by Paul Li Wai Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Includes bibliographical references. / Acknowledgements --- p.i / Summary --- p.ii / Index to Figures --- p.V / Index to Tables --- p.vii / Abbreviations --- p.viii / CONTENTS / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter Chapter 2 --- LITERATURE REVIEW : SALT AND HYPERTENSION / Chapter 2.1. --- Summary of evidence linking salt and hypertension --- p.4 / Chapter 2.1.1. --- Epidemiological studies --- p.4 / Chapter 2.1.2. --- Dietary intervention studies --- p.7 / Chapter 2.1.3. --- Experimental studies --- p.9 / Chapter 2.2. --- Cellular sodium transport --- p.10 / Chapter 2.2.1. --- The Sodium Pump --- p.10 / Chapter 2.2.2. --- Defects in sodium transport in hypertension --- p.14 / Chapter 2.3. --- Hypothesis linking salt to the pathogenesis of hypertension --- p.15 / Chapter 2.4. --- Evidence for the presence of natriuretic Hormone --- p.18 / Chapter 2.4.1. --- Indirect evidence --- p.18 / Chapter 2.4.2. --- Direct evidence --- p.18 / Chapter 2.4.3. --- The source and properties of natriuretic hormone --- p.20 / Chapter 2.4.4. --- Other natriuretic factors --- p.21 / Chapter Chapter 3 --- REGULATION OF THE SODIUM PUMP / Chapter 3.1. --- General introduction --- p.24 / Chapter 3.2. --- Regulation of the sodium pump by intracellular sodium --- p.24 / Chapter 3.3. --- "Effects of ethanol on Na+,K+-ATPase activity" --- p.26 / Chapter 3.4. --- "Effects of potassium depletion on Na+,K+-ATPase activity" --- p.27 / Chapter 3.4.1. --- In vivo studies of sodium pump regulation by potassium --- p.27 / Chapter 3.4.2. --- In vitro studies of sodium pump regulation by potassium --- p.29 / Chapter 3.5. --- Effects of cardiac glycosides on the sodium pump --- p.30 / Chapter 3.5.1. --- In vivo studies of sodium pump regulation by cardiac glycosides --- p.31 / Chapter 3.5.2. --- In vitro studies of sodium pump regulation by cardiac glycosides --- p.33 / Chapter 3.6. --- Effects of dietary salt on the sodium pump --- p.35 / Chapter 3.6.1. --- Acute effects of salt-loading --- p.35 / Chapter 3.6.2. --- Chronic effects of salt-loading --- p.36 / Chapter Chapter 4 --- AIMS OF THE STUDY --- p.39 / Chapter Chapter 5 --- MEASUREMENT OF THE SODIUM PUMP ACTIVITY / Chapter 5.1. --- General introduction --- p.41 / Chapter 5.2. --- The measurement of sodium pump activity --- p.42 / Chapter 5.2.1. --- The sodium pump transport activity --- p.42 / Chapter 5.2.2. --- Quantitation of the number of sodium pump sites --- p.45 / Chapter 5.2.3. --- The measurement of enzyme activity --- p.47 / Chapter (a) --- Introduction --- p.47 / Chapter (b) --- Preparation of tissues and detergent treatment --- p.48 / Chapter (c) --- Measurement of ATPase activity by measuring the rate of release of inorganic phosphate --- p.49 / Chapter (d) --- The coupled-enzyme assay --- p.53 / Chapter (e) --- The K+-stimulated 3-0-MFPase assay --- p.54 / Chapter Chapter 6 --- METHODS - ESTABLISHMENT AND EVALUATION / Chapter 6.1. --- Chemicals --- p.57 / Chapter 6.2. --- "Measurement of Na+,K+-ATPase activity by the rate of release of inorganic phosphate" --- p.58 / Chapter 