Large-scale bisulfite treatment and short reads sequencing technology allows comprehensive estimation of methylation states of Cs in the genomes of different tissues, cell types, and developmental stages. Accurate characterization of DNA methylation is essential for understanding genotype phenotype association, gene and environment interaction, diseases, and cancer. The thesis work first evaluates the performance of several commonly used bisulfite short read mappers and investigates how pre-processing data might affect the performance. Aligning bisulfite short reads to a reference genome remains a challenging task. In practice, only a limited proportion of bisulfite treated DNA reads can be mapped uniquely (around 50-70%) while a significant proportion of reads (called multireads) are aligned to multiple genomic locations. The thesis outlines a strategy to improve the mapping efficiencies of the existing bisulfite short reads software by finding unique locations for multireads. Analyses of both simulated data and real hairpin bisulfite sequencing data show that our strategy can effectively assign approximately 70% of the multireads to their best locations with up to 90% accuracy, leading to a significant increase in the overall mapping efficiency. The most common and essential downstream task in DNA methylation analysis is to detect differential methylated cytosines (DMCs). Although many statistical methods have been applied to detect DMCs, inconsistency in detecting differential methylated sites among statistical tools remains. We adapt the wavelet-based functional mixed models (WFMM) to detect DMCs. Analyses of simulated Arabidopsis data show that WFMM has higher sensitivities and specificities in detecting DMCs compared to existing methods especially when methylation differences are small. Analyses of monozygotic twin data who have different pain sensitivity also show that WFMM can find more relevant DMCs related to pain sensitivity compared to methylKit. In addition, we provide a strategy to modify the default settings in both WFMM and methylKit to be more tailored to a given methylation profile, thus improving the accuracy of detecting DMCs. Population growth and climate change leave billions of people around the world living in water scarcity conditions. Therefore, utility of reclaimed water (treated wastewater) is pivotal for water sustainability. Recently, researchers discovered microbial regrowth problems in reclaimed water distribution systems (RWDs). The third part of the thesis involves: 1) identifying fundamental conditions that affect proliferation of antibiotic resistance genes (ARGs), 2) identifying the effect of water chemistry and water age on microbial regrowth, and 3) characterizing co-occurrence of ARGs and/or mobile genetics elements (MGEs), i.e., plasmids in simulated RWDs. Analyses of preliminary results from simulated RWDs show that biofilms, bulk water environment, temperature, and disinfectant types have significant influence on shaping antibiotic resistant bacteria (ARB) communities. In particular, biofilms create a favorable environment for ARGs to diversify but with lower total ARG populations. ARGs are the least diverse at 300C and the most diverse at 220C. Disinfectants reduce ARG populations as well as ARG diversity. Chloramines keep ARG populations and diversity at the lowest rate. Disinfectants work better in bulk water environment than in biofilms in terms of shaping resistome. Network analysis on assembly data is done to determine which ARG pairs are the most co-occurred. Bayesian network is more consistent with the co-occurrence network constructed from assembly data than the network based on Spearman's correlation network of ARG abundance profiles. / Ph. D.

Yes / We report the draft genome sequence of Pandoraea sp. strain E26 isolated from a former landfill site, sequenced by the Illumina MiSeq platform. This genome sequence will be useful to further understand the quorum-sensing system of this isolate. / University of Malaya High-Impact Research (HIR) grants (UM C/625/1/HIR/MOHE/CHAN/01, grant A-000001- 50001 and UM-MOHE HIR UM C/625/1/HIR/MOHE/CHAN/14/1, grant H-50001-A000027)

Analysis of gene expression has undergone a technological revolution. What was impossible 6 years ago is now routine. High-throughput DNA sequencing machines capable of generating hundreds of millions of reads allow, indeed force, a major revision toward the study of the genome’s functional output—the transcriptome. This thesis examines the history of DNA sequencing, measurement of gene expression by sequencing, isoform complexity driven by alternative splicing and mammalian piRNA precursor biogenesis. Examination of these topics is framed around development of a novel RNA-templated DNA-DNA ligation assay (SeqZip) that allows for efficient analysis of abundant, complex, and functional long RNAs. The discussion focuses on the future of transcriptome analysis, development and applications of SeqZip, and challenges presented to biomedical researchers by extremely large and rich datasets.

