Liu, Ke. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 175-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Meng, Fanbiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

"Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to dementia in adults. The mechanism of neurodegeneration is thought to involve the extracellular production of a highly toxic A-beta peptide that engages cell surface receptors to induce cellular oxidative stress and apoptosis, but the signal transduction pathways that lead to A-beta induced cell death are unknown. We previously showed that a human ependymin neurotrophic peptide mimetic (hEPN-1) can promote cell survival in an in vitro AD model system. This initial observation was extended in this thesis by investigating the mechanism of A-beta induced apoptosis and hEPN-1 induced survival. Immunoblots were used to assay the total cellular levels of specific caspase proteins. The results show that A-beta induced apoptosis uses an extrinsic caspase pathway involving caspases-2 and -3, and that hEPN-1 treatment can reduce those caspase levels. A caspase activity assay showed that A-beta increased caspase-3/7 activity, while hEPN-1 treatment lowered it. Moreover, in vivo studies with AD transgenic mice showed that hEPN-1 treatment increased antioxidative superoxide dismutase levels in brain. Thus, hEPN-1 holds potential as a therapeutic to treat the underlying neurodegenerative cause of AD, not merely its symptoms as with other currently approved AD drugs."

"Alzheimer’s disease (AD) is a neurodegenerative disorder that currently affects an estimated 4.2 million to 5.8 million Americans. Although the cause of AD is not fully known, the current working model proposes that amyloid precursor protein (APP) is unnaturally cleaved by beta and gamma secretases to form the highly neurotoxic peptide beta-amyloid (Aâ) which engages cell surface receptors to cause cell death through a series of events involving oxidative stress and apoptosis. An in vitro model for AD uses cultured human SHSY-5Y (commonly abbreviated SHSY) neuroblastoma cells treated with Yankner peptide, an 11 amino acid peptide representing Aâ residues 25-35 that strongly binds receptor. Treatment of SHSY cells with 20 µM Yankner peptide strongly induces cellular apoptosis. Synthetic peptide human ependymin-1 (hEPN-1) is a derivative of a naturally occurring protein within the human brain, previously shown by our laboratory to upregulate antioxidative enzymes in SHSY cells, and AP-1 transcription factor associated with long-term memory formation. Since hEPN-1 has anti-oxidative potential as a therapeutic, we hypothesized that hEPN-1 can reverse the neurotoxic effects of Yankner peptide treatment of cultured human SHSY neuronal cells. Microtiter dishes were plated with SHSY cells under control conditions (no Yankner peptide), in the presence of 20 µM Yankner peptide, or in the presence of Yankner peptide plus various concentrations of hEPN-1 therapeutic, then cultured for 3 days to 80% confluency. Unattached dying cells were gently washed away, then the residual cells were monitored by measuring cell number, cell viability (Trypan blue exclusion), LDH activity per mg protein (an indirect measure of cell viability), and nuclear blebbing (a measure of apoptosis). Statistical significance was determined using a One Way ANOVA under the LSD stringency, using SPSS. In three independent trials, average cell numbers per microtiter well decreased 44.7% (from 3.11 x 105 to 1.72 x 105) in the presence of 20 µM Yankner peptide (p < 0.05 compared to control), were 2.73 x 105 when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.05 compared to Yankner), and were 2.96 x 105 when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.05 relative to Yankner alone). The control mean was not statistically distinguishable from either of the hEPN-1-treated samples (p = 0.220 and p = 0.671, respectively). With respect to the trypan blue data, in three independent trials, the mean percent viable cells (excluding trypan blue) decreased 41.0% (from 68.7% to 40.5%) in the presence of 20 µM Yankner peptide (p < 0.001 relative to control), was 60.7% when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.001 relative to Yankner alone), and was 61.4% when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.001 relative to Yankner alone). The control mean was not statistically distinguishable from either of the hEPN-1-treated samples (p = 0.013 and 0.03, respectively). In the LDH activity experiments, in four independent trials, the average LDH OD decreased 80.8% (from 0.47 to 0.09) in the presence of 20 µM Yankner peptide (p < 0.001 relative to control), was 0.47 when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.001 relative to Yankner alone), and was 0.48 when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.001 relative to Yankner alone). The control mean was not statistically distinguishable from either of the hEPN-1-treated samples (p = 0.174 and 0.479, respectively). Although previous reports in the literature indicated LDH expression is constitutive in SHSY cells (thus its activity is an indirect measure of cell numbers or viability), it was possible the hEPN-1 treatments upregulated LDH activity. So to ensure our observed changes in LDH activity levels did not represent changes per unit protein, the LDH activity values were divided by the mg of protein present in the sample, and all four experimental samples were statistically indistinguishable (p values = 0.184, 0.995, 0.872, respectively, relative to control). In the nuclear blebbing experiments, in five independent trials, the mean percent blebbed nuclei (a measure of apoptosis) doubled from 7.5% to 16.0% in the presence of 20 µM Yankner peptide (p < 0.001 relative to control), was 6.7% when 75 µM hEPN-1 was added simultaneously with Yankner (p < 0.001 relative to Yankner alone), and was 6.5% when 75 µM hEPN-1 was added 24 hrs post-Yankner (p < 0.001 relative to Yankner alone). The decreased apoptosis observed in the hEPN-1-treated samples was however, not statistically significant (p = 0.381 and 0.279, respectively). Overall, the data suggest that hEPN-1 can protect human neuronal cells from Yankner-induced cell death, whether added simultaneous to the insult, or 24 hrs post. Because the therapeutic can act 24 hrs post-insult, it may interfere with a late-stage apoptotic event. As there is currently no known drug that blocks Yankner-induced toxicity, the hEPN-1 therapeutic shows potential in combating the underlying apoptosis of Alzheimer’s disease."

