Shing, Ka Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 158-188). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- CELL-FREE NUCLEIC ACIDS IN HUMAN BODILY FLUIDS --- p.2 / Chapter 1.1 --- Early studies on the presence of cell-free nucleic acids in human bodily fluids --- p.2 / Chapter 1.2 --- Circulating nucleic acids in plasma and serum --- p.2 / Chapter 1.2.1 --- Cancer Detection --- p.3 / Chapter 1.2.1.1 --- Circulating tumor-derived DNA --- p.3 / Chapter 1.2.1.2 --- Circulating tumor-derived RNA --- p.5 / Chapter 1.2.2 --- Prenatal diagnosis --- p.7 / Chapter 1.2.2.1 --- Circulating fetal DNA --- p.7 / Chapter 1.2.2.2 --- Circulating fetal messenger RNA --- p.11 / Chapter 1.2.2.3 --- Circulating placental microRNA --- p.13 / Chapter 1.3 --- Cell-free nucleic acids in urine --- p.14 / Chapter 1.3.1 --- Transrenal DNA (Tr-DNA) --- p.15 / Chapter 1.3.1.1 --- Biology of Tr-DNA --- p.15 / Chapter 1.3.1.2 --- Detection of fetal-derived Tr-DNA --- p.15 / Chapter 1.3.1.3 --- Potential problems associated with the detection of Tr-DNA --- p.16 / Chapter 1.3.2 --- Cell-free DNA in urine as released from the urinary tract --- p.17 / Chapter 1.4 --- Other bodily fluids with cell-free nucleic acids --- p.18 / Chapter 1.4.1 --- Amniotic fluid --- p.19 / Chapter 1.4.2 --- Cerebrospinal fluid (CSF) --- p.20 / Chapter 1.4.3 --- Peritoneal fluid --- p.20 / Chapter CHAPTER 2: --- MICRORNA IN HUMANS --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Biogenesis --- p.21 / Chapter 2.2.1 --- Transcription of microRNA genes --- p.21 / Chapter 2.2.2 --- Processing and maturation of microRNA precursors --- p.23 / Chapter 2.3 --- Mechanisms of gene regulation --- p.24 / Chapter 2.3.1 --- Cleavage of target mRNA --- p.24 / Chapter 2.3.2 --- Translational repression of mRNA --- p.25 / Chapter 2.4 --- Functional roles of microRNAs --- p.25 / Chapter 2.4.1 --- Oncogenesis --- p.25 / Chapter 2.4.2 --- Programmed cell death --- p.26 / Chapter 2.4.3 --- Cellular differentiation and development --- p.27 / Chapter 2.4.4 --- Regulation of physiological and cellular processes --- p.28 / Chapter 2.5 --- Aim of this thesis --- p.28 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.30 / Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF CIRCULATING AND URINARY NUCLEIC ACIDS --- p.31 / Chapter 3.1 --- Preparation of samples --- p.31 / Chapter 3.1.1 --- Preparation of plasma --- p.31 / Chapter 3.1.2 --- Preparation of blood cells --- p.32 / Chapter 3.1.3 --- Preparation of placental tissue --- p.32 / Chapter 3.1.4 --- Preparation of urine and urine cell pellet --- p.32 / Chapter 3.2 --- Nucleic acid extraction --- p.33 / Chapter 3.2.1 --- "Extraction of small RNA-containing total RNA from plasma, blood cells and placental tissue" --- p.33 / Chapter 3.2.2 --- Extraction of DNA from urine --- p.37 / Chapter 3.3 --- Quantitative measurements of nucleic acids --- p.38 / Chapter 3.3.1 --- Principle of real-time quantitative PCR --- p.38 / Chapter 3.3.2 --- One-step QRT-PCR assays for mRNA quantification --- p.40 / Chapter 3.3.2.1 --- Principle --- p.40 / Chapter 3.3.2.2 --- Quantification of human placental lactogen （hPL) mRNA --- p.40 / Chapter 3.3.3 --- Two-step QRT-PCR assays for microRNA quantification --- p.45 / Chapter 3.3.3.1 --- Principle --- p.45 / Chapter 3.3.3.2 --- Advantages --- p.46 / Chapter 3.3.3.3 --- TaqMan® MicroRNA Assays --- p.47 / Chapter 3.3.4 --- QPCR assays for DNA quantification --- p.53 / Chapter 3.3.4.1 --- Principle --- p.53 / Chapter 3.3.4.2 --- Quantification of the leptin gene and the sex-determining region on Ychromosome gene --- p.53 / Chapter 3.4 --- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.57 / Chapter 3.4.1 --- Principle --- p.57 / Chapter 3.4.2 --- Zinc finger protein gene assay for determining the fractional concentration of male DNA --- p.58 / Chapter 3.5 --- Statistical analyses --- p.65 / Chapter SECTION III: --- CIRCULATING PLACENTAL MICRORNAS IN MATERNAL PLASMA AS MARKERS FOR PRENATAL DIAGNOSIS　 --- p.66 / Chapter CHAPTER 4: --- THE EXISTENCE AND QUANTITATIVE DETECTION OF CELL-FREE MICRORNAS IN PLASMA --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.69 / Chapter 4.2.1 --- Sample collection --- p.69 / Chapter 4.2.2 --- Experimental design --- p.69 / Chapter 4.2.3 --- RNA extraction and quantification --- p.72 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Validation of two-step QRT-PCR system for miRNA quantification --- p.75 / Chapter 4.3.2 --- Detection of cell-free miRNA in maternal plasma --- p.82 / Chapter 4.4 --- Discussion --- p.82 / Chapter CHAPTER 5: --- SYSTEMATIC IDENTIFICATION AND CHARACTERIZATION OF PLACENTAL MICRORNAS IN MATERNAL PLASMA --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and methods --- p.88 / Chapter 5.2.1 --- Sample collection --- p.88 / Chapter 5.2.2 --- Experimental design --- p.88 / Chapter 5.2.3 --- RNA extraction and miRNA quantification --- p.91 / Chapter 5.3 --- Results --- p.93 / Chapter 5.3.1 --- A systematic search for placental miRNAs in maternal plasma using two-step QRT-PCR assays --- p.93 / Chapter 5.3.2 --- Detection rate and clearance kinetics of placental miRNAs in maternal plasma --- p.97 / Chapter 5.3.3 --- Effects of filtering maternal plasma on the concentration of placental miRNA and mRNA --- p.99 / Chapter 5.3.5 --- Temporal profile of placental miRNA concentrations in maternal plasma across different trimesters of pregnancies --- p.103 / Chapter 5.4 --- Discussion --- p.115 / Chapter SECTION IV: --- DETECTION OF CELL-FREE DNA IN URINE --- p.119 / Chapter CHAPTER 6: --- HEMATOPOIETIC STEM CELL TRANSPLANTATION RECIPIENTS AS A MODEL TO STUDY CELL-FREE DNA IN URINE --- p.120 / Chapter 6.1 --- Introduction --- p.120 / Chapter 6.2 --- Materials and methods --- p.123 / Chapter 6.2.1 --- Sample collection --- p.123 / Chapter 6.2.2 --- Experimental design --- p.124 / Chapter 6.2.3 --- DNA extraction and quantification --- p.125 / Chapter 6.3 --- Results --- p.128 / Chapter 6.3.1 --- Validation of the zinc finger protein gene assay --- p.128 / Chapter 6.3.2 --- Fractional concentration of male DNA in blood cells and plasma of sex-mismatched HSCT patients --- p.129 / Chapter 6.3.3 --- Fractional concentration of male DNA in the urine and the urine cell pellets of sex-mismatched HSCT patients --- p.131 / Chapter 6.3.4 --- Size distribution of cell-free DNA in peripheral blood and urine samples of sex-mismatched HSCT patients --- p.132 / Amplicon size --- p.138 / Chapter 6.4 --- Discussion --- p.143 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.147 / Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.148 / Chapter 7.1 --- Circulating miRNA is a valuable resource for molecular analysis --- p.148 / Chapter 7.2 --- The presence of donor-derived DNA in the urine of HSCT recipients --- p.150 / Chapter 7.3 --- Prospects for future work --- p.152 / APPENDIX 1 --- p.154 / REFERENCES --- p.158

by Ho, Wai Fun. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 79-87). / LIST OF TABLES --- p.iv / LIST OF FIGURES --- p.vi / ACKNOWLEDGEMENTS --- p.vii / ABSTRACT --- p.viii / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- METABOLIC DERANGEMENTS AND CACHEXIA IN CANCER --- p.1 / Chapter 1.2 --- PROTEIN METABOLISM IN MALIGNANCY --- p.4 / Chapter 1.3 --- REVIEW OF REPORTS ON AMINO ACID DISTURBANCES IN MALIGNANCY --- p.5 / Chapter 1.4 --- AMINO ACID ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY --- p.10 / Chapter 1.4.1 --- Amino Acid Analysis by Ion-Exchange HPLC --- p.11 / Chapter 1.4.2 --- Amino Acid Analysis by Reversed-Phase HPLC --- p.13 / Chapter 1.4.3 --- Derivatizing Agents --- p.15 / Chapter 1.5 --- CHOICE OF CANCER PATIENTS AND METHODOLOGY FOR THIS STUDY --- p.19 / Chapter 1.5.1 --- Choice of Cancer Patients --- p.19 / Chapter 1.5.2 --- Methodology Chosen and Its Principle --- p.20 / Chapter 2. --- OBJECTIVES --- p.23 / Chapter 3. --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- STUDY SUBJECTS --- p.24 / Chapter 3.1.1 --- Patients --- p.24 / Chapter 3.1.2 --- Control Subjects --- p.25 / Chapter 3.2 --- CLINICAL FEATURES --- p.25 / Chapter 3.3 --- BLOOD COLLECTION --- p.25 / Chapter 3.4 --- GENERAL BIOCHEMICAL TESTS --- p.26 / Chapter 3.5 --- PLASMA AMINO ACID ANALYSIS BY HPLC --- p.26 / Chapter 3.5.1 --- Apparatus --- p.26 / Chapter 3.5.2 --- Reagents --- p.27 / Chapter 3.5.3 --- Reagent Preparation --- p.28 / Chapter 3.5.3.1 --- Mobile phase --- p.28 / Chapter 3.5.3.2 --- Derivatizing reagent --- p.29 / Chapter 3.5.4 --- Standard Preparation --- p.29 / Chapter 3.5.4.1 --- Internal standard solution --- p.29 / Chapter 3.5.4.2 --- Composite standard solution --- p.30 / Chapter 3.5.4.3 --- Composite standard-internal standard mixture --- p.32 / Chapter 3.5.5 --- Sample Preparation --- p.32 / Chapter 3.5.5.1 --- Protein removal --- p.32 / Chapter 3.5.5.2 --- Addition of internal standard --- p.32 / Chapter 3.5.6 --- Preparation of Samples for the WISP Sample Processor --- p.33 / Chapter 3.5.7 --- Sample Queue for the WISP Sample Processor --- p.33 / Chapter 3.5.8 --- Automated Derivatization Procedure --- p.36 / Chapter 3.5.9 --- Chromatographic Conditions --- p.36 / Chapter 3.6 --- STATISTICAL STUDIES --- p.38 / Chapter 4. --- RESULTS --- p.40 / Chapter 4.1 --- ANALYTICAL PERFORMANCES --- p.40 / Chapter 4.1.1 --- Chromatograms --- p.40 / Chapter 4.1.2 --- Precision --- p.47 / Chapter 4.1.3 --- Linearity --- p.47 / Chapter 4.1.4 --- Analytical Recovery --- p.51 / Chapter 4.2 --- DATA DISTRIBUTION STUDIES --- p.53 / Chapter 4.3 --- PATIENTS' ANTHROPOMETRIC DATA AND BLOOD BIOCHEMISTRY --- p.55 / Chapter 4.4 --- FREE PLASMA AMINO ACID CONCENTRATIONS IN NORMAL CONTROLS --- p.59 / Chapter 4.5 --- FREE PLASMA AMINO ACID CONCENTRATIONS IN CANCER PATIENTS --- p.59 / Chapter 5. --- DISCUSSION --- p.68 / Chapter 5.1 --- METHOD ESTABLISHMENT --- p.68 / Chapter 5.2 --- NORMAL CONTROLS --- p.68 / Chapter 5.3 --- CANCER PATIENTS --- p.71 / Chapter 5.3.1 --- Nasopharyngeal Cancer --- p.71 / Chapter 5.3.2 --- Lung Cancer --- p.71 / Chapter 5.3.3 --- Breast Cancer --- p.72 / Chapter 5.3.4 --- Colorectal Cancer --- p.74 / Chapter 5.4 --- SUMMARY OF THE PLASMA AMINO ACID PROFILES IN CANCER --- p.75 / Chapter 5.5 --- FURTHER STUDIES --- p.76 / Chapter 6. --- CONCLUSION --- p.78 / Chapter 7. --- REFERENCES --- p.79

The present thesis addresses two research areas related to film growth in a highly ionized magnetron sputtering system: plasma characterization, and thin film growth and analysis. The deposition technique used is called high power pulsed magnetron sputtering (HPPMS). Characteristic for this technique are high energy pulses (a few Joules) of length 50-100 µs that are applied to the target (cathode) with a duty time of less than 1 % of the total pulse time. This results in a high electron density in the discharge (>1x1019 m-3) and leads to an increase of the ionization fraction of the sputtered material reaching up to 70 % for Cu. In this work the spatial and temporal evolution of the plasma parameters, including the electron energy distribution function (EEDF), the electron density and the electron temperature are determined using electrostatic Langmuir probes. Electron temperature measurements reveal a low effective temperature of 2-3 eV. The degree of ionization in the HPPMS discharge is explained in light of the self-sputtering yield of the target material. A simple model is therefore provided in order to compare the sputtering yield in HPPMS and that in dc magnetron sputtering (dcMS) for the same average power. Thin Ta films are grown using HPPMS and dcMS and their properties are studied. It is shown that enhanced microstructure and morphology of the deposited films is achieved by HPPMS. The Ta films are also deposited at a number of substrate inclination angles ranging from 0o (i.e., facing the target surface) up to 180 o (i.e., facing away from the target). Deposition rate measurements performed at all inclination angles for both techniques, reveal that growth made using HPPMS resulted in an improved film thickness at higher inclination. Furthermore, the high ionization of the Ta atoms in HPPMS discharge is found to allow for phase tailoring of the deposited films at all inclination angles by applying a bias voltage to the substrate. Finally, highly ionized magnetron sputtering of a compound MAX-phase material (Ti3SiC2) is performed, demonstrating that the HPPMS discharge could also be used to tailor the composition of the growing Ti-Si-C films. / On the day of the public defence of the doctoral thesis, the status of articles III and IV was Submitted. The titles of papers VI and VII changed between their manuscript forms and when they were published.

