by Yung, Kim Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 135-153). / Abstract --- p.i / Acknowledgments --- p.iv / List of content --- p.v / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Neural crest cells and cardiac neural crest cells --- p.1 / Chapter 1.2 --- The role of cardiac neural crest cells in the septation of the outflow tract --- p.5 / Chapter 1.3 --- Neural crest-related malformations --- p.8 / Chapter 1.4 --- Early changes in cardiovascular development induced by neural crest ablation --- p.11 / Chapter 1.5 --- Experimental strategies commonly employed in tracing the premigratory neural crest cells --- p.14 / Chapter 1.6 --- Objectives of the present study --- p.21 / Chapter Chapter 2 --- Location of the cardiac neural crest along the neural axis in the mouse embryo --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.29 / Chapter 2.2.1 --- Preparation of DiI --- p.29 / Chapter 2.2.2 --- Embryo collection --- p.29 / Chapter 2.2.3 --- Microinjection of DiI --- p.30 / Chapter 2.2.4 --- Isolation of tissue fragments from the lateral neural epithelium --- p.31 / Chapter 2.2.5 --- Dil labelling of the donor fragment isolated from the lateral neural epithelium --- p.32 / Chapter 2.2.6 --- Grafting of DiI labelled fragments from the lateral neural epithelium --- p.32 / Chapter 2.2.7 --- Embryo culture --- p.33 / Chapter 2.2.8 --- Examination of cultured embryos --- p.34 / Chapter 2.2.9 --- Cryosection --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Development of the cultured embryos in control and experimental groups --- p.36 / Chapter 2.3.2 --- Location of the cardiac neural crest region along the neural axis --- p.38 / Chapter 2.4 --- Discussion --- p.44 / Chapter 2.4.1 --- Development of embryos in vitro --- p.44 / Chapter 2.4.2 --- Comparison of the two methods for tracing cell migration: focal labelling and orthotopic grafting --- p.49 / Chapter 2.4.3 --- Location of the cardiac neural crest region along the neural tube --- p.53 / Chapter Chapter 3 --- Initial and terminal stages of cardiac neural crest cell migration --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Examination of the initial and terminal stages of migration of cardiac neural crest cells by haematoxylin and eosin (H&E) staining --- p.62 / Chapter 3.2.2 --- Preparation of WGA-Au --- p.62 / Chapter 3.2.3 --- Collection of embryos for microinjection of WGA-Au --- p.63 / Chapter 3.2.4 --- WGA-Au labelling of the presumptive cardiac neural crest region --- p.64 / Chapter 3.2.5 --- Embryo culture --- p.65 / Chapter 3.2.6 --- Examination of cultured embryos --- p.66 / Chapter 3.2.7 --- Silver enhancement staining --- p.66 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Initial stage of cardiac neural crest migration studied by haematoxylin and eosin staining and silver enhancement staining --- p.67 / Chapter 3.3.2 --- Terminal stage of cardiac neural crest migration studied by haematoxylin and eosin staining and silver enhancement staining --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Wheat germ agglutinin-gold conjugate (WGA-Au) as a cell marker --- p.71 / Chapter 3.4.2 --- Initial stage for cardiac neural crest cell migration --- p.72 / Chapter 3.4.3 --- Terminal stage for cardiac neural crest cell migration --- p.74 / Chapter Chapter 4 --- Migration pathways of cardiac neural crest cells… --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and Methods --- p.82 / Chapter 4.2.1 --- Preparation of DiI --- p.82 / Chapter 4.2.2 --- Preparation of WGA-Au --- p.82 / Chapter 4.2.3 --- Embryo collection --- p.82 / Chapter 4.2.4 --- Microinjection of WGA-Au and DiI --- p.82 / Chapter 4.2.5 --- Isolation of tissue fragments from the lateral neural epithelium --- p.83 / Chapter 4.2.6 --- WGA-Au labelling of the donor fragments from the lateral neural epithelium --- p.83 / Chapter 4.2.7 --- DiI labelling of the donor neural epithelium --- p.83 / Chapter 4.2.8 --- Grafting of WGA-Au or DiI-labelled donor tissues from the lateral neural epithelium --- p.83 / Chapter 4.2.9 --- Coating