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Exposures to ionising radiation resulting from natural sources or medical diagnostics are generally very low. In contrast, exposures to therapeutic radiation, although, they are often partial exposures, represent much higher doses. Similar levels of exposure may also occur as a consequence of a radiological accident, where it would be necessary to quickly separate individuals requiring urgent medical attention from the “worried-well”. The well-established biodosimetry techniques based on cytogenetics, particularly scoring dicentric chromosomes, are accurate and sensitive, yet, they are unsuitable for mass screening due to limited capacity and the time required for providing dose estimates. Measuring gene expression changes following radiation exposure was suggested over a decade ago to be an alternative method for dose estimation, as it is a quick, sensitive and suitable technique for high throughput application. The qPCR protocol was extensively optimised for increased reproducibility and sensitivity in order to be suitable for biodosimetry purposes. Radiation-responsive transcripts were identified and characterised in terms of temporal- and dose-responses. Finally, candidate transcripts were validated in human blood irradiated ex vivo and in vivo in blood samples obtained from cancer patients undergoing radiotherapy treatment. The data generated here serve as a proof of principle that qPCR-based gene expression assays can be used for radiation biodosimetry purposes to aid classical cytogenetics tools in an event of mass causality.

Thesis (M. Phil.)--Chinese University of Hong Kong. / Bibliography: leaves 158-185.

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Chum Wing Yan, Winnie. / Thesis submitted in 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 157-178). / Abstracts in English and Chinese. / English Abstract --- p.i / Chinese Abstract --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Life cycle --- p.2 / Chapter 1.3 --- Nutritional value --- p.4 / Chapter 1.4 --- Medicinal value --- p.4 / Chapter 1.4.1 --- Antitumor ability --- p.5 / Chapter 1.4.2 --- Antimicrobial ability --- p.5 / Chapter 1.4.3 --- Hypocholesterolaemic effect --- p.6 / Chapter 1.4.4 --- Anti-viral effect --- p.6 / Chapter 1.4.5 --- Anticaries effects --- p.7 / Chapter 1.6 --- Commercial value --- p.7 / Chapter 1.6.1 --- Biodecolorization --- p.7 / Chapter 1.6.2 --- Bioconversion --- p.7 / Chapter 1.6.3 --- Biodegradation --- p.8 / Chapter 1.6.4 --- Indicator --- p.9 / Chapter 1.7 --- Cultivation --- p.9 / Chapter 1.8 --- Content --- p.10 / Chapter 1.9 --- Transformation --- p.11 / Chapter 1.10 --- Gene regulation for growth and fruiting body development --- p.13 / Chapter 1.11 --- Serial Analysis of Gene Expression --- p.18 / Chapter 1.11.1 --- Introduction --- p.18 / Chapter 1.11.2 --- Principles --- p.20 / Chapter 1.11.3 --- SAGE Application in Cancer and Immunology Studies --- p.22 / Chapter 1.11.4 --- Improvement of SAGE --- p.23 / Chapter 1.11.5 --- Bioinformatics --- p.24 / Chapter 1.12 --- DNA Microarray --- p.27 / Chapter 1.12.1 --- Introduction --- p.27 / Chapter 1.12.2 --- Application --- p.28 / Chapter 1.12.3 --- Method of cDNA Microarray --- p.28 / Chapter Chapter Two --- Serial Analysis of Gene Expression / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Material and Methods --- p.32 / Chapter 2.2.1 --- Mushroom cultivation and RNA extraction --- p.32 / Chapter 2.2.2 --- RNA Quality Estimation --- p.33 / Chapter 2.2.3 --- mRNA Isolation --- p.34 / Chapter 2.2.4 --- Serial Analysis of Gene Analysis (SAGE) --- p.34 / Chapter 2.2.4.1 --- Binding mRNA to magnetic beads for cDNA synthesis --- p.34 / Chapter 