Studies have shown that tumor cells are susceptible to pharmacological targeting of their altered glycolytic metabolism with a variety of compounds that result in apoptosis. One such compound, 3-bromopyruvate (3-BP), has been shown to eradicate cancer in an animal model. However, no studies have shown whether the apoptotic fragments resulting from 3-BP treatment have the capacity to elicit an immunogenic cell death that activates dendritic cells, the primary antigen presenting cell in the immune system. Immunogenic cell death is critical to eliciting an effective adaptive immune response that selectively kills additional target cells and generates immunological memory. We demonstrated that 3-bromopyruvate induced apoptosis in a number of different murine breast cancer cell lines, including the highly metastatic 4T1 line. The dying tumor cells stimulated immature dendritic cells (DCs) of the immortal JAWS II cell line to produce high levels of the pro-inflammatory cytokine IL-12, and increased their expression of key co-stimulatory molecules CD80 and CD86. The activated dendritic cells showed increased uptake of fragments from dying tumor cells that correlated with the increased levels of calreticulin on the surface and release of high group motility box 1 (HMGB1) of the latter following 3-BP treatment. Additionally, the anti-phagocytic signal CD47 present on breast cancer cells was reduced by treatment with 3-bromopyruvate when compared to the levels on untreated 4T1 cells. 3-BP treated breast cancer cells were able to activate dendritic cells through TLR4 signaling. Signaling was dependent on both the expression of surface calreticulin and on the extracellular release of high mobility group box 1 protein (HMGB1) during the process of immunogenic cell death. Killing by 3-BP was compared to mitoxantrone and doxorubicin, among the few chemotherapeutics that induce immunogenic cell death. 3-BP killing was likewise compared to camptothecin, a compound that fails to induce immunogenic cell death. Importantly, 3-BP did not markedly decrease the levels of the key peptide presenting molecule MHC I on DCs that were co-cultivated with dying tumor cells. Treatment of the highly aggressive triple negative BT-20 human breast cancer cell line with 3-BP also induced an immunogenic cell death, activating human dendritic cells in vitro. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Extracellular stimuli may influence the M1/M2 phenotypic polarization of macrophages. We examined M1/M2 biomarkers, phagocytic activity, and tumoricidal activity in RAW 264.7 mouse macrophages. Macrophages were treated with conditioned media (CM) from 4T1 breast cancer cells, curcumin, 22-oxacalcitriol, LPS, or a combination of the previously listed. Arginase activity, a M2 phenotypic biomarker, was upregulated by the treatment of macrophages with conditioned media. Curcumin, 22- oxacalcitriol, and LPS partially inhibited RAW 264.7 arginase activity in the presence of 4T1 breast cancer media. 22-oxacalcitriol increased the phagocytic ability of RAW 264.7 macrophages in the presence of M2 polarizing substances produced by the 4T1 breast cancer cells. Also, LPS increased RAW 264.7 phagocytic ability in the presence of 4T1 breast cancer CM. This study looked at the potential substances that would possibly reverse the M2 tumor promoting macrophage phenotype seen in the breast cancer tumor environment. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

We attempted to understand the molecular regulators that impact inflammation using a rat model of human sensation-seeking/risk-taking trait for drug and stress vulnerability, based on their exploratory behavior displaying high rates (HRs) or low rates of locomotor reactivity (LRs) to environmental stress. We found that HRs have a pro-inflammatory phenotype as indicated by increased protein expression of the inflammatory cytokine TNF-(Sa(B. Furthermore, we found that HRs have a lower gene expression of the glucocorticoid receptor and histone deacetylase 2 which are known to play an immunosuppressive role. Autophagy (macroautophagy) is a homeostatic process needed for cell maintenance, growth and proliferation and known to assist in tumor survival. FYVE and coiled-coil domain containing 1 (FYCO1) is a novel protein implicated to assist in the plus-end directed trafficking and fusion of autophagosomes. In these studies, we show that FYCO1 gene expression among human breast cell lines of varying degrees of malignancy. / Lillian C. Onwuka-Ekpete. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Cancer is the second leading cause of death in the western world. In order to treat various types of cancer, platinum-based drugs are most widely employed as metal-containing chemotherapeutic agents. However, their clinical usage is hindered by toxic side effects, and by the emergence of drug resistance. Our focus was to replace platinum with less toxic metal like tin which can give better alternatives for cancer treatment. The major aim of our study was to synthesize novel organotin polyethers (Sn-O) which can be used to combat cancer. Preliminary results from our laboratory using organotin polyethers, that were synthesized by varying the structure of diols showed growth inhibition in Balb-3T3 cells. This study directly led us to hypothesize the two structural windows, first by changing the distance between diol and second, by presence of unsaturation in diols, the biological activity of organotin polyethers (Sn-O) can be enhanced significantly. Different series of polymeric compounds were synthesized based upon these two structural windows and the formation of products was validated using standard techniques like infrared spectroscopy (IR), light scattering photometer, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nuclear magnetic resonance (NMR). The synthesized polymers arrested the growth of cancer cell lines including bone, prostate, colon, breast, pancreas and lung cancer derived cell lines in vitro. In number of instances where chemotherapeutic index values of two and greater were found that these polymers are significantly more active against cancer cells than non-cancerous cells in culture. / These results support the starting premise that the polymers may exhibit cancer cell selectivity. In general, it was found that the presence of unsaturation increased the probability that the polyether would inhibit the growth of various cancer cell lines. Further, in some cases, polyethers with short distances between the oxygen atoms showed a superior ability to inhibit the growth of various cancer cell lines in comparison to those with longer distances between the oxygen atoms. These results provide a framework for the discovery of novel cancer therapeutics. / by Girish Vallabhbhai Barot. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

He, Jun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 136-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Fan, Dahua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 117-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

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.

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

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences