Wong, Hon-Kit. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 119-139). / Abstract in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vii / General abbreviations --- p.ix / Abbreviations of chemicals --- p.x / List of figures --- p.xii / Table of contents --- p.xvi / Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Nitrogen assimilation and regulation in plants --- p.1 / Chapter 1.2 --- Asparagine metabolism and its gene regulation in plants --- p.2 / Chapter 1.2.1 --- A brief introduction of asparagine --- p.2 / Chapter 1.2.2 --- Asparagine synthetase gene family in A. thaliana --- p.3 / Chapter 1.2.3 --- Reciprocal regulation of ASN1 and ASN2 gene --- p.3 / Chapter 1.2.4 --- Primary structure difference of ASN1 and ASN2 protein --- p.4 / Chapter 1.2.5 --- "ASN1 overexpressor support the notion that it is a major gene regulating the free asparagine levels in plant tissues, while ASN may play different physiological function(s)" --- p.2 / Chapter 1.2.6 --- Evidence support ammonium-dependent AS in plant --- p.6 / Chapter 1.3 --- Ammonium toxicity and mechanism of ammonium toxicity to plant --- p.7 / Chapter 1.3.1 --- Ammonium toxicity --- p.7 / Chapter 1.3.2 --- Mechanism of ammonium toxicity --- p.9 / Chapter 1.4 --- "Relationship among asparagine, ammonium, and stress physiology" --- p.12 / Chapter 1.4.1 --- Ammonium accumulates under stress conditions --- p.12 / Chapter 1.4.2 --- Asparagine accumulates under stress conditions --- p.14 / Chapter 1.5 --- Relationship of asparagine metabolism and photorespiration --- p.17 / Chapter 1.5.1 --- A brief introduction of photorespiratory pathway --- p.17 / Chapter 1.5.2 --- Involvement of Asn in the photorespiration nitrogen cycle --- p.18 / Chapter 1.5.3 --- Reassimilation of ammonium released from photorespiration --- p.19 / Chapter 1.5.4 --- Photorespiration and stress physiology --- p.21 / Chapter 1.6 --- Role of amino acids in abiotic stress resistance --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Proline accumulation and plant adaptation to water deficits and salinity stress --- p.24 / Chapter 1.6.3 --- Role of amino acids as precursors of quaternary ammonium compounds serving as compatible osmolytes --- p.28 / Chapter 1.7 --- A brief history of protoplast transient expression systems --- p.35 / Chapter 1.8 --- Advantages of mesophyll protoplast transient expression systems --- p.37 / Chapter 1.9 --- Hypothesis and main idea of this study --- p.38 / Chapter 2 --- Methods and Materials --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Plants --- p.39 / Chapter 2.1.2 --- Bacterial strains and plasmid vector --- p.39 / Chapter 2.1.3 --- Primer used --- p.39 / Chapter 2.1.4 --- Chemicals and reagents used --- p.40 / Chapter 2.1.5 --- Solution used --- p.40 / Chapter 2.1.6 --- Commercial kits used --- p.40 / Chapter 2.1.7 --- Equipment and facilities used --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Growth medium and condition --- p.41 / Chapter 2.2.1.1 --- Normal growth condition --- p.41 / Chapter 2.2.1.2 --- Growth medium and stresses treatments --- p.41 / Chapter 2.2.1.3 --- Plant growth in Azaserine medium --- p.43 / Chapter 2.2.2 --- Biochemical Assay --- p.44 / Chapter 2.2.2.1 --- Ammonium assay --- p.44 / Chapter 2.2.2.2 --- Ammonium extraction for ammonium assay --- p.46 / Chapter 2.2.2.3 --- Soluble protein determination --- p.46 / Chapter 2.2.2.4 --- Detection of chlorophyll content --- p.47 / Chapter 2.2.3 --- Molecular techniques --- p.47 / Chapter 2.2.3.1 --- Bacterial cultures for recombinant DNA --- p.47 / Chapter 2.2.3.