Poon Yin-tat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 101-111). / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / LIST OF ABBREVIATIONS --- p.IV / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter CHAPTER 2: --- PURIFICATION OF α- AND β-MOMORCHARINS --- p.26 / Chapter CHAPTER 3: --- N-GLYCOSIDASE ACTIVITY OF α- AND β-MOMORCHARINS --- p.45 / REFERENCES --- p.101

by Kwong Joseph. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 142-164). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prostatic Secretory Proteins --- p.1 / Chapter 1.2 --- Rat Prostatic Secretory Proteins --- p.1 / Chapter 1.2.1 --- Prostatic Secretory Proteins in Ventral Prostate --- p.2 / Chapter 1.2.1.1 --- Prostatic Binding Protein (PBP) --- p.2 / Chapter 1.2.1.2 --- Androgen-Suppressed Proteins of Rat Ventral Prostate --- p.6 / Chapter 1.2.1.3 --- The 20-kDa Protein --- p.8 / Chapter 1.2.1.4 --- Spermine-Binding Proteins --- p.9 / Chapter 1.2.1.5 --- Prostatic Acid Phosphatase (PAP) --- p.10 / Chapter 1.2.2 --- Prostatic Secretory Proteins in Dorsal Prostate --- p.12 / Chapter 1.2.2.1 --- Dorsal Proteins I and II (DP I and DPII) --- p.12 / Chapter 1.2.2.2 --- Seminal Vesicle Secretion II (SVSII) --- p.14 / Chapter 1.2.2.3 --- Probasin --- p.16 / Chapter 1.2.3 --- Prostatic Secretory Proteins in Lateral Prostate --- p.18 / Chapter 1.3 --- Human Prostatic Secretion --- p.18 / Chapter 1.4 --- Human Prostatic Secretory Proteins --- p.18 / Chapter 1.4.1 --- Prostatic Acid Phosphatase (PAP) --- p.19 / Chapter 1.4.2 --- Prostate Specific Antigen (PSA) --- p.22 / Chapter 1.4.2.1 --- Molecular Biology of PSA --- p.22 / Chapter 1.4.2.2 --- Synthesis of PSA --- p.23 / Chapter 1.4.2.3 --- Kallikrein Gene Family --- p.23 / Chapter 1.4.2.4 --- Physiological Function of PSA --- p.24 / Chapter 1.4.2.5 --- PSA as an Immunohistochemical Marker --- p.25 / Chapter 1.4.2.6 --- PSA is not a Prostate-Specific Molecule --- p.26 / Chapter 1.4.3 --- Prostatic Secretory Protein of 94 Amino Acids (PSP94) --- p.27 / Chapter 1.4.3.1 --- Nucleotide Sequence of the PSP94 cDNA --- p.28 / Chapter 1.4.3.2 --- Amino Acid sequence of PSP94 --- p.28 / Chapter 1.4.3.3 --- Biological Properties of PSP94 --- p.29 / Chapter 1.4.3.4 --- Physiological Roles of PSP94 --- p.31 / Chapter 1.4.3.5 --- PSP94 and Its mRNA in Other Non-Prostatic Tissue --- p.31 / Chapter 1.4.3.6 --- PSP94 as a Tumor Marker of Prostate Cancer --- p.32 / Chapter 1.4.3.7 --- Homologous Proteins of PSP94 --- p.34 / Chapter 1.5 --- Aim of Study --- p.35 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and Supply of Noble Rat --- p.37 / Chapter 2.2 --- Chemicals --- p.37 / Chapter 2.3 --- Bilateral Ochidectomy of Animals --- p.37 / Chapter 2.4 --- Androgen Replacement --- p.38 / Chapter 2.5 --- Hormonal and Drug Treatments on Castrated Animals --- p.38 / Chapter 2.6 --- Induction of Prostatic Intraepithelial Neoplasia in Noble Rat Prostate Gland by Long-Term Treatment with Steroids --- p.39 / Chapter 2.6.1 --- Preparation of Steroid Hormone-Filled Silastic® Tubings --- p.39 / Chapter 2.6.2 --- Surgical Implantation of Silastic® Tubings --- p.39 / Chapter 2.6.3 --- Protocols of Hormonal Treatments --- p.40 / Chapter 2.7 --- Androgen-Dependent Rat Dunning Prostatic Adenocarcinoma --- p.40 / Chapter 2.8 --- Androgen-Independent Prostatic Carcinoma Line (AIT) of Noble Rat --- p.41 / Chapter 2.9 --- Plasmids --- p.41 / Chapter 2.10 --- Restriction Enzyme Digestions of pLvB10 and cM-403 --- p.42 / Chapter 2.11 --- Amplification of Rat SVSII cDNA Fragment by RT-PCR and Subcloning --- p.42 / Chapter 2.12 --- Purification of DNA Fragment from Agarose Gel --- p.43 / Chapter 2.13 --- Subcloning of DNA into Vector --- p.44 / Chapter 2.14 --- Tissue Preparation for In-situ Hybridization --- p.47 / Chapter 2.15 --- Synthesis of Digoxigenin (DIG)-Labeled RNA Probe --- p.47 / Chapter 2.16 --- In-situ Hybridization --- p.48 / Chapter 2.17 --- Total RNA Extraction --- p.50 / Chapter 2.18 --- Northern Blotting Analysis --- p.51 / Chapter 2.19 --- Primers and Cycling Conditions --- p.53 / Chapter 2.20 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.54 / Chapter 2.21 --- Southern Blotting Analysis --- p.56 / Chapter 2.21.1 --- Southern Blotting --- p.56 / Chapter 2.21.2 --- Preparation of DIG-dUTP Labeled Rat PSP94 cDNA Probe --- p.56 / Chapter 2.21.3 --- Hybridization --- p.57 / Chapter 2.22 --- Restriction Mapping --- p.58 / Chapter 2.23 --- Semi-Quantitative RT-PCR --- p.59 / Chapter 2.24 --- Statistical Analysis --- p.59 / Chapter 2.25 --- "Protein Extraction, SDS-PAGE and Western Blotting Analysis" --- p.60 / Chapter 2.26 --- Immunohistochemistry --- p.63 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Subcloning of DNAs into Vector --- p.65 / Chapter 3.1.1 --- Subcloning of 18s Ribosomal RNA cDNA Fragment --- p.65 / Chapter 3.1.2 --- Subcloning of Probasin cDNA Fragment --- p.66 / Chapter 3.1.3 --- Subcloning of SVSII cDNA Fragment --- p.66 / Chapter 3.1.4 --- Restriction Enzyme Mapping for PCR Product of SVSII --- p.67 / Chapter 3.2 --- Detection of mRNA and Protein Expression of PSP94 in Normal Rat Prostates --- p.68 / Chapter 3.2.1 --- In-situ Hybridization --- p.68 / Chapter 3.2.2 --- Northern Blotting --- p.68 / Chapter 3.2.3 --- RT-PCR Amplification --- p.69 / Chapter 3.2.4 --- Immunohistochemistry --- p.69 / Chapter 3.2.5 --- Western Blotting --- p.70 / Chapter 3.3 --- Detection of mRNA Expression of Probasin and SVSII in Normal Rat Prostates --- p.71 / Chapter 3.3.1 --- In-situ Hybridization --- p.71 / Chapter 3.3.2 --- RT-PCR Amplification --- p.71 / Chapter 3.4 --- "Androgen Regulation of PSP94,Probasin and SVSII mRNA Expression" --- p.72 / Chapter 3.4.1 --- In-situ Hybridization --- p.72 / Chapter 3.4.2 --- "Relative Expression Levels of PSP94, Probasin and SVSII mRNA in Normal, Castrated and Androgen Replaced Rat Lateral Prostates as Measured by a Semiquantitative RT-PCR Method" --- p.73 / Chapter 3.4.2.1 --- Determination of Exponential Range of PCR --- p.73 / Chapter 3.4.2.2 --- Semi-Quantitative RT-PCR --- p.74 / Chapter 3.4.3 --- Western Blot Analysis --- p.75 / Chapter 3.5 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Prostates" --- p.76 / Chapter 3.5.1 --- Semi-Quantitative RT-PCR --- p.76 / Chapter 3.5.2 --- Western Blot Analysis --- p.77 / Chapter 3.6 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.78 / Chapter 3.6.1 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in T+E2-Induced Prostatic Intraepithelial Neoplasia (PIN) of the Lateral Prostate of Noble Rats by In-situ Hybridization" --- p.78 / Chapter 3.6.2 --- "Detection of PSP94,Probasin and SVSII mRNA Expression in Dunning Tumor and AIT Prostatic Tumor" --- p.79 / Chapter 3.6.2.1 --- In-situ Hybridization --- p.79 / Chapter 3.6.2.2 --- RT-PCR Amplification --- p.79 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Specific Expression of PSP94 in the Lateral Lobe of Rat Prostate --- p.114 / Chapter 4.2 --- Androgen Regulation of PSP94 --- p.118 / Chapter 4.2.1 --- Molecular Mechanism of Androgen Action --- p.118 / Chapter 4.2.2 --- Androgen Regulation of PSP94 in Rat Lateral Prostate --- p.121 / Chapter 4.3 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Lateral Prostate" --- p.126 / Chapter 4.4 --- "Detection of PSP94, Probasin, SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.133 / Chapter 4.5 --- Gene Therapy --- p.139 / Chapter Chapter 5 --- Conclusions --- p.141 / References --- p.142 / Appendixes --- p.165

Medulloblastoma (MB) is the commonest intracranial childhood malignancy and despite recent advances, current therapeutic approaches are still associated with high morbidity and mortality. A novel molecular classification has recently been proposed for these tumours – WNT Group (best prognosis), SHH Group (intermediate prognosis), Group 3 (worst prognosis) and Group 4 (intermediate prognosis). BMI1, a transcriptional repressor of the Polycomb group genes, is overexpressed in MB, most significantly in those of Group 4 MBs. Bone Morphogenetic Proteins (BMPs) are morphogens belonging to TGF-β superfamily of growth factors, and are known to inhibit MB cell proliferation and induce apoptosis in vitro, and to inhibit tumour growth in vivo. Our team have recently demonstrated that Bmi1 regulates cell adhesion properties during cerebellar development through repression of the BMP pathway. The aim of this project is to assess whether BMI1 overexpression may contribute to MB pathogenesis through repression of the BMP pathway. Here we demonstrate that BMI1 knock down derepresses BMP pathway, and using a novel xenograft model of human MB of Group 4, we show that BMI1 controls tumour volume and intraparenchymal invasion. In in vitro assays on MB cell lines we show that cell adhesion and motility is controlled by BMI1 in a BMP dependent manner and that deregulation of extracellular matrix proteins are key mediators of this effect. Furthermore, we demonstrate that BMP treatment to BMI1 overexpressing MB cells reduces cell proliferation and invasion, suggesting BMI1 as a possible biomarker for those tumours that could benefit from treatment with BMP agonist small molecules.