of latex beads by WGA-Au --- p.83 / Chapter 4.2.10 --- Microinjection of WGA-Au-coated latex beads --- p.84 / Chapter 4.2.11 --- Embryo culture --- p.84 / Chapter 4.2.12 --- Examination of cultured embryos --- p.85 / Chapter 4.2.13 --- Silver enhancement staining of the WGA-Au labelled sections --- p.85 / Chapter 4.2.14 --- Cryosection --- p.85 / Chapter 4.3 --- Results --- p.86 / Chapter 4.3.1 --- Distribution of labelled cells after WGA-Au labelling or orthotopic grafting --- p.86 / Chapter 4.3.2 --- Distribution of labelled cells after DiI labelling or orthotopic grafting --- p.88 / Chapter 4.3.3 --- Distribution of latex beads --- p.90 / Chapter 4.4 --- Discussion --- p.92 / Chapter 4.4.1 --- Methodology --- p.92 / Chapter 4.4.2 --- Migration pathways of the cardiac neural crest cells --- p.94 / Chapter 4.4.3 --- Migration of latex beads --- p.98 / Chapter Chapter 5 --- Derivatives of cardiac neural crest cells in the developing mouse heart --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Materials and Methods --- p.110 / Chapter 5.2.1 --- DiI labelling of the cardiac neural crest region of the mouse embryo --- p.110 / Chapter 5.2.2 --- Collection of the embryonic hearts --- p.111 / Chapter 5.2.3 --- Heart organ culture --- p.111 / Chapter 5.2.4 --- Cryosectioning --- p.112 / Chapter 5.2.5 --- Paraffin wax sectioning --- p.113 / Chapter 5.2.6 --- Immunohistochemical staining --- p.113 / Chapter 5.3 --- Results --- p.118 / Chapter 5.3.1 --- Distribution of 2H3 positive cells in the heart developedin vivo --- p.118 / Chapter 5.3.2 --- Development of the heart at 10.5 d.p.c. in organ culture --- p.119 / Chapter 5.3.3 --- Distribution of DiI labelled cells in the heart one day after organ culture --- p.119 / Chapter 5.3.4 --- Distribution of 2H3 positive cells in the hearts one day after organ culture --- p.120 / Chapter 5.4 --- Discussion --- p.121 / Chapter 5.4.1 --- Relationship between 2H3 positive cells and cardiac conduction system --- p.121 / Chapter 5.4.2 --- Development of the mouse embryonic hearts in vitro --- p.123 / Chapter 5.4.3 --- Distribution patterns of the 2H3 immunopositive cellsin the hearts developed in vitro and in vivo --- p.125 / Chapter 5.4.4 --- Relationship between the DiI labelled cells and2H3 immunopositive cells --- p.125 / Chapter 5.4.5 --- Genes that express in the cardiac neural crest cells --- p.127 / Chapter Chapter 6 --- Conclusion --- p.129 / References --- p.135 / Appendix --- p.154

by Chiu Pui Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 86-118). / Abstracts in English and Chinese. / Title Page --- p.i / Abstract --- p.ii-iv / Acknowledgement --- p.v / Table of Contents --- p.vi-viii / List of Tables --- p.ix / List of Figures --- p.x-xii / List of Abbreviations --- p.xiii / Chapter Section I: --- Introduction / Chapter Chapter 1: --- Overview --- p.1-2 / Chapter Chapter 2: --- Teratogenic Effects of Hyperglycemia / Chapter 2.1 --- What is Hyperglycemia --- p.3 / Chapter 2.2 --- Teratogenic Effects of Hyperglycemia --- p.4-6 / Chapter 2.2.1 --- Human Studies / Chapter 2.2.2 --- Animal Studies / Chapter 2.3 --- Timetables for Embryogenesis: Rats versus Humans --- p.7 / Chapter 2.4 --- Mechanisms of Hyperglycemia Induced Teratogenesis --- p.8-12 / Chapter 2.4.1 --- What are Free Radicals? / Chapter 2.4.2 --- Major Free Radical Species Involvedin Hyperglycemic Teratogenesis / Chapter 2.4.3 --- Molecular Damage Induced by Reactive Oxygen Species / Chapter 2.4.4 --- Supporting Evidence of Reactive Oxygen Species Causing Anomalies / Chapter 2.4.5. --- Hyperglycemia and Formation of Free Radicals / Chapter Chapter 3: --- Caffeine as Teratogen and Antioxidant / Chapter 3.1 --- Popularity of Caffeine --- p.13 / Chapter 3.2 --- Basic Metabolism of Caffeine --- p.14 / Chapter 3.3 --- Biological Actions of Caffeine --- p.15 / Chapter 3.4 --- Teratogenicity of Caffeine --- p.16-20 / Chapter 3.4.1 --- Animal Studies / Chapter 3.4.1.1 --- Teratogenic Effects of Caffeine in Animals / Chapter 3.4.1.2 --- Teratogenic Dose of Caffeine / Chapter 3.4.1.3 --- Interspecies Sensitivity / Chapter 3.4.2 --- Human Studies / Chapter 3.5 --- Possible Mechanisms for the Teratogenic Actions of Caffeine --- p.21 / Chapter 3.6 --- Caffeine as an Antioxidant --- p.22 / Chapter 3.7 --- Combined Effects of Caffeine with Other Substances --- p.23 / Chapter Chapter 4: --- Combined Effects of Hyperglycemia and Caffeine on Early Embryogenesis- A Question to be Answered / Chapter 4.1 --- Possible Links between Hyperglycemia and Caffeine --- p.24 / Chapter 4.2 --- Objectives of the Present Study --- p.25 / Chapter 4.3 --- Hypothesis --- p.26 / Chapter Section II: --- Research Designs and Methods / Chapter Chapter 5: --- Materials and Methods / Chapter 5.1 --- Licenses --- p.27 / Chapter 5.2 --- Overall Study Design --- p.28-40 / Chapter 5.2.1 --- Whole Embryo Culture Model / Chapter 5.2.1.1 --- Animals / Chapter 5.2.1.2 --- Explantation of Embryos and Serum Collection / Chapter 5.2.1.3 --- Preparation of Serum / Chapter 5.2.1.4 --- Culture Media / Chapter 5.2.1.5 --- Embryo Culture / Chapter 5.2.2 --- Experimental Groups / Chapter 5.2.3 --- Morphological Assessment / Chapter 5.2.4 --- Quantitation of Oxidative Stress / Chapter 5.2.5 --- Protein Assay / Chapter 5.3 --- Statistical Evaluation --- p.41 / Chapter Chapter 6: --- Laboratory Considerations / Chapter 6.1 --- Whole Embryo Culture Model --- p.42-43 / Chapter 6.1.1 --- Subjects / Chapter 6.1.2 --- Time Mating / Chapter 6.1.3 --- Culture Medium / Chapter 6.1.4 --- Gas Phase and Rotating Bottle Culture Method / Chapter 6.2 --- Quantification of Oxidative Stress --- p.47-49 / Chapter 6.2.1 --- 8-Isoprostaglandins F2a as a Marker / Chapter 6.2.2 --- Assay for 8-Isoprostaglandins F2a / Chapter 6.2.2.1 --- Enzyme Immunoassay versus Gas Chromatography/ Mass Spectrometry / Chapter Section III: --- Results / Chapter Chapter 7: --- Results / Chapter 7.1 --- Justifications of Methods of Statistical Analysis --- p.50 / Chapter 7.2 --- Effects of Hyperglycemia on Early Embryogenesis --- p.51-56 / Chapter 7.2.1 --- Effects of Hyperglycemia on Morphological Development / Chapter 7.2.2 --- Effects of Hyperglycemia on Production of 8-isoprostaglandins F2a / Chapter 7.2.3 --- Effects of Hyperglycemia on Total Protein Content / Chapter 7.3 --- Effects of Caffeine on Early Embryogenesis --- p.57-61 / Chapter 7.3.1 --- Effects of Caffeine on Morphological Development / Chapter 7.3.2 --- Effects of Caffeine on Total Protein Content / Chapter 7.4 --- Combined Effects of Hyperglycemia and Caffeine on Early Embryogenesis --- p.62-66 / Chapter 7.4.1 --- Combined Effects of Hyperglycemia and Caffeine on Morphological Development / Chapter 7.4.2 --- Combined Effects of Hyperglycemia and Caffeine on Production of 8-isoprostaglandins F2a / Chapter 7.4.3 --- Combined Effects of Hyperglycemia and Caffeine on Total Protein Content / Chapter Section IV: --- Discussion and Conclusions / Chapter Chapter 8: --- Discussion --- p.67-83 / Chapter Chapter 9: --- Conclusions and Future Directions --- p.84 / Appendices --- p.85 / References --- p.86-118

Typescript, etc. / Digitized by Kansas Correctional Industries

In 1897 Davenport measured the wet and dry weights of amphibian embryos from the stage of hatching onwards. He observed that there was a continuous increase in the wet weight but that the dry weight remained constant until the embryo began feeding. From this he concluded that "growth is due chiefly to imbibed water". Schaper (1902) noted a similar constancy of dry weight from the early tail bud stages until the time of feeding in embryos of Rana fusca. These early observations have been confirmed by Dempster (1933) who, working with Amblystoma punctatwn, extended his experiments to include the earliest developmental stages. The increase in volume, and hence the growth, of amphibian embryos is therefore due to the uptake of water from the environment. Many embryologists have attempted to correlate this water uptake with the osmotic pressure of the embryos. The early work in this field has been extensively reviewed by Needham (1931).