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.3.3 --- Transformation of DH5a Competent cell --- p.48 / Chapter 2.2.3.4 --- Gel electrophoresis --- p.49 / Chapter 2.2.3.5 --- DNA and RNA extraction from plant tissues --- p.50 / Chapter 2.2.3.6 --- Generation of cRNA probes for Northern blot analyses --- p.52 / Chapter 2.2.3.7 --- Northern blot analysis --- p.53 / Chapter 2.2.3.8 --- PCR techniques --- p.54 / Chapter 2.2.3.9 --- Sequencing --- p.55 / Chapter 2.2.4 --- Genetic techniques --- p.56 / Chapter 2.2.4.1 --- Backcross of Azaserine resistant mutant --- p.56 / Chapter 2.2.4.2 --- Phenotype screening of backcross progenies --- p.56 / Chapter 2.2.5 --- Transient gene expression --- p.57 / Chapter 2.2.5.1 --- Protoplast isolation from Arabidopsis leave --- p.57 / Chapter 2.2.5.2 --- Protoplast transformation --- p.58 / Chapter 2.2.5.3 --- Gus protein extraction from protoplasts --- p.59 / Chapter 2.2.5.4 --- Gus assay --- p.60 / Chapter 2.2.5.5 --- MU calibration standard --- p.60 / Chapter 2.2.5.6 --- Sample assay --- p.60 / Chapter 3 --- Result --- p.61 / Chapter 3.1 --- Expression of ASN2 and ammonium assay in Arabidopsis thaliana under various stress conditions and senescence --- p.61 / Chapter 3.1.1 --- Ammonium assay of wild type seedlings under stress conditions --- p.61 / Chapter 3.1.2 --- Kinetic studies of ASN2 expression under different stresses treatments --- p.65 / Chapter 3.1.3 --- Ammonium assay of wild type seedlings under stress conditions --- p.70 / Chapter 3.2 --- NH4+ regulation on expression of ASN2 promoter --- p.73 / Chapter 3.2.1 --- The cloning ASN2 promoter --- p.73 / Chapter 3.2.1.1 --- Defining of ASN2 promoter region --- p.73 / Chapter 3.2.1.2 --- PCR amplification of ASN2 promoter from genomic sequence --- p.77 / Chapter 3.2.1.3 --- Cloning ASN2 promoter into transient gene expression vector (pBI221 vector) --- p.80 / Chapter 3.2.2 --- Transient gene expression --- p.84 / Chapter 3.2.2.1 --- Arabidopsis leave mesophyll protoplasts isolation --- p.84 / Chapter 3.2.2.2 --- Transformation and GUS expression assay --- p.87 / Chapter 3.3 --- Characterization ASN2 transgenic plants under stress conditions --- p.91 / Chapter 3.3.1 --- Construction of ASN2 transgenic plants --- p.91 / Chapter 3.3.2 --- Characterization of ASN2 transgenic plants --- p.93 / Chapter 3.3.2.1 --- Ammonium assay of ASN2 transgenic plant under different concentration of ammonium --- p.93 / Chapter 3.3.2.2 --- Ammonium assay of ASN2 transgenic plant under high light irradiance --- p.93 / Chapter 3.4 --- Characterization of mutant plants (AzaR) that showed altered ASN2 expression --- p.97 / Chapter 3.4.1 --- Phenotype of azaserine resistant mutant --- p.97 / Chapter 3.4.2 --- ASN2 expression level up-regulated in azaserine resistant mutant --- p.99 / Chapter 3.4.3 --- Checking for linkage between azaserine resistance and ASN2 overexpression --- p.101 / Chapter 3.4.4 --- Crossing the mutant with Landsberg for mapping the azaserine resistant mutant --- p.106 / Chapter 4 --- Discussion --- p.108 / Chapter 4.1 --- ASN2 may relate to ammonium metabolism --- p.108 / Chapter 4.2 --- ASN2 transgenic plants and their response under stresses conditions --- p.111 / Chapter 4.3 --- ASN2 promoter studies by transient gene expression method --- p.112 / Chapter 4.3.1 --- Identification of promoter region --- p.113 / Chapter 4.3.2 --- Isolation of protoplasts from Arabidopsis leaf --- p.114 / Chapter 4.3.3 --- Studies of ASN2 promoter transient gene expression in A thaliana protoplasts --- p.114 / Chapter 4.4 --- Azaserine Resistant Mutant --- p.115 / Chapter 4.4.1 --- Overexpression of ASN2 gene in Azaserine resistant mutant and checking for linkage --- p.115 / Chapter 4.4.2 --- Cross of Azaserine Resistant mutants with Lersberg ecotype for mapping --- p.116 / Chapter 5 --- Conclusion and prospective --- p.118 / References --- p.119 / Appendix --- p.140