6.3. --- "Automated coupled-enzyme assay of Na+,K+-ATPase activity" --- p.62 / Chapter 6.4. --- "The measurement of Na+,K+-ATPase activity by the potassium-stimulated 3-0-MFPase assay" --- p.67 / Chapter 6.5. --- Determination of protein concentration --- p.70 / Chapter 6.6. --- Statistical analysis --- p.73 / Chapter 6.7. --- Results --- p.73 / Chapter 6.7.1. --- Evaluation of the inorganic phosphate release method --- p.73 / Chapter 6.7.2. --- Evaluation of the coupled-enzyme method --- p.78 / Chapter 6.7.3. --- Evaluation of the K+-stimulated 3-0-MFPase method --- p.89 / Chapter 6.7.4. --- Evaluation of the protein determination method --- p.94 / Chapter 6.8. --- Discussion --- p.96 / Chapter Chapter 7 --- "THE EFFECTS OF DIGOXIN TREATMENT ON Na+,K+-ATPase ACTIVITY OF DIFFERENT TISSUES" / Chapter 7.1. --- Introduction --- p.101 / Chapter 7.2. --- Materials and Methods --- p.103 / Chapter 7.2.1. --- Animals and diets --- p.103 / Chapter 7.2.2. --- Drugs --- p.103 / Chapter 7.2.3. --- Pharmacokinetics of digoxin --- p.103 / Chapter 7.2.4. --- The digoxin regimes --- p.104 / Chapter 7.2.5. --- Preparation and deoxycholate treatment of tissue homogenates --- p.105 / Chapter 7.2.6. --- "Measurement of Na+,K+-ATPase activity" --- p.107 / Chapter 7.2.7. --- Digoxin radioimmunoassay --- p.107 / Chapter 7.2.8. --- Measurement of plasma electrolytes --- p.109 / Chapter 7.3. --- Results --- p.110 / Chapter 7.3.1. --- The pharmacokinetics of digoxin in the rat --- p.110 / Chapter 7.3.2. --- Plasma digoxin levels --- p.110 / Chapter 7.3.3. --- Effects of digoxin treatment on body weight --- p.113 / Chapter 7.3.4. --- Effects of digoxin treatment on plasma electrolytes --- p.117 / Chapter 7.3.5. --- "Effects of digoxin treatment on tissue Na+,K+-ATPase activity" --- p.119 / Chapter 7.4. --- Discussion --- p.129 / Chapter Chapter 8 --- THE SALT-LOADING EXPERIMENT / Chapter 8.1. --- Introduction --- p.140 / Chapter 8.2. --- Materials and Methods --- p.142 / Chapter 8.2.1. --- Animals --- p.142 / Chapter 8.2.2. --- The salt-loading protocol --- p.142 / Chapter 8.2.3. --- Preparation of crude tissue homogenates --- p.143 / Chapter 8.2.4. --- "Measurement of Na+,K+-ATPase activity" --- p.144 / Chapter 8.2.5. --- Analysis of urinary electrolytes --- p.144 / Chapter 8.2.6. --- Measurements of body weight and wet weight of kidney --- p.145 / Chapter 8.3. --- Results --- p.145 / Chapter 8.3.1. --- Effects of salt loading on the body weight --- p.145 / Chapter 8.3.2. --- Effects of salt loading on 24-hour urinary sodium excretion --- p.148 / Chapter 8.3.3. --- Effects of salt loading on the wet weight of kidney --- p.152 / Chapter 8.3.4. --- "Effects of salt loading on tissue Na+,K+-ATPase activity" --- p.152 / Chapter 8.4. --- Discussion --- p.163 / Chapter Chapter 9 --- CONCLUSIONS AND FUTURE WORK --- p.175 / REFERENCES --- p.183

Thesis (Ph. D.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Non-Latin script record. Includes bibliographical references (p. 116-139).

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Title from title page of PDF document. Bibliography: pages 64-70, 104-108, 121-158.