Chemerin是一種新近發現的脂肪因數，其與肥胖，代謝綜合症和炎症密切相關。Chemerin通過G蛋白偶聯受體ChemR23 發揮生物學效應。脂肪組織為分泌型Chemerin的最主要來源。最近的研究顯示Chemerin 可能與腫瘤的發生相關。本研究的目的在於確定胃癌細胞上是否存在ChemR23,以及Chemerin/ChemR23 在腫瘤侵襲過程中的作用。 / 我們首先用western blot和免疫組織化學技術在人腫瘤組織及兩株腫瘤細胞AGS和MKN28上尋找ChemR23。我們首次證實ChemR23 在胃癌細胞上表達。 / 既往研究表明，炎症因數IL-6 可上調人類內皮細胞上表達的ChemR23。為了證明IL-6 對胃癌細胞表達ChemR23的調節作用，我們將兩株胃癌細胞培養於含有不同濃度的Chemerin 的溶液中。研究證明IL-6 對於ChemR23 的表達具有濃度和時間依賴性的上調作用。 / 接下來，我們關注於Chemrin/ChemR23 在VEGF，MMP-7和IL-6 表達中的作用。我們將胃癌細胞用含有或者不含有Chemerin的溶液培養，進而測量VEGF ，MMP-7和IL-6 的表達量。進而我們將研究Chemerin 對mammalian family of mitogen-activated protein kinases (MAPKs)即：Erk，P38，Jnk的調節作用，MAPKs對於VEGF，MMP-7和IL-6 的產生起著重要的作用。我們發現Chemerin對VEGF，MMP-7和IL-6 的上調作用具有濃度和時間依賴性。Chemrin對MAPKs的啟動亦具有時間依賴性。MAPKs抑制劑可減弱Chemein相關的VEGF, MMP-7和IL-6的生成。 / 因為VEGF，MMP-7和IL-6與腫瘤的侵襲相關，我們進一步對Chemerin 在腫瘤轉移中的作用進行探索。我們使用transwell實驗工具盒來檢測腫瘤的侵襲能力。我們將AGS或MKN28細胞懸液裡加入不同濃度的Chemerin,結果是Chemerin增強腫瘤的侵襲能力（P<0.001）。為了驗證chemeri 引發的腫瘤侵襲是否通過MAPKs信號通路，我們將AGS和MKN28細胞用MAPK抑制劑處理2小時候，再加入Chemerin 0.1ng/ml。結果是Chemerin引發的腫瘤細胞侵襲可被MAPK抑制劑所抑制。 / 隨後，我們將探索人血清Chemerin濃度是否與胃癌TNM分型及組織學分型相關。研究包括36例胃癌患者的血清。TNM分型與組織病理檢查及臨床評估為依據。對於組織分型的分析，患者分為兩組，腸型和非腸型。人血清Chemerin濃度用Elisa方法測定。用one way ANOVA 進行統計學分析， II，III+IV型胃癌患者血清 Chemrin濃度明顯高於I型患者. 用獨立樣本 T test 分析，結果是非腸型胃癌患者血清Chemerin 濃度 明顯高於腸型患者 (P<0.05) / 總之，我們首次證明了胃癌細胞表達ChemR23。Chemerin /ChemR23 對腫瘤細胞分泌VEGF，MMP-7 和IL-6具有重要上調作用，該作用通過MAPKs信號通路實現。人類血清Chemerin濃度與胃癌患者TNM分析及組織性分型相關。Chemerin 可被看作是促進腫瘤侵襲的因素之一。 / Chemerin is a newly discovered adipokine which is closely associated with obesity, metabolic syndrome, and inflammatory conditions. It acts via its distinct G protein-coupled receptor ChemR23. While Chemerin is predominantly released by adipocytes, recent studies have also shown its possible correlation with carcinogenesis. This thesis aims to determine whether ChemR23 is expressed in gastric cancer, and to clarify the roles of Chemerin/ChemR23 in the invasive capacity of gastric carcinoma. / We first started by investigating the native expression of ChemR23 in human gastric cancer tissue samples and two gastric cancer cell lines, namely AGS and MKN28, using immunohistochemistry and Western blot techniques. In this set of initial experiments, ChemR23 was found to be present in gastric cancer cells with different magnitudes of expression. / Previous studies have also shown that ChemR23 synthesis in human endothelial cells was upregulatable by proinflammatory cytokines, such as IL-6. We thus investigated the biological effect of IL-6 on ChemeR23 expression in gastric cancer. By incubating the two cell lines with different concentration of recombinant IL-6, a dose- and time- dependent upregulation of ChemR23 expression was seen in both MKN28 and AGS. / We next sought to investigate the effect of Chemerin/ChemR23 on VEGF，MMP-7 and IL-6 expression. Gastric cancer cells were cultured with or without recombinant Chemerin. Corresponding VEGF, MMP-7 and IL-6 expressions were measured at sequential time points. In addition, the effect of Chemrerin on activation of mammalian family of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), P38MAPK, and c-Jun NH(2)-terminal kinase (JNK), which play a key role in VEGF, MMP-7 and IL-6 expression, was determined. Our Results revealed that Chemerin induced VEGF, MMP-7 and IL-6 expressions in gastric cancer cells in a dose- and time- dependent manner, and such an effect was reversible by MAPK inhibitors. / As VEGF, MMP-7, IL-6 are known to be associated with invasion of cancer, we therefore proceeded to determine whether Chemerin had any effect on invasiveness of gastric cancer in vitro. Commercial transwell