Luk Chun-yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iv / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6 / Chapter 2.1 --- Introduction --- p.7 / Chapter 2.2 --- Ammonia chemistry --- p.10 / Chapter 2.3 --- Ammonia metabolism and excretion --- p.11 / Chapter 2.3.1 --- Ammonia production --- p.11 / Chapter 2.3.2 --- Blood levels of ammonia --- p.12 / Chapter 2.3.3 --- Ammonia Excretion --- p.17 / Chapter 2.4 --- Urea metabolism and excretion --- p.23 / Chapter 2.4.1 --- Urea Chemistry --- p.23 / Chapter 2.4.2 --- Urea production in fishes --- p.24 / Chapter 2.4.3 --- Argininolysis --- p.25 / Chapter 2.4.4 --- Uricolysis --- p.26 / Chapter 2.4.5 --- Ornithine-urea Cycle (OUC) --- p.28 / Chapter 2.4.5.1 --- Tilapia inhabiting the highly alkaline Lake Magadi --- p.32 / Chapter 2.4.5.2 --- High Ambient Ammonia --- p.33 / Chapter 2.4.5.3 --- Air Exposure --- p.34 / Chapter 2.4.5.4 --- Toadfishes --- p.34 / Chapter 2.4.6 --- Blood urea concentration --- p.35 / Chapter 2.4.7 --- Urea excretion in fishes --- p.37 / Chapter 2.4.7.1 --- Branchial urea excretion in fishes --- p.37 / Chapter 2.4.7.2 --- Mechanisms of renal excretion in fishes --- p.40 / Chapter 2.5 --- Influence of environmental salinity on nitrogen excretion in teleosts --- p.42 / Chapter CHAPTER 3 --- BODY COMPOSITION AND UREA BIOSYNTHESIS OF SPAR US SARBA IN DIFFERENT SALINITIES --- p.46 / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Materials and Methods --- p.49 / Chapter 3.2.1 --- Experimental animals --- p.49 / Chapter 3.2.2 --- Tissue sampling --- p.49 / Chapter 3.2.3 --- Water chemistry analysis --- p.50 / Chapter 3.2.4 --- Hematological parameters --- p.50 / Chapter 3.2.5 --- Metabolite and electrolyte contents --- p.51 / Chapter 3.2.6 --- Hepatic enzymes activities --- p.51 / Chapter 3.2.6.1 --- Tissue preparation --- p.51 / Chapter 3.2.6.2 --- Carbamyl phosphate synthetases (CPSases; E.C. 2.7.2.5) --- p.52 / Chapter 3.2.6.3 --- Ornithine carbamoyl transferase (OCTase; E.C. 2.1.3.3) --- p.53 / Chapter 3.2.6.4 --- Argininosuccinate synthetase (ASS; E.C. 6.3.4.5) --- p.54 / Chapter 3.2.6.5 --- Argininosuccinate lyase (ASL; E.C. 4.3.2.1) --- p.54 / Chapter 3.2.6.6 --- Arginase (ARG; 3.5.3.1) --- p.55 / Chapter 3.2.6.7 --- Glutamate dehydrogenase (EC 1.4.1.3) --- p.55 / Chapter 3.2.6.8 --- Uricase (E.C. 1.7.3.3) --- p.56 / Chapter 3.2.6.9 --- Allantoinase --- p.57 / Chapter 3.2.6.10 --- Allantoicase --- p.57 / Chapter 3.2.7 --- Statistical analysis --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- "Changes in hepatosmatic index, renal somatic index, muscle water and lipid content and hematological parametersin response to different salinity acclimation" --- p.59 / Chapter 3.3.2 --- Changes in serum chemistry in response to different salinity acclimation --- p.60 / Chapter 3.3.3 --- Changes in hepatic ornithine-urea cycle enzyme activitiesin response to different salinity acclimation --- p.61 / Chapter 3.3.4 --- Changes in GDHase and uricolytic enzyme activitiesin response to different salinity acclimation --- p.62 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Hematological responses --- p.72 / Chapter 3.4.2 --- Muscle moisture content --- p.74 / Chapter 3.4.3 --- Circulating electrolyte levels --- p.75 / Chapter 3.4.4 --- Circulating metabolites levels --- p.77 / Chapter 3.4.5 --- Urea metabolism --- p.80 / Chapter 3.4.5.1 --- Ornithine-urea cycle enzymes --- p.80 / Chapter 3.4.5.2 --- Carbamoyl phosphate synthetase isozymes --- p.81 / Chapter 3.4.5.3 --- Uricolytic pathway and argininolysis --- p.85 / Chapter 3.4.5.4 --- Influence of salinity on urea metabolism --- p.86 / Chapter 3.4.6 --- Conclusion --- p.87 / Chapter CHAPTER 4 --- EFFECT OF SALINITY ON NITROGEN EXCRETION OF SPARUS SARBA --- p.88 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and Methods --- p.91 / Chapter 4.2.1 --- Experimental animals --- p.91 / Chapter 4.2.2 --- Experimental protocol --- p.92 / Chapter 4.2.3 --- Determination of net ammonia and urea excretion rates --- p.94 / Chapter 4.2.4 --- Statistical analysis --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Net ammonia-N and urea-N excretion rates --- p.95 / Chapter 4.3.2 --- Changes in net ammonia-N and urea-N excretion ratesin response to abrupt hyposmotic exposure --- p.95 / Chapter 4.3.3 --- Changes in net ammonia-N and urea-N excretion rates after exposure to amiloride for 3 hours --- p.96 / Chapter 4.3.4 --- Changes in net urea-N excretion rates in response to elevated body urea levels --- p.96 / Chapter 4.3.5 --- Changes in net ammonia-N excretion rates in response to elevated body ammonia levels --- p.97 / Chapter 4.4 --- Discussion --- p.106 / Chapter 4.4.1 --- Influence of environmental salinity on net ammonia-N and urea-N excretion rates --- p.106 / Chapter 4.4.2 --- Effects of amiloride on nitrogen excretion --- p.109 / Chapter 4.4.3 --- Effect of increased body ammonia on ammonia excretion --- p.113 / Chapter 4.4.4 --- Changes in net urea-N excretion rates in response to elevated body urea levels --- p.113 / Chapter 4.5 --- Conclusion --- p.114 / Chapter CHAPTER 5 --- GENERAL CONCLUSION --- p.115 / references --- p.119

Research presented in this dissertation falls into two parts: fragmentation mechanisms of peptide and fragmentation mechanism of amino acid derivatives. The study of peptide fragmentation may help to improve protein identification by incorporating the rules governing this process into search algorithms. This study elucidates the chemical 'rules' governing peptide dissociation. It is believed that these 'rules' can be incorporated into searching algorithms to achieve better protein identification. The present study focuses on the effects of different amino acids on fragmentation. Amino acids with a wide range of different chemical and physical properties are investigated, including amino acids with hydrophilic side chains, amino acids with aliphatic side chains and amino acids without side chains. It can be concluded from the present studies that the different amino acid properties have great influence on the peptide fragmentation and spectrum appearance.The study of fragmentation mechanisms of amino acid derivatives is another focus of this dissertation. Based on the fragmentation mechanism study, a quantification method was developed. The method can distinguish glutamine with 15N-label at N-terminal amine vs the side chain even if they have same molecular weight. Ammonia metabolism was successfully monitored by feeding mosquitoes with isotope-labeled compounds and subsequently measuring the amount of the labeled amino acids. This method demonstrates the power of mass spectrometry in metabolism studies.