by Albert Yan Wo Chan. / Thesis (M.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 203-242). / Chapter CHAPTER1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Brief History of thyroid diseases --- p.2 / Chapter 1.2 --- Thyrotoxicosis and muscle diseases --- p.7 / Chapter 1.2.1 --- Thyrotoxic myopathy --- p.8 / Chapter 1.2.2 --- Exophthalmic ophthalmoplegia (Grave's ophthalmopathy) --- p.10 / Chapter 1.2.3 --- Myasthenia gravis --- p.10 / Chapter 1.2.4 --- Thyrotoxic periodic paralysis (TPP) --- p.11 / Chapter 1.2.4.1 --- Overview --- p.11 / Chapter 1.2.4.2 --- Prevalence --- p.13 / Chapter 1.3 --- Periodic paralysis syndromes in the Chinese --- p.16 / Chapter 1.4 --- Potassium homeostasis in TPP --- p.19 / Chapter 1.5 --- Cellular potassium transport --- p.24 / Chapter 1.5.1 --- Role of the sodium pump --- p.24 / Chapter 1.5.2 --- Hormonal control of the sodium pump --- p.26 / Chapter 1.5.3 --- Molecular biology of the sodium pump --- p.27 / Chapter 1.5.4 --- Na-K-Cl transporter --- p.30 / Chapter 1.5.5 --- Summary --- p.32 / Chapter 1.6 --- Mechanism of paralysis --- p.33 / Chapter 1.7 --- Aetiology of TPP --- p.37 / Chapter 1.7.1 --- Genetic predisposition --- p.37 / Chapter 1.7.2 --- Possible membrane defect --- p.38 / Chapter 1.7.3 --- The central role of the sodium pump in the pathogenesis of TPP --- p.39 / Chapter 1.7.4 --- Environmental factor --- p.40 / Chapter 1.7.5 --- Summary --- p.40 / Chapter 1.8 --- Aims of the thesis --- p.41 / Chapter CHAPTER2 --- A PILOT STUDY ON THE PATHOPHYSIOLOGY OF TPP --- p.44 / Chapter 2.1 --- Aim --- p.45 / Chapter 2.2 --- Background --- p.45 / Chapter 2.2.1 --- The measurement of Na-K ATPase/ sodium pump activity --- p.45 / Chapter 2.2.2 --- In vitro decline in plasma potassium concentration --- p.48 / Chapter 2.2.3 --- Catecholamines --- p.49 / Chapter 2.3 --- Subjects & methods --- p.50 / Chapter 2.4 --- Results --- p.51 / Chapter 2.5 --- Discussion --- p.56 / Chapter 2.6 --- Conclusion --- p.58 / Chapter CHAPTER3 --- PLATELET NA-K ATPASE AS A TISSUE MARKER OF HYPERTHYROIDISM --- p.59 / Chapter 3.1 --- Aim --- p.60 / Chapter 3.2 --- Background --- p.60 / Chapter 3.2.1 --- Thyroid function tests (TFTs) --- p.60 / Chapter 3.2.2 --- TFTs vs tissue markers as an index of hyperthyroidism --- p.61 / Chapter 3.2.3 --- Sodium pump activity as a tissue marker and TPP --- p.63 / Chapter 3.2.4 --- Choice of tissue for sodium pump study --- p.64 / Chapter 3.2.5 --- Rationale behind the aim of study --- p.65 / Chapter 3.3 --- Subjects & methods --- p.68 / Chapter 3.3.1 --- Chemicals --- p.68 / Chapter 3.3.2 --- Subjects --- p.68 / Chapter 3.3.3 --- Plasma thyroid hormones analysis --- p.69 / Chapter 3.3.4 --- Determination of platelet Na-K ATPase activity --- p.71 / Chapter 3.3.4.1 --- Principle --- p.71 / Chapter 3.3.4.2 --- Preparation of platelets --- p.73 / Chapter 3.3.4.3 --- Preparation of platelet lysate --- p.73 / Chapter 3.3.4.4 --- Measurement of Na-K ATPase activity --- p.74 / Chapter 3.3.4.5 --- Measurement of Pi --- p.76 / Chapter 3.3.4.6 --- Measurement of protein --- p.78 / Chapter 3.4 --- Statistics & data handling --- p.80 / Chapter 3.5 --- Results --- p.81 / Chapter 3.5.1 --- Development of the platelet Na-K ATPase assay --- p.81 / Chapter 3.5.1.1 --- Introduction --- p.81 / Chapter 3.5.1.2 --- Effect of saponin concentration on the Na-K ATPase activity --- p.81 / Chapter 3.5.1.3 --- Linearity of the Na-K ATPase assay --- p.83 / Chapter 3.5.1.4 --- Imprecision of the Na-K ATPase assay --- p.83 / Chapter 3.5.1.5 --- Linearity of the Pi assay --- p.86 / Chapter 3.5.1.6 --- Linearity of the protein assay --- p.86 / Chapter 3.5.2 --- Thyroid function tests --- p.89 / Chapter 3.5.3 --- Platelet Na-K ATPase activity --- p.92 / Chapter 3.5.4 --- Correlation between thyroid hormones concentrations and