2.2.4.2 --- DNA synthesis verification --- p.35 / Chapter 2.2.4.3 --- NlaIII digestion --- p.36 / Chapter 2.2.4.4 --- NlaIII digestion verification --- p.36 / Chapter 2.2.4.5 --- Adapters ligation --- p.37 / Chapter 2.2.4.6 --- Cleaving with tagging enzyme --- p.37 / Chapter 2.2.4.7 --- Ditags creation --- p.38 / Chapter 2.2.4.8 --- PCR optimization and scale-up --- p.38 / Chapter 2.2.4.9 --- Polyacrylamide gel electrophoresis --- p.39 / Chapter 2.2.4.10 --- Eluting DNA from the gel --- p.40 / Chapter 2.2.4.11 --- NlaIII Cleavage and polyacrylamide gel electrophoresis --- p.40 / Chapter 2.2.4.12 --- Concatemers Ligation --- p.41 / Chapter 2.2.4.13 --- Cloning Concatemers into pZErO®-1 --- p.42 / Chapter 2.2.5 --- One Shot® TOP 10 Electrocomp´ёØ E. Coli transformation --- p.42 / Chapter 2.2.6 --- PCR Screening and Sequencing --- p.44 / Chapter 2.2.7 --- Sequence Analysis --- p.44 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- RNA Extraction --- p.45 / Chapter 2.3.2 --- cDNA Synthesis --- p.45 / Chapter 2.3.3 --- NlaIII digestion --- p.45 / Chapter 2.3.4 --- PCR amplification --- p.46 / Chapter 2.3.5 --- Gel-purification of the 100bp Ditags --- p.46 / Chapter 2.3.6 --- Isolation of the 26bp Ditags --- p.46 / Chapter 2.3.7 --- Concatemers Generation --- p.47 / Chapter 2.3.8 --- PCR Screening --- p.47 / Chapter 2.3.9 --- The Abundance and Identity of SAGE Tags --- p.48 / Chapter 2.4 --- Discussion --- p.86 / Chapter 2.4.1 --- RNA Extraction --- p.86 / Chapter 2.4.2 --- cDNA Synthesis and NlaIII digestion --- p.86 / Chapter 2.4.3 --- PCP amplification --- p.87 / Chapter 2.4.4 --- SAGE Tags Analysis --- p.88 / Chapter Chapter Three --- Microarray / Chapter 3.1 --- Introduction --- p.96 / Chapter 3.2 --- Materials and Methods --- p.97 / Chapter 3.2.1 --- Microarray chip preparation --- p.97 / Chapter 3.3.2 --- Sample preparation --- p.97 / Chapter 3.2.3 --- cDNA Synthesis and Sample labeling --- p.98 / Chapter 3.2.4 --- cDNA Purification --- p.99 / Chapter 3.2.5 --- cDNA analysis --- p.99 / Chapter 3.2.6 --- Array Hybridization --- p.102 / Chapter 3.2.6.1 --- Sample Preparation --- p.102 / Chapter 3.2.6.2 --- Hybridization Procedure --- p.102 / Chapter 3.2.7 --- Stringency Washes --- p.103 / Chapter 3.2.8 --- Detection with TSA --- p.103 / Chapter 3.2.9 --- Scanning and Analysis --- p.105 / Chapter 3.3 --- Results --- p.109 / Chapter 3.4 --- Discussion --- p.120 / Chapter Chapter Four --- Full Length Sequencing / Chapter 4.1 --- Introduction --- p.124 / Chapter 4.2 --- Material and Methods --- p.124 / Chapter 4.3 --- Results and Discussion --- p.125 / Chapter Chapter Five --- General Discussion --- p.149 / Appendix --- p.155 / References --- p.157

Duan Meijuan. / Thesis submitted in: August 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 170-194). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Chinese abstract --- p.vi / List of Tables --- p.viii / List of Figures --- p.ix / List of Abbreviations --- p.xiv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.3 / Chapter 2.1 --- Hybrid rice: Genetics and breeding --- p.3 / Chapter 2.1.1 --- Classification in rice --- p.5 / Chapter 2.1.2 --- Heterosis in rice --- p.6 / Chapter 2.1.2.1 --- Performance of heterosis in rice --- p.6 / Chapter 2.1.2.2. --- Genetic mechanism of heterosis in rice --- p.7 / Chapter 2.1.3 --- Utilization of heterosis in rice --- p.9 / Chapter 2.2 --- Grain quality in rice --- p.11 / Chapter 2.2.1 --- Composition of rice grain quality --- p.11 / Chapter 2.2.1.1 --- Appearance quality --- p.11 / Chapter 2.2.1.2 --- Milling quality --- p.11 / Chapter 2.2.1.3 --- Nutritional quality --- p.12 / Chapter 2.2.1.4 --- Cooking and eating quality --- p.17 / Chapter 2.2.2 --- Genetic and breeding