In the animal feed industries, there is a global need for adding certain nutritional ingredients to augment deficits usually associated with plant-based materials. As a result, the industrial practices require direct addition of ingredients such as amino acids and vitamins. One of the key ingredients in this context is lysine. Alternately, the same goal can be achieved indirectly through in situ co-culturing of microorgan-isms. The focus of this thesis was genetic improvement of bacterial and /or fungal mutants, which could over-produce lysine. The accumulation of free lysine during microbial growth serves this end based on de-regulation of the lysine biosynthetic pathway. Microorganisms used in this thesis were nine species of lactobacilli and <i>Aspergillus ficuum</i>. Having in mind the highly complex nutritional requirements of lacto-bacilli, the assessment of possible lysine auxotrophy was performed. No lysine auxotrophs were found and the choice of <i>Lactobacillus plantarum</i> as the working species among nine others was based on its higher growth rate in minimal medium. Selection of mutants that overproduced lysine was carried out in the minimal medium supplemented with the following lysine analogs: S-aminoethyl-L-cysteine (AEC), DL-aspartic acid-Ò-hydroxamate (DL-ASP), Ò -fluoropyruvic-acid (FPA), L-lysine hydroxamate (LHX) and diaminopimelic acid (DAP). In L. plantarum, LHX was shown to be the most potent inhibitor; although, the bacterium demonstrated high resistance to all the analogs tested. The inhibition by LHX was obtained only after significant alteration of the minimal medium M3. Furthermore, the mutant # 34, resistant to 2 mM of LHX, secreted only 4.52 £gM of lysine in M3. To address the question of low lysine yield obtained by L. plantarum, thorough study of the regulation of aspartokinase (AK) was performed. It was found that AK exists as four isozymes, threonine sensitive, methionine sensitive and two lysine sensitive isozymes. Activity differed with respect to the growth stage of L. plantarum. Beside lysine, threonine and methionine have influenced the repression of AK isozymes, which suggested that effective lysine over-production could be obtained only if AK is simultaneously resistant to threonine and methionine analogs. In the case of <i>A. ficuum</i>, mutant #5-10 secreted 29.25 £gM of lysine in the minimal medium, which was approximately 30 % higher than that of the wild type. DL-ASP was found as the most potent inhibitor only after the conidia were soaked for 8 h in 0.03 % Tween 80. Ammonium phosphate as a nitrogen source enhanced lysine secretion in <i>A. ficuum</i> compared to five other nitrogen sources tested.

In the animal feed industries, there is a global need for adding certain nutritional ingredients to augment deficits usually associated with plant-based materials. As a result, the industrial practices require direct addition of ingredients such as amino acids and vitamins. One of the key ingredients in this context is lysine. Alternately, the same goal can be achieved indirectly through in situ co-culturing of microorgan-isms. The focus of this thesis was genetic improvement of bacterial and /or fungal mutants, which could over-produce lysine. The accumulation of free lysine during microbial growth serves this end based on de-regulation of the lysine biosynthetic pathway. Microorganisms used in this thesis were nine species of lactobacilli and <i>Aspergillus ficuum</i>. Having in mind the highly complex nutritional requirements of lacto-bacilli, the assessment of possible lysine auxotrophy was performed. No lysine auxotrophs were found and the choice of <i>Lactobacillus plantarum</i> as the working species among nine others was based on its higher growth rate in minimal medium. Selection of mutants that overproduced lysine was carried out in the minimal medium supplemented with the following lysine analogs: S-aminoethyl-L-cysteine (AEC), DL-aspartic acid-Ò-hydroxamate (DL-ASP), Ò -fluoropyruvic-acid (FPA), L-lysine hydroxamate (LHX) and diaminopimelic acid (DAP). In L. plantarum, LHX was shown to be the most potent inhibitor; although, the bacterium demonstrated high resistance to all the analogs tested. The inhibition by LHX was obtained only after significant alteration of the minimal medium M3. Furthermore, the mutant # 34, resistant to 2 mM of LHX, secreted only 4.52 £gM of lysine in M3. To address the question of low lysine yield obtained by L. plantarum, thorough study of the regulation of aspartokinase (AK) was performed. It was found that AK exists as four isozymes, threonine sensitive, methionine sensitive and two lysine sensitive isozymes. Activity differed with respect to the growth stage of L. plantarum. Beside lysine, threonine and methionine have influenced the repression of AK isozymes, which suggested that effective lysine over-production could be obtained only if AK is simultaneously resistant to threonine and methionine analogs. In the case of <i>A. ficuum</i>, mutant #5-10 secreted 29.25 £gM of lysine in the minimal medium, which was approximately 30 % higher than that of the wild type. DL-ASP was found as the most potent inhibitor only after the conidia were soaked for 8 h in 0.03 % Tween 80. Ammonium phosphate as a nitrogen source enhanced lysine secretion in <i>A. ficuum</i> compared to five other nitrogen sources tested.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 189-242). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 189-242). Also available on the Internet.