Baixos níveis séricos de 25(OH)D3 e 1,25(OH)2D3 (calcitriol) podem estar associados à incidência e prognóstico do câncer de mama. Além disso, vários estudos indicam que a vitamina D tenha um efeito antiproliferativo em linhagens celulares de câncer de mama expostas a concentrações supra-fisiológicas de calcitriol (1,25(OH)2D3, 100nM). A suplementação com vitamina D é indicada a mulheres pós-menopausadas para prevenção de osteoporose e observamos previamente que a suplementação de calcitirol a pacientes pós-menopausadas com câncer de mama causa redução do índice proliferativo tumoral. Entretanto, não há estudos até o momento que avaliam o efeito da vitamina D na expressão gênica global in vivo. Incluímos 31 pacientes pós-menopausadas com câncer de mama. Estas pacientes realizaram suplementação com calcitriol (0,5g/dia, dose indicada para prevenção de osteoporose) por um curto período de tempo (mediana de 32 dias). A amostra tumoral foi coletada por ocasião da biópsia (présuplementação) e da ressecção tumoral (pós-suplementação). Os perfis de expressão gênica de 16 pacientes foram analisados a partir de 100ng de RNA total no gene chip U133 Plus 2.0 Affymetrix. Observamos redução na expressão de Ki-67 após a suplementação. Dentre os genes diferencialmente expressos encontram-se EGR1, FOS, DUSP1, MMP12 e RGS1, os quais foram mais expressos em amostras pós-suplementadas. Genes modulados pela vitamina D estão associados à resposta inflamatória e à membrana. Nossos resultados indicam que a suplementação com vitamina D reduz o índice de proliferação tumoral, sendo a mesma envolvida em vias importantes na regulação da resposta inflamatória / Low 25(OH)2D3 or 1,25(OH)2D3 serum levels may be associated with breast cancer incidence and prognosis. Additionally, the antiproliferative effects of vitamin D are observed in breast cancer cell lines exposed to phamacological doses of calcitriol (1,25(OH)2D3, 100nM). Vitamin D supplementation is indicated for post-menopausal women to prevent osteoporosis and a previous study from our group observed a reduced tumor proliferative index after calcitriol supplementation on post menopausal breast cancer patients. However, there is no study that verifies the effect of vitamin D on gene expression profile in vivo so far. Thirty one post menopausal breast cancer patients were included on our analysis. They were supplemented with calcitriol after tumor biopsy (0.50g/day, indicated dose for osteoporosis prevention) for a short period of time (median 32 days). Tumor samples were collected during biopsy (before supplementation) and breast surgery (after supplementation). Gene expression profile of 16 patients was analyzed using the U133 Plus 2.0 Affymetrix Gene Chips from 100ng of total RNA. After supplementation, a reduced expression of Ki-67 was observed. Among the differentially expressed genes, EGR1, FOS, DUSP1, MMP12 and RGS1 were upregulated after calcitriol supplementation. Differentially expressed genes were involved in inflammatory response or were associated with the membrane. Our results indicate that calcitriol supplementation diminish tumor proliferation index regulating inflammatory pathways .

In the first part of this thesis, two chromosome Y specific genes ( SRYand TSPY) were chosen as the molecular targets to investigate the characteristics of fetal-specific DNA fragments in maternal plasma. By employing the touch down ligation-mediated PCR coupled with cloning and sequencing, the end property and the fragment species of fetal DNA were studied. / Noninvasive prenatal detection of fetal chromosomal aneuploidies is a much sought-after goal in fetomaternal medicine. The discovery of fetal DNA in the plasma of pregnant women has offered new opportunities for this purpose. However, the fact that fetal DNA amounts to just a minor fraction of all DNA in maternal plasma makes it challenging for locus-specific DNA assays to detect the small increase in sequences derived from a trisomic chromosome. On the other hand, although the clinical applications of plasma DNA for prenatal diagnosis are expanding rapidly, the biological properties of circulating DNA in plasma remain unclear. Recently, next-generation sequencing technologies have transformed the landscape of biomedical research through the ultra-high-throughput sequence information generated in a single run. Massively parallel sequencing allows us to study plasma DNA at an unprecedented resolution and also precisely detect fetal chromosomal aneuploidies in a locus-independent way. / Our group has demonstrated the use of massively parallel sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. In