Leung, Chiu Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 219-235). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / 摘要 --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xiv / LIST OF TABLES --- p.xviii / LIST OF ABBREVIATIONS --- p.xix / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- Introduction to carotenoids --- p.5 / Chapter 2.1.1 --- Structures and general chemical properties --- p.6 / Chapter 2.1.2 --- Dietary source of carotenoids --- p.8 / Chapter 2.2 --- Biosynthesis of carotenoids in plants --- p.8 / Chapter 2.2.1 --- Formation of isopretenyl diphosphate (IPP) --- p.12 / Chapter 2.2.2 --- "Formation of C40 backbone, phytoene" --- p.13 / Chapter 2.2.3 --- Desaturation reactions --- p.16 / Chapter 2.2.4 --- Isomerization --- p.18 / Chapter 2.2.5 --- Cyclization --- p.18 / Chapter 2.2.6 --- Xanthophylls synthesis --- p.19 / Chapter 2.2.6.1 --- Hydroxylation --- p.19 / Chapter 2.2.6.2 --- Epoxidation and de-epoxidation --- p.19 / Chapter 2.2.6.3 --- Neoxanthin formation --- p.20 / Chapter 2.2.7 --- Carotenoids catabolism by cleavage enzymes --- p.20 / Chapter 2.2.8 --- Carotenoids sequestration --- p.20 / Chapter 2.2.9 --- Regulations of Carotenogenesis in plant --- p.21 / Chapter 2.3 --- Roles of carotenoids in plants --- p.24 / Chapter 2.3.1 --- Precursor of abscisic acid (ABA) production --- p.24 / Chapter 2.3.2 --- Photomorphogenesis: Prolamella body formation --- p.27 / Chapter 2.3.3 --- "Light-harvesting, energy transfer and photoprotection" --- p.30 / Chapter 2.4 --- Importance of carotenoids to human --- p.34 / Chapter 2.4.1 --- Provitamin A activity and conversion --- p.34 / Chapter 2.4.2 --- Roles of vitamin A and carotenoids in diseases prevention --- p.36 / Chapter 2.4.2.1 --- Visual cycle and related diseases --- p.36 / Chapter 2.4.2.2 --- Cardiovascular diseases --- p.37 / Chapter 2.4.2.3 --- Cancer --- p.38 / Chapter 2.4.3 --- Roles of vitamin A in gene regulation --- p.39 / Chapter 2.4.4 --- Bioavailability and daily intake recommendation --- p.39 / Chapter 2.5 --- Vitamin A deficiency (VAD) --- p.42 / Chapter 2.5.1 --- Clinicopathological features --- p.44 / Chapter 2.5.1.1 --- Visual problems --- p.44 / Chapter 2.5.1.2 --- Infection --- p.45 / Chapter 2.6 --- Global efforts in combating VAD --- p.45 / Chapter 2.6.1 --- Dietary diversification --- p.46 / Chapter 2.6.2 --- Supplementation --- p.48 / Chapter 2.6.3 --- Pro-vitamin A enriched crops by genetical engineering --- p.49 / Chapter 2.6.3.1 --- Tomato --- p.49 / Chapter 2.6.3.2 --- Potato --- p.50 / Chapter 2.6.3.3 --- Canola --- p.50 / Chapter 2.6.3.4 --- The Golden Rice (GR) project --- p.51 / Chapter 2.6.3.4.1 --- The 1st generation (GR1) --- p.52 / Chapter 2.6.3.4.2 --- The 2nd generation (GR2) --- p.54 / Chapter 2.7 --- Rice phytoene synthase as a GR candidate enzyme --- p.55 / Chapter 2.7.1 --- General properties of phytoene synthase in higher plant --- p.55 / Chapter 2.7.1.1 --- Gene duplication and structure --- p.55 / Chapter 2.7.1.2 --- Membrane association and cation requirement --- p.57 / Chapter 2.7.1.3 --- Expression pattern and tissue-specificity --- p.58 / Chapter 2.7.2 --- "Rice phytoene synthases: Ospsy1, Ospsy2 and Ospsy3" --- p.60 / Chapter 2.7.2.1 --- Gene duplication and structure --- p.60 / Chapter 2.7.2.2 --- Protein structures and phylogenetic analysis --- p.60 / Chapter 2.7.2.3 --- Expression pattern --- p.62 / Chapter 2.7.2.4 --- Light- and stress-induced expression --- p.63 / Chapter 