I present the characterization and applications of a technique to modify the surface of the conducting polymer, polypyrrole, via a novel, 12-amino acid peptide, THRTSTLDYFVI (T59). This peptide non-covalently binds to the chlorine-doped conducting polymer polypyrrole, allowing it to be used in tethering molecules to polypyrrole for uses such as a scaffold for the treatment of peripheral nerve injury or in surface coatings of neural recording electrodes. I have quantified the binding of this peptide as well as investigating the mechanism of the binding. The equilibrium constant of the binding interaction of PPyCl and the T59 peptide was found through a binding assay to be 92.6 nM, and the off rate was found to be approximately 2.49 s⁻¹, via AFM force spectroscopy. The maximum observed surface density of the peptide was 1.27 +/- 0.42 femtomoles/cm². Furthermore, my studies suggest that the eighth residue, aspartic acid, is the main contributor of the binding, by interacting with the partially positive charge on the backbone of polypyrrole. I have demonstrated practical applications of the technique in the successful modification of a PPyCl surface with the laminin fragment IKVAV, as well as the so-called stealth molecule poly(ethylene glycol) (PEG). A subcutaneous implant study was performed to confirm that the T59 peptide did not induce any significant reaction in vivo. Significantly, the conductivity of a PPyCl surface was unaffected by this surface modification technique. / text

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

<p> DL-Mimosine has been synthesized by debenzylation and detosylation of the product obtained by condensation of 3-benzyloxy-4-pyrone with β-amino-α-tosylaminopropionic acid. A new method for the isolation of mimosine from Leucaena glauca Benth. is described. The biosynthesis of mimosine was studied by feeding radioactive aspartates, glycerol, glycerate and ribose to Mimosa pudica L. . Mimosine-C^14 was isolated and partially degraded, and it was shown that the carbon-3 of aspartic acid is specifically incorporated into the pyridone ring.</p> / Thesis / Doctor of Philosophy (PhD)

Chiao Ying Ann. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 127-140). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VII / Abbreviations --- p.IX / Abbreviation of chemicals --- p.XI / Table of Contents --- p.XII / List of figures and tables --- p.XVIII / Chapter Chapter 1. --- Literature review / Chapter 1.1 --- Significances of manipulation of nitrogen sink-source relationship --- p.1 / Chapter 1.2 --- Nitrogen sink-source relationship in plants --- p.2 / Chapter 1.3 --- Aspartate family amino acid metabolism --- p.5 / Chapter 1.3.1 --- Asparagine metabolism --- p.9 / Chapter 1.3.1.1 --- "Asparagine synthetase (AS, EC 6.3.5.4)" --- p.9 / Chapter 1.3.1.2 --- "Asparaginase (ANS, EC 3.5.1.1)" --- p.10 / Chapter 1.3.2 --- Metabolism of aspartate-derived essential amino acids --- p.10 / Chapter 1.3.2.1 --- "Aspartate kinase (AK, EC 2.7.2.4)" --- p.10 / Chapter 1.3.2.2 --- "Homoserine dehydrogenase (HSD, EC 1.1.1.3)" --- p.12 / Chapter 1.3.2.3 --- "Dihydrodipicolinate synthase (DHPS, EC 4.2.1.52)" --- p.13 / Chapter 1.3.2.4 --- "Lysine a-ketoglutarate reductase (LKR, EC 1.5.1.7)" --- p.14 / Chapter 1.3.2.5 --- "Threonine synthase (TS, EC 4.2.3.1)" --- p.15 / Chapter 1.3.2.6 --- Cystathionine γ-synthase (CGS，EC 2.5.1.48) --- p.16 / Chapter 1.3.2.7 --- Threonine deaminase (TD，EC 4.3.1.19) --- p.17 / Chapter 1.4 --- Previous attempts to manipulate seed protein quantity and quality --- p.18 / Chapter 1.4.1 --- Enhancement of