Zhao Zheng. / "December 2000." / On t.p. "2" is subscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 187-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Le but de ce travail de thèse est d’étudier les propriétés d’un plasma réactif ainsi que les caractéristiques structurales, optiques et électriques de couches minces d’oxynitrure de tantale (TaOxNy) élaborées par pulvérisation cathodique radiofréquence. L’élaboration de ce matériau ternaire par pulvérisation d’une cible de tantale au moyen d’un plasma contenant à la fois de l’argon, de l’oxygène et de l’azote est complexe en raison de phénomènes d’empoisonnement de la cible. L’analyse de la composition du plasma par spectroscopie d’émission optique et le suivi de l’évolution de certaines raies représentatives d’espèces excitées dans le milieu, nous ont permis de déterminer les conditions optimales au dépôt de films de types TaOxNy sur une large gamme de compositions. Grâce à une étude par diffraction des rayons X et spectroscopie de photoélectrons X, nous avons suivi les évolutions structurales de couches ayant subi ou non un recuit thermique. Nous avons montré de quoi étaient constituées les parties amorphes et cristallisées de ces films et déterminé la taille des domaines de cohérence. Enfin, les propriétés optiques (indice de réfraction, gap optique, paramètre d’Urbach) et diélectriques ont été corrélées à la structure des matériaux. / The aim of this thesis is to study the properties of a reactive plasma as well as the structural, optical and electrical properties of tantalum oxynitride thin films (TaOxNy) prepared by radiofrequency sputtering. The elaboration of this ternary material by sputtering a pure tantalum target using plasma containing both of argon, oxygen and nitrogen is complex due to the target-poisoning phenomenon. The analysis of the composition of the plasma by optical emission spectroscopy and monitoring the evolution of some representative line of excited species in this environment, allow us to determine the optimal conditions to deposit TaOxNy films over a wide range of composition. Thanks to a study by X-ray diffraction and X-ray photoelectron spectroscopy, we followed the structural evolution of the films subjected or not to a rapid thermal annealing. We showed by what were constituted the amorphous and crystalline parts of the films and determined the size of the crystalline domains. Finally, the optical properties (refractive index, optical gap, Urbach parameter) and dielectric behavior have been correlated with the structure of materials.

Lysophosphatidic acid (LPA) influences many physiological processes, such as brain and vascular development. It is associated with several diseases including ovarian cancer, breast cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, multiple myeloma atherosclerotic diseases, cardiovascular diseases, pulmonary inflammatory diseases and renal diseases. LPA plasma and serum levels have been reported to be important values in diagnosing ovarian cancer and other diseases. However, the extraction and quantification of LPA in plasma are very challenging because of the low physiological concentration and similar structures of LPA to other phospholipids. Many previous studies have not described the separation of LPA from other phospholipids, which may make analyses more challenging than necessary. We developed an SPE extraction method for plasma LPA that can extract LPA at high purity. We also developed an HPLC post-column fluorescence detection method that allows the efficient quantification of LPA. These methods were used in a clinical study for ovarian cancer diagnosis to help validate LPA as a biomarker of ovarian cancer. Moreover, molecular imprinted polymers (MIPs) were designed and synthesized as material for the improved extraction of LPA. Compared to the commercially available materials, the MIP developed shows enhanced selectivity for LPA. The extraction was overall relatively more efficient and less labor-intensive.