2.7.3 --- Rice phytoene synthases activity in rice seeds --- p.64 / Chapter 2.7.3.1 --- Previous study in rice seed carotenogenic capacity --- p.64 / Chapter 2.8 --- Seed-specific rice promoters --- p.66 / Chapter 2.8.1 --- Previous studies on seed-specific expression in rice --- p.66 / Chapter 2.8.1.1 --- Endosperm-specific cis-acting regulatory elements --- p.67 / Chapter 2.8.1.2 --- Requirements to confer endosperm-specific expression in rice --- p.69 / Chapter 2.9 --- Project overview and hypothesis --- p.71 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.73 / Chapter 3.1 --- Chemicals --- p.73 / Chapter 3.2 --- Vectors and bacterial strains in regular cloning --- p.73 / Chapter 3.3 --- Plant materials --- p.74 / Chapter 3.4 --- Construction of gene cassettes for plant transformation --- p.74 / Chapter 3.4.1 --- Construction of gene cassettes for Ospsyl and Ospsy2 study --- p.74 / Chapter 3.4.1.1 --- Cloning of Ospsyl and Ospsy2 from rice --- p.76 / Chapter 4.1.1.1 --- Total RNA extraction --- p.75 / Chapter 3.4.1.1.2 --- Amplification of cDNA of Ospsyl by RT-PCR --- p.77 / Chapter 3.4.1.1.3 --- Amplification of cDNA of Ospsy2 by PCR --- p.80 / Chapter 3.4.1.2 --- Cloning of constitutive CaMV35S promoter --- p.82 / Chapter 3.4.1.2.1 --- Preparation of pBI121 vector --- p.82 / Chapter 3.4.1.2.2 --- Amplification of CaMV35S promoter by PCR --- p.82 / Chapter 3.4.1.3 --- Cloning of endosperm-specific rice glutelin-1 (Gt-1) promoter --- p.84 / Chapter 3. 4.1.3.1 --- Genomic DNA extraction --- p.84 / Chapter 3.4.1.3.2 --- Amplification of Gt-1 promoter by PCR --- p.84 / Chapter 3.4.1.4 --- Construction of gene cassettes for Ospsyl and Ospsy2 driven by CaMV35S and rice Gt-1 promoter --- p.87 / Chapter 3.4.2 --- Construction of gene cassettes for promoter analysis --- p.90 / Chapter 3.4.2.1 --- Cloning of full length and fragments of Ospsyl promoter --- p.92 / Chapter 3.4.2.1.1 --- Genomic DNA extraction --- p.95 / Chapter 3.4.2.1.2 --- Amplification of full length and fragments of Ospsy1 promoter --- p.95 / Chapter 3.4.2.2 --- Cloning of CaMV35S minimal promoter --- p.96 / Chapter 3.4.2.2.1 --- Amplification of CaMV35S minimal promoter --- p.97 / Chapter 3.4.2.3 --- Cloning of rice Gt-1 promoter --- p.97 / Chapter 3.4.2.3.1 --- Genomic DNA extraction --- p.97 / Chapter 3.4.2.3.2 --- Amplification of Gt-1 promoter --- p.97 / Chapter 3.4.2.4 --- Annealing of linker --- p.93 / Chapter 3.4.3.5 --- Making of rice GCN4 multimers --- p.98 / Chapter 3.4.2.6 --- Construction of gene cassettes for promoter analysis --- p.99 / Chapter 3.4.2.7 --- Construction of gene cassettes for Gt-1 promoter analysis --- p.104 / Chapter 3.4.3 --- Confirmation of sequence fidelity --- p.106 / Chapter 3.5 --- Rice transformation --- p.107 / Chapter 3.5.1 --- Plant materials --- p.107 / Chapter 3.5.2 --- Preparation of Agrobacterium --- p.107 / Chapter 3.5.3 --- Agrobacterium mediated transformation --- p.108 / Chapter 3.5.4 --- Callus induction from mature rice seeds --- p.109 / Chapter 3.5.6 --- Co-cultivation and selection --- p.109 / Chapter 3.5.7 --- Pre-regeneration and regeneration of transgenic rice --- p.110 / Chapter 3.5.8 --- Plantation of transgenic rice --- p.110 / Chapter 3.6 --- Detection of transgene expression --- p.112 / Chapter 3.6.1 --- Detection at DNA level --- p.112 / Chapter 3.6.1.1 --- Genomic DNA extraction --- p.112 / Chapter 3.6.1.2 --- PCR screening --- p.122 / Chapter 3.6.1.3 --- Synthesis of DIG-labeled