Glucocorticoid activation of two types of adrenocorticosteroid receptors (ACRs), themineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), influences hippocampalneuron vulnerability to injury. Excessive activation of GR may compromise hippocampalneuron survival after several types of challenge including ischemic, metabolic, and excitotoxicinsults. In contrast, MR prevents adrenalectomy-induced loss of granule neurons in the dentategyrus. The present thesis addresses the respective roles of MR and GR in modulating neuronalsurvival following two forms of neuronal injury, excitotoxicity and traumatic brain injury. MaleSprague-Dawley rats were pretreated with MR antagonist spironolactone or GR antagonistmifepristone (RU486) and subsequently injected with kainic acid, an excitotoxic glutamateanalog, or injured with a controlled cortical impact. Twenty-four hours following injury,hippocampal neuron survival was measured to test the hypotheses that MR blockade wouldendanger and GR blockade would protect hippocampal neurons following injury. MessengerRNA levels of viability-related genes including bcl-2, bax, p53, BDNF, and NT-3 were alsomeasured to test the hypothesis that ACR regulation of these genes wouldcorrelate with neuronal survival. In addition, ACR mRNA levels were measured followingreceptor blockade and injury to test the hypothesis that glucocorticoid signaling is alteredfollowing neuronal injury via regulation of ACR expression.Mineralocorticoid receptor blockade with spironolactone increased neuronal vulnerability toexcitotoxic insult in hippocampal field CA3, and GR blockade with RU486 prevented neuronalloss after traumatic brain injury in field CA1. These results are consistent with the hypothesesthat MR protects and GR endangers hippocampal neurons. Adrenocorticosteroid receptorblockade decreased mRNA levels of the anti-apoptotic gene bcl-2 in select regions of uninjuredhippocampus, yet ACR regulation of bcl-2 did not consistently correspond with measures ofneuronal survival after injury. Kainic acid decreased MR mRNA levels in CA1 and CA3, whileboth kainic acid and controlled cortical impact dramatically decreased GR mRNA levels indentate gyrus. These data suggest that injury modulation of glucocorticoid signaling throughregulation of ACR expression may influence hippocampal neuron viability following injury.

Células dendríticas (DCs) são células apresentadoras de antígenos usadas em estratégias imunoterapêuticas, como as que utilizam híbridos de DCs e células tumorais. Este projeto avaliou a possibilidade de utilizar, como parceiro de fusão das DCs, células de linhagem tumoral previamente transfectadas com mRNA amplificado de células tumorais contra as quais se pretende induzir resposta. Os híbridos foram capazes de induzir uma resposta antígeno-específica e foi possível enriquecer uma célula com antígenos de outra, uma vez que células transfectadas passaram a apresentar mRNAs da célula doadora, e expressar GFP quando a célula doadora era positiva para GFP. Foi possível amplificar integralmente o mRNA para GFP e amplificar os mRNAs de uma célula, gerando um material contendo mRNAs do preparado inicial mas incapaz de aumentar a expressão das moléculas avaliadas nas células transfectadas, mostrando que o protocolo ainda precisa ser aperfeiçoado. Em conjunto, os resultados mostraram que a estratégia de imunoterapia aqui explorada é promissora e merece maior investigação. / Dendritic cells (DCs) are antigen presenting cells widely used in immunotherapy strategies, as the ones that utilize dendritic cell tumor cell hybrids. The present project evaluated the possibility of using cells from tumor cell lines, which were previously transfected with mRNA amplified from tumor cells against which a response is aimed, to fuse with DCs. The hybrids were able to induce a specific immune response. Also, it was possible to enriched one cell with antigens from another one, since transfected cells increased the amount of mRNAs from the donator cell, and expressed GFP when the donator cells expressed this protein. It was possible to successfully amplify the GFP specific mRNA. The mRNAs amplified from RNA of different cell lines contained mRNAs that were present in the total extracted RNA, but were not able to increase the expression of the molecules we attempted to detect in the transfected cells, showing that the protocol still need to be improved. Together, these results show that this new strategy is promising and deserves further investigation.