invasion chambers were used. We found that Chemerin up-regulated the ability of invasion of gastric cancer (P<0.001). To test whether Chemerin induced gastric cancer cells invasion was mediated through MAPKs pathway, we pretreated AGS and MKN28 with or without MAPKs inhibitors 2 hours before adding Chemerin. This experiment proved that Chemerin enhanced cancer invasions were reversible by MAPK inhibitor. / In the last part of this project, the correlation between human serum Chemerin level and TNM stage/ histology of clinically collected gastric cancer samples was analyzed. This study included 36 gastric cancer patients. The cancer staging was based on a routine histopathological assessment according to the 6th UICC TNM (tumor-nodulus-metastases) system. One way ANOVA analysis showed that the mean Chemerin levels of stage II and III+IV groups were significantly higher than that of stage I cases. When serum Chemerin level was analyzed according to Lauren’s histological subtypes of the tumours, it was found to be significantly higher in the non-intestinal group when compared to the intestinal group. (P<0.05) / In conclusion, I have demonstrated for the first time that ChemR23 is expressed by gastric cancer cells. Chemerin/ChemR23 has upregulating effect on VEGF, MMP-7and IL-6 expression through the MAPK pathways. The invasive capacity of gastric cancer was significantly potentiated by Chemerin in vitro. The serum Chemerin level also has correlation with TNM stage and histology of gastric cancer. So Chemerin may be a functional onco-protein modulating the invasiveness of human gastric cancer. / 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. / Detailed summary in vernacular field only. / Wang, Chunhu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 129-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.II / ACKNOWLEDGEMENTS --- p.VII / LIST OF ABBREVIATIONS --- p.VIII / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.XI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction of Gastric Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Risk Factors --- p.2 / Chapter 1.1.3 --- Therapy --- p.6 / Chapter 1.2 --- Gastric cancer invasion --- p.8 / Chapter 1.2.1 --- The role of mitogen activated protein kinases (MAPKs) and other signaling pathways in gastric cancer invasion --- p.9 / Chapter 1.2.2 --- VEGF, MMP-7 and IL-6 and their roles in gastric cancer invasion --- p.14 / Chapter 1.3 --- Novel mediators: Chemerin/ChemR23 --- p.16 / Chapter 1.3.1 --- What is Chemerin and ChemR23 --- p.17 / Chapter 1.3.2 --- The role of Chemerin/ChemR23 in pathological conditions --- p.20 / Chapter 2.1 --- Experiment 1 :ChemR23 expression in gastric cancer --- p.24 / Chapter 2.1.1 --- Materials and methods --- p.24 / Chapter 2.1.2 --- Results --- p.26 / Chapter 2.2 --- Experiment 2: In vitro ChemR23 expression in Gastric Cancer cell culture and its interaction with IL-6 --- p.32 / Chapter 2.2.1 --- Materials and methods --- p.32 / Chapter 2.2.2 --- Results: --- p.35 / Chapter 2.3 --- Discussion --- p.45 / Chapter Chapter 3 --- Chemerin activates FAK, MAPK and Akt signal pathway on gastric cancer --- p.50 / Chapter 3.1 --- Materials and methods --- p.50 / Chapter 3.1.1 --- Reagents and antibodies： --- p.51 / Chapter 3.1.2 --- Cell cultures and treatment --- p.51 / Chapter 3.1.3 --- MTT assay for the effect of Chemerin on gastric cancer proliferation --- p.52 / Chapter 3.1.4 --- ChemR23 siRNA transfection --- p.52 / Chapter 3.1.5 --- Western blot analysis. --- p.53 / Chapter 3.1.5 --- Statistical analysis. --- p.54 