DNA probes --- p.116 / Chapter 3.6.1.4 --- Southern blot analysis --- p.118 / Chapter 3.6.2 --- Detection at RNA level --- p.119 / Chapter 3.6.2.1 --- Total RNA extraction --- p.119 / Chapter 3.6.2.2 --- Northern blot analysis --- p.119 / Chapter 3.6.3 --- Detection at protein level --- p.119 / Chapter 3.6.3.1 --- Antibody production --- p.119 / Chapter 3.6.3.1 --- Ospsyl and Ospsy2 induction in pET system --- p.120 / Chapter 3.63.1.2 --- Immunization of rabbit and serum collection --- p.123 / Chapter 3.6.3.2 --- Total protein extraction from plant materials --- p.124 / Chapter 3.6.3.2.1 --- Protein extraction from rice calli and leaves --- p.124 / Chapter 3.6.3.2.2 --- Protein extraction from immature and mature rice seeds --- p.124 / Chapter 3.6.3.3 --- Tricine SDS-PAGE --- p.125 / Chapter 3.6.3.4 --- Western blot analysis --- p.125 / Chapter 3.6.4 --- Detection at metabolite level --- p.126 / Chapter 3.6.4.1 --- Isoprenoids extraction from plant materials --- p.126 / Chapter 3.6.4.2 --- UPLC analysis for isoprenoid identification --- p.127 / Chapter 3.6.5 --- Detection of promoter activity --- p.128 / Chapter 3.6.5.1 --- Histochemical staining of GUS activity --- p.128 / Chapter 3.6.5.1.1 --- Histochemical staining --- p.128 / Chapter 3.6.5.1.2 --- Plant tissue fixation for microscopic observation --- p.128 / Chapter 3.6.5.2 --- GUS activity assay --- p.129 / Chapter 3.6.5.2.1 --- Protein extraction and quantitation with Bio-Rad protein assay --- p.129 / Chapter 3.6.5.2.2 --- G US activity assay --- p.130 / Chapter CHAPTER 4. --- RESULTS --- p.131 / Chapter 4.1 --- Tissue-specificity and endosperm specific expression of rice psy1 and Psy2 --- p.131 / Chapter 4.1.1 --- Construction of gene cassettes for study in Ospsy1 and Ospsy2 --- p.133 / Chapter 4.1.2 --- Rice transformation --- p.135 / Chapter 4.1.3 --- Transgene detection --- p.137 / Chapter 4.1.3.1 --- Genomic DNA PCR screening --- p.137 / Chapter 4.1.3.2 --- Southern blot analysis --- p.139 / Chapter 4.1.3.2.1 --- Southern blot analysis on transgenic rice calli --- p.141 / Chapter 4.1.3.2.2 --- Southern blot analysis on regenerated rice --- p.143 / Chapter 4.1.4 --- Detection of transgene expression --- p.149 / Chapter 4.1.4.1 --- Northern blot analysis on immature transgenic seed --- p.149 / Chapter 4.1.4.2 --- Western blot analysis on transgenic rice tissues --- p.152 / Chapter 4.1.4.2.1 --- Antibody production --- p.152 / Chapter 4.1.4.2.2 --- Western blot analysis of transgenic rice calli --- p.155 / Chapter 4.1.4.2.3 --- Western blot analysis of transgenic rice leaves --- p.157 / Chapter 4.1.4.2.4 --- Western blot analysis of immature transgenic rice seeds --- p.160 / Chapter 4.1.5 --- Detection of OsPSYs activity at metabolite level --- p.163 / Chapter 4.1.5.1 --- UPLC analysis on transgenic rice tissues --- p.163 / Chapter 4.1.5.1.1 --- Carotenoid profiling of transgenic rice calli --- p.163 / Chapter 4.1.5.1.2 --- Carotenoid profiling of transgenic rice leaves --- p.168 / Chapter "4.1.5.1,3" --- Carotenoid profiling of mature transgenic rice seeds --- p.172 / Chapter 4.2 --- Promoter analysis of modified rice psy1 promoter --- p.176 / Chapter 4.2.1 --- Construction of gene cassettes for promoter analysis --- p.178 / Chapter 4.2.2 --- Rice transformation --- p.180 / Chapter 4.2.3 --- Transgene detection --- p.180 / Chapter 4.2.3.1 --- Genomic DNA PCR screening --- p.180 / Chapter 4.2.3.2 --- Southern blot analysis --- p.185 / Chapter 4.2.3.2.1 --- Southern blot analysis of regenerated rice --- p.186 / Chapter 4.2.4 --- Detection of promoter activity --- p.196 / Chapter 4.2.4.1 --- Promoter activity in transgenic rice leaves --- p.196 / Chapter 4.2.4.1.1 --- Histochemical staining of GUS --- p.196 / Chapter 4.2.4.1.2 --- G US activity assay --- p.199 / Chapter 4.2.4.2 --- Promoter activity in transgenic immature seeds --- p.202 / Chapter 4.2.4.2.1 --- Histochemical staining of GUS --- p.202 / Chapter 4.2.4.2.2 --- GUS activity assay --- p.206 / Chapter CHAPTER 5. --- DISCUSSIONS --- p.209 / Chapter 5.1 --- Tissue-specificity and endosperm specific expression of rice psyl and psy2 --- p.209 / Chapter 5.1.1 --- OsPSYl and OsPSY2 activities in rice calli --- p.209 / Chapter 5.1.2 --- OsPSYl and OsPSY2 activities in rice leaves --- p.210 / Chapter 5.1.3 --- OsPSYl and OsPSY2 activities in rice seeds --- p.211 / Chapter 5.2 --- Analysis of modified rice psyl promoter --- p.213 / Chapter 5.3 --- Future prospects of Golden Rice --- p.214 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.217 / REFERENCES --- p.219

Chan, Ka Ki Alex. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 245-263). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General introduction --- p.1 / Chapter 1.1 --- Structure and function of the enteric nervous system --- p.1 / Chapter 1.2 --- Neural crest cells (NCC) --- p.5 / Chapter 1.2.1 --- Vagal neural crest cells --- p.7 / Chapter 1.2.2 --- Sacral neural crest cells --- p.10 / Chapter 1.3 --- Prespecialization of the neural crest cells to form ENS --- p.15 / Chapter 1.4 --- Signaling pathways involved in ENS development --- p.19 / Chapter 1.4.1 --- Endothelin signaling pathway --- p.20 / Chapter 1.4.2 --- Ret signaling pathway: GDNF/Ret/GFRa1 --- p.22 / Chapter 1.4.3 --- Ret signaling pathway: NRTN/Ret/GFRa2 --- p.26 / Chapter 1.4.4 --- Phox2b --- p.28 / Chapter 1.4.5 --- Sox10 --- p.29 / Chapter 1.5 --- Hirschsprung's Disease (HSCR) --- p.31 / Chapter 1.6 --- Objective of studies --- p.32 / Figures and legends --- p.35 / Chapter Chapter Two --- The early migratory pathways of mouse sacral neural crest cells --- p.39 / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.2 --- Materials and Methods --- p.46 / Chapter 2.2.1 --- Animals --- p.46 / Chapter 2.2.2 --- Isolation of the mouse embryos at E95 --- p.46 / Chapter 2.2.3 --- Preparation ofWGA-Au --- p.47 / Chapter 2.2.4 --- Preparation of Dil --- p.48 / Chapter 2.2.5 --- Microinjection ofWGA-Au or Dil --- p.48 / Chapter 2.2.6 --- Preparation of rat serum --- p.49 / Chapter 2.2.7 --- Preparation of culture medium --- p.50 / Chapter 2.2.8 --- in vitro whole embryo culture system --- p.50 / Chapter 2.2.9 --- Examination of embryo after culture --- p.51 / Chapter 2.2.10 --- Histological preparation of WGA-Au labelled embryos --- p.51 / Chapter 2.2.11 --- Silver enhancement staining on sections of WGA-Au labelled embryo --- p.52 / Chapter 2.2.12 --- Histological preparation of Dil labelled embryos --- p.53 / Chapter 2.2.13 --- Reconstruction of the mouse embryos --- p.53 / Chapter 2.2.14 --- Cell counting on labelled sacral NCC between the anterior and posterior halves of the somite --- p.54 / Chapter 2.2.15 --- Cell counting on migrating labelled sacral NCC for each somite at different developmental stages --- p.55 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Development of E9.5 mouse embryo in vitro and in vivo --- p.57 / Chapter 2.3.2 --- Labelling of sacral