Apart from carrying genetic information, RNAs also act as effectors of cellular processes through folding into intricate secondary and tertiary structures. The ubiquitous RNA structures in eukaryotic mRNAs, in collaboration with specific RNA-binding proteins, control many aspects of the post-transcriptional regulation of gene expression. However, the molecular bases for the recognition of these mRNA structures by their protein partners remain poorly understood due to the lack of structural information. This dissertation presents our structural studies on two protein-RNA complexes that both include regulatory mRNA stem-loop structures. We first describe the crystal structure of a ternary complex including the highly conserved human histone mRNA stem-loop (SL), the stem-loop binding protein (SLBP) and the 3′ to 5′ exonuclease 3′hExo. This structure identifies a single sequence-specific interaction between the SL and SLBP, and the mostly shape-dependent RNA-recognition mode by both proteins. In addition to explaining the large body of biochemical and biophysical data on this complex accumulated over the last two decades, we also for the first time elucidate the induced-fit mechanism underlying the cooperativity between SLBP and 3′hExo. We next shift our focus to a class of less conserved mRNA stem-loop structures named constitutive decay elements (CDE). The RNA-binding ROQ domain of Roquin recognizes the various CDEs and mediates the decay of CDE-containing mRNAs, which predominantly encode proteins responsible for inflammation and autoimmunity. Structural and biochemical studies of the ROQ domain in complex with two different CDE RNAs unexpectedly reveal two distinct RNA binding sites on this protein, one recognizing CDE stem-loops and the other binding to double-stranded RNAs. The stuctures are also in agreement with the versatility of Roquin and have opened up new avenues to investigating its functions in modulating the stability of target mRNAs.

During mRNA synthesis, pre-mRNAs must be cleaved and polyadenylated at their 3'-end to be fully mature, before being exported from the nucleus. In yeast, there is a large protein machinery comprised of dozens of proteins that work together to perform these two reactions. Some of these proteins are capable of recognizing and binding key sequence elements in the pre-mRNA, effectively directing where in the transcript the cleavage and polyadenylation occur. In this thesis, recently reported structural findings related to the pre-mRNA 3'-end processing machinery are summarized. Within this machinery, the Cleavage Factor IA (CF-IA) complex is comprised of the Rna14, Rna15, and Pcf11 and Clp1 proteins. Results reported here include the crystal structure of the Rna14-Rna15 complex, which indicates that the Rna14 protein forms a dimer that has inherent conformational variability. The Rna15 protein binds to the C-terminal domain of Rna14, and is connected to the Rna14 HAT domain by a flexible linker, which may indicate that Rna15 functions somewhat independently of the Rna14 HAT domain. The complete CF-IA complex is explored in detail, including protein-protein interactions within the complex and the stoichiometric ratios of CF-IA components. Unlike previous reports, results indicate that CF-IA may form a dimer with a 2:2:2:2 stoichiometry of Rna14:Rna15:Clp1:Pcf11. Also reported are projects unrelated to CF-IA, including the crystal structure of the biotin-dependent alpha(6)beta(6) geranyl-CoA carboxylase (GCC) holoenzyme. Comparison of GCC to the closely related 3-methylcrotonyl CoA carboxylase (MCC) holoenzyme reveals a conserved domain swap in the carboxyltransferase (CT) domains of both enzymes. This domain swap is not present in the related biotin-dependent carboxylases propionyl-CoA carboxylase (PCC) and acetyl-CoA carboxylase (ACC), which may indicate a distinct lineage for biotin-dependent carboxylases that target the γ-carbon. In addition, comparison of the two structures also reveals a conserved Phe191 in MCC that is absent in GCC. Phe191 blocks a key substrate-binding pocket and explains the differences in substrate-specificities between MCC and GCC. The role of Phe191 is tested by site-directed mutagenesis to a Glycine to open the pocket in MCC and by mutating a structurally equivalent Glycine to Phe to close the pocket in GCC. These mutations can convert MCC to a GCC and vice versa.