for high nutritional quality rice --- p.17 / Chapter 2.2.3 --- "Structural, physiological and biochemical changes during rice seed development" --- p.18 / Chapter 2.3 --- Molecular biological characteristics of rice seed storage protein --- p.20 / Chapter 2.3.1 --- "Property, classification and structure" --- p.20 / Chapter 2.3.1.1 --- Property and classification --- p.20 / Chapter 2.3.1.2 --- Composition structure --- p.20 / Chapter 2.3.1.2.1 --- Glutelin --- p.21 / Chapter 2.3.1.2.2 --- Prolamin --- p.22 / Chapter 2.3.1.2.3 --- Globulin and Albumin --- p.23 / Chapter 2.3.2 --- "Structure, expression and regulation of genes encoding rice seed storage protein genes" --- p.24 / Chapter 2.3.2.1 --- Structure --- p.24 / Chapter 2.3.2.1.1 --- Glutelin gene family --- p.24 / Chapter 2.3.2.1.2 --- Prolamin gene family --- p.26 / Chapter 2.3.2.1.3 --- Albumin and globulin gene family --- p.27 / Chapter 2.3.2.2 --- Expression of storage proteins in rice seed development --- p.28 / Chapter 2.3.2.3 --- Regulation of expression of seed storage protein genes --- p.29 / Chapter 2.3.2.3.1 --- Regulation at transcriptional level --- p.29 / Chapter 2.3.2.3.2 --- Regulation at post-transcriptional level --- p.31 / Chapter 2.3.2.3.3 --- Regulation at translational level --- p.31 / Chapter 2.3.3 --- "Synthesis, processing and deposition of rice seed storage proteins" --- p.32 / Chapter 2.4 --- Molecular characteristics of starch in rice grain --- p.34 / Chapter 2.4.1 --- Property of rice starch --- p.34 / Chapter 2.4.2 --- Starch biosynthesis in rice --- p.37 / Chapter 2.4.3 --- Enzymes involved in starch biosynthesis in rice --- p.39 / Chapter 2.4.3.1 --- ADP-glucose pyrophosphorylase (AGPase) --- p.39 / Chapter 2.4.3.2 --- Soluble starch synthase (SSS) --- p.41 / Chapter 2.4.3.3 --- Granular-bound starch synthase (GBSS) --- p.42 / Chapter 2.4.3.4 --- Starch branching enzyme (SBE) --- p.44 / Chapter 2.4.3.5 --- Starch debranching enzyme (SDE) --- p.46 / Chapter 2.5 --- Aspartate family amino acid biosynthetic pathway in rice --- p.48 / Chapter 2.5.1 --- Introduction --- p.48 / Chapter 2.5.2 --- Two key regulatory enzymes involved in lysine synthesis pathway --- p.50 / Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.50 / Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.51 / Chapter 2.5.2.3. --- Regulation of lysine and the other end products of AK pathway --- p.52 / Chapter 2.6 --- Proteomics in plants --- p.53 / Chapter 2.7 --- Approaches for grain quality improvement in rice --- p.56 / Chapter 2.7.1 --- Improvement of nutrition quality --- p.56 / Chapter 2.7.2 --- Improvement of eating and cooking quality --- p.57 / Chapter 2.8 --- Objectives of my project --- p.58 / Chapter Chapter 3. --- Materials and Methods --- p.60 / Chapter 3.1 --- Materials --- p.60 / Chapter 3.1.1 --- Chemicals --- p.60 / Chapter 3.1.2 --- Apparatus and commercial kits --- p.60 / Chapter 3.1.3 --- Plant materials --- p.61 / Chapter 3.1.4 --- DNA sequencing --- p.61 / Chapter 3.1.5 --- Software --- p.61 / Chapter 3.2 --- Methods --- p.62 / Chapter 3.2.1 --- Search for protein and DNA sequences of all genes --- p.62 / Chapter 3.2.1.1 --- Genes encoding rice glutelin family --- p.62 / Chapter 3.2.1.2 --- Genes encoding rice prolamin family --- p.63 / Chapter 3.2.1.3 --- Genes encoding rice albumin family --- p.63 / Chapter 3.2.1.4 --- Genes encoding rice globulin family --- p.64 / Chapter 3.2.1.5 --- Genes encoding rice starch synthesis enzymes --- p.64 / Chapter 3.2.2 --- Alignment of homologous DNA sequence between family member genes --- p.65 / Chapter 3.2.2.1 --- Seeds storage protein gene families of rice seeds --- p.65 / Chapter 3.2.2.2 --- Rice starch synthase gene families --- p.67 / Chapter 