The mining industry has provided researchers access to the deep subsurface. The deep subsurface is known to harbor a treasure of novel genes and proteins that can be exploited in the biotechnology industry. It has been established that microorganisms in the deep subsurface are potentially novel and are able to endure high temperatures and extreme pH with limited nutrients for survival. Unfortunately most of these organisms are unculturable. Due to the lack of nutrients these microorganisms utilize reduced metals and minerals from the environment as a source of survival in a process known as biogeochemical cycling. Two fissure water samples were collected from two borehole sites at the Northam platinum mine and analyzed through molecular approaches. Microbial biodiversity was determined for borehole NO24FW030908 fissure water sample. The microbial biodiversity was based on the 16S rRNA and 18S rRNA gene clone libraries determined through phylogenetic clustering analyses using ARB software and a comparative analysis was done using DGGE profiling. The prokaryote and eukaryote diversity revealed low diversity at the species level but a high intraspecies diversity probably associated with the novelty of the biome. Unique isolates were cultured from borehole NO212FW050508 fissure water sample. Five isolates showed novelty at the species level and one isolate showed novelty at the genus level. Two isolates, Geobacillus sp. A8 and Geobacillus sp. A12 were sent for characterization at the DSMZ, Germany. Both isolates exhibited similarities at the genus level but significant differences at the species level based on the type strain to warrant different taxonomical positions. These isolates along with Thermus scotoductus SA-01 were analyzed for platinum reduction and the possible formation of metallic platinum. All isolates showed the ability to reduce platinum (IV) to platinum (0). Geobacillus sp. A8 was selected for further characterizations of platinum nanoparticle formation. Platinum nanoparticles were characterized with various tools to show the size and shape using TEM and SEM, to show the composition using XRD and EDS, and to show the particle size distribution using the NanoTrac and NiComp 380 ZLS systems. It has been proposed that a classical hydrogenase activated by a cytochrome c3 is responsible for the two-step reduction of platinum. Therefore, hydrogenase inhibition tests and the TTC test for the presence of an active hydrogenase were done to confirm the presence of a hydrogenase in Geobacillus sp. A8. It was then selected for the construction of the metabolic pathway genome database to study the metabolism of the microorganism in order to identify alternate or novel pathway(s) associated with the genome. Platinum reduction activity tests based on subcellular fractionation revealed platinum reduction and deposition that occurred in the periplasm; therefore, the putative protein involved in platinum reduction was probably periplasmic. The periplasmic fraction was separated into three fraction sizes, greater than 30 kDa, between 10 and 30 kDa and less that 10 kDa. The 10 â 30 kDa fraction revealed positive platinum reduction. The active fraction was analyzed on a SDS-PAGE and revealed three bands that were digested by trypsin and the peptides were analyzed by protein mass spectrometry. Two proteins were identified, an oxidoreductase commonly known as the old yellow enzyme previously shown to reduce chromate (VI), and a hypothetical YajQ protein. Both proteins were expressed and purified and both proteins showed the ability to reduce platinum. Further work would be to elucidate the in situ mechanism involved in the reduction of platinum with hydrogen as the electron donor.

The consumption of meat is increasingly linked to various diseases and this has already affected the growth of this sector of the food industry in some countries. Pork is seen as one of the major contributors to this problem. The meat industry reacted by using strategies such as dietary supplementation and direct addition of healthier lipids to manipulate the nutritional value of meat. The positive effects of CLA on human health are well documented and various strategies have been successfully employed in increasing the levels of CLA in different animal models such as pigs and eventually pork products. The effects CLA may have on a fermented meat product like salami has not been studied yet. No research have been reported where it was attempted to increase the nutritional value of salami, maintain acceptable product quality and include a therapeutically high level of CLA with the belief that it will benefit human health. In the first experiment of this study, 40 Duroc X Landrace gilts weighing on average 35 kg were randomly divided into two groups fed either a diet containing 0.5% sunflower oil (SFO) or a diet containing 0.5% conjugated linoleic acid (Luta-CLA® 60, BASF). These groups were further divided into two slaughter weight groups of ±70 kg and ±90 kg. After slaughter the lean meat and backfat from the loins of these animals were pooled by treatment group and utilized to manufacture salami. The aim was to determine if salami quality is influenced by slaughter weight and dietary supplementation of CLA. Both variables had major effects on the fatty acid composition and fatty acid ratios of the muscle and fat raw material as well as salami. The fatty acids and fatty acid ratios of technological importance were mostly positively influenced while the fatty acids and fatty acid ratios of nutritional and health concern were mostly negatively influenced by increased slaughter weight and dietary CLA supplementation. The microbial, physical, sensory and lipid stability parameters of salami were unaffected or inconsistently affected by both variables. Although dietary CLA was deposited successfully in muscle and fat, the deposition level was low. Consumption of a 28 g portion of salami manufactured from CLA enriched pork could only supply in 1% of the RDA for CLA. It could be concluded that although dietary supplementation of pork with CLA improved the technological properties of fat tissue it could not be considered a very successful approach to increase human consumption of CLA. In the second experiment of this study the aim was to increase the CLA content of salami to three different percentages (25%, 50% and 100%) of the RDA for CLA per 28 g portion of salami. This was accomplished through the direct addition of CLA (Tonalin® TG 80) in a pre-emulsified form with proportional decreases in the normally used pork BF content of the salamis. The salamis from these three treatment groups were then compared to a 100% pork BF control group for any possible effects on the microbial, physical and lipid stability parameters as well as fatty acid composition and fatty acid ratios. Microbial and sensory parameters were largely unaffected with varying effects on the physical and lipid stability parameters. Major effects on the fatty acid composition and fatty acid ratios of the salamis were observed. The partial replacement of pork BF and direct addition of CLA to salami proved to be an effective method of increasing CLA levels in salami in an attempt to improve the health aspects of salami to the point where it could be regarded as a functional food.