In this series of work, we examined the role of measuring cytokine mRNA expression in the urinary sediments for the assessment of SLE disease activity. The Th-1/Th-2 imbalance observed in patients with active lupus nephritis supports its relevance in pathogenesis. Our results also suggest that urinary T-BET-to-GATA-3 expression ratio may predict lupus flare. The measurement of mRNA expression in the urinary sediment may provide valuable information for the assessment and risk stratification of SLE patients. (Abstract shortened by UMI.) / In this series of work, we investigated (i) the pattern of cytokine gene expression in the urinary sediment of lupus patients, (ii) the relation between the gene expression profile in the urinary sediment and the clinical and histological disease activity of lupus patients; and (iii) the application of this non-invasive method on the assessment and monitoring of SLE disease activity. / Systemic lupus erythematosus (SLE) is a relapsing autoimmune disease with clinical manifestations that affect multiple organ systems. Lupus nephritis (LN) is recognized as one of the most severe organ involvement in SLE and affects half of the lupus patients. LN is characterized by intra-renal lymphocyte activation and inflammation. Since most of the cytokines exert their effects in a paracrine fashion, measuring their expression at the site of pathology should be of biologic relevance. Although kidney biopsy is widely used to determine the histology and severity of LN, this invasive procedure has its own risk and is not practical for serial monitoring. We hypothesize that measurement of messenger RNA (mRNA) expression in the urinary sediment may provide a non-invasive means to assess the disease activity of lupus patients. / Chan Wing Yan. / "August 2005." / Adviser: Cheuk Chun Szeto. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3692. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 302-333). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Ectopic and over-expression of G protein-coupled receptor (GPCR) have been reported to induce tumor formation. Mas protein is a member of GPCR family and was originally isolated from human epidermoid carcinoma. It was demonstrated that mas mRNA was abundantly expressed in human and rat brains by in situ hybridization and RNase protection assays. However, cellular mechanism that leads to such tumorigenic transformation is still an open question. / In order to identify the cellular mechanism of mas-induced tumor formation, a full-length mas cDNA was cloned into a mammalian expression vector pFRSV with dihydrofolate reductase gene as a selection marker. Detailed analyses of mas-transfected cell lines by Southern blot, Northern blot and tumorigenicity assay indicated that tumorigenicity of mas-transfected cells depended on the sites of chromosomal integration and the levels of mas expression. These results suggest that overexpression of mas is not sufficient to induce tumor formation. In line with the ability of mas-transfected cells Mc0M80 to form solid tumor in nude mice, MTT cell proliferation assay indicated that the mas-transfected cells Mc0M80 proliferated faster than vector-transfected cells. Moreover, mas-transfected cells Mc0M80 exhibited significantly increased anchorage-independent growth. Furthermore, mas-transfected cells Mc0M80 showed higher percentage cells in G2/M phase but lower in S-phase in comparison with vector-transfected