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That Lactobacillus arabinosus 17-5, ATCC 8014, can supply its own requirement for the amino acid, lysine, is demonstrated by the fact that the organism is capable of growth in media devoid of lysine. Since the final biosynthetic step in lysine formation in all bacteria studied to date involves the decarboxylation of meso-dlaminopimelic acid (DAP) to produce lysine, it was of interest to determine whether an enzyme catalyzing such a reaction (DAP decarboxylase) is present in L. arabinosus.

Chan Man Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 102-114). / Abstracts in English and Chinese. / Table of Contents / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- The importance of rice --- p.4 / Chapter 2.2 --- The prevalence of hunger and malnutrition --- p.4 / Chapter 2.3 --- Limitation of essential amino acids in crop plants --- p.5 / Chapter 2.4 --- Lysine biosynthesis and catabolism --- p.7 / Chapter 2.5 --- Lysine biosynthetic pathway --- p.7 / Chapter 2.5.1 --- The biosynthesis of aspartate --- p.7 / Chapter 2.5.2 --- The aspartate family pathway --- p.9 / Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.11 / Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.13 / Chapter 2.5.2.3 --- Threonine Synthase (TS) and other enzymes --- p.15 / Chapter 2.6 --- The lysine catabolic pathway --- p.16 / Chapter 2.6.1 --- "LKR-SDHproteins, mRNAs and genes" --- p.18 / Chapter 2.6.2 --- Regulation of lysine catabolic pathway --- p.21 / Chapter 2.6.2.1 --- Regulation at biochemical level --- p.21 / Chapter 2.6.2.2 --- Regulation through linkage between LKR and SDH --- p.22 / Chapter 2.6.2.3 --- Regulation through LKR/SDH gene expression --- p.24 / Chapter 2.6.2.4 --- Implication of regulatory mechanism of saccharopine pathway --- p.26 / Chapter 2.7 --- Overall regulation of lysine content in plants --- p.27 / Chapter 2.8 --- Increasing lysine content in plants --- p.28 / Chapter 2.8.1 --- "Breeding, selection and naturally occurring mutants" --- p.28 / Chapter 2.8.2 --- Induced biochemical mutants --- p.29 / Chapter 2.8.3 --- Transgenic plants --- p.31 / Chapter 2.8.4 --- Insight into the way of lysine accumulation --- p.35 / Chapter 2.9 --- Gene silencing in plant --- p.36 / Chapter 2.9.1 --- Mechanism of antisense RNA and RNAi --- p.36 / Chapter 2.9.2 --- Application of antisense technology to produce transgenic plants --- p.39 / Chapter 2.10 --- Hypothesis --- p.41 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.43 / Chapter 3.1 --- Chemicals --- p.43 / Chapter 3.2 --- Bacterial strains --- p.43 / Chapter 3.3 --- Chimeric gene construction for rice transformation --- p.43 / Chapter 3.3.1 --- Plasmids and genetic materials --- p.43 / Chapter 3.3.2 --- Construction of chimeric genes with seed-specific promoters --- p.46 / Chapter 3.3.3 --- Construction of chimeric gene with 35S promoter --- p.51 / Chapter 3.3.4 --- Construction of antisense and RNAi constructs --- p.53 / Chapter 3.3.5 --- "Construction of chimeric genes expressing AK, DHPS and RNAi synchronously" --- p.58 / Chapter 3.3.6 --- Confirmation of sequence fidelity of chimeric genes --- p.59 / Chapter 3.4 --- Rice transformation --- p.59 / Chapter 3.4.1 --- Plant materials --- p.59 / Chapter 3.4.2 --- Preparation of Agrobacterium --- p.59 / Chapter 3.4.3 --- Agrobacterium-mediated rice transformation --- p.60 / Chapter 3.4.3.1 --- Callus induction from mature seed embryos --- p.60 / Chapter 3.4.3.2 --- Callus induction from immature seed embryos --- p.60 / Chapter 3.4.3.3 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.60 / Chapter 3.5 --- Analysis of transgenic expression --- p.62 / Chapter 3.5.1 --- Genomic DNA extraction --- p.62 / Chapter 3.5.2 --- Total RNA extraction --- p.62 / Chapter 3.5.3 --- Synthesis of DIG-labeled DNA probe / Chapter 3.5.4 --- Southern blot analysis --- p.65 / Chapter 3.5.5 --- Northern blot analysis --- p.65 / Chapter 3.5.6 --- Extraction of immature seed protein --- p.65 / Chapter 3.5.7 --- Tricine SDS-PAGE --- p.66 / Chapter 3.5.8 --- Western blot analysis --- p.66 / Chapter 3.6 --- Free amino acid analysis --- p.67 / Chapter CHAPTER 4. --- RESULTS --- p.68 / Chapter 4.1 --- Construction of chimeric genes --- p.68 / Chapter 4.2 --- Rice transformation --- p.70 / Chapter 4.3 --- Detection of target genes in transgenic rice lines --- p.72 / Chapter 4.3.1 --- PCR of Genomic DNA --- p.72 / Chapter 4.3.2 --- Southern blot analysis --- p.75 / Chapter 4.4 --- Northern blot analysis --- p.77 / Chapter 4.5 --- "Western blot analysis ofAK, DHPS and LKR protein" --- p.80 / Chapter 4.6 --- Free amino acid analysis --- p.82 / Chapter 4.6.1 --- Free lysine content --- p.82 / Chapter 4.6.2 --- Changes of other amino acids --- p.84 / Chapter CHAPTER 5. --- DISCUSSION --- p.93 / Chapter 5.1 --- Rice transformation and transgene expression --- p.93 / Chapter 5.2 --- Co-expression of E. coli feedback-insensitive AK and DHPS --- p.94 / Chapter 5.3 --- Enhancing free Lys through down-regulation of LKR --- p.95 / Chapter 5.4 --- Co-expression of AK and DHPS together with down-regulation of LKR --- p.96 / Chapter 5.5 --- Free amino acid changes in different genotypes --- p.97 / Chapter 5.6 --- Future perspectives --- p.98 / Chapter CHAPTER 6. --- CONCLUSION --- p.100 / REFERENCES --- p.102