platelet Na-K ATPase activity --- p.95 / Chapter 3.5.5 --- Correlation between age and platelet Na-K ATPase activity --- p.95 / Chapter 3.5.6 --- Performance of platelet ATPase as an indicator of hyperthyroidism --- p.99 / Chapter 3.6 --- Discussion --- p.102 / Chapter CHAPTER4 --- BASAL NA-K ATPASE ACTIVITY IN THYROTOXIC SUBJECTS WITH AND WITHOUT HYPOKALAEMIC PERIODIC PARALYSIS --- p.107 / Chapter 4.1 --- Aim --- p.108 / Chapter 4.2 --- Introduction --- p.108 / Chapter 4.2.1 --- Background --- p.108 / Chapter 4.2.2 --- Difficulties and limitations in TPP study --- p.109 / Chapter 4.3 --- Subjects & methods --- p.112 / Chapter 4.3.1 --- Platelet Na-K ATPase --- p.112 / Chapter 4.3.2 --- Rubidium loading test --- p.114 / Chapter 4.4 --- Statistics & data handling --- p.115 / Chapter 4.5 --- Results --- p.117 / Chapter 4.5.1 --- Platelet Na-K ATPase activity --- p.117 / Chapter 4.5.1a --- Thyrotoxic vs TPP --- p.121 / Chapter 4.5.1b --- Thyrotoxic vs euthyroid and TPP vs EuTPP --- p.124 / Chapter 4.5.1c --- Control vs euthyroid and EuTPP --- p.126 / Chapter 4.5.2 --- Rubidium loading test --- p.127 / Chapter 4.6 --- Discussion --- p.129 / Chapter 4.6.1 --- Clinical marker of TPP --- p.129 / Chapter 4.6.2 --- RBC/ lymphocyte sodium pump activity --- p.130 / Chapter 4.6.3 --- Platelet Na-K ATPase activity --- p.135 / Chapter CHAPTER5 --- VALIDATION OF THE ORAL GLUCOSE TOLERANCE TEST --- p.142 / Chapter 5.1 --- Aim --- p.143 / Chapter 5.2 --- Background --- p.143 / Chapter 5.2.1 --- Need for a validated protocol for OGTT --- p.143 / Chapter 5.2.2 --- Effectiveness of sodium fluoride as a preservative of glucose in blood sample --- p.144 / Chapter 5.2.3 --- Effect of delay in sample handling --- p.146 / Chapter 5.2.4 --- Ideal concentration of NaF --- p.147 / Chapter 5.2.5 --- D-mannose as a preservative of blood glucose --- p.147 / Chapter 5.2.6 --- Rationale behind the aim of study --- p.148 / Chapter 5.3 --- Subjects & methods --- p.149 / Chapter 5.3.1 --- Effectiveness of NaF --- p.149 / Chapter 5.3.2 --- Effect of delay in sample handling --- p.150 / Chapter 5.3.3 --- Ideal concentration of NaF --- p.151 / Chapter 5.3.4 --- Evaluation of D-mannose as a preservative of blood glucose --- p.151 / Chapter 5.4 --- Results and discussion --- p.153 / Chapter 5.4.1 --- Effectiveness of NaF --- p.153 / Chapter 5.4.2 --- Effect of delay in sample handling --- p.157 / Chapter 5.4.3 --- Ideal concentration of NaF --- p.159 / Chapter 5.4.4 --- D-mannose as a preservative of glucosein blood --- p.161 / Chapter 5.4.5 --- Summary --- p.167 / Chapter CHAPTER6 --- HYPERINSULINAEMIA AND NA-K ATPASE ACTIVITY IN TPP --- p.168 / Chapter 6.1 --- Aim --- p.169 / Chapter 6.2 --- Background --- p.169 / Chapter 6.2.1 --- "Insulin, hypokalaemia and sodium pump" --- p.169 / Chapter 6.2.2 --- Insulin and skeletal muscle membrane potential --- p.170 / Chapter 6.2.3 --- Potential role of insulin in the pathogenesis of TPP --- p.172 / Chapter 6.2.4 --- Hyperinsulinaemia and thyrotoxicosis --- p.173 / Chapter 6.2.5 --- TPP vs uncomplicating thyrotoxic patients --- p.173 / Chapter 6.2.6 --- Catecholamines and insulin secretion --- p.175 / Chapter 6.3 --- Subjects and methods --- p.177 / Chapter 6.4 --- Statistics and data handling --- p.179 / Chapter 6.5 --- Results --- p.180 / Chapter 6.6 --- Discussion --- p.187 / Chapter CHAPTER7 --- OVERALL DISCUSSION AND CONCLUSION --- p.192 / Chapter 7.1 --- General discussion --- p.193 / Chapter 7.2 --- Role of sodium pump in the pathogenesis of TPP --- p.196 / Chapter 7.3 --- Strategy for further study --- p.201 / Chapter 7.4 --- Conclusion --- p.202 / REFERENCES --- p.203 / APPENDIX --- p.239 / (Selected publications)

Thesis (M.S.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Science." Title from title page of PDF document. Bibliography: p. 39-48.