cells. / Interestingly, Southern blot analysis of individual xenografted tumor tissue indicated that tumor was composed of cells not only derived from injected mas-transfected CHO cells but also cells from mouse tissues. The presence of mouse stromal cells in the tumor was confirmed by immunohistochemistry and in situ hybridization. Previously our laboratory had identified some up- and down-regulated genes in mas-transfected cells by fluorescent differential display (FluoroDD). Northern blot showed that these differential expressed genes were up- or down-regulated in mas-transfected cells and tumor samples, which might play an important role in cancerous growth. / Taken together, these results suggest that over-expression of GPCR mas up-regulated tumor-related genes, resulting in promoting excessive cell growth and tumorigenic transformation. In addition, when the tumor mass formed they secreted some growth factor(s) which altered the migration of mouse stromal cells into tumor mass. The interactions of transformed cells and stromal cells further aggravate the tumorigenicity process. / To complement our fluorescent differential display study and to compare changes of gene expression when transformed cells were exposed to the microenvironment in nude mice, protein expression profiles of mas over-expressing cells as well as tumor tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The 2D-PAGE analysis showed that a similar but distinct protein expression profiles in mas-transfected cells and in mas-induced tumor. Mass spectrometry analysis identified several cancerous growth-related proteins and they are involved in processes such as cell signaling, energy metabolism, transcription and translation and cytoskeleton organization. / Lin Wenzhen. / "December 2005." / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6381. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 222-240). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Yuen, Wing Sum. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-155). / Abstract also in Chinese. / Chapter I --- Title page --- p.i / Chapter II --- Acknowledgements --- p.ii / Chapter III --- Abstract --- p.iii / Chapter IV --- Abstract (Chinese version) --- p.vi / Chapter V --- Table of contents --- p.viii / Chapter VI --- List of abbreviations --- p.xv / Chapter VII --- List of figures --- p.xvi / Chapter Chapter 1 --- General introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.5 / Chapter 2.1 --- Cystic fibrosis transmembrane conductance regulator in human --- p.5 / Chapter 2.1.1. --- Pathology of cystic fibrosis --- p.5 / Chapter 2.1.2. --- CFTR gene and the encoded protein --- p.6 / Chapter 2.1.3. --- Hypothetical model for CFTR function --- p.7 / Chapter 2.1.4. --- Functions of CFTR --- p.7 / Chapter 2.1.5. --- Regulation of CFTR gene expression --- p.8 / Chapter 2.1.6 --- Regulation of CFTR protein --- p.9 / Chapter 2.1.7. --- Discovery of CFTR homologues in other vertebrates --- p.10 / Chapter 2.2 --- Cystic fibrosis transmembrane conductance regulator in teleosts --- p.10 / Chapter 2.2.1. --- Evidence for the presence of CFTR homologue in teleosts --- p.10 / Chapter 2.2.2. --- Molecular cloning of teleost CFTR genes --- p.11 / Chapter 2.2.3. --- Role of teleost CFTR in osmoregulation --- p.13 / Chapter 2.2.3.1. --- Tissue distribution of CFTR --- p.13 / Chapter 2.2.3.2. --- Changes in CFTR expression in response to ambient salinities --- p.14 / Chapter 2.2.3.3. --- Immunocytochemical studies of CFTR --- p.15 / Chapter 2.2.3.4. --- Regulation of CFTR --- p.17 / Chapter 2.3 --- Osmoregulation in teleosts --- p.19 / Chapter 2.3.1. --- Importance of osmoregulation --- p.19 / Chapter 2.3.2. --- Major components of chloride cells in marine teleosts --- p.20 / Chapter 2.3.2.1. --- Overview --- p.20 / Chapter 2.3.2.2. --- Sodium-potassium adenosine triphosphatase (Na+，K+-ATPase) --- p.21 / Chapter 2.3.2.3. --- Cystic fibrosis transmembrane conductance regulator (CFTR) --- p.22 / Chapter 2.3.2.4. --- Na+/K+/2Cr cotransporter (NKCC) --- p.23 / Chapter 2.3.2.5. --- Potassium (K+) channel --- p.25 / Chapter 2.4 --- Endocrine control of osmoregulation --- p.26 / Chapter 2.4.1. --- Overview --- p.26 / Chapter 2.4.2. --- Growth hormone (GH) and insulin-like growth factor I (IGF-I) --- p.27 / Chapter 2.4.2.1. --- Role of GH in osmoregulation --- p.27 / Chapter 2.4.2.2. --- Mediation through IGF-I --- p.29 / Chapter 2.4.2.3. --- Synergic effect with cortisol --- p.30 / Chapter 2.4.3. --- Prolactin (PRL) --- p.30 / Chapter 2.4.3.1. --- Role of PRL in osmoregulation --- p.30 / Chapter 2.4.3.2. --- Synergic effect with cortisol --- p.33 / Chapter 2.4.4. --- Cortisol --- p.33 / Chapter 2.4.4.1. --- Role of cortisol in osmoregulation --- p.33 / Chapter 2.4.4.2. --- Dual functions of cortisol --- p.34 / Chapter Chapter 3 --- Cloning and tissue distribution of silver sea bream CFTR gene --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and methods --- p.38 / Chapter 3.2.1. --- Part A: Cloning of silver sea bream CFTR gene --- p.38 / Chapter 3.2.1.1. --- Fish and culture conditions --- p.38 / Chapter 3.2.1.2. --- Sampling of fish --- p.38 / Chapter 3.2.1.3. --- Preparation of first strand cDNA --- p.38 / Chapter 3.2.1.4. --- Design of primers --- p.39 / Chapter 3.2.1.5. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.40 / Chapter 3.2.1.6 --- Cloning --- p.41 / Chapter 3.2.2. --- Part B: Tissue distribution of CFTR in silver sea bream --- p.41 / Chapter 3.2.2.1. --- Fish and culture conditions --- p.41 / Chapter 3.2.2.2. --- Tissue sampling --- p.42 / Chapter 3.2.2.3. --- Preparation of first strand cDNA --- p.42 / Chapter 3.2.2.4 --- Design of primers --- p.42 / Chapter 3.2.2.5. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.43 / Chapter 3.3 --- Results --- p.44 / Chapter 3.3.1. --- Part A: Cloning of silver sea bream CFTR gene --- p.44 / Chapter 3.3.2. --- Part B: Tissue distribution of CFTR in silver sea bream --- p.60 / Chapter 3.4 --- Discussion --- p.62 / Chapter 3.4.1. --- Part A: Cloning of silver sea bream CFTR --- p.62 / Chapter 3.4.2. --- Part B: Tissue distribution of CFTR in silver sea bream --- p.64 / Chapter Chapter 4 --- Effect of salinity on CFTR mRNA expression in gill and posterior intestine of silver sea bream --- p.68 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Materials and methods --- p.70 / Chapter 4.2.1. --- Part A: Effect of long-term exposure to different salinities on CFTR expression --- p.70 / Chapter 4.2.1.1. --- Experimental fish and salinity adaptation --- p.70 / Chapter 4.2.1.2. --- Tissue sampling --- p.70 / Chapter 4.2.1.3. --- Serum ion levels --- p.71 / Chapter 4.2.1.4. --- Preparation of first strand cDNA --- p.71 / Chapter 4.2.1.5. --- Design of primers --- p.71 / Chapter 4.2.1.6. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.71 / Chapter 4.2.1.7. --- Statistical analysis --- p.72 / Chapter 4.2.2. --- Part B: Effect of abrupt transfer on CFTR expression --- p.73 / Chapter 4.2.2.1. --- Experimental fish --- p.73 / Chapter 4.2.2.2. --- Experimental design --- p.73 / Chapter 4.2.2.2.1 --- Experiment 1: Abrupt transfer from seawater (SW) to 6 ppt --- p.73 / Chapter 4.2.2.2.2. --- Experiment 2: Abrupt transfer from 6 ppt to SW --- p.73 / Chapter 4.2.2.3. --- Tissue sampling --- p.74 / Chapter 4.2.2.4. --- Serum ion levels --- p.74 / Chapter 4.2.2.5. --- Preparation of first strand cDNA --- p.74 / Chapter 4.2.2.6. --- Design of primers --- p.75 / Chapter 4.2.2.7. --- Semi-quantitative reverse transcriptase (RT)-PCR --- p.75 / Chapter 4.2.2.8. --- Statistical analysis --- p.75 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1. --- Part A: Effect of long-term exposure to different salinities on CFTR expression --- p.76 / Chapter 4.3.1.1. --- Serum ion levels --- p.76 / Chapter 4.3.1.2. --- CFTR expression in gill --- p.76 / Chapter 4.3.1.3. --- CFTR expression in posterior intestine --- p.76 / Chapter 4.3.2. --- Part B: Effect of abrupt salinity transfer on CFTR expression --- p.83 / Chapter 4.3.2.1. --- Experiment 1: Abrupt transfer from SW to 6 ppt --- p.83 / Chapter 4.3.2.1.1. --- Serum ion levels --- p.83 / Chapter 4.3.2.1.2. --- CFTR in gill --- p.83 / Chapter 4.3.2.1.3. --- CFTR in posterior intestine --- p.83 / Chapter 4.3.2.2. --- Experiment 2: Abrupt transfer from 6 ppt to SW --- p.89 / Chapter 4.3.2.2.1. --- Serum ion levels --- p.89 / Chapter 4.3.2.2.2. --- CFTR in gill --- p.89 / Chapter 4.3.2.2.3. --- CFTR in posterior intestine --- p.89 / Chapter 4.4 --- Discussion --- p.95 / Chapter 4.4.1. --- Long-term exposure to various salinities --- p.95 / Chapter 4.4.2. --- Abrupt salinity transfer --- p.98 / Chapter 4.4.2.1. --- Abrupt hypo-osmotic transfer (33 ppt to 6 ppt) --- p.98 / Chapter 4.4.2.2. --- Abrupt seawater transfer (6 ppt to 33 ppt) --- p.99 / Chapter 4.4.3. --- CFTR mRNA expression in posterior intestine --- p.101 / Chapter 4.4.4. --- Conclusion --- p.101 / Chapter Chapter 5 --- Effect of hormones on CFTR expression in gill and posterior intestine of silver sea bream --- p.102 / Chapter 5.1 --- Introduction --- p.102 / Chapter 5.2 --- Materials and methods --- p.104 / Chapter 5.2.1. --- Part A: In vivo effect of hormones on CFTR expression --- p.104 / Chapter 5.2.1.1. --- Experimental fish and salinity adaptation --- p.104 / Chapter 5.2.1.2. --- Hormone treatment --- p.104 / Chapter 5.2.1.3. --- Tissue sampling --- p.105 / Chapter 5.2.1.4. --- "Serum ion levels, preparation of first strand cDNA, design of primers and semi-quantitative reverse transcriptase (RT)-PCR" --- p.105 / Chapter 5.2.1.5. --- Statistical analysis --- p.105 / Chapter 5.2.2. --- Part B: In vitro effect of hormones on CFTR expression --- p.106 / Chapter 5.2.2.1. --- Fish and culture conditions --- p.106 / Chapter 5.2.2.2. --- Gill and posterior intestine preparations --- p.106 / Chapter 5.2.2.3. --- Hormone treatment --- p.106 / Chapter 5.2.2.4. --- "Preparation of first strand cDNA, design of primers and semi-quantitative reverse transcriptase (RT)-PCR" --- p.107 / Chapter 5.2.2.5. --- Statistical analysis --- p.107 / Chapter 5.3 --- Results --- p.108 / Chapter 5.3.1. --- Part A: In vivo effect of hormones on CFTR expression --- p.108 / Chapter 5.3.1.1. --- Serum ion levels --- p.108 / Chapter 5.3.1.1.1. --- Serum [Na+] level --- p.108 / Chapter 5.3.1.1.2. --- Serum [K+] level --- p.108 / Chapter 5.3.1.1.3. --- Serum [Cl' ] level --- p.108 / Chapter 5.3.1.2. --- CFTR expression in gill --- p.109 / Chapter 5.3.1.3. --- CFTR expression in posterior intestine --- p.109 / Chapter 5.3.2. --- Part B: In vitro effect of hormones on CFTR expression --- p.115 / Chapter 5.3.2.1. --- CFTR expression in gill --- p.115 / Chapter 5.3.2.2. --- CFTR expression in posterior intestine --- p.115 / Chapter 5.4 --- Discussion --- p.122 / Chapter 5.4.1. --- Effects of cortisol on CFTR expression --- p.122 / Chapter 5.4.2. --- Effects of growth hormone on CFTR expression --- p.124 / Chapter 5.4.3. --- Effects of prolactin on CFTR expression --- p.127 / Chapter 5.4.4. --- "Overall effect of cortisol, growth hormone and prolactin on CFTR expression" --- p.128 / Chapter 5.4.5 --- Conclusion --- p.130 / Chapter Chapter 6 --- General discussion and conclusion --- p.132 / References --- p.136

Pang, Weng I. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 116-148). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xv / LIST OF ABBREVIATIONS --- p.xvi / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- CIRCULATING NUCLEIC ACIDS IN PRENATAL DIAGNOSIS --- p.2 / Chapter 1.1 --- Prenatal diagnosis --- p.2 / Chapter 1.2 --- Circulating fetal DNA in maternal plasma --- p.2 / Chapter 1.2.1 --- Biology of circulating fetal DNA --- p.2 / Chapter 1.2.2 --- Clinical applications of circulating fetal DNA --- p.3 / Chapter 1.2.2.1 --- Qualitative fetal-specific sequence detection --- p.4 / Chapter 1.2.2.2 --- Quantitative aberration detection --- p.4 / Chapter 1.2.3 --- Circulating fetal epigenetic markers --- p.5 / Chapter 1.3 --- Circulating fetal RNA in maternal plasma --- p.6 / Chapter 1.3.1 --- Biology of circulating fetal RNA --- p.6 / Chapter 1.3.2 --- Clinical applications of circulating fetal RNA --- p.8 / Chapter 1.3.2.1 --- Quantitative aberration detection --- p.8 / Chapter 1.3.2.2 --- Chromosomal aneuploidy detection --- p.9 / Chapter 1.3.3 --- Enrichment of fetal RNA --- p.10 / Chapter 1.4 --- Circulating microRNA in maternal plasma --- p.10 / Chapter CHAPTER 2: --- FETAL GROWTH AND WELL-BEING --- p.12 / Chapter 2.1 --- Normal fetal growth --- p.12 / Chapter 2.1.1 --- Role of the mother --- p.12 / Chapter 2.1.2 --- Role of the placenta --- p.12 / Chapter 2.1.3 --- Role of the fetus --- p.13 / Chapter 2.1.4 --- Role of the somatotrophic axis --- p.15 / Chapter 2.2 --- Abnormal fetal growth --- p.15 / Chapter 2.2.1 --- Intrauterine growth restriction --- p.16 / Chapter 2.1.2 --- Definition of IUGR --- p.16 / Chapter 2.2.3 --- Risk factors of IUGR --- p.17 / Chapter 2.2.4 --- Diagnosis of IUGR --- p.20 / Chapter 2.2.4.1 --- Biometric tests --- p.20 / Chapter 2.2.4.2 --- Biophysical tests --- p.21 / Chapter 2.2.4.3 --- Biochemical tests --- p.22 / Chapter 2.2.4.4 --- Others --- p.22 / Chapter 2.3 --- Limitations of current modalities in fetal growth assessment --- p.23 / Chapter 2.4 --- Aims of this thesis --- p.24 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.26 / Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF CIRCULATING RNA --- p.27 / Chapter 3.1 --- Sample collection and processing --- p.27 / Chapter 3.1.1 --- Preparation of plasm a --- p.27 / Chapter 3.1.2 --- Preparation of blood cells --- p.27 / Chapter 3.1.3 --- Preparation of placental tissues --- p.27 / Chapter 3.2 --- Total RNA extraction --- p.28 / Chapter 3.2.1 --- Plasma and blood cells --- p.28 / Chapter 3.2.2 --- Placental tissues --- p.32 / Chapter 