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2009. / Includes bibliographical references. / Iron availability and primary productivity in the oceans are intricately linked through photosynthesis. At the global scale we understand how iron addition induces phytoplankton blooms through meso-scale iron-addition experiments. At the atomic scale, we can describe the length and type of bonds that connect iron atoms to components of photosystem I, the most efficient light-harvesting complex in nature. Yet, we know little of how iron influences microbial diversity and distribution in the open ocean. In this study, we assess the influence of iron on the ecology of the numerically abundant marine cyanobacterium, Prochlorococcus. With its minimal genome and ubiquity in the global ocean, Prochlorococcus represents a model system in which to study the dynamics of the link between iron and primary productivity. To this end, we tested the iron physiology of two closely-related Prochlorococcus ecotypes. MED4 is adapted to high-light environments while MIT9313 lives best in low-light conditions. We determined that MIT9313 is capable of surviving at low iron concentrations that completely inhibit MED4. Furthermore, concentrations of Fe' that inhibit growth in culture are sufficient to support Prochlorococcus growth in the field, which raises questions about the species of iron available to Prochlorococcus. We then examined the molecular basis for the ability of MIT9313 to grow at lower iron concentrations than MED4 by assessing whole-genome transcription in response to changes in iron availability in the two ecotypes. / Genes that were differentially expressed fell into two categories: those that are shared by all (Prochlorococcus core genome) and those that are not (non-core genome). Only three genes shared between MED4 and MIT9313 were iron-responsive in both strains. We then tested the iron physiology of picocyanobacteria in the field and found that Synechococcus is iron-stressed in samples where Prochlorococcus is not. Finally, we propose a method to measure how iron stress in Prochlorococcus changes over natural gradients of iron in the oligotrophic ocean by quantifying transcription of the iron-stress induced gene, isiB. Taken together, our studies demonstrate that iron metabolism influences the ecology of Prochlorococcus both by contributing to its diversity and distinguishing it from other marine cyanobacteria. / by Anne Williford Thompson. / Ph.D.

Dendritic cells (DCs) comprise a distinct lineage of potent antigen-presenting mononuclear phagocytes that serve as both mediators of innate immune responses and key facilitators of the adaptive immune response. DCs play both immunogenic and tolerogenic roles through their dual ability to elicit pathogen-specific T cell immunity as well as induce regulatory T cell (Treg) responses to promote tolerance in the steady state. The aim of the work presented here is to examine the normal regulatory mechanisms of DC development and function, starting with the dissection of mechanisms behind an aberrantly activated developmental pathway, followed by the exploration of new mechanisms governed by two candidate transcription factors. The first chapter of the thesis focuses on the growth factor receptor Flt3, an essential regulator of normal DC development in both mice and humans, and concurrently one of the most commonly mutated proteins found in acute myeloid leukemia (AML). We investigated the effect of its most common activating mutation in AML, the Flt3 internal tandem duplication (Flt3-ITD), and found that this mutation caused a significant cell-intrinsic expansion of all DC populations. This effect was associated with an expansion of Tregs and the ability to dampen self-reactivity, with an inability to control autoimmunity in the absence of Tregs. Thus, we describe a potential mechanism by which leukemia can modulate T cell responses and support Treg expansion indirectly through DCs, which may compromise immunosurveillance and promote leukemogenesis. The subsequent chapters explore the basic molecular mechanisms of DC development by using Flt3 expression as a guide to uncover new candidates involved in the DC transcriptional program. We show that Myc family transcription factor, Mycl1, is largely dispensable for DC development and function, contrary to recent published findings that propose a role in proliferation and T cell priming. On the other hand, we find that conditional deletion of our second candidate gene, an Ets family transcription factor, has diverse effects on DC development, monocyte homeostasis, and cytokine production. Overall, our studies highlight an unexpected molecular link between DC development and leukemogenesis, and elucidate novel mechanisms controlling DC differentiation and function.

The discovery of novel members of tumorigenic pathways remains a critical step to fully dissect the molecular biology of cancer. Indeed, because a number of cancer drivers are themselves undruggable, elucidating the signaling apparatuses in which they participate is essential for discovering novel therapeutic targets that will allow the treatment of aggressive neoplastic growth. In the context of oncoproteins and tumor suppressors, novel participants may be upstream regulators, downstream effectors, or physical cognate binding partners. In this work, we develop in silico approaches to more fully elucidate the tumorigenic signaling machinery used by tumor suppressors and oncoproteins. We first report applications of machine-learning algorithms to integrate diverse networkbased information to generate testable hypotheses of proteins involved in canonical oncogenic pathways. We develop the OncoSig algorithm to elucidate novel members of protein-centric maps to elucidate upstream modulators, cognate binding partners, and downstream effectors for any tumor suppressor or oncogene in a tumor-specific fashion. We specifically apply OncoSig to elucidate the oncogenic KRAS regulatory map in Lung adenocarcinoma (LUAD). Oncogenic KRAS is a key driver of aggressive tumor growth in many LUAD patients, yet has no FDA-approved drugs targeting it. Thus, elucidating members of the KRAS protein-centric map is critical for discovering synthetic lethal interactions that may be subject to therapeutic targeting. Critically, 18/22 of novel predicted KRAS interactors elicited synthetic lethality in LUAD organoid cultures that harbored an activating KRAS mutation. We then extend the OncoSig algorithm to 10 oncogenic/tumor suppressor pathways (such as TP53, EGFR, and PI3K), and show that OncoSig is able to recover known regulators and downstream effectors of these critical mediators of tumorigenesis. We then focus specifically on dissecting KRAS’s physical protein-protein interactions. Many cognate binding partners bind to KRAS via a structurally conserved RAS-Binding Domain (RBD), thus propagating KRAS signal transduction. Thus, for example, CRAF, PI3K, and RALGDS, all bind to KRAS via an RBD. To elucidate novel KRAS protein-protein interactors, we use structural and sequence based approaches to discover biophysical properties of known RBDs. We apply the PrePPI algorithm, which predicts novel protein-protein interactions based on structural similarity, and find that PrePPI successfully recovers known RBDs while discriminating from domains structurally similar to the RBD that do not bind to KRAS. Using this information, we develop biophysical features to computationally predict novel KRAS binding partners. Finally, we report computational and experimental work addressing whether KRAS forms a homodimer. The precise mechanism for how KRAS propagates signal transduction after binding to the RBD remains elusive, and KRAS homo-dimerization, for example, may play a key role in KRAS induced tumorigenesis. Using Analytical Utracentrifugation to measure binding affinity, we find that KRAS forms either a weak dimer or a large non-specific multimer. Furthermore, analysis of KRAS protein structures deposited in the Protein Data Bank reveals key regions that have a propensity to form homodimer contacts in the crystal complexes, and may mediate KRAS homo-dimerization in a biological setting as well. These results provide mechanistic insight into how KRAS dimerization may facilitate cellular signal transduction.