amino acids transported from source to sink --- p.18 / Chapter 1.4.2 --- Redirection of metabolic pathways to increase target amino acids --- p.19 / Chapter 1.4.2.1 --- Production of aspartate by Aspartate Aminotransferase (AAT) --- p.24 / Chapter 1.4.2.2 --- Deregulation of AK to increase the common substrate for all essential aspartate family amino acids --- p.25 / Chapter 1.4.2.3 --- Inhibition of TS and enhancement of CGS to increase Met biosynthesis --- p.25 / Chapter 1.4.2.3.1 --- Inhibition of TS --- p.26 / Chapter 1.4.2.3.2 --- Enhancement of CGS --- p.26 / Chapter 1.4.2.4 --- Deregulation of DHPS and reduction of lysine catabolism to increase lysine content --- p.27 / Chapter 1.4.2.4.1 --- Deregulation of DHPS --- p.28 / Chapter 1.4.2.4.2 --- Reduction of Lys catabolism --- p.29 / Chapter 1.4.2.3.3 --- Deregulation of DHPS and reduction of LKR --- p.29 / Chapter 1.4.3 --- Expression of seed storage proteins to entrap the free amino acids --- p.30 / Chapter 1.5 --- Expression of multiple transgenes in plants --- p.34 / Chapter 1.5.1 --- Significance of multiple genes manipulation in seed quality improvement --- p.34 / Chapter 1.5.2 --- Difficulties in introduction of multiple genes into plant genomes --- p.34 / Chapter 1.5.3 --- Recent advances in introduction of multiple genes into plant genome --- p.35 / Chapter 1.6 --- Global nitrogen regulators in plants --- p.36 / Chapter 1.6.1 --- Global regulation of nitrogen metabolism --- p.36 / Chapter 1.6.2 --- General amino acid control by GCN system --- p.38 / Chapter 1.6.3 --- General amino acid control in plants --- p.39 / Chapter 1.6.4 --- GCN system in plants --- p.41 / Chapter 1.7 --- Hypothesis and specific objectives of this study --- p.42 / Chapter Chapter 2 --- Materials and methods --- p.46 / Chapter 2.1 --- Materials --- p.46 / Chapter 2.1.1 --- "Vectors, bacterial strains and plants" --- p.46 / Chapter 2.1.2 --- Chemicals and reagents used --- p.49 / Chapter 2.1.3 --- "Buffer, solution, gel and medium" --- p.49 / Chapter 2.1.4 --- Commercial kits used --- p.49 / Chapter 2.1.5 --- Equipments and facilities used --- p.49 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Molecular techniques --- p.50 / Chapter 2.2.1.1 --- DNA gel electrophoresis --- p.59 / Chapter 2.2.1.2 --- PCR technique --- p.50 / Chapter 2.2.1.3 --- Restriction digestion --- p.50 / Chapter 2.2.1.4 --- Ligation (for sticky-end ligation) --- p.51 / Chapter 2.2.1.5 --- DNA purification --- p.51 / Chapter 2.2.1.6 --- DNA sequencing --- p.51 / Chapter 2.2.1.7 --- Transformation of competent E. coli cells --- p.52 / Chapter 2.2.1.8 --- Preparation of plasmid from bacterial cells --- p.53 / Chapter 2.2.1.9 --- Transformation of competent Agrobacterium tumefaciens cells --- p.53 / Chapter 2.2.1.10 --- DNA extraction from plant tissue (Small-scale) --- p.54 / Chapter 2.2.1.11 --- RNA extraction from plant tissue --- p.55 / Chapter 2.2.2 --- Growth conditions of A. thaliana --- p.55 / Chapter 2.2.2.1 --- Surface sterilization of A. thaliana seeds --- p.55 / Chapter 2.2.2.2 --- Growing A. thaliana --- p.55 / Chapter 2.2.3 --- Characterization of transgenic A. thaliana with altered sink-source relationship --- p.57 / Chapter 2.2.3.1. --- Determination of amino acid contents in seeds --- p.57 / Chapter 2.2.3.2. --- Expression study of developing siliques of transgenic lines --- p.58 / Chapter 2.2.3.2.1 --- Tagging siliques of different developmental stages --- p.58 / Chapter 2.2.3.2.2 --- Extraction of silique RNA --- p.58 / Chapter 2.2.3.2.3 --- cDNA synthesis --- p.58 / Chapter 2.2.3.2.4 --- Real-time PCR --- p.59 / Chapter 2.2.4 --- Characterization of transgenic A. thaliana overexpressing GCN2 --- p.60 / Chapter 2.2.4.1 --- Gene expression study of vegetative tissues by real-time PCR --- p.60 / Chapter 2.2.4.2 --- Gene expression study of developing siliques by real-time PCR --- p.61 / Chapter 2.2.5 --- Making transgenic A. thaliana --- p.61 / Chapter 2.2.5.1 --- Cloning of multigene construct --- p.61 / Chapter 2.2.5.1.1 --- Subcloning of target genes into donor vectors --- p.61 / Chapter 2.2.5.1.1.1 --- Cloning of LRP into donor vector VS --- p.61 / Chapter 2.2.5.1.1.2 --- Cloning of dapA into donor vector SV --- p.64 / Chapter 2.2.5.1.1.3 --- Cloning of ansB into donor vector VS --- p.67 / Chapter 2.2.5.1.1.4 --- Cloning of antisense LKR fragment into donor vector SV --- p.70 / Chapter 2.2.5.1.2 --- Preparation of phosphorylated linkers --- p.73 / Chapter 2.2.5.1.3 --- Introduction of target genes to acceptor vector --- p.73 / Chapter 2.2.5.2 --- Agrobacterium-mediated transformation of A. thaliana via Vacuum infiltration --- p.78 / Chapter 2.2.5.3 --- Screening of transformants --- p.79 / Chapter Chapter 3. --- Results --- p.80 / Chapter 3.1 --- Characterization of transgenic lines with altered sink-source relationship --- p.80 / Chapter 3.1.1 --- Amino acid analysis of mature seeds of transgenic lines --- p.80 / Chapter 3.1.1.1 --- Aspartate family amino acids levels remain steady in seeds of transgenic plants --- p.83 / Chapter 3.1.1.2 --- Increase in seed Met content in Met-rich protein expressing transgenic plants --- p.85 / Chapter 3.1.1.3 --- Increase in seed Lys content in phas-dapA/phas-LRP transgenic plants --- p.87 / Chapter 3.1.2 --- Gene expression study of transgenic line --- p.89 / Chapter 3.1.2.1 --- Down-regulation of akthr1 and akthr2 in transgenic plants with altered N sink-source relationship --- p.89 / Chapter 3.1.2.2 --- Down regulation of GCN2 in transgenic plants with altered N sink-source relationship --- p.90 / Chapter 3.1.2.4 --- Expression study of other genes in aspartate family pathway --- p.90 / Chapter 3.2 --- Characterization of GCN2 overexpressing line --- p.93 / Chapter 3.2.1 --- Gene expression study of seedlings of GCN2 overexpressing plants --- p.93 / Chapter 3.2.1.1 --- Increased GCN2 expression by azaserine treatment --- p.93 / Chapter 3.2.1.2 --- Increased akthrl and akthr2 expression in GCN2 overexpressing plants --- p.96 / Chapter 3.2.1.3 --- Expression study of other genes in aspartate family pathway --- p.96 / Chapter 3.2.2 --- Gene expression study of GCN2 overexpressing plants during seed development --- p.98 / Chapter 3.3 --- Construction of transgenic plants by multigene assembly system --- p.100 / Chapter 3.3.1 --- Successful construction of recombinant plasmid carrying four target genes --- p.100 / Chapter 3.3.2 --- Transformation of A. thaliana with multigene vector --- p.103 / Chapter Chapter 4 --- Discussion --- p.104 / Chapter 4.1 --- Characterization of transgenic plants with altered sink-source relationship of aspartate family amino acid metabolism --- p.104 / Chapter 4.1.1 --- Total content of aspartate family amino acids remains steady in transgenic lines --- p.105 / Chapter 4.1.2 --- Methionine content increases in phas-PN2S and phas-MetL transgenic plants --- p.106 / Chapter 4.1.3 --- Relative lysine content increases in phas-dapA/phas-LRP transgenic plants --- p.107 / Chapter 4.1.4 --- Coordinated regulation of gene expressions of akthrl and akthr2 with GCN2 expression in transgenic plants with altered sink-source relationship --- p.109 / Chapter 4.2 --- GCN system in plants --- p.110 / Chapter 4.2.1 --- Transcriptional regulation of GCN2 in A. thaliana --- p.110 / Chapter 4.2.2 --- Regulation of amino acid biosynthesis by GCN system --- p.111 / Chapter 4.2.2.1 --- Regulation of akthrl and akthr2 by GCN2 --- p.111 / Chapter 4.2.2.2 --- GCN4 homolog in plants? --- p.112 / Chapter 4.2.2.3 --- Regulation of amino acid metabolism by GCN system --- p.113 / Chapter 4.3 --- Generation of transgenic plants with a combination of altered sink- source relationship --- p.114 / Chapter Chapter 5. --- Conclusion and Future Prospective --- p.116 / Appendix I: The major chemicals and reagents used in this research --- p.118 / "Appendix II: Major buffers, solutions and mediums used in this research" --- p.120 / Appendix III: Commercial kits used in this research --- p.125 / Appendix IV: Major equipment and facilities used in this research --- p.126 / References --- p.127

Ip Lai Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 81-92). / Abstracts in English and Chinese. / Table of Contents --- p.1 / Abbreviations --- p.4 / Chapter Chapter 1 --- Introduction and Background --- p.5 / Chapter 1.1 --- Endothelin system --- p.5 / Chapter 1.1.1 --- Endothelins --- p.5 / Chapter 1.1.2 --- Endothelin converting enzyme (ECE) isoforms --- p.12 / Chapter 1.1.3 --- Endothelin receptors --- p.24 / Chapter 1.2 --- Signal-transduction mechanisms in ET system --- p.27 / Chapter 1.3 --- The aim of the present thesis --- p.31 / Chapter Chapter 2 --- Materials and Methods --- p.32 / Chapter 2.1 --- Primer Design --- p.32 / Chapter 2.2 --- Total RNA Isolation --- p.33 / Chapter 2.3 --- Reverse transcriptase polymerase chain reaction (RT-PCR) --- p.34 / Chapter 2.3.1 --- First Strand cDNA Synthesis --- p.34 / Chapter 2.3.2 --- PCR reaction --- p.34 / Chapter 2.4 --- Agarose gel electrophoresis --- p.35 / Chapter 2.5 --- Ligation of PCR inserts to cloning vector by TA cloning method --- p.35 / Chapter 2.6 --- Competent cell preparation --- p.36 / Chapter 2.7 --- Transformation and Screening --- p.37 / Chapter 2.8 --- Plasmid DNA Extraction --- p.38 / Chapter 2.9 --- DNA sequencing --- p.38 / Chapter 2.10 --- DIG RNA Labeling --- p.38 / Chapter 2.10.1 --- Plasmid Linearization --- p.38 / Chapter 2.10.2 --- Transcription --- p.39 / Chapter 2.10.3 --- Probe purification --- p.39 / Chapter 2.11 --- In situ hybridizaion --- p.40 / Chapter 2.11.1 --- Tissue preparation and slide mounting --- p.40 / Chapter 2.11.2 --- Non-radioactive in situ hybridization --- p.41 / Chapter 2.12 --- Whole Mount non-radioactive in situ hybridization --- p.42 / Chapter 2.12.1 --- Dissection and fixation --- p.42 / Chapter 2.12.2 --- Hybridization --- p.43 / Chapter 2.12.3 --- Antibody incubation --- p.43 / Chapter 2.12.4 --- Histochemistry --- p.44 / Chapter Chapter 3 --- Results --- p.46 / Chapter 3.1 --- The molecular cloning of ECE-2 from rat brain --- p.46 / Chapter 3.2 --- Sequence characteristics of rat ECE-2 --- p.52 / Chapter 3.3 --- Comparison of rat ECE-2 with bovine and human ECE-2 and with the rat ECE-1 --- p.53 / Chapter 3.4 --- Tissue distribution of ECE-2 in rat and localization in C6 glial cells by RT-PCR --- p.60 / Chapter 3.5 --- ECE-2 in rat embryos at different gestation stages by RT-PCR --- p.60 / Chapter 3.6 --- ECE-2 distribution in C6 glioma cells --- p.63 / Chapter 3.7 --- ECE-2 distribution in rat embryo E15.5 --- p.63 / Chapter 3.8 --- ECE-2 distribution in rat brain sections --- p.63 / Chapter Chapter 4 --- p.74 / Discussion --- p.74 / References --- p.81

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 143-164). Also available on the Internet.