Remediation of heavy metals from industrial effluents and ground water sources poses a significant challenge. Hexavalent chromium is one such heavy metal, prevalent in industrial wastewaters, which has been proven to be toxic to humans and other living organisms. Most of the conventional methods available for dealing with chromium are either cost prohibitive or generate secondary effluents which are difficult to deal with. The idea of bioremediation has gained much momentum over the last few decades because of its potential low cost and minimum impact on the environment. This study explored the potential for hexavalent chromium bioremediation using a synthetic cationic biopolymer alpha-poly-l-lysine (alpha-PLL) as a biosorbent. In the present research work, equilibrium batch studies were performed in a specially designed dialysis apparatus to obtain preliminary information about the adsorption capacity of the polymer. Metal uptake by the polymer was found to be maximum when the pH of chromium solution (pH 4.6) and that of poly-lysine (pH 5.7) was not changed at the beginning of the experiment. Applying the Langmuir adsorption isotherm model showed that alpha-PLL has a maximum uptake capacity of 42.2 microgram Cr/mg alpha-PLL, and a binding constant of 1.2 microgram/mL +/- 10%. The metal uptake performance of the polymer was also evaluated in a Polymer Enhanced Diafiltration (PEDF) system. The polymer-metal complex was retained and concentrated by the PEDF set up using a tangential flow filtration membrane, while the clean filtrate flowed through. When 3.4 L of 10 mg/L chromium solution in the Cr2O72- form was processed using 300 mL of 2 gm/L PLL, the concentration of chromium in the permeate reached a maximum of 0.79 mg/L. When 30 mg/L chromium solution was used, 2 L could be processed using 300 mL of 2gm/L PLL, and 7.8 mg/L chromium could be detected in the permeate in the end.