Dissertation (Ph.D.)--University of Toledo, 2008. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title in Ohio LINK ETD Center record : Na/K-ATPase signaling : from bench to bedside. Title from title page of PDF document. Bibliography: p. 99-120.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 6, 2010). Vita. Thesis advisor: Mark Milanick "June 2008" Includes bibliographical references

強心苷是一類多年普遍用於心力衰竭治療的化合物,包括蟾蜍靈和地高辛。鈉泵（也可稱為鈉鉀ATP酶）是強心苷的受體。最近流行病學研究，體外實驗，動物實驗和臨床試驗表明，強心苷具有癌症治療的強大潛力。 / 大腸癌是全球第三大殺手，約有一半的大腸癌患者需要手術切除後的輔助治療。因此，通過化療殺死腫瘤細胞，是一個可行的辦法來治療大腸癌患者。在本課題的研究中，強心苷抗人結腸癌的作用在HT-29和Caco-2細胞上進行了評價與闡釋。在結腸癌細胞研究模型中，蟾蜍靈誘導caspase非依賴性的細胞死亡，伴隨沒有早期凋亡，沒有聚（ADP-核糖）聚合酶(PARP)與caspase-3裂解，這些發現與強心苷誘發其它類腫瘤細胞凋亡的機製完全不同。相反，蟾蜍靈激活自噬途徑，促進LC3-II積累和自噬流動。此外，其它強心苷如地高辛與烏本苷也促使LC3-II在HT-29細胞內聚集。沉默ATG5和Beclin-1顯著降低蟾蜍靈誘導的LC3- II積累和細胞死亡。蟾蜍靈誘導的自噬與活性氧（ROS）產生和JNK活化相關。我們的研究結果揭示了蟾蜍靈藥物對抗結腸癌細胞的一種新的機制，開闢了強心苷通過自噬途徑來治療大腸癌的可能性。 / 最近的研究表明，強心苷誘導多種癌細胞系的細胞包括促使凋亡與自噬的細胞週期阻滯在G2／M期。然而，沒有詳細的信息闡述強心苷如何阻滯細胞週期進展。在本課題研究中，我們研究了強心苷介導的細胞週期阻滯的分子機制。蟾蜍靈處理的HeLa H2B-YFP細胞被阻滯在前中期，伴隨姐妹染色單體凝聚，染色體未排列在赤道板，未退出有絲分裂期。這一結果被蟾蜍靈誘導的四倍DNA含量細胞既不在四倍體G1期也不在胞質分裂期進一步證明。此後，我們檢測了紡錘體組裝和染色體分離所需的Aurora激酶和Polo-like kinase 1 (Plk1)。結果發現，在HT-29和HeLa細胞上，蟾蜍靈和其它強心苷能顯著降低總蛋白質和磷酸化的Aurora激酶與Plk1。此外，我們還發現，蟾蜍靈通過PI3K下調有絲分裂酶的活性。這些結果已經通過沉默鈉泵α做了驗證。總之，我們的結果表明, 蟾蜍靈和其它強心苷鈉鉀泵抑製劑強有力的抑制細胞在前中期是通過PI3K/HIF-1α/NF-κB途徑下調Aurora激酶的蛋白質和磷酸化水平和Plk1的蛋白質水平。我們的研究發現在了解如何利用強心苷的潛能治療癌症以及認知鈉泵在細胞週期中的功能方面提供了有用的信息。 / The sodium pump (also known as Na+/K+-ATPase) is the receptor for cardiac glycosides, a group of compounds including bufalin and digoxin which have been commonly used for heart failure treatment for many years. Recent epidemiological studies, in vitro studies, animal studies and clinical trials have shown that cardiac glycosides have potential applications for cancer treatment. / Colorectal cancer is the third leading cause of cancer death worldwide and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treat patients with colorectal cancer. In this study, the effects of cardiac glycosides were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to their well documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis, as well as poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. Moreover, other cardiac glycosides digoxin and ouabain could also induce the accumulation of LC3-II in HT-29 cells. The silencing of ATG5 and Beclin-1 significantly reduced bufalin-induced LC3-II accumulation and cell death. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS) and JNK activation. My findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer by cardiac glycosides through an autophagy pathway. / Recent studies have revealed that cardiac glycosides induce G2/M phase arrest in many cancer cells, which include apoptosis- and autophagy-promoting cells. However, no detailed information is available on how cardiac glycosides arrest cell cycle progression. In this study, I studied the molecular mechanisms of cell cycle arrest mediated by cardiac glycosides. Bufalin-treated HeLa H2B-YFP cells were arrested at prometaphase, as characterized by the presence of sister chromatid cohesion, absence of chromosomes alignment on the metaphase plate, and failure to exit mitosis. This result was further confirmed by bufalin-induced cells with 4N DNA content in neither tetraploid G1 phase nor cytokinesis. Thereafter, I detected the Aurora kinases and Polo-like kinase 1 (Plk1), which are required for both spindle assembly and chromosome segregation. It was found that bufalin and other cardiac glycosides could significantly reduce the total protein and phosphorylation of Aurora kinases and