the second part of this thesis, the clinical utility of this new sequencing approach was extended to the prenatal detection of fetal trisomy 18 and 13. A region-selection method was developed to minimize the effects of GC content on the diagnostic sensitivity and precision for the prenatal diagnosis of trisomy 13. To facilitate the next-generation sequencing-based maternal plasma DNA analysis for clinical implementation, two measures, i.e., lowering the starting volume of maternal plasma and barcoding multiple maternal plasma samples, were investigated. / Taken together, the results presented in this thesis have demonstrated the clinical utility of massively parallel sequencing of maternal plasma DNA and have also provided us a better understanding of the biology of circulating DNA molecules. / The third part of this thesis focuses on the massively parallel paired-end sequencing of plasma DNA. By analyzing millions of sequenced DNA fragments, the biological properties of maternal plasma DNA were elucidated, such as the size distribution of fetal-derived and maternally-contributed DNA molecules and the potential effect of epigenetic modification on DNA fragmentation. Moreover, the plasma DNA from hematopoietic stem cell transplant patients was characterized by paired-end sequencing approach. These sequencing data not only confirmed the predominant hematopoietic origin of cell-free DNA but also revealed the size difference between hematologically-derived and other tissue-derived DNA molecules in plasma. / Zheng, Wenli. / Adviser: Lo Yu Ming Dennis. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 261-275). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Shuk-kei Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 141-148). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.vi / 論文摘要 --- p.viii / Abbreviations --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- HBV and its role in hepatocarcinogenesis --- p.3 / Chapter 1.2.1 --- Current situation of HBV infection and the HCC incidencein the world --- p.3 / Chapter 1.2.2 --- Current situation of HBV infection and the HCC incidencein Hong Kong --- p.4 / Chapter 1.2.3 --- Genetic organization of HBV --- p.4 / Chapter 1.2.4 --- Principle of hepatocarcinogenesis induced by HBV --- p.5 / Chapter 1.2.4.1 --- Role of chronic hepatitis in hepatocarcinogenesis --- p.5 / Chapter 1.2.4.2 --- Role of HBV in hepatocarcinogenesis --- p.6 / Chapter 1.2.5 --- Current screening tests for HCC --- p.7 / Chapter 1.2.6 --- Current therapies for HCC --- p.9 / Chapter 1.3 --- Aim of the present study --- p.13 / Chapter 1.4 --- "Combining Expressed Sequence Tag (EST), Suppression Subtractive Hybridization and cDNA microarray for rapid differentially by expressed genes screening" --- p.14 / Chapter 1.4.1 --- Expressed Sequence Tag (EST) --- p.14 / Chapter 1.4.2 --- cDNA subtraction --- p.15 / Chapter 1.4.3 --- cDNA microarray --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- PCR-select cDNA subtraction --- p.17 / Chapter 2.1.1 --- Amplification of subtracted cDNA clones by PCR --- p.17 / Chapter 2.1.2 --- Cycle sequencing of subtracted cDNA clones --- p.18 / Chapter 2.1.3 --- Sequence analysis using BLAST server and Stanford Online Universal Resource for Clones and ESTs (SOURCE) --- p.19 / Chapter 2.2 --- cDNA microarray analysis --- p.20 / Chapter 2.2.1 --- Array fabrication --- p.20 / Chapter 2.2.1.1 --- Amplification of cDNA clones by PCR --- p.20 / Chapter 2.2.1.2 --- Purification of PCR products --- p.21 / Chapter 2.2.1.3 --- Cycle sequencing for clones checking --- p.22 / Chapter 2.2.2 --- Microarray printing --- p.22 / Chapter 2.2.2.1 --- Preparation of cDNA target --- p.22 / Chapter 2.2.2.2 --- Arraying --- p.22 / Chapter 2.2.3 --- Screening of differentially expressed genes in hepatocellular carcinoma and its surrounding normal counterpart by cDNA microarray --- p.23 / Chapter 2.2.3.1 --- Extraction of RNA --- p.23 / Chapter 2.2.3.2 --- RNA labeling --- p.24 / Chapter 2.2.3.3 --- Microarray hybridization --- p.26 / Chapter 2.2.3.4 --- Collection of data --- p.27 / Chapter 2.2.3.5 --- Data normalization and analysis --- p.28 / Chapter 2.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.30 / Chapter 2.3.1 --- Tissue distribution of T2L522 gene --- p.30 / Chapter 2.3.1.1 --- Northern hybridization --- p.30 / Chapter 2.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.2 --- Expression level of T2L522 in HCC and its surrounding normal counterpart --- p.33 / Chapter 2.