Bacteria adhere despite severe mechanical perturbations. In Gram-positive bacteria, this adhesion is dependent upon a set of extracellular proteins, most notably pili, that have a unique abundance of internal disulfide, isopeptide, and thioester bonds. How these cell adhesion proteins manage to withstand such mechanical assaults, and what role these internal covalent bonds play to that end, remain open questions. Herein, we apply single-molecule force spectroscopy to delve into the mechanical behavior of three Gram-positive pilus proteins. We find that structural components of the Actinomyces oris and Corynebacterium diphtheriae pili have isopeptide-delimited extensions at extreme mechanical forces. This behavior enables efficient energy dissipation under high mechanical loads. Meanwhile, the pilus tip adhesin of Streptococcus pyogenes can covalently bind to targets via its internal thioester bond. We find that reactions with this internal thioester bond are reversible, and that both the nucleophilic bond cleavage and its spontaneous reformation are negatively force-dependent, inhibited at forces above ~30 pN and above ~7 pN, respectively. Based on these observations, we propose a model of shear-enhanced covalent adhesion for Gram-positive bacteria. Finally, we move from single-molecule characterization to application as we explore the potential of a peptide competitors to modulate the folding and function of bacterial virulence factors.

腎細胞癌是一種成人惡性腫瘤，治療效果不理想且常發生腫瘤轉移。目前對腎細胞癌的研究主要集中於鑒定並驗證可用於癌癥早期診斷和預後判斷的新型潛在生物標誌物。擬遺傳學變化尤其是啟動子CpG二核苷酸甲基化所導致的抑癌基因功能喪失已被廣泛認為是腫瘤發生的一個主要機理。迄今為止，已有許多抑癌基因在腎細胞癌中被報道出現啟動子甲基化。這些發現為腎癌發生的分子機制及潛在生物標誌物提供了新的思路。本課題旨在探索ZNF382和BCL6B這兩個鋅指蛋白抑癌基因在腎細胞癌中的啟動子甲基化情況，及其與腫瘤抑制有關的生物學功能和可能的分子機制。 / 鋅指蛋白轉錄抑制子ZNF382已在多種癌癥中被報道為功能性抑癌基因， 且常伴隨有啟動子甲基化導致的基因沈默，但其在腎細胞癌中尚未被報道。我們發現ZNF382在腎癌細胞系中由於啟動子CpG甲基化而致表達下調或基因沈默，並且其表達下調或沈默可被去甲基化藥物逆轉，在正常細胞系中則觀察不到這一現象。腎癌原發腫瘤組織中也廣泛檢測到ZNF382異常甲基化。在ZNF382沈默的腎細胞癌細胞系中，外源表達的ZNF382顯著地抑制了腫瘤細胞集落形成和細胞遷移，並且誘導細胞發生雕亡。而且，我們發現ZNF382在腎癌細胞系中可抑制多種致瘤基因和幹細胞標誌基因的表達。因此，本研究證明ZNF382通過抑制下遊致癌基因和幹細胞標誌基因的表達從而發揮抑制腫瘤的功能，並且其在腎細胞癌中常因啟動子高度甲基化而導致基因失活。 / 另一個鋅指蛋白基因BCL6B（ZNF62）已被證實可通過招募組蛋白去乙酰化酶抑制靶基因的轉錄，但其在腎細胞癌中的表達情況和生物學功能尚不清楚。我們發現，BCL6B基因在正常腎組織和正常細胞系中穩定表達， 但在腎癌細胞系中由於啟動子甲基化其表達下調或沈默。去甲基化藥物可以重新激活BCL6B的表達，同時伴隨其啟動子的去甲基化。BCL6B甲基化在腎癌原發腫瘤組織中也被頻繁檢測到。在腎癌細胞系中，外源表達BCL6B顯著抑制了腫瘤細胞集落形成和細胞遷移，並且誘導腫瘤細胞雕亡。我們進一步發現，BCL6B作為功能性轉錄抑制子在腎癌細胞系中抑制多種致癌基因和幹細胞標誌基因的表達。這些結果表明BCL6B是腎細胞癌的一個抑癌基因且其在腎癌中常被甲基化。 / 綜上所述，本課題從擬遺傳學和生物學功能兩個方面分別鑒定了腎癌中的兩個鋅指蛋白抑癌基因，ZNF382 和BCL6B。此研究可以幫助更好地了解腎癌發生的分子機理，並且為發展新的腎癌標誌物提供了更多思路。 / Renal cell carcinoma (RCC) is a malignant cancer in adults, often with poor outcome and frequent metastasis. Recent studies on this disease focus on the identification and verification of novel potential biomarkers for early detection and prognostic prediction of cancer. Epigenetic alterations, especially promoter CpG methylation, leading to the loss of tumor suppressor gene (TSG) function have been widely recognized as a major cause for tumor pathogenesis. To date, a number of TSGs with aberrant promoter methylation have been reported in RCC, which provides new insights into the molecular mechanism of renal cancer and the potential as biomarkers. The aim of this study is to characterize promoter methylation of two zinc finger tumor suppressors, ZNF382 and BCL6B, their biological functions and underlying molecular mechanisms in RCC. / Transcription repressor ZNF382 (zinc finger protein 382) was reported as a functional TSG with frequent inactivation by promoter methylation in multiple carcinomas, but not studied in RCC yet. I found that ZNF382 was silenced or downregulated in RCC cell lines due to promoter CpG methylation which could be reversed by pharmacologic demethylation treatment, but not in normal renal cell lines. Aberrant methylation of ZNF382 was also frequently detected in the RCC primary tumors. Ectopic expression of ZNF382 in the silenced RCC cells strongly inhibited their clonogenicity and migration, as well as promoted cell apoptosis. Moreover, I found that ZNF382 repressed the expression of multiple oncogenes and stem cell markers in RCC cells. Therefore, my results demonstrate ZNF382 exerts the tumor suppressive function through repressing the downstream target oncogenes and stem cell markers, and is often epigenetically inactivated by promoter methylation in RCC. / Another zinc finger protein, B cell CLL/lymphoma 6 member B (BCL6B, ZNF62) has been identified to repress transcription of its target genes by recruiting histone deacetylases, but its expression and biological function in RCC remain largely unknown. BCL6B was readily expressed in normal kidney tissue and renal cell line. BCL6B was silenced or downregulated by promoter CpG methylation in RCC cell lines. Pharmacologic demethylation