neural crest cells by means of different cell markers --- p.58 / Chapter 2.3.3 --- Migration of sacral neural crest cells at different developmental stages --- p.59 / Chapter 2.3.3.1 --- Distribution of sacral NCC at the 26th somite stage --- p.60 / Chapter 2.3.3.2 --- Distribution of sacral NCC at the 28th somite stage --- p.61 / Chapter 2.3.3.3 --- Distribution of sacral NCC at the 30th somite stage --- p.61 / Chapter 2.3.3.4 --- Distribution of sacral NCC at the 32nd somite stage --- p.63 / Chapter 2.3.3.5 --- Distribution of sacral NCC at the 34th somite stage --- p.64 / Chapter 2.3.4 --- Defined migration pathways of the sacral neural crest cells --- p.65 / Chapter 2.3.5 --- Quantification of migrating sacral NCC at different somite axial levels at different developmental stages --- p.66 / Chapter 2.4 --- Discussion --- p.68 / Chapter 2.4.1 --- E9.5 mouse embryo grew normally in vitro using whole embryo culture --- p.69 / Chapter 2.4.2 --- Migration of sacral neural crest cells at 26th somite stage --- p.70 / Chapter 2.4.3 --- Migration of sacral neural crest cells at 28th somite stage --- p.72 / Chapter 2.4.4 --- Migration or sacral neural crest cells at 30th somite stage --- p.73 / Chapter 2.4.5 --- Migration of sacral neural crest cells at 32nd somite --- p.75 / Chapter 2.4.6 --- Migration of sacral neural crest cells at 34th somite stage --- p.77 / Chapter 2.4.7 --- Majority of sacral neural crest cells migrate along the dorsomedial pathway --- p.80 / Figures and Legends --- p.82 / Tables --- p.136 / Chapter Chapter Three --- The early migratory pathways of Dom mouse sacral neural crest cells --- p.139 / Chapter 3.1 --- Introduction --- p.139 / Chapter 3.2 --- Materials and Methods --- p.145 / Chapter 3.2.1 --- Animals --- p.145 / Chapter 3.2.2 --- In vitro culture of Dom mouse embryos --- p.145 / Chapter 3.2.3 --- Genotyping by polymerase chain reaction (PCR) --- p.146 / Chapter 3.2.4 --- Treatment of the harvested Dom mouse embryos --- p.147 / Chapter 3.2.5 --- Reconstruction of images and cell counting --- p.148 / Chapter 3.2.6 --- Percentage of migrating sacral neural crest cells reduction in Dom mouse embryo --- p.148 / Chapter 3.3 --- Results --- p.150 / Chapter 3.3.1 --- Migration of sacral neural crest cells in Dom mouse embryos at different developmental stages --- p.150 / Chapter 3.3.1.1 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 26th somite stage --- p.150 / Chapter 3.3.1.2 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 28th somite stage --- p.151 / Chapter 3.3.1.3 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 30th somite stage --- p.152 / Chapter 3.3.1.4 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 32nd somite stage --- p.154 / Chapter 3.3.1.5 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 34th somite stage --- p.156 / Chapter 3.3.2 --- Number of migrating sacral NCC of different genotypes of Dom mouse embryos at different developmental stage --- p.158 / Chapter 3.4 --- Discussion --- p.160 / Chapter 3.4.1 --- The use of Dom mouse model to study the etiology of Hirschsprung's disease (HSCR) --- p.161 / Chapter 3.4.2 --- Migration of sacral NCC in Dom mouse embryos --- p.164 / Figures and legends --- p.169 / Tables --- p.230 / Chapter Chapter Four --- General discussion and conclusions --- p.236 / Appendix --- p.241 / References --- p.245

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1 v. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1993