3.3 --- Quantitative measurements of nucleic acids --- p.32 / Chapter 3.3.1 --- Principles of real-time quantitative PCR --- p.33 / Chapter 3.3.2 --- One-step QR T-PCR assays for placental mRNA quantification --- p.3 7 / Chapter 3.3.3 --- QPCR assays for checking genomic DNA contamination --- p.43 / Chapter 3.4 --- Statistical analysis --- p.45 / Chapter SECTION III: --- EVALUATION OF PLACENTA-DERIVED MRNA AS POSSIBLE MARKERS FOR FETAL GROWTH ASSESSMENT --- p.46 / Chapter CHAPTER 4: --- SELECTION OF POTENTIAL FETAL GROWTH MRNA MARKERS FOR MATERNAL PLASMA DETECTION --- p.47 / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Materials and methods --- p.49 / Chapter 4.2.1 --- Sample collection and processing --- p.49 / Chapter 4.2.2 --- Experimental design --- p.49 / Chapter 4.2.3 --- RNA extraction and quantification --- p.51 / Chapter 4.2.4 --- Statistical analysis --- p.51 / Chapter 4.3 --- Results --- p.52 / Chapter 4.3.1 --- Identification of potential fetal growth mRNA markers in maternal plasma --- p.52 / Chapter 4.3.2 --- Development of real-time QR T-PCR assays --- p.56 / Chapter 4.3.3 --- Validation of maternal plasma detectability and pregnancy-specificity --- p.58 / Chapter 4.3.4 --- Assessment of the gestational trend in maternal plasma --- p.64 / Chapter 4.4 --- Discussion --- p.68 / Chapter CHAPTER 5: --- RELATIONSHIP BETWEEN CIRCULATING PLACENTAL MRNA AND FETAL GROWTH --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and methods --- p.73 / Chapter 5.2.1 --- Sample collection and processing --- p.73 / Chapter 5.2.2 --- "Ultrasound measurement, placental weight and birth weight.…" --- p.74 / Chapter 5.2.3 --- Experimental design --- p.74 / Chapter 5.2.4 --- RNA extraction and quantification --- p.75 / Chapter 5.2.5 --- Statistical analysis --- p.75 / Chapter 5.3 --- Results --- p.75 / Chapter 5.3.1 --- Expression of potential growth markers in placental tissues --- p.76 / Chapter 5.3.2 --- Relationship between circulating placental mRNA and birth measurements --- p.76 / Chapter 5.3.3 --- Relationship between circulating placental mRNA and fetal biometric measurements --- p.77 / Chapter 5.4 --- Discussion --- p.85 / Chapter SECTION IV: --- CLINICAL APPLICATION OF POTENTIAL FETAL GROWTH MARKERS IN THE ASSESSMENT OF IUGR --- p.93 / Chapter CHAPTER 6: --- QUANTITATIVE ANALYSIS OF PLACENTAL MRNA IN IUGR WITH OR WITHOUT PET --- p.94 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Materials and methods --- p.95 / Chapter 6.2.1 --- Sample collection and processing --- p.95 / Chapter 6.2.2 --- Experimental design --- p.96 / Chapter 6.2.3 --- RNA extraction and quantification --- p.96 / Chapter 6.2.4 --- Statistical analysis --- p.97 / Chapter 6.3 --- Results --- p.97 / Chapter 6.3.1 --- Cross-sectional comparison of placental mRNA concentrations --- p.97 / Chapter 6.3.2 --- Longitudinal comparison of placental mRNA concentrations --- p.102 / Chapter 6.4 --- Discussion --- p.103 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.107 / Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.108 / Chapter 7.1 --- A strategy for identifying circulating placental MRNA markers for fetal growth assessment --- p.108 / Chapter 7.2 --- Implications of mRNA marker development strategy --- p.111 / Chapter 7.3 --- Prospects for future work --- p.112 / REFERENCES --- p.116

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 141-152). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 152-162).