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- MTT assay result --- p.54 / Chapter 3.2.2 --- Chemerin activates FAK signal pathways --- p.56 / Chapter 3.2.3 --- Chemerin activate MAPK signal pathways --- p.58 / Chapter 3.2.4 --- Chemerin induced Akt activation --- p.63 / Chapter 3.2.5 --- Chemerin activates MAPK pathways through ChemR23 --- p.65 / Chapter 3.3 --- Discussion --- p.69 / Chapter Chapter 4 --- Chemerin up-regulates VEGF, MMP-7 and IL-6 expression throug MAPK pathway on gastric cancer --- p.73 / Chapter 4.1 --- Materials and methods --- p.73 / Chapter 4.1.1 --- Reagents and antibodies： --- p.73 / Chapter 4.1.2 --- Cell cultures and treatment --- p.74 / Chapter 4.1.3 --- ChemR23 siRNA transfection --- p.74 / Chapter 4.1.4 --- Treatment MAPK inhibitors --- p.75 / Chapter 4.1.5 --- uantitative reverse transcript (RT)-PCR assay --- p.75 / Chapter 4.1.6 --- Western blot analysis --- p.76 / Chapter 4.1.7 --- Statistical analysis. --- p.77 / Chapter 4.2 --- Results --- p.78 / Chapter 4.2.1 --- Chemerin up-regulated VEGF, MMP-7, IL-6 expression --- p.78 / Chapter 4.2.2 --- Chemerin induced VEGF and MMP-7expression through ChemR23 --- p.87 / Chapter 4.2.3 --- Chemerin induced VEGF, MMP-7 and IL-6 expression through MAPK pathways --- p.91 / Chapter 4.3 --- Discussion: --- p.96 / Chapter Chapter 5 --- Chemerin enhances gastric cancer invasiveness through the MAPK pathways --- p.100 / Chapter 5.1 --- Materials and methods --- p.100 / Chapter 5.1.1 --- Reagents and antibodies： --- p.100 / Chapter 5.1.2 --- Cell culture and treatments --- p.101 / Chapter 5.1.3 --- Tumor invasion potential evaluated with Transwell invasion chambers --- p.101 / Chapter 5.1.4 --- Statistical analysis. --- p.102 / Chapter 5.2 --- Results: --- p.103 / Chapter 5.2.1 --- Chemerin increased gastric cancer cells invasion abilities --- p.103 / Chapter 5.2.2 --- Chemerin increased gastric cancer cells invasion abilities through MAPK pathways --- p.104 / Chapter 5.3 --- Discussion: --- p.109 / Chapter Chapter 6 --- Chemerin relates with TNM stage and histology of gastric cancer --- p.113 / Chapter 6.1 --- Materials and methods --- p.113 / Chapter 6.1.1 --- Study population --- p.113 / Chapter 6.1.2 --- Serum Chemerin level measurements --- p.114 / Chapter 6.1.3 --- Statistical analysis --- p.115 / Chapter 6.2 --- Results: --- p.115 / Chapter 6.2.1 --- Serum Chemerin level in healthy subject plus gastric cancer patients followed normal distribution but did not follow homogeneity of variances --- p.115 / Chapter 6.2.2 --- Serum Chemerin level in gastric cancer patients was higher than healthy subjects --- p.116 / Chapter 6.2.3 --- Serum Chemerin level in gastric cancer followed normal distribution and homogeneity of variances --- p.120 / Chapter 6.2.4 --- Human serum Chemerin level was related to gastric cancer TNM stage --- p.121 / Chapter 6.2.5 --- Association between human serum Chemerin level and histology --- p.123 / Chapter 6.3 --- Discussion --- p.125 / Chapter Chapter 7: --- Conclusion and future plan --- p.127 / Reference --- p.129