3.2.3 --- Primer design --- p.69 / Chapter 3.2.4 --- Collection of developing hybrid rice seeds --- p.71 / Chapter 3.2.5 --- Total RNA extraction --- p.75 / Chapter 3.2.6 --- Quantification of total RNA and determination of internal control --- p.75 / Chapter 3.2.7 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.77 / Chapter 3.2.8 --- Northern blot analysis --- p.78 / Chapter 3.2.9 --- DNA sequencing --- p.79 / Chapter 3.2.10 --- Protein extraction --- p.80 / Chapter 3.2.10.1 --- Extraction of four kinds of storage proteins --- p.80 / Chapter 3.2.10.2 --- Extraction of the Wx protein --- p.81 / Chapter 3.2.11 --- Tricine SDS-PAGE --- p.82 / Chapter 3.2.12 --- "Determination of crude protein and amylose content in P64S,9311 and F1 hybrid" --- p.83 / Chapter 3.2.12.1 --- Determination of crude protein --- p.83 / Chapter 3.2.12.2 --- Determination of amylose content --- p.84 / Chapter 3.2.13 --- Two-dimension gel electrophoresis --- p.85 / Chapter 3.2.13.1 --- Clean up of protein sample for 2-D gel --- p.85 / Chapter 3.2.13.2 --- Quantification of protein samples --- p.86 / Chapter 3.2.13.3 --- First-dimension IEF (isoelectric focusing) --- p.86 / Chapter 3.2.13.4 --- IPG strips equilibration --- p.87 / Chapter 3.2.13.5 --- Second-dimension SDS PAGE --- p.87 / Chapter 3.2.13.6 --- Silver staining of 2-D gel --- p.88 / Chapter 3.2.14 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.88 / Chapter 3.2.14.1 --- Sample destaining --- p.88 / Chapter 3.2.14.2 --- In-gel digestion with trypsin enzyme --- p.89 / Chapter 3.2.14.3 --- Desalination of the digested sample with Zip Tip --- p.90 / Chapter 3.2.14.4 --- Mass spectrometry --- p.90 / Chapter Chapter 4. --- Results --- p.91 / Chapter 4.1 --- Quantification of the total RNA from developing seeds at different developingstages --- p.91 / Chapter 4.2 --- Determination of internal control --- p.92 / Chapter 4.3 --- DNA sequence analysis --- p.95 / Chapter 4.4 --- Profiling the expression of genes encoding rice seed storage proteins --- p.97 / Chapter 4.4.1 --- The glutelin genes --- p.97 / Chapter 4.4.1.1 --- The Gtl (GluA-2) gene --- p.100 / Chapter 4.4.1.2 --- The Gt2 (GluA-1) gene --- p.100 / Chapter 4.4.1.3 --- The Gt3 (GluA-3) gene --- p.101 / Chapter 4.4.1.4 --- Comparison of the expression profiles of GluA subfamily genes --- p.101 / Chapter 4.4.1.5 --- The GluB-1 gene --- p.101 / Chapter 4.4.1.6 --- The GluB-2 gene --- p.102 / Chapter 4.4.1.7 --- The GluB-4 gene --- p.102 / Chapter 4.4.1.8 --- Comparing of the expression profiles of GluB subfamily genes --- p.102 / Chapter 4.4.1.9 --- Profiling the expression of glutilin family genes in developing hybrid rice seeds --- p.103 / Chapter 4.4.1.10 --- Profiling glutelin gene expression in developing seeds at protein level --- p.103 / Chapter 4.4.2 --- Profiling the expression of genes encoding prolamin familyin developing hybrid rice seeds --- p.105 / Chapter 4.4.2.1 --- The 10-kDa prolamin gene --- p.105 / Chapter 4.4.2.2 --- The RP5 gene --- p.108 / Chapter 4.4.2.3 --- The RP6 gene --- p.108 / Chapter 4.4.2.4 --- The Prol 7 gene --- p.109 / Chapter 4.4.2.5 --- The Prol 14 gene --- p.109 / Chapter 4.4.2.6 --- The Prol 17 gene --- p.109 / Chapter 4.4.2.7 --- Expression profiles of prolamin family genes --- p.110 / Chapter 4.4.2.8 --- Expression profiles of prolamin genes in developing hybrid rice seeds at protein level --- p.111 / Chapter 4.4.3 --- Profiling the expression of genes encoding globulin familyin developing hybrid rice seed --- p.113 / Chapter 4.4.3.1 --- The 26-kDa globulin (alpha-globulin) gene --- p.113 / Chapter 4.4.3.2 --- The globulin 1 gene --- p.113 / Chapter 4.4.3.3 --- The globulin 2 gene --- p.115 / Chapter 4.4.3.4 --- The Low molecular