / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Human papillomavirus infection at the larynx --- p.2 / Chapter 1.2.1 --- Biology of human papillomavirus --- p.4 / Chapter 1.2.2 --- HPV E6 protein --- p.5 / Chapter 1.2.3 --- HPV E7 protein --- p.7 / Chapter 1.3 --- Apoptosis --- p.9 / Chapter 1.3.1 --- Apoptosis signaling pathways --- p.11 / Chapter 1.4 --- Transcription factor: Nuclear factor -kB --- p.14 / Chapter 1.4.1 --- Overview of the NF-kB signaling pathway --- p.14 / Chapter 1.4.2 --- Regulation of NF-kB signaling --- p.16 / Chapter 1.4.3 --- Roles of NF-kB in cancers --- p.19 / Chapter 1.5 --- Pteris semipinnata L extract: ent-11 -hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) --- p.21 / Chapter 1.6 --- Objectives --- p.22 / Chapter Chapter Two --- Materials and Methods / Chapter 2.1 --- Cell culture --- p.24 / Chapter 2.2 --- Cell proliferation analysis --- p.24 / Chapter 2.3 --- Western Blotting --- p.26 / Chapter 2.3.1 --- Total protein extraction --- p.26 / Chapter 2.3.2 --- Nuclear and cytoplasmic protein extraction --- p.26 / Chapter 2.3.3 --- Quantification of protein concentration --- p.27 / Chapter 2.3.4 --- Sodium dodecyl sulfate - polyacylamide gel electrophoresis (SDS-PAGE) and protein transfer --- p.28 / Chapter 2.3.5 --- Immunoblotting --- p.29 / Chapter 2.4 --- NF-kB Luciferase Assay --- p.29 / Chapter 2.5 --- Annexin V apoptosis assay --- p.31 / Chapter 2.6 --- mRNA expression analyses --- p.33 / Chapter 2.6.1 --- RNA extraction --- p.33 / Chapter 2.6.2 --- Reverse Transcription --- p.33 / Chapter 2.6.3 --- Polymerase Chain Reaction --- p.34 / Chapter 2.7 --- Antibodies --- p.35 / Chapter Chapter Three --- Results / Chapter 3.1 --- "Anti-proliferation effect of 5F on laryngeal cancer cells UMSCC11A, UMSCC12 and HEp-2 cells" --- p.36 / Chapter 3.2 --- Suppression by 5F in HEp-2 of mRNA and protein expression levels in HPV18 E7 while the expression level of HPV18 E6 was not altered --- p.38 / Chapter 3.3 --- Quantification of 5F-induced apoptosis in laryngeal cancer cells by Annexin V assay --- p.40 / Chapter 3.4 --- Morphological changes in laryngeal cancer cells induced by 5F --- p.41 / Chapter 3.5 --- "Cleavage of poly (ADP-ribose) polymerase (PARP) and pro-caspase-3 induced by 5F in UMSCC11 A, UMSCC12 and HEp-2 cell lines" --- p.47 / Chapter 3.6 --- "Down-regulation of TNF-α-induced NF-kB subunit p65 and p50 nuclear translocations in UMSCC11 A, UMSCC12 and HEp-2 by 5F" --- p.47 / Chapter 3.7 --- Dose-dependent inhibition of 5F on NF-kB transcriptional activity measured by luciferase assay --- p.53 / Chapter 3.8 --- Partial inhibition of TNF-α induced kBα degradation by 5F in UMSCC11A but not in UMSCC12 and HEp-2 --- p.56 / Chapter 3.9 --- Cell proliferation inhibition and apoptosis induction by Bay (11-7082) in laryngeal cancer cells --- p.56 / Chapter 3.10 --- Differential basal nuclear translocation of p65 and p50 in laryngeal cancer cell lines --- p.57 / Chapter 3.11 --- 5F regulated NF-kB target gene expression --- p.58 / Chapter Chapter Four --- Discussions --- p.64 / Reference --- p.71 / Appendix / Appendix 1 Map of pLuc- NF-kB plasmid --- p.81

Abstract not available

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 191-209).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 161-184).