The ability to therapeutically manipulate regulatory T (Treg) cell survival/function would have far reaching implications; with the potential to limit immune pathology in autoimmune disease, allergy and transplantation; and to reduce regulation of anti-tumour responses in cancer. This study has established a method to study the survival and function of antigen specific Treg cells in vivo, adapting an existing approach in which an endogenous naïve T cell population is expanded and tracked. Multiple immunisation of antigen, and an agonistic anti-DR3 antibody were used to ensure a sufficient number and proportion of Treg cells could be expanded. Further to this, an assay for investigating Treg cell function in vivo was applied to this system. This approach revealed that the tumour necrosis factor receptors OX40 and CD30 may play a role in Treg function, as well as expansion. Unexpectedly, these data also revealed that in the absence of OX40 there is a gross defect in the function of CD4 T cells. A regulatory role of group 3 Innate Lymphoid cells is emerging in the literature, and in accordance with this, this study demonstrates that Treg cell expansion is grossly impaired in mice which lack RORγt, their lineage defining transcription factor.

Cytomegalovirus (CMV) is a β-herpes virus that infects the majority of the world’s population. Tyrosine kinase inhibitors (in particular imatinib, dasatinib and nilotinib) have been successfully used in the treatment of chronic myeloid leukaemia, as they target the \(Abl\) kinase, which is constitutively activated in the disease. Although thought of as targeted therapies, they have significant “off target” effects including inhibition of \(Src\) family kinases important in T cell receptor mediated activation. I demonstrated that CMV infection is associated with significant alterations in the immune repertoire in imatinib-treated patients; in particular with expansions of differentiated CD8 T cells and Vδ1 γδ T cells. Furthermore, dasatinib treatment is associated with evidence of subclinical CMV reactivation and marked expansions of terminally differentiated CD8 T cells and Vδ1 γδ T cells. These atypical Vδ1 γδ T cells have activity against CMV infected fibroblasts, and sequencing of their TCRs demonstrated remarkable oligoclonality suggestive of antigen driven proliferation. In a second group of patients that underwent reduced intensity allogeneic stem cell transplant for myeloid malignancies, CMV seropositivity of patient or donor is associated with increased lymphocyte counts at 3 months post transpant, particularly of CD8 and Vδ1 γδ T cell subsets. Survival analysis of these patients revealed that CMV seropositivity is associated with improved overall survival, due to a decreased relapse risk.

This thesis investigates the role of CD151 in modulating the form and function of its integrin partners, α3β1 and α6β1/β4. Stable depletion of CD151 in the MDA-MB-231 (MDA-231) cell line, changed the glycosylation profile of α3β1, but not α6β1/β4 integrins. Glycosylation of CD151, tight association between CD151 and α3β1 integrin and recruitment into tetraspanin enriched microdomains (TERM) are all required for this effect and the intervention occurs during the first mannose trimming step within the ER. Further analysis showed that CD151 preferentially associates with α3β1 over α6β1/β4. CD151 mediated changes to glycosylation of α3β1 integrin decreased their ability to migrate towards Lm332 by ~90%. Sucrose density gradient assays showed α3β1 and α6β1/β4 are recruited by CD151 into the light fractions as part of a TERM as well as separate entities. Glyco-analysis of the tetraspanins themselves showed CD9 and possibly CD63 and CD82 to be phospho-mannosylated. Castanospermine treatment dramatically reduced the level of CD151, α3β1 and α6β1/β4 in these cells, indicating a novel therapeutic use. Depletion of various tetraspanins in MDA-231 altered their cell surface glycotope presentation and the cleavage profile of α6β1/β4 integrin; highlighting the role played by tetraspanins in post-translational modification of their partners.