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.35 / Chapter 2.3.3.1 --- "Cloning of T2L522 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.35 / Chapter 2.3.3.2 --- Transformation of yeast competent cells --- p.39 / Chapter 2.3.3.3 --- Mating of T2L522-BD with pretransformed human liver cDNA library --- p.40 / Chapter 2.3.3.4 --- Colony lift p-galactosidase filter assay --- p.42 / Chapter 2.3.4 --- Subcellular localization of T2L522 gene by tagging with green fluorescence protein (GFP) --- p.43 / Chapter 2.3.4.1 --- "Cloning of T2L522 gene into the eukaryotic GFP expression vector, pEGFP-Cl" --- p.43 / Chapter 2.3.4.2 --- Transfection of pEGFP-T2L522 into HepG2 cell --- p.43 / Chapter Chapter 3 --- Results / Chapter 3.1 --- PCR-select cDNA subtraction --- p.45 / Chapter 3.1.1 --- The sequencing results of subtracted-HCC cDNA clones --- p.45 / Chapter 3.1.2 --- Categorization of ESTs sequenced from subtracted-HCC library --- p.45 / Chapter 3.2 --- Microarray analysis --- p.49 / Chapter 3.2.1 --- Array fabrication --- p.49 / Chapter 3.2.1.1 --- Amplification of cDNA microarray targets --- p.49 / Chapter 3.2.2 --- Microarray printing --- p.52 / Chapter 3.2.3 --- Microarray analysis of differentially expressed genesin hepatocellular carcinoma and its surrounding normal counterpart --- p.55 / Chapter 3.2.4 --- Data collection --- p.57 / Chapter 3.2.5 --- Image processing: spots finding and quantitation --- p.61 / Chapter 3.2.6 --- Data normalization and analysis --- p.61 / Chapter 3.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.73 / Chapter 3.3.1 --- Tissue distribution of T2L522 --- p.77 / Chapter 3.3.1.1 --- Northern hybridization --- p.77 / Chapter 3.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.79 / Chapter 3.3.2 --- Expression level of T2L522 in hepatocellular carcinoma and its surrounding normal counterpart --- p.81 / Chapter 3.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.85 / Chapter 3.3.4 --- Subcellular localization of GFP tagged T2L522 --- p.87 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- EST analysis on subtracted-HCC cDNA library --- p.89 / Chapter 4.2 --- cDNA microarray analysis --- p.92 / Chapter 4.2.1 --- Generation of reliable data using cDNA microarray --- p.92 / Chapter 4.2.1.1 --- Reproducibility of signal and normalized ratio --- p.92 / Chapter 4.2.2 --- Comparison of data between multiple slides --- p.96 / Chapter 4.2.2.1 --- Assession of data quality and statistical significance --- p.96 / Chapter 4.2.2.2 --- Interpretation of gene expression data from single and multiple hybridizarion --- p.97 / Chapter 4.3 --- Candidate genes differentially expressed in HCC and its surrounding normal counterpart --- p.99 / Chapter 4.3.1 --- Protein up-regulated in HCC --- p.99 / Chapter 4.3.1.1 --- Extracellular matrix protein --- p.99 / Chapter 4.3.1.2 --- Protein involved in other metabolism --- p.100 / Chapter 4.3.1.3 --- Protein involved in transcription and translation --- p.100 / Chapter 4.3.2 --- Protein down-regulated in HCC --- p.101 / Chapter 4.3.2.1 --- Membrane associated protein --- p.101 / Chapter 4.3.2.2 --- Protein involved in other metabolism --- p.102 / Chapter 4.3.2.2 --- Secretory protein --- p.104 / Chapter 4.3.3 --- Novel protein differentially expressed in HCC --- p.107 / Chapter 4.4 --- "TBC1 domain containing protein, T2L522" --- p.108 / Chapter 4.4.1 --- Possible involvement of T2L522 gene in HCC --- p.109 / Chapter 4.4.2 --- Tissue distribution and expression pattern of T2L522 --- p.110 / Chapter 4.4.3 --- Potential interacting partner of T2L522 --- p.110 / Chapter 4.4.4 --- Subcellular localization of T2L522 --- p.112 / Chapter 4.5 --- Summary --- p.113 / Appendix --- p.114 / References --- p.141

Lau, Wai Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 141-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Le phénomène de massification des données de santé constitue une opportunité de répondre aux questions des vigilances et de qualité des soins. Dans les travaux effectués au cours de cette thèse, nous présenterons des approches permettant d’exploiter la richesse et le volume des données intra hospitalières pour des cas d’usage de pharmacovigilance et de surveillance de bon usage du médicament. Cette approche reposera sur la modélisation de trajectoires de soins intra hospitalières adaptées aux besoins spécifiques de la pharmacovigilance. Il s’agira, à partir des données d’un entrepôt hospitalier de caractériser les événements d’intérêt et d’identifier un lien entre l’administration de ces produits de santé et l’apparition des effets indésirables, ou encore de rechercher les cas de mésusage du médicament. L’hypothèse posée dans cette thèse est qu’une approche visuelle interactive serait adaptée pour