weight (LMW) globulin gene --- p.115 / Chapter 4.4.3.5 --- Profiling the expression of the globulin family genes --- p.115 / Chapter 4.4.3.6 --- Expression profiles of globulin proteins in developing hybrid rice seeds at protein level --- p.117 / Chapter 4.4.4 --- Profiling the expression of genes encoding rice albumin familyin developing hybrid rice seeds --- p.118 / Chapter 4.4.4.1 --- The RA5 gene --- p.118 / Chapter 4.4.4.2 --- The RA 14 gene --- p.119 / Chapter 4.4.4.3 --- The RA 17 gene --- p.119 / Chapter 4.4.4.4 --- Profiling the expression of the albumin family genes --- p.121 / Chapter 4.4.4.5 --- Albumin gene expression in developing hybrid rice seeds at protein level --- p.121 / Chapter 4.4.5 --- Comparison of expression pattern of all genes encoding rice seed storage proteins in developing hybrid rice seeds --- p.123 / Chapter 4.4.6 --- Profiling the total proteins in developing hybrid rice seeds --- p.126 / Chapter 4.5 --- Profiling the expression of genes encoding rice starch synthasesin developing hybrid rice seeds --- p.127 / Chapter 4.5.1 --- Rice ADP-glucose pyrophosphorylase (AGPase) genes --- p.127 / Chapter 4.5.1.1 --- The AGPase large subunit gene --- p.127 / Chapter 4.5.1.2 --- The AGPase small subunit gene --- p.127 / Chapter 4.5.2 --- "The Wx (Granule bound starch synthase, GBSS) gene" --- p.129 / Chapter 4.5.3 --- Genes encoding rice SSS (Soluble starch synthase) family --- p.132 / Chapter 4.5.3.1 --- The SSS1 gene --- p.132 / Chapter 4.5.3.2 --- The SSS II-1 gene --- p.132 / Chapter 4.5.3.3 --- The SSS II-2 gene --- p.132 / Chapter 4.5.3.4 --- The SSS II-3 gene --- p.135 / Chapter 4.5.3.5 --- The SSS III-2 gene --- p.135 / Chapter 4.5.3.6 --- The SSS IV-1 gene --- p.135 / Chapter 4.5.3.7 --- The SSS IV-2 gene --- p.135 / Chapter 4.5.3.8 --- Profiling the expression of SSS family genes --- p.136 / Chapter 4.5.4 --- Genes encoding rice starch branching enzyme (SBE) family --- p.138 / Chapter 4.5.4.1 --- The SBE-1 gene --- p.138 / Chapter 4.5.4.2 --- The SBE-3 gene --- p.138 / Chapter 4.5.4.3 --- The SBE-4 gene --- p.138 / Chapter 4.5.4.4 --- Profiling the expression of SBE family genes --- p.140 / Chapter 4.5.5 --- Genes encoding rice starch debranching enzyme (SDE) family --- p.141 / Chapter 4.5.5.1 --- The isoamylase gene --- p.141 / Chapter 4.5.5.2 --- The pullulanase gene --- p.141 / Chapter 4.5.5.3 --- Difference between isoamylose and pullulanase --- p.141 / Chapter 4.5.6 --- Comparison of the expression patterns of genes encoding the enzymes involved in starch synthesis in developing hybrid rice seeds --- p.143 / Chapter 4.6 --- Profiling the expression of genes encoding aspartate family amino acid biosynthetic pathway in rice in developing hybrid rice seeds --- p.145 / Chapter 4.6.1 --- Rice AK (aspartate kinase) gene --- p.145 / Chapter 4.6.2 --- The DHPS gene --- p.145 / Chapter 4.7 --- Two-dimension gel electrophoresis and MALDI-ToF seed proteins analysis of rice --- p.147 / Chapter Chapter 5. --- Discussion --- p.152 / Chapter 5.1 --- Super hybrid rice as experimental material and its significance --- p.152 / Chapter 5.2 --- RT-PCR and northern blotting as methods to profile gene expression --- p.153 / Chapter 5.3 --- Regulation of genes related to nutritional quality in rice --- p.155 / Chapter 5.3.1 --- Storage protein genes --- p.155 / Chapter 5.3.2 --- Lysine synthesis enzymes --- p.158 / Chapter 5.4 --- Regulation of genes related to cooking and eating quality in rice --- p.159 / Chapter 5.5 --- Heredity of genes expression in F1 hybrid --- p.161 / Chapter 5.6 --- Application of 2-D gel electrophoresis --- p.162 / Chapter 5.7 --- Future perspectives --- p.163 / Chapter Chapter 6. --- Conclusion --- p.164 / References --- p.170