The incidence of oesophageal adenocarcinoma (OAC) is increasing at an alarming rate in the Western World. Despite advances in surgical technique and patient selection, overall survival remains abysmal. Understanding the molecular and genetic events in the evolution of OAC is crucial to improving outcome. The role of iron in the carcinogenesis of OAC is supported by epidemiological and experimental evidence. Oesophageal cancers are iron loaded and aggressively capture systemic iron to potentiate a malignant phenotype. This project aimed to establish that OAC cells are also capable of acquiring haem and to explore the potential of iron chelation therapy in the treatment of oesophageal cancer. Haem import proteins are sequentially over expressed in the evolution of OAC. Culturing OAC cells with supplementary haem significantly enhances viability, proliferation, migration and anchorage independent growth. Abrogation of haem import protein expression reverses the stimulatory effect of supplementary haem and significantly reduces tumour burden in-vivo. Different classes of iron chelators exhibit potent in-vitro and in-vivo anti-neoplastic action in oesophageal malignancy. Iron chelators demonstrate chemo-sensitising properties and are able to overcome chemo-resistance. Haem import proteins are potential therapeutic targets in the treatment of oesophageal malignancy. Iron chelation therapy represents an effective, predictable and well tolerated adjunct to standard chemotherapy and should be considered for clinical trial.

The N-terminus of BRCA1 is clinically important as inheritance of a mutation in this region correlates to an increased risk is breast and ovarian cancer. Whilst this is fairly clear, what specific mutations do and the aetiology of the disease is not clear. This thesis investigates N-terminal BRCA1 mutations using both in vitro and cell-based methods with a focus on DNA repair, mainly double-strand break and DNA crosslink repair. The use of chemotherapy agents is used with specific mutations to look at the individual phenotypes these create to each drug or radiation. This thesis also provides evidence on haploinsufficient or dominant negative effects of N-terminal mutations. Overall, the N-terminus of BRCA1 can affect DNA repair and increase genome instability that may lead to tumour development.

CD4+ T-cells play a pivotal role within the immune response, and multiple studies have highlighted their importance in anti-tumour immunity. TCR gene transfer is a successful method of specifically redirecting T-cell specificity. We have therefore investigated the anti tumour potential of EBV-specific MHC class II restricted T-cells, generated by this approach. We have identified and cloned a DR52b-restricted TCR, specific for an EBNA2 derived peptide (PRS), which is expressed in Post-Transplant Lymphoproliferative Disease (PTLD) and some other EBV-associated malignancies. We have shown that the TCR is functional in both CD4+ and CD8+ T-cells, with transduced T-cells specifically recognising the PRS-peptide with a high avidity. Transduced T-cells have been shown to proliferate, produce multiple cytokines and have direct cytotoxic capacity in response to physiological levels of EBNA2 processed and presented by EBV-infected B-cells. Additionally to this direct response, CD4+ T-cells retain helper functions. Importantly, transduced T-cells have shown hints of tumour control in vivo. Results from this study highlight that TCR gene transfer with EBV-specific MHC class II restricted TCRs can generate polyclonal T-cells with functional capacity against virus infected cells. PRS specific TCR gene transfer may thus be useful in rapid generation of T cells for treatment of PTLD. Given the importance of CD4+ T-cells for anti-tumour responses, this study also highlights the potential for using TCR gene transfer to target these cells towards other MHC class II-positive tumours.