reactivated BCL6B expression along with concomitant promoter demethylation. BCL6B methylation was also frequently detected in RCC primary tumors. Ectopic expression of BCL6B in RCC cells significantly inhibited tumor clonogenicity and migration of RCC cells, and induced tumor cell apoptosis. We further found that BCL6B as functional repressor suppressed the expression of multiple oncogenes and stem cell markers. These data indicated BCL6B was a functional tumor suppressor frequently methylated in RCC. / In summary, my study identified two zinc finger tumor suppressors, ZNF382 and BCL6B, in RCC from both epigenetical and functional aspects. This work may contribute to a better understanding of the molecular mechanisms of renal cancer pathogenesis and also give more clues to the discovery of novel biomarkers for RCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Rong, Rong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 110-128). / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of abbreviations --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / List of Publications --- p.xviii / Chapter Chapter 1 --- Literature Reviews --- p.1 / Chapter 1.1 --- Molecular basis of cancer --- p.1 / Chapter 1.1.1 --- Oncogenes and TSGs --- p.2 / Chapter 1.1.2 --- Cancer genetics --- p.3 / Chapter 1.1.3 --- Cancer epigenetics --- p.4 / Chapter 1.1.4 --- DNA methylation --- p.4 / Chapter 1.1.4.1 --- Mechanism of DNA methylation --- p.5 / Chapter 1.1.4.2 --- DNA methylation and gene transcription --- p.6 / Chapter 1.1.4.3 --- Types of DNA methylation in human cancers --- p.7 / Chapter 1.1.4.3.1 --- Hypomethylation in cancer genome --- p.8 / Chapter 1.1.4.3.2 --- Hypermethylation of TSGs in cancer --- p.8 / Chapter 1.1.5 --- The link between cancer genetics and cancer epigenetics --- p.9 / Chapter 1.2 --- Renal cell carcinoma (RCC) --- p.10 / Chapter 1.2.1 --- Epidemiology of RCC --- p.10 / Chapter 1.2.2 --- Histopathology of RCC --- p.14 / Chapter 1.2.3 --- Genetic and epigenetic alterations in RCC --- p.16 / Chapter 1.2.3.1 --- Genetic alterations --- p.17 / Chapter 1.2.3.2 --- Epigenetic alterations --- p.22 / Chapter 1.2.3.2.1 --- Aberrant DNA hypermethylation in RCC --- p.22 / Chapter 1.2.3.2.2 --- Histone and chromatin regulations in RCC --- p.24 / Chapter 1.2.4 --- Signaling pathways associated with RCC --- p.25 / Chapter 1.2.4.1 --- VHL/HIF signaling in RCC --- p.26 / Chapter 1.2.4.2 --- PI3K/AKT/mTOR signaling in RCC --- p.27 / Chapter 1.2.4.3 --- Wnt/β-catenin signaling in RCC --- p.28 / Chapter 1.2.4.4 --- HGF/MET signaling in RCC --- p.31 / Chapter 1.3 --- Transcription factor family of zinc finger proteins --- p.32 / Chapter 1.3.1 --- Zinc Finger Protein 382 (ZNF382) --- p.33 / Chapter 1.3.2 --- B cell CLL/lymphoma 6, member B (BCL6B) --- p.34 / Chapter Chapter 2 --- Aim of Study --- p.36 / Chapter 2.1 --- Identify two zinc finger repressors as TSGs for RCC --- p.37 / Chapter 2.2 --- Study their tumor suppressor roles in RCC --- p.37 / Chapter 2.3 --- Explore the mechanisms of their tumor suppressor function --- p.38 / Chapter Chapter 3 --- Materials and Methods --- p.39 / Chapter 3.1 --- Cell lines and tissue samples --- p.39 / Chapter 3.1.1 --- Cell lines, tumors and normal tissue samples --- p.39 / Chapter 3.1.2 --- Maintenance of cell lines --- p.39 / Chapter 3.1.3 --- Drug treatment of cell lines --- p.40 / Chapter 3.1.4 --- Total RNA extraction --- p.40 / Chapter 3.1.5 --- Genomic DNA extraction --- p.41 / Chapter 3.2 --- General techniques --- p.42 / Chapter 3.2.1 --- Agarose gel electrophoresis --- p.42 / Chapter 3.2.2 --- TA cloning --- p.43 / Chapter 3.2.3 --- Transformation of cloning vectors into E. coli competent cells --- p.43 / Chapter 3.2.4 --- Plasmid DNA extraction --- p.44 / Chapter 3.2.4.1 --- Mini-prep of plasmid DNA --- p.44 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.45 / Chapter 3.2.5 --- Measurement of DNA and RNA concentrations --- p.45 / Chapter 3.2.6 --- Preparation of reagents and medium --- p.46 / Chapter 3.2.6.1 --- Reagents for agarose gel electrophoresis --- p.46 / Chapter 3.2.6.2 --- Reagents for mini-prep of plasmid DNA --- p.46 / Chapter 3.2.6.3 --- LB medium and LB plates --- p.46 / Chapter 3.3 --- Semi-quantitative Reverse transcription (RT)-PCR --- p.47 / Chapter 3.3.1 --- Reverse Transcription --- p.47 / Chapter 3.3.2 --- Semi-quantitative PCR --- p.48 / Chapter 3.3.2.1 --- Primer design --- p.48 / Chapter 3.3.2.2 --- PCR reaction --- p.49 / Chapter 3.4 --- Real-time PCR --- p.49 / Chapter 3.5 --- Methylation analysis --- p.50 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.50 / Chapter 3.5.2 --- Bioinformatical analysis of CpG island --- p.51 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.51 / Chapter 3.5.3.1 --- Primers design --- p.51 / Chapter 3.5.3.2 --- PCR reaction --- p.53 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.53 / Chapter 3.5.4.1 --- Primers design --- p.53 / Chapter 3.5.4.2 --- PCR amplification and TA-cloning --- p.54 / Chapter 3.6 --- Construction of expression plasmids for studied genes --- p.54 / Chapter 3.6.1 --- Construction of the ZNF382-expressing vector --- p.54 / Chapter 3.6.2 --- Construction of the BCL6B-expressing vector --- p.55 / Chapter 3.7 --- Functional Study --- p.56 / Chapter 3.7.1 --- Colony formation assay on monolayer culture --- p.56 / Chapter 3.7.2 --- Wound healing assay --- p.57 / Chapter 3.7.3 --- TUNEL assay --- p.58 / Chapter 3.8 --- Western blot --- p.58 / Chapter 3.9 --- Statistical analysis --- p.58 / Chapter Chapter 4 --- Results --- p.60 / Chapter 4.1 --- Epigenetic and Functional study of ZNF382 in RCC --- p.60 / Chapter 4.1.1 --- Expression profiling of ZNF382 in human adult tissues --- p.60 / Chapter 4.1.2 --- Expression profiling of ZNF382 in RCC cell lines --- p.61 / Chapter 4.1.3 --- Dense promoter CpG methylation of ZNF382 correlated with its reduced expression in RCC --- p.62 / Chapter 4.1.4 --- Restoration of ZNF382 expression by pharmacologic demethylation --- p.65 / Chapter 4.1.5 --- Frequent methylation of ZNF382 in RCC primary tumors --- p.67 / Chapter 4.1.6 --- Functional study of ZNF382 in RCC --- p.68 / Chapter 4.1.6.1 --- Ectopic expression of ZNF382 inhibits clonogencity of RCC cells --- p.68 / Chapter 4.1.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.71 / Chapter 4.1.7 --- ZNF382 induces apoptosis of RCC cells --- p.72 / Chapter 4.1.8 --- ZNF382 represses the expression of multiple oncogenes and stem cell markers in RCC --- p.73 / Chapter 4.1.9 --- Discussion --- p.76 / Chapter 4.2 --- Epigenetic and Functional study of BCL6B in RCC --- p.82 / Chapter 4.2.1 --- Expression profiling of BCL6B in human adult tissues --- p.82 / Chapter 4.2.2 --- Expression profiling of BCL6B in RCC cell lines --- p.83 / Chapter 4.2.3 --- Correlation of the methylation status of ZNF382 promoter CpG island with its aborted expression in RCC --- p.84 / Chapter 4.2.4 --- Restoration of BCL6B expression by pharmacological demethylation --- p.87 / Chapter 4.2.5 --- Frequent BCL6B methylation in RCC primary tumors --- p.88 / Chapter 4.2.6 --- Functional study of BCL6B in RCC --- p.90 / Chapter 4.2.6.1 --- Ectopic expression of BCL6B inhibits clonogencity of RCC cells --- p.90 / Chapter 4.2.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.92 / Chapter 4.2.7 --- BCL6B induces apoptosis of RCC cells --- p.93 / Chapter 4.2.8 --- BCL6B represses the expression of multiple oncogenes and stem cell markers in RCC --- p.94 / Chapter 4.2.9 --- Discussion --- p.97 / Chapter Chapter 5 --- General discussion --- p.103 / Chapter Chapter 6 --- Summary --- p.106 / Chapter Chapter 7 --- Future Study --- p.108 / Chapter 7.1 --- Identification of key responsive elements in gene promoter --- p.108 / Chapter 7.2 --- Study of genetic alterations leading to gene inactivation --- p.109 / Chapter 7.3 --- Elucidation of the transcription-repressor activity in RCC --- p.109 / Reference List --- p.110

大腸癌是全世界最常見的癌症之一，亦是一個癌症死亡率的首要原因。大腸癌患者約50%在病程中會出現轉移病灶。近十年來，雖然多種被批准用於臨床治療的新化療藥顯著提高了大腸癌的治療效果，但是轉移性大腸癌病人的預後仍然很差。隨著各種分子生物技術的進步，新的治療標靶可能在大腸癌細胞株中被發現，並得以在病人標本中驗證。 / 在本研究中，我們採用即時定量多聚酶鏈反應(qPCR)陣列分析，比較大腸癌細胞株和正常大腸細胞株基因表達譜，試圖識別潛在的新的治療標靶。結果提示，與正常大腸細胞株 CCD-18Co 比較，Jagged 2 (JAG2) 和 Frizzled-3 (FZD3)基因 在大腸癌細胞株 SW480 和 SW620 中表達升高。病人大腸癌組織的免疫組織化學染色 (IS) 檢查進一步證實了上述結果，大腸癌組織較其癌旁正常組織表達3.1倍JAG2和6.6倍FZD3蛋白。因此， 我們假設JAG2和FZD3在大腸癌的發生中起重要作用。 / 為了檢驗該假設的真偽，我們運用RNA 干擾的方法進行功能缺失研究。通過該方法，大腸癌細胞株中JAG2 信使RNA和蛋白均能夠被下調，但是FZD3蛋白卻沒有顯示降低。為了弄清JAG2基因的功能，我們進行了單層細胞劃痕傷口癒合試驗和Matrigel 侵襲試驗。結果提示，JAG2 基因下調顯著抑制大腸癌細胞遷移和侵襲的能力。 / 為了調查參與上述功能的機制，我們利用腫瘤轉移相關基因的qPCR陣列分析，試圖檢測出JAG2基因敲除後上調或下調表達的轉移相關基因。結果顯示組織蛋白酶K (CTSK)，一種溶酶體半胱氨酸蛋白酶，在JAG2基因沉默的大腸癌細胞株中表達下調。為了闡明CTSK 活性在大腸癌細胞株侵襲能力中起到的作用，我們採用CTSK抑制劑處理大腸癌細胞株HCT116和DLD-1，發現這兩種細胞株的侵襲能力分別下降了36%和59%。總之, 這些發現表明CTSK可能是JAG2的下游靶基因，活性CTSK可能參與了JAG2介導的大腸癌細胞株侵襲能力。 / 以前的研究表明p38 MAPK通路參與癌細胞遷和侵襲能力的調控。通過Western blot方法，磷酸化的p38和磷酸化的STAT3被發現在JAG2基因沉默的大腸癌細胞中表達降低。p38抑制劑處理的 HCT116和DLD-1細胞降低了侵襲能力下降，同時遷移能力也由於p38抑制劑的處理而降低，支持p38可調控癌細胞遷移和侵襲能力的事實。 / 總之，我們的結果顯示JAG2高表達通過啟動CTSK和p38 MAPK通路，可能促進大腸癌轉移。因此，JAG2可能成為轉移性大腸癌治療的潛在標靶。 / Colorectal cancer (CRC) is one of the most frequent cancers worldwide and is a leading cause of cancer mortality. Around 50% of patients with CRC will experience metastases. Although significant progress has been made in CRC treatment within the last decade with the approval of multiple new chemotherapeutic agents, the prognosis for patients with metastatic CRC remains poor. With the advancement of molecular techniques, novel therapeutic targets are able to be discovered in CRC cell lines and validated in patient samples. / Therefore in this project, I aim to identify potential novel therapeutic targets by comparing the gene expression profile of colon cancer cell lines and a normal colon cell line using quantitative polymerase chain reaction (qPCR) arrays. Results showed that Jagged 2 (JAG2) and Frizzled-3 (FZD3) were up-regulated in the CRC cell lines SW480 and SW620 as compared to the normal colon cell line CCD-18Co. Those results were further validated by immunohistochemical staining (IS), which detected up-regulated JAG2 (3.1-fold) and FZD3 (6.6-fold) proteins expression in CRC tissues as compared to adjacent normal tissues. Thus I hypothesized that JAG2 and FZD3 may play an important role in CRC carcinogenesis. / In order to study the roles of FZD3 and JAG2 in CRC, loss-of-function studies by RNA interference (RNAi) were carried out. While the expression of FZD3 protein failed to be down-regulated by RNAi, JAG2 expression was successfully knocked down in CRC cell lines at both the mRNA and protein levels. Functional analyses using the monolayer scratch wound-healing assay and Matrigel invasion assay showed that JAG2 knockdown significantly inhibited migration and invasion in CRC cell lines. / To investigate the mechanisms involved, a tumour metastasis qPCR array was used to examine the changes in the expression level of metastasis-related genes after JAG2 gene knockdown. Results showed that the expression of Cathepsin K (CTSK), a lysosomal cystein protease, was found to be down-regulated in CRC cell lines following JAG2 silencing. To demonstrate the importance of CTSK activity in CRC cell invasion, HCT116 and DLD-1 CRC cell lines were treated with a CTSK inhibitor and its effect were assessed by the Matrigel invasion assay. Results showed that CTSK inhibition led to a 36% and 59% reduction in number of invaded cells in HCT116 and DLD-1 cell lines, respectively. Taken together, these findings show that CTSK may be a downstream target of JAG2 and that active CTSK may involve in JAG2 mediated invasion in CRC cell lines. / Previous works by others have shown that the p38 MAPK pathway is involved in the regulation of migration and invasive activity of cancer cell lines. Using Western blot analysis, the expression of phosphorylated p38 MAPK and phosphorylated STAT3 were found to be down-regulated following JAG2 depletion in CRC cell lines. In support of a role for p38 MAPK in the regulation of cancer cell migration and invasive capability, treatment with a p38 MAPK inhibitor was found to reduce the percentage of invasive cells and distance moved by migratory cells in HCT116 and DLD-1 cell lines. / In conclusion, my results show that JAG2 over-expression in CRC may promote cancer cell migration and invasion through activation of CTSK and the p38 MAPK pathway. Therefore, JAG2 may be a potential therapeutic target for treatment of metastatic CRC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / He, Wan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 164-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Colorectal Cancer (CRC) --- p.1 / Chapter 1.1.1 --- Epidemiology and Incidence --- p.1 / Chapter 1.1.2 --- Histology --- p.2 / Chapter 1.1.3 --- Gender and Age --- p.4 / Chapter 1.1.4 --- Etiology of CRC --- p.4 / Chapter 1.1.4.1 --- Environment --- p.4 / Chapter 1.1.4.2 --- Hereditary Factors --- p.5 / Chapter 1.1.4.3 --- Dietary Factors --- p.6 / Chapter 1.1.4.4 --- Obesity --- p.6 / Chapter 1.1.4.5 --- Tobacco and alcoho --- p.7 / Chapter 1.1.4.6 --- Inflammatory bowel disease (IBC) --- p.7 / Chapter 1.1.5 --- Genetic Changes in CRC --- p.8 / Chapter 1.1.5.1 --- Chromosomal Aberration --- p.8 / Chapter 1.1.5.2 --- Tumor Suppressor Genes --- p.10 / Chapter 1.1.5.2.1 --- APC gene --- p.10 / Chapter 1.1.5.2.2 --- P53 gene --- p.11 / Chapter 1.1.5.2.3 --- SMAD4 gene --- p.11 / Chapter 1.1.5.3 --- Oncogenes --- p.12 / Chapter 1.1.5.3.1 --- Epidermal Growth Factor Receptor (EGFR) gene --- p.12 / Chapter 1.1.5.3.2 --- RAS gene and BRAF gene --- p.13 / Chapter 1.1.5.4 --- Proposed Two-hit Model for the Multistep Pathogenesis of CRC --- p.15 / Chapter 1.1.6 --- Clinical Presentation and Diagnosis --- p.16 / Chapter 1.1.7 --- Theatment --- p.16 / Chapter 1.1.7.1 --- Surgery --- p.16 / Chapter 1.1.7.2 --- Radiotherapy (RT) --- p.17 / Chapter 1.1.7.3 --- Concurrent Chemotherapy --- p.17 / Chapter 1.1.7.4 --- Target Therapy --- p.18 / Chapter 1.1.7.5 --- Colorectal Cancer Treatment by Stage --- p.19 / Chapter 1.1.7.6 --- Novel Strategies --- p.20 / Chapter 1.1.7.6.1 --- Epigenetic therapy --- p.20 / Chapter 1.1.7.6.2 --- Immunotherapy --- p.21 / Chapter 1.2 --- Pathways Involved in CRC Carcinogenesisand