l’exploitation de ces données biomédicales hétérogènes et multi-domaines dans le champ de la pharmacovigilance. Nous avons développé deux prototypes permettant la visualisation et l’analyse des trajectoires de soins. Le premier prototype est un outil de visualisation du dossier patient sous forme de frise chronologique. La deuxième application est un outil de visualisation et fouille d’une cohorte de séquences d’événements. Ce dernier outil repose sur la mise en œuvre d’algorithme d’analyse de séquences (Smith-Waterman, Apriori, GSP) pour la recherche de similarité ou de motifs d’événements récurrents. Ces interfaces homme-machine ont fait l’objet d’études d’utilisabilité sur des cas d’usage tirées de la pratique réelle qui ont prouvé leur potentiel pour un usage en routine. / The massification of health data is an opportunity to answer questions about vigilance and quality of care. The emergence of big data in health is an opportunity to answer questions about vigilance and quality of care. In this thesis work, we will present approaches to exploit the diversity and volume of intra-hospital data for pharmacovigilance use and monitoring the proper use of drugs. This approach will be based on the modelling of intra-hospital care trajectories adapted to the specific needs of pharmacovigilance. Using data from a hospital warehouse, it will be necessary to characterize events of interest and identify a link between the administration of these health products and the occurrence of adverse reactions, or to look for cases of misuse of the drug. The hypothesis put forward in this thesis is that an interactive visual approach would be suitable for the exploitation of these heterogeneous and multi-domain biomedical data in the field of pharmacovigilance. We have developed two prototypes allowing the visualization and analysis of care trajectories. The first prototype is a tool for visualizing the patient file in the form of a timeline. The second application is a tool for visualizing and searching a cohort of event sequences The latter tool is based on the implementation of sequence analysis algorithms (Smith-Waterman, Apriori, GSP) for the search for similarity or patterns of recurring events. These human-machine interfaces have been the subject of usability studies on use cases from actual practice that have proven their potential for routine use.

Coronaviruses are a diverse group of large, single-stranded RNA virus that cause respiratory and enteric diseases in mammalian and avian species. Phylogenetic analysis shows that SARS-CoV is an unique branch of coronavirus showing no close relationship to other groups of coronaviruses. The genome size of SARS-CoV is about 30 kilobase and the genome, like other coronaviruses, is composed of replicase (rep), spike (S), envelope (E), membrane (M) and nucleocapsid (N) and 8 additional unknown open reading frames (ORFs) (ORF 3a, ORF 3b, ORF 6, ORF 7a, ORF 7b, ORF8a, ORF 8b and ORF 9b). The 3a gene, the largest unknown ORF, encodes a viral protein which is predicted to be a transmembrane protein. In this study, we showed that the 3a protein was expressed in SARS patients' lung and intestinal tissues, and it is localized to the endoplasmic reticulum (ER) in 3a-transfected monkey kidney Vero E6 cells. Results from experiments including chromatin condensation, DNA fragmentation and antibody microarray suggest that the 3a protein may trigger apoptosis through a caspase-8-dependent pathway and possibly a PKR-mediated FADD-caspase-8 pathway. Our data show that over-expression of the SARS-CoV protein can induce apoptosis in vitro. / Severe acute respiratory syndrome (SARS), an atypical form of pneumonia, is first recognized in Guangdong Province, China in November 2002 and later spread to Hong Kong in mid February 2003. It is believed that the etiological agent of SARS is a previously unknown coronavirus - SARS-CoV. Over 8,400 cases and 789 deaths were reported to World Health Organization (WHO) from over 28 countries around the world including Hong Kong. Up to now, there are still no efficient antiviral drugs to treat the disease, and the detailed pathology of SARS-CoV infection and the host response to the viral infection are still unknown. During the epidemic, we have done complete genome sequencing for five SARS-CoV isolates and we postulate that at least two SARS-CoV strains with distinct etiological origins exist in the environment during the epidemic. / Law Tit-wan Patrick. / "Aug 2005." / Adviser: Stephen K. W. Tsui. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 156-172). / 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.