Yu, Wai Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-101). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / 摘要 --- p.vi / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- The importance of rice --- p.3 / Chapter 2.2 --- Limitation of essential amino acids in rice --- p.4 / Chapter 2.3 --- Lysine biosynthetic pathway --- p.6 / Chapter 2.3.1 --- The biosynthesis of aspartate --- p.6 / Chapter 2.3.2 --- Aspartate family pathway --- p.3 / Chapter 2.3.2.1 --- Aspartate kinase (AK) --- p.10 / Chapter 2.3.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.12 / Chapter 2.3.2.3 --- Other enzymes --- p.14 / Chapter 2.4 --- Regulation of lysine content in plant --- p.15 / Chapter 2.5 --- Enhancement of lysine content in plants --- p.16 / Chapter 2.5.1 --- "Breeding, selection and naturally occuring muatnts" --- p.17 / Chapter 2.5.2 --- Induced biochemical mutants --- p.18 / Chapter 2.5.3 --- Transgenic plants --- p.19 / Chapter 2.6 --- Hypothesis --- p.24 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.25 / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Chemicals --- p.25 / Chapter 3.3 --- Bacterial strains --- p.25 / Chapter 3.4 --- Cloning of AK and DHPS cDNAs --- p.25 / Chapter 3.4.1 --- Plant materials --- p.25 / Chapter 3.4.2 --- RNA extraction --- p.26 / Chapter 3.4.3 --- RT-PCR amplification of AK and DHPS cDNAs --- p.26 / Chapter 3.4.4 --- Sequence modification of AK and DHPS cDNAs --- p.27 / Chapter 3.4.5 --- DNA sequencing of AK and DHPS cDNAs --- p.32 / Chapter 3.5 --- Chimeric gene construction for rice transformation --- p.32 / Chapter 3.5.1 --- Plasmid and genetic material --- p.32 / Chapter 3.5.2 --- Construction of chimeric genes with seed-specific promoter --- p.35 / Chapter 3.5.3 --- Sequence fidelity of chimeric genes --- p.37 / Chapter 3.6 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.37 / Chapter 3.7 --- Rice transformation --- p.38 / Chapter 3.7.1 --- Plant materials --- p.38 / Chapter 3.7.2 --- Preparation of agrobacterium --- p.33 / Chapter 3.7.3 --- Agrobacterium-mediated rice transformation --- p.39 / Chapter 3.7.3.1 --- Callus induction from mature rice seed embryos --- p.39 / Chapter 7.3.2 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.39 / Chapter 3.8 --- Analysis of transgenic expression --- p.41 / Chapter 3.8.1 --- Genomic DNA extraction --- p.41 / Chapter 3.8.2 --- Total RNA extraction --- p.41 / Chapter 3.8.3 --- Synthesis of DIG-labeled DNA probe --- p.42 / Chapter 3.8.4 --- Southern blot analysis --- p.43 / Chapter 3.8.5 --- Northern blot analysis --- p.43 / Chapter 3.8.6 --- Extraction of rice seed protein --- p.43 / Chapter 3.8.7 --- Tricine SDS-PAGE --- p.44 / Chapter 3.8.8 --- Raising AK and DHPS antibody --- p.44 / Chapter 3.8.9 --- Western blot analysis --- p.46 / Chapter 3.9 --- Free amino acid analysis --- p.46 / Chapter CHAPTER 4. --- RESULTS --- p.48 / Chapter 4.1 --- Cloning of AK and DHPS cDNAs from rice --- p.48 / Chapter 4.1.1 --- RNA extraction and cDNAs amplification --- p.43 / Chapter 4.1.2 --- Sequencing of AK and DHPS cDNAs --- p.50 / Chapter 4.2 --- Sequence modification of AK and DHPS cDNAs --- p.50 / Chapter 4.3 --- Construction of chimeric genes --- p.50 / Chapter 4.4 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.56 / Chapter 4.5 --- Rice transformation --- p.58 / Chapter 4.6 --- Detection of target genes in transgenic rice lines --- p.60 / Chapter 4.6.1 --- PCR of genomic DNA --- p.60 / Chapter 4.6.2 --- Southern blot analysis --- p.63 / Chapter 4.7 --- Northern blot analysis --- p.65 / Chapter 4.8 --- Western blot analysis of AK and DHPS proteins --- p.66 / Chapter 4.9 --- Free amino acid analysis --- p.68 / Chapter 4.9.1 --- Free lysine content --- p.68 / Chapter 4.9.2 --- Changes in other amino acids --- p.69 / Chapter CHAPTER 5. --- DISCUSSION --- p.82 / Chapter 5.1 --- Cloning and modification of AK and DHPS cDNAs --- p.82 / Chapter 5.2 --- Seed-specific expression of modified AK and DHPS in rice --- p.82 / Chapter 5.3 --- Free amino acid changes in transgenic rice lines --- p.83 / Chapter 5.4 --- Future perspectives --- p.85 / Chapter CHAPTER 6. --- CONCLUSION --- p.86 / REFERENCES --- p.87 / APPENDIX --- p.102

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

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.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

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.

Glucocorticoid receptor (GR) is known to associate with KATs and KDACs to regulate transcription. The current model of GR-mediated transcription focuses on agonist-dependent recruitment of KATs to acetylate histones and casts KDACs as corepressors in the presence of antagonist. Recent studies have shown KDACs to function as coactivators in the GR-mediated activation of the MMTV promoter and inhibition of KDACs impairs this activation. Nevertheless, the effect of KDAC inhibition on the GR-regulated transcriptome is unknown. Our expression profiling studies in a glucocorticoid (GC) responsive hepatoma-derived cell line, show that the class I-selective KDACi, VPA, has a profound impact on the GR-regulated hepatic transcriptome. VPA treatment alone mimics GC signaling at some GR-target genes and cooperates with GC to activate a small number of genes. However, the predominant effect of VPA, seen in more than 50% of the GR-target genes, is impairment of normal GR-mediated activation. This suggests that KDACs play a significant role in facilitating GR signaling. We have shown that VPA does not impair GR processing and that the inhibitory effects of VPA are due to impaired transcription. We have also determined that apicidin, a structurally distinct class I-selective KDACi, impairs GR-transactivation similar to VPA, while valpromide, a structural analog of VPA without KDACi activity, does not. In addition, siRNA-mediated depletion of KDAC1 fully or partially mimics the effects of VPA at most of the VPA impaired GR-target genes and co-depletion of KDACs 1 and 2 caused full or partial impairment of Dex-activation at a few other genes. Collectively, our results show that class-I KDACs facilitate GR-mediated transcription at most of the GR-target genes and that KDAC1 alone or in co-operation with KDAC2 is required for efficient GR-mediated transactivation. Furthermore, ChIP assays have shown that active KDACs are constitutively present at the gene promoters and that KDAC inhibition does not affect GR binding to the DNA. Thus KDACs could potentially deacetylate the coregulators necessary for transcriptional activation. Finally, KDACs are known targets of a group of drugs either being used or evaluated in the treatment of cancer and other diseases. These results also pose ramifications for the clinical use of these drugs.