Plk1 in HT-29 and HeLa cells. In addition, I found that PI3K was responsible for the bufalin-induced downregulation of the activities of mitotic kinases. This result was validated by silencing of sodium pump alpha. Taken together, my results demonstrate that bufalin and other cardiac glycoside inhibitors of the sodium pump potently arrest cancer cells at prometaphase by downregulating the total protein and phosphorylation of Aurora kinases and the total protein of Plk1 through the PI3K/HIF-1α/NF-κB pathway. My findings provide useful information in understanding how cardiac glycosides could be exploited for their potentials in treating cancer and in identifying the function of sodium pump in cell cycle progression. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Chuanming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.iii / Abstract --- p.vi / Abstract (in Chinese) --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- The chemical structure of cardiac glycosides --- p.2 / Chapter 1.3 --- The traditional use of cardiac glycosides in cardiology --- p.4 / Chapter 1.4 --- The role of cardiac glycosides in cancer treatment --- p.4 / Chapter 1.5 --- The mechanisms of action by cardiac glycosides in cancer --- p.5 / Chapter 1.5.1 --- The structure and functions of cardiac glycosides receptor sodium pump --- p.5 / Chapter 1.5.2 --- Sodium pump as anticancer target --- p.6 / Chapter 1.5.3 --- The signal pathways involved in anticancer effect of cardiac glycosides --- p.7 / Chapter 1.6 --- The role of cardiac glycosides in apoptosis and autophagy --- p.8 / Chapter 1.7 --- Objectives of this project --- p.12 / Chapter Chapter 2 --- Bufalin induces autophagy but not apoptosis in human colon cancer cells --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Reagents and antibodies --- p.19 / Chapter 2.2.2 --- Cell culture --- p.19 / Chapter 2.2.3 --- Cell viability and cell death assay --- p.20 / Chapter 2.2.4 --- Annexin V and PI staining --- p.20 / Chapter 2.2.5 --- Cell cycle analysis --- p.21 / Chapter 2.2.6 --- Analysis of cleaved caspase-3-positive cells by flow cytometry --- p.21 / Chapter 2.2.7 --- Western blot analysis --- p.21 / Chapter 2.2.8 --- Immunofluorescence analysis of LC3 distribution --- p.22 / Chapter 2.2.9 --- RNA isolation and RT-PCR --- p.22 / Chapter 2.2.10 --- siRNAs transfection and treatments --- p.23 / Chapter 2.2.11 --- Transmission electron microscopy --- p.23 / Chapter 2.2.12 --- Statistical analysis --- p.24 / Chapter 2.3 --- Results --- p.24 / Chapter 2.3.1 --- Bufalin induces cell death and cell cycle arrest at G2/M phase in colon cancer cells --- p.24 / Chapter 2.3.2 --- Bufalin induces caspase-independent cell death in colon cancer cells --- p.28 / Chapter 2.3.3 --- Bufalin induces autophagy in colon cancer cells --- p.30 / Chapter 2.3.4 --- Bufalin-induced autophagy is dependent on ATG5 and Beclin-1 --- p.37 / Chapter 2.3.5 --- Increased autophagy is responsible for bufalin-induced cell death --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Bufalin mediates autophagic cell death through ROS generation and JNK activation --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Reagents and antibodies --- p.46 / Chapter 3.2.2 --- Cell culture --- p.47 / Chapter 3.2.3 --- Cell viability and cell death assay --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.47 / Chapter 3.2.5 --- Quantification of cells with > 5 LC3 punctate staining --- p.47 / Chapter 3.2.6 --- siRNAs transfection and treatments --- p.48 / Chapter 3.2.7 --- RNA isolation and RT-PCR --- p.48 / Chapter 3.2.8 --- ROS analysis --- p.48 / Chapter 3.2.9 --- JC-1 staining --- p.49 / Chapter 3.2.10 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- Bufalin induces autophagy-mediated cell death via ROS generation --- p.50 / Chapter 3.3.2 --- Activation of JNK is required for the upregulation of ATG5 and Beclin-1, and subsequent autophagy-mediated cell death in response to bufalin --- p.54 / Chapter 3.3.3 --- ROS generation is upstream of JNK activation in bufalin-induced cell death --- p.59 / Chapter 3.3.4 --- Bufalin-induced ROS generation is derived from mitochondria --- p.62 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- Bufalin arrests cells at prometaphase --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Reagents and antibodies --- p.70 / Chapter 4.2.2 --- Cell synchronization --- p.70 / Chapter 4.2.3 --- Mitotic index analysis of phosphorylation of MPM2 --- p.71 / Chapter 4.2.4 --- Cell cycle analysis --- p.71 / Chapter 4.2.5 --- Time-lapse experiments --- p.71 / Chapter 4.2.6 --- Immunofluorescence analysis of phospho-histone H3 (Ser10) --- p.72 / Chapter 4.2.7 --- Western blot analysis --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.3.1 --- Bufalin reduces mitotic marker phosphorylation of histone H3 and MPM2 and increases cells with 4N DNA content --- p.73 / Chapter 4.3.2 --- Increased cells with 4N DNA content after bufalin treatment are in neither a tetraploid G1 phase nor a cytokinesis arrest --- p.77 / Chapter 4.3.3 --- Bufalin-treated cells can enter prophase, but fail to pass through metaphase --- p.80 / Chapter 4.4 --- Discussion --- p.83 / Chapter Chapter 5 --- Bufalin induces prometaphase arrest through downregulating mitotic kinases --- p.87 / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.89 / Chapter 5.2.1 --- Reagents and antibodies --- p.89 / Chapter 5.2.2 --- Cell synchronization --- p.90 / Chapter 5.2.3 --- Immunofluorescence staining --- p.90 / Chapter 5.2.4 --- siRNAs transfection and treatments --- p.91 / Chapter 5.2.5 --- Western blot analysis --- p.91 / Chapter 5.2.6 --- Statistic analysis --- p.91 / Chapter 5.3 --- Results --- p.92 / Chapter 5.3.1 --- Bufalin downregulates Aurora A and B in protein and phosphorylation levels --- p.92 / Chapter 5.3.2 --- Bufalin prevents Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores --- p.97 / Chapter 5.3.3 --- Bufalin prevents Plk1 recruitment to mitotic centrosomes and unattached kinetochores through downregulation of protein levels of Plk1 --- p.101 / Chapter 5.3.4 --- Bufalin decreases the activities of Aurora A, Aurora B and Plk1 through PI3K pathway --- p.105 / Chapter 5.3.5 --- HIF-1α and NF-κB pathways are involved in sodium pump-mediated the regulation of mitotic kinases --- p.109 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General discussion --- p.115 / Chapter 6.1 --- Potential toxicity of bufalin --- p.115 / Chapter 6.2 --- Cardiac glycosides induced programmed cell death --- p.115 / Chapter 6.3 --- Signal pathways involved in cardiac glycosides-mediated autophagy --- p.117 / Chapter 6.4 --- The relationship between ROS and JNK in cardiac glycosides-induced autophagy --- p.120 / Chapter 6.5 --- The role of ROS in apoptosis and autophagy --- p.121 / Chapter 6.6 --- The role of cardiac glycosides in cell cycle arrest --- p.122 / Chapter 6.7 --- Application of cardiac glycosides in combination with chemotherapy and radiotherapy --- p.125 / Chapter Chapter 7 --- Conclusions and future perspectives --- p.127 / References --- p.133 / Appendices --- p.153 / Publication --- p.153

Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 86-96). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;