Les facteurs de transcription de la famille Rel/NF-κB régulent l’expression d’un grand nombre de gènes impliqués dans les réponses immunitaires et inflammatoires ainsi que dans la régulation de la prolifération et de la survie cellulaire. Le caractère transitoire de l’activation de NF-κB est donc crucial pour poterger les cellules de l’autoxicité due à une trop forte expression des gènes cibles de ce facteur de transcription. Dans le cadre de notre thèse de doctorat, nous avons étudié les mécanismes moléculaires régulant la cinétique d’activation de NF-κB, en accordant une attention toute particulière au complexe kinase IKK, qui semble être le regulateur clef de l’activation de NF-κB. Nos résultats suggèrent que p300 pourrait réguler la durée d’activation des IKKs d’une part par acétylation directe, et d’autre part, indépendamment de son activité HAT, en stabilisant les IKKs et donc en prolongeant leur demie-vie et par conséquent leur activation.<p>Certains virus utilisent la voie de signalisation NF-κB afin de promouvoir leur propre réplication. C’est le cas du virus HIV-1 (Human Immunodeficiency Virus type 1), qui contient dans son promoteur deux sites de liaison pour NF-κB. Notre laboratoire a précédemment montré que l’utilisation du TNFα en combinaison avec la TSA, active l’expression virale de manière synergique. L’administration combinée d’un activateur du facteur NF-κB et d’un inhibiteur de désacétylases pourrait, en présence d’une thérapie anti-HIV-1 efficace, être envisagée dans le but d’éliminer les cellules réservoirs infectées de manière latente. L’utilisation thérapeutique du TNFα ou de la TSA étant inenvisageable en raison de leur toxicité, nous avons étudié l’effet d’autres substances ayant un plus grand potentiel thérapeutique et nous avons apporté une preuve de principe du potentiel thérapeutique de la coadministration de plusieurs activateurs viraux (inhibiteurs de HDACs[HDACIs]+inducteurs de la voie NF-κB) pour réduire le pool des réservoirs cellulaires infectés de manière latente.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.

Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. The nuclear factor κB (NF-κB) is an important family of transcription factors whose aberrant activation has been found in many types of cancer, including CRC. Therefore, understanding the regulation of NF-κB is of ultimate importance for cancer therapy. Using a novel validation-based insertional mutagenesis (VBIM) strategy, our lab has identified the novel adenomatous polyposis coli (APC) like protein (ALP) gene as a negative regulator of NF-κB. Preliminary studies from our lab demonstrated that overexpression of ALP led to decreased NF-κB activity by κB reporter assay and electrophoresis mobility gel shift assay (EMSA). The current project aims to further evaluate the role of ALP in the regulation of NF-κB signaling in CRC cells. We found that overexpression of ALP in human CRC HT29 cells greatly reduced both the number and the size of colonies that were formed in a soft agar assay. ALP overexpression also decreased the cell growth rate and cell migration ability, while shRNA mediated knockdown of ALP showed opposite effects, confirming that ALP is a tumor suppressor in CRC HT29 cells. Overexpression of ALP led to decreased NF-κB activity by κB reporter assay and condition media assay in CRC HT29 cells. Furthermore, immunohistochemical analysis with human colon vii tissues revealed that there is a gradual loss of ALP protein with tumor progression. We also found that ALP predominantly localizes in the cytoplasm, and binds to the p65 subunit of NF-κB, and might be functioning downstream of IκB kinase (IKK). In summary, in this study, we provide evidence regarding the tumor suppressor role of ALP in CRC by functioning as novel negative regulator of NF-κB. This discovery could lead to the establishment of ALP as a potential biomarker and therapeutic target in CRC.