Progression --- p.22 / Chapter 1.2.1 --- Wnt Signaling Pathway --- p.22 / Chapter 1.2.2 --- Notch Signaling --- p.23 / Chapter 1.2.3 --- Nuclear Factor-kappa B (NF-κB) Signaling Pathway --- p.23 / Chapter 1.2.4 --- Phosphatidylinositol 3-kinase (PI3K) Signaling Pathway --- p.24 / Chapter 1.2.5 --- Crosstalk Among WNT, NOTCH, NF-κB and PI3K Signaling Pathway in CRC --- p.24 / Chapter 1.3 --- Hypothesis and Objectives of this Study --- p.28 / Chapter Chapter 2 --- Identification of Differentially Expressed Genes between Colorectal Cancer Cell Lines and A Normal Colon Cell Line --- p.29 / Chapter 2.1 --- Background --- p.29 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Cell Lines --- p.33 / Chapter 2.2.2 --- Identification of Differetially Expressed Genes by qPCR Arrays --- p.33 / Chapter 2.2.2.1 --- Total RNA Extraction --- p.33 / Chapter 2.2.2.2 --- RNA Quality Contol --- p.34 / Chapter 2.2.2.3 --- Reverse Transcription (RT) --- p.34 / Chapter 2.2.2.4 --- PCR Arrays --- p.34 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Differentially Expressed Genes in WNT Signaling Pathway --- p.36 / Chapter 2.3.2 --- Differentially Expressed Genes in Notch Signaling Pathway --- p.40 / Chapter 2.3.3 --- Differentially Expressed Genes in NF-κB Signaling Pathway --- p.43 / Chapter 2.3.4 --- Differentially Expressed Genes in PI3K-AKT Signaling Pathway --- p.46 / Chapter 2.3.5 --- Choice of over-expressed genes for further validation and characterization --- p.49 / Chapter 2.4 --- Discussions --- p.53 / Chapter 2.4.1 --- WNT Signaling Pathway --- p.53 / Chapter 2.4.2 --- NOTCH Signaling Pathway --- p.54 / Chapter 2.4.3 --- NF-κB Signaling Pathway --- p.55 / Chapter 2.4.4 --- PI3K-AKT Signaling Pathway --- p.56 / Chapter 2.4.5 --- Choice of over-expressed genes for further validation and characterization --- p.56 / Chapter Chapter 3 --- JAG2, FZD3 and NOTCH4 Expression in Colorectal Cancer Cell Lines and Colorectal Cancer Tissues --- p.59 / Chapter 3.1 --- Background --- p.59 / Chapter 3.1.1 --- JAG2 Ligand --- p.59 / Chapter 3.1.2 --- FZD3 Receptor --- p.61 / Chapter 3.1.3 --- NOTCH4 Receptor --- p.62 / Chapter 3.2 --- Materials and Methods --- p.64 / Chapter 3.2.1 --- CRC Cell Lines --- p.65 / Chapter 3.2.2 --- CRC Tissues --- p.65 / Chapter 3.2.3 --- Quantitative RT-PCR --- p.66 / Chapter 3.2.4 --- Detection of JAG2, FZD3 and NOTCH4 Protein Expression in CRC Tissues by Immunohistochemical Staining (IS) --- p.67 / Chapter 3.2.5 --- Western Blot Assays --- p.68 / Chapter 3.2.5.1 --- Protein extraction --- p.68 / Chapter 3.2.5.2 --- SDS-PAGE gel electrophroresis --- p.68 / Chapter 3.2.5.3 --- Protein blotting --- p.68 / Chapter 3.2.6 --- Detection of JAG2 and FZD3 Protein Expression in CRC and Normal Colon Cell Lines by Western Blotting --- p.69 / Chapter 3.2.7 --- Statistical Analysis --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- JAG2 and FZD3 but not NOTCH4 mRNA were Over -expressed in CRC Cell Lines --- p.71 / Chapter 3.3.2 --- Over-expression of JAG2 and FZD3 Proteins in CRC Tissues --- p.72 / Chapter 3.3.3 --- FZD3 Over-expression Correlated with Tumour-Node Metastasis (TNM) stages --- p.76 / Chapter 3.3.4 --- JAG2 and FZD3 Protein Expression in Colorectal Cancer and Normal Cell Lines --- p.77 / Chapter 3.4 --- Discussions --- p.78 / Chapter Chapter 4 --- Functional Analyses of JAG2 and FZD3 in CRC Cell Lines by RNA Interference --- p.81 / Chapter 4.1 --- Background --- p.81 / Chapter 4.2 --- Materials and Methods --- p.84 / Chapter 4.2.1 --- Transfection of siRNA into CRC Cell Lines --- p.84 / Chapter 4.2.2 --- Cell Proliferation Assay --- p.85 / Chapter 4.2.3 --- Monolayer Scratch Wound Healing Assay --- p.85 / Chapter 4.2.4 --- Matrigel Invasion Assay --- p.86 / Chapter 4.2.5 --- Statistical Analysis --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Knockdown of JAG2 and FZD3 Expression by RNA Interference --- p.88 / Chapter 4.3.2 --- Effect of JAG2 Knockdown on Migration of CRC Cell Lines --- p.91 / Chapter 4.3.3 --- JAG2 Knockdown by siRNA 2 Transfection Reduced Migratory Capability of HCT116, DLD-1and HT29 cell lines --- p.94 / Chapter 4.3.4 --- JAG2 Knockdown Impaired the Invasiveness of HCT116 and DLD-1 Cell Lines --- p.97 / Chapter 4.3.5 --- Decreased Migratory and Invasive Capabilities Induced by JAG2 Knockdown was not Due to Reduced Cell Proliferation --- p.100 / Chapter 4.4 --- Discussions --- p.102 / Chapter Chapter 5 --- NOTCH Pathway Inactivation by JAG2 Silencing Reduces Oncogenic Properties of HT29 but not HCT116 andDLD-1 CRC Cell Lines --- p.106 / Chapter 5.1 --- Background --- p.106 / Chapter 5.2 --- Materials and Methods --- p.109 / Chapter 5.2.1 --- CRC Cell lines --- p.109 / Chapter 5.2.2 --- Pharmacological Inhibition of