Wong, Ho Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 66-77). / Abstracts in English and Chinese. / Acknowledgement --- p.i / List of abbreviations --- p.ii / List of tables and figures --- p.iv / Abstract in English --- p.vi / Abstract in Chinese --- p.viii / Table of content --- p.x / Chapter Chapter 1 --- Literature review / Chapter 1.1 --- Nasopharyngeal carcinoma (NPC) and treatments --- p.1 / Chapter 1.2 --- EBV and NF-kB signaling in NPC / Chapter 1.2.1 --- Role of EBV and NF-kB in NPC --- p.2 / Chapter 1.2.2 --- NF-kB signaling in cancer --- p.4 / Chapter 1.2.3 --- NF-kB activation in NPC --- p.7 / Chapter 1.2.3.1 --- NF-kB activation by LMP1 --- p.8 / Chapter 1.2.3.2 --- NF-kB and LMP2A --- p.10 / Chapter 1.2.3.3 --- NF-kB activation by non-viral factors --- p.10 / Chapter 1.2.4 --- NF-kB target genes in NPC --- p.11 / Chapter 1.3 --- NF-kB targeting / Chapter 1.3.1 --- NF-kB targeting agents --- p.14 / Chapter 1.3.2 --- "DHMEQ, a novel blocker of NF-kB Transactivation" --- p.15 / Chapter Chapter 2 --- Aim of study and Research plan --- p.18 / Chapter Chapter 3 --- Materials and Methods / Chapter 3.1 --- Cell lines and Reagents --- p.20 / Chapter 3.2 --- Cell viability assay --- p.21 / Chapter 3.3 --- Cell apoptosis detection / Chapter 3.3.1 --- PARP cleavage --- p.22 / Chapter 3.3.2 --- DNA fragmentation --- p.22 / Chapter 3.4 --- Cell cycle analysis --- p.22 / Chapter 3.5 --- Transwell migration or Matrigel invasion assay --- p.23 / Chapter 3.6 --- Soft agar colony formation assay --- p.24 / Chapter 3.7 --- Drug treatment for western blotting --- p.25 / Chapter 3.8 --- "Protein extraction and quantification, SDS-PAGE and western blotting" / Chapter 3.8.1 --- Protein extraction and quantification --- p.25 / Chapter 3.8.2 --- SDS-PAGE and western blotting --- p.26 / Chapter 3.9 --- Fractionation --- p.28 / Chapter 3.10 --- NF-kB transcriptional activity assay / Chapter 3.10.1 --- Construction of NF-kB reporter system --- p.29 / Chapter 3.10.2 --- Luciferase assay --- p.29 / Chapter 3.11 --- Statistical Analysis --- p.30 / Chapter Chapter 4 --- Results / Chapter 4.1 --- Anti-tumor activity of DHMEQ in NPC / Chapter 4.1.1 --- Growth inhibition in NPC cell lines --- p.31 / Chapter 4.1.2 --- Apoptotic induction in NPC cell lines --- p.35 / Chapter 4.1.3 --- Cell cycle arrest in NPC cell lines --- p.38 / Chapter 4.1.4 --- Inhibition of migration and invasive behavior of NPC cell lines --- p.38 / Chapter 4.1.5 --- Abrogation of soft agar colony formation ability of NPC cell lines --- p.43 / Chapter 4.2 --- Mechanistic study of DHMEQ in NPC / Chapter 4.2.1 --- Blockade of p65 nuclear translocation --- p.48 / Chapter 4.2.2 --- Attenuation of NF-kB transcriptional activity --- p.48 / Chapter 4.2.3 --- Downregulation of NF-kB target genes --- p.53 / Chapter Chapter 5 --- Discussion --- p.54 / Chapter Chapter 6 --- Summary --- p.60 / Chapter Chapter 7 --- Future Study --- p.63 / Reference List --- p.66 / Appendix / Chapter Appendix 1 --- Construction of NF-kb report plasm id --- p.78 / Chapter Appendix 2 --- Wound healing assay --- p.86 / Chapter Appendix 3 --- Reverse-phase protein Array --- p.88

published_or_final_version / Anaesthesiology / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

TAL-1, ectopically expressed in 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients, may contribute to poor chemotherapy response. This research sought to determine if TAL-1 influences expression of proteins involved in the NF-kB signaling pathway and thus, resistance to cell death. NF-kB, IKKy, and TRAF-2 expression levels were found to be TAL-1 dependent. Cell death levels were higher in staurosporine-treated cells compared to tumor necrosis factor a-treated or dual-treated cells. TAL-1, NF-kB, IKKy, and TRAF-2 expression levels were elevated in tumor necrosis factor a-treated cells and reduced in staurosporine-treated or dual treated cells compared to untreated cells. These results suggest TAL-1 influences expression of proteins involved in the NF-kB signaling pathway, thus inducing an anti-apoptotic response in the cell. / Department of Biology

Thesis (M.S.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains ix, 70 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 63-68).

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 95 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 82-95).

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains x, 202 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

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.

ATRX is a chromatin remodeling protein associated with X-linked Alpha-Thalassemia Mental Retardation syndrome and cancers that use the Alternative Lengthening of Telomere pathway. In the absence of ATRX there is a DNA damage response associated with telomeres and the expression of certain genes are perturbed. Recent findings (Law et al, 2010 Cell) have shown that ATRX is preferentially enriched at GC-rich tandem repeats in the genome. The mechanism for this localisation is unknown but may be related to the potential for these GC-rich tandem repeats to adopt non-B form DNA structures; ATRX has been shown to bind such structures (G4) in vitro. This study aims to understand the specific factors of the repeats that signal ATRX targeting. To address the research questions, an experimental system was developed, in which known targets, the ψζ VNTR and telomere repeats, were inserted into an inducible ectopic gene in the 293T-Rex cell line by site-directed recombination. ATRX was found to be enriched at the ectopic repeats compared to an endogenous negative control suggesting that it is recruited by the repeats independent of its original context. Furthermore, ATRX enrichment increased upon transcription of the ectopic gene, and this was dependent on the orientation of the repeat with the non-template strand being G-rich. Interestingly, when the repeat was transcribed, the distribution of ATRX across the repeats was asymmetrical with most ATRX binding downstream of the repeat. Moreover, there was a direct correlation between the repeat size and level of ATRX bound: the longer the repeat the higher the increase in ATRX enrichment. To determine the signal for ATRX binding, assays were performed to look for features which reflected the distribution of ATRX including H3K9me3, RNA polII, G4, R loops and DNA supercoiling. R loops look to be a strong candidate for the signaling of ATRX binding.

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