by Peter, Wei Gong Chang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 103-124). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Human genome project --- p.5 / Chapter 1.3 --- Human adult heart cDNA library --- p.7 / Chapter 1.4 --- Human fetal heart cDNA library --- p.8 / Chapter 1.5 --- Sequencing of a human heart cDNA clone --- p.9 / Chapter 1.6 --- Knowledge of the role of selenium --- p.13 / Chapter 1.7 --- Mouse 56kDa selenium binding protein and acetaminophen-binding protein --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Plating out the cDNA library --- p.20 / Chapter 2.1.1 --- "Mediums, buffers and solutions" --- p.20 / Chapter 2.1.2 --- Bacteriophage clones preparation --- p.21 / Chapter 2.2 --- cDNA clone amplification by PCR --- p.23 / Chapter 2.3 --- Cycle sequencing of PCR products --- p.25 / Chapter 2.3.1 --- "Media, buffers and solutions" --- p.25 / Chapter 2.3.2 --- Preparation of sequencing reaction --- p.25 / Chapter 2.4 --- Gel electrophoresis using an automated A.L.F sequencer --- p.27 / Chapter 2.5 --- DNA sequence analysis --- p.29 / Chapter 2.6 --- Preparation of competent E. coli for transformation --- p.30 / Chapter 2.7 --- Transformation of plasmid into competent E. coll --- p.31 / Chapter 2.8 --- Mini-preparation of plasmid DNA --- p.32 / Chapter 2.9 --- Large scale plasmid DNA preparation by QIAGEN --- p.34 / Chapter 2.10 --- Cloning the human 56 kDa selenium binding protein (hSP56) into the pAED4 vector --- p.36 / Chapter 2.10.1 --- Bacterial strains and vector --- p.36 / Chapter 2.10.2 --- "Media, buffers and solutions" --- p.38 / Chapter 2.10.3 --- Primers design and PCR --- p.42 / Chapter 2.10.4 --- Purification of PCR products by Geneclean --- p.43 / Chapter 2.10.5 --- Restriction digestion of purified PCR product and pAED4 --- p.44 / Chapter 2.10.6 --- Ligation and transformation of hSP56 --- p.45 / Chapter 2.10.7 --- Screening and purification ofpAED4hSP56. --- p.47 / Chapter 2.11 --- Expression of hsp56 --- p.50 / Chapter 2.11.1 --- Induction of hSP56 expression --- p.50 / Chapter 2.11.2 --- SDS-PAGE and protein detection --- p.51 / Chapter 2.12 --- Northern hybriddization of hSP56 --- p.53 / Chapter 2.12.1 --- Animals & human tissue --- p.53 / Chapter 2.12.2 --- "Mediums, buffers and solutions" --- p.53 / Chapter 2.12.3 --- Preparation of total RNA --- p.56 / Chapter 2.12.4 --- Formaldehyde agarose gel electrophoresis --- p.57 / Chapter 2.12.5 --- Preparation of radioactive probe --- p.58 / Chapter 2.12.6 --- RNA transfer and Northern hybridization --- p.59 / Chapter 2.13 --- Chromosomal mapping of the hSP56 gene --- p.62 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- The sequencing results of 553 cDNA clones --- p.63 / Chapter 3.2 --- Catalogues of genes expressed --- p.65 / Chapter 3.3 --- Sequence analysis of hSP56 --- p.71 / Chapter 3.4 --- Northern hybridization of hSP56 --- p.84 / Chapter 3.5 --- Cloning of hSP56 into pAED4 --- p.87 / Chapter 3.6 --- Expression of the hSP56 in E. coli --- p.89 / Chapter 3.7 --- Chromosomal mapping of the hSP56 gene --- p.92 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- General discussion --- p.94 / Chapter 4.2 --- The possible roles of hSP56 and mSP56 --- p.101 / Chapter 4.3 --- Future prospects --- p.102 / REFERENCES --- p.103 / APPENDIX 1 --- p.125 / APPENDIX.2 --- p.127