NOTCH signaling by DAPT --- p.109 / Chapter 5.2.3 --- Combination of DAPT Treatment and JAG2 Silencing by siRNA --- p.109 / Chapter 5.2.4 --- Western Blotting --- p.109 / Chapter 5.2.5 --- Cell Proliferation Assay (MTS Assay) --- p.110 / Chapter 5.2.6 --- Monolayer Scratch Wound Healing Assay --- p.110 / Chapter 5.2.7 --- Matrigel Invasion Assay --- p.111 / Chapter 5.2.8 --- Statistical Analysis --- p.111 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- JAG2 Silencing Down-regulates Notch Pathway Signaling in CRC Cell Lines --- p.112 / Chapter 5.3.2 --- Inhibition of NOTCH Signaling by DAPT Treatment in CRC Cell Lines --- p.112 / Chapter 5.3.3 --- NOTCH Inhibition Does not Significantly Affect Cell Proliferation in CRC Cell Lines --- p.114 / Chapter 5.3.4 --- Suppression of NOTCH Signaling by DAPT Inhibits Migration in HT29 but not in HCT116 and DLD-1 CRC Cell Lines --- p.115 / Chapter 5.3.5 --- Suppression of NOTCH Signaling by DAPT does not Significantly Affect Invasiveness of HCT116 and DLD-1 CRC Cell Lines --- p.117 / Chapter 5.4 --- Discussions --- p.118 / Chapter Chapter 6 --- JAG2 Knockdown Inhibits Invasion in CRC Cell Lines through Inactivation of Cathepsin K --- p.121 / Chapter 6.1 --- Background --- p.121 / Chapter 6.2 --- Materials and Methods --- p.123 / Chapter 6.2.1 --- Human Tumour Metastasis RT2 Profiler[superscript TM] PCR Array --- p.123 / Chapter 6.2.2 --- Measurement of CTSK Gene expression level by Quantitative Real-Time PCR --- p.123 / Chapter 6.2.3 --- Immunohistochemical Staining (IS) of CTSK in CRC Tissues --- p.124 / Chapter 6.2.4 --- Pharmacological Inhibitior of CTSK in CRC Cell Lines --- p.124 / Chapter 6.2.5 --- Inhibition of CTSK in CRC Cell Lines for Migration Study --- p.124 / Chapter 6.2.6 --- Inhibition of CTSK in CRC Cell Lines for Invasion Study --- p.125 / Chapter 6.2.7 --- Western Blotting --- p.125 / Chapter 6.2.8 --- Statistical Analysis --- p.125 / Chapter 6.3 --- Results --- p.126 / Chapter 6.3.1 --- Identification of Metastasis Related Genes Which were Down-regulated by JAG2 Knockdown in HCT116 Cells --- p.126 / Chapter 6.3.2 --- Validation of Down-regulation of CTSK Gene by JAG2 Knockdown in HCT116 Cell Line by qRT-PCR --- p.126 / Chapter 6.3.3 --- JAG2 Knockdown Reduced Expression of Active CTSK Protein in CRC Cell Lines --- p.128 / Chapter 6.3.4 --- CTSK Protein Expression in CRC Tissue Samples --- p.130 / Chapter 6.3.5 --- Pharmacological Inhibition of CTSK Suppressed Invasiveness of CRC Cell Lines --- p.131 / Chapter 6.3.6 --- Pharmacological Inhibition of CTSK did not Affect Migration of CRC Cell Lines --- p.132 / Chapter 6.4 --- Discussions --- p.133 / Chapter Chapter 7 --- Depletion of JAG2 Inhibits Migration and Invasion in CRC Cell Lines through Inactivation of p38 MAPK/HSP27 Pathway --- p.137 / Chapter 7.1 --- Background --- p.137 / Chapter 7.2 --- Materials and Methods --- p.140 / Chapter 7.2.1 --- Pharmocological Inhibition of p38 MAPK Phosphorylation CRC Cell Lines --- p.140 / Chapter 7.2.2 --- Inhibition of p38 MAPK Phosphorylation for Migration Study in CRC Cell Lines --- p.140 / Chapter 7.2.3 --- Inhibition of p38 MAPK Phosphorylation for Invasion Study in CRC Cell Lines --- p.140 / Chapter 7.2.4 --- Knockdown of STAT3 by RNA interference --- p.141 / Chapter 7.2.5 --- Knockdown of STAT3 for Migration Study in CRC Cell Lines --- p.141 / Chapter 7.2.6 --- Knockdown of STAT3 for Invasion Study in CRC Cell Lines --- p.141 / Chapter 7.2.7 --- Western Blotting --- p.141 / Chapter 7.2.8 --- Statistical Analysis --- p.142 / Chapter 7.3 --- Results --- p.143 / Chapter 7.3.1 --- JAG2 Knockdown Inhibits p38 MAPK / HSP27 Pathway in CRC Cell Lines --- p.143 / Chapter 7.3.2 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Invasive Capability of CRC Cell Line --- p.145 / Chapter 7.3.3 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Migration of CRC Cell lines --- p.147 / Chapter 7.3.4 --- JAG2 Knockdown Inactivated p38 MAPK / HSP27 Pathway Independently of NOTCH Pathway in CRC Cell Lines --- p.149 / Chapter 7.3.5 --- JAG2 Knockdown Inhibits STAT3 Activation in CRC Cell Lines --- p.151 / Chapter 7.3.6 --- STAT3 Silencing Reduced Invasive Capability in CRC Cell Lines --- p.152 / Chapter 7.3.7 --- STAT3 Silencing Reduced Migratory Capability in CRC Cell Lines --- p.154 / Chapter 7.3.8 --- Inhibition of p38 MAPK Activity Suppressed STAT3 Activation in HCT116 Cells --- p.156 / Chapter 7.4 --- Discussions --- p.157 / Chapter Chapter 8 --- Conclusions and Future Works --- p.161 / Chapter 8.1 --- Conclusions --- p.161 / Chapter 8.2 --- Future work --- p.163 / References --- p.164 / Chapter Appendix 1 --- List of Figures and Tables --- p.208 / Chapter Appendix 2 --- Abbrevations used in this thesis --- p.212