Litteraturstudier har genomförts med syftet att utveckla minst en ny detektionsmetod som skulle kunna ersätta den metod som företaget Cavidi använder sig av idag. Cavidi hade specificerat krav som metodförslaget skulle uppfylla. Dessa krav var att metoden skulle vara snabb, lättanvänd, billig, ha hög känslighet och kunna förvaras i rumstemperatur. Två lovande metoder som baseras på två olika principer valdes ut. Den ena metoden bygger på att enkelsträngat DNA med en specifik nukleinsyrakomposition syntetiseras. Denna sekvens har egenskapen att spontant bilda en sekundärstruktur som kan bilda komplex med en fluorofor. Detta ökar dess fluorescens. Ökningen detekteras med fluorescensspektroskopi. Den andra metoden baseras på ett optomagnetiskt fenomen vilket innebär att ett magnetiskt fält påverkas av polariserat ljus. Metoden går ut på att virioner först renas fram från blodplasma och att de fäster på jonbytarkulor under rådande buffertförhållanden. Magnetiska nanopartiklar tillsätts som binder till jonbytarkulornas lediga ytor. Om många virioner har bundit till jonbytarkulorna finns det en större mängd fria nanopartiklar i lösningen. Antalet fria nanopartiklar i lösningen är proportionellt mot mängden HIV i provet och kan då detekteras med en fotodetektor. Fördelarna med dessa metoder är att processen blir billigare, snabbare och har en hög känslighet. Metoderna är lättanvända och använder färre komponenter jämfört med Cavidis nuvarande metod. Därmed blir Cavidis produkter billigare och tillgängliga för fler människor.

MSc., Faculty of Science, University of the Witwatersrand, 2011 / Gold has for many years been regarded as being inert and catalytically inactive compared to the PGMs (platinum group metals). However, in the past decade it has attracted a lot of interest as both a heterogeneous and a homogenous catalyst and has been shown to catalyse a wide range of reactions e.g. oxidation, hydrogenation and reduction among others. Highly dispersed gold nanoparticles on metal oxides, like titanium oxide (Degussa, P25) have predominantly been studied because they yield some of the most active and stable catalysts. Modification of the catalysts and/or supports has been shown to affect their catalytic properties. Likewise, perovskites, which can be manipulated by partial substitution, are reported to be active supports for CO oxidation, but only at high temperatures with no activity shown for temperatures below 200°C. In this study, these perovskites were investigated at low temperatures (below 100°C) with improved activity found upon gold deposition. The presence of gold nanoparticles therefore significantly enhanced the catalytic activity, while the support itself was suspected to be involved in the reaction mechanism. A series of perovskites of the type ABO3 (LaMnO3, LaFeO3, LaCoO3 and LaCuO3) were prepared using the citrate method, while the gold was deposited on them using the deposition-precipitation method. The supports were calcined at different temperatures for optimisation. The catalysts were tested for carbon monoxide oxidation and the active catalysts characterised by XRF, XPS, XRD, Raman spectroscopy and BET surface area measurements. With the support calcined at 800ºC, the best catalyst was then modified and compared with the unmodified catalyst. The 1-wt%Au supported on LaFeO3 was found to give the best catalytic performance. This support was then modified with various weight loadings of calcium to determine the effect of calcium on the catalytic activity. Calcium-doped materials showed decreased surface area, poorer crystallinity and a drop in catalytic activity relative to the Au-LaFeO3 which indicated the best results for CO oxidation. In addition, Au-LaFeO3 showed online stability over 21 hours. Calcining the support improved the incorporation of gold nanoparticles into the perovskite lattice, resulting in superior catalytic activity. Nevertheless, at higher calcination temperatures, the catalytic activity of Au-CaTiO3 was depressed while that of Au-LaFeO3 was enhanced. The activity of perovskites increased upon gold deposition. XPS, revealed that in the active catalysts, both cationic and metallic gold co-existed, whilst in the inactive catalysts the gold existed predominantly either as cationic or metallic gold.

Rapid and accurate species identification and antibiotic susceptibility testing are of great importance for patients with sepsis and to stop over- and misuse of antibiotics contributing to antibiotic resistance. QuickMIC™ is a rapid antibiotic susceptibility testig system based on a microfluidic technology solution developed by Gradientech that measure MICs on bacteria from positive blood culture bottles. By combining QuickMIC™ with a rapid system for detection and identification, the time to detection, identification and antibiotic susceptibiolity testing could be shortened with days compared to pipelines used today which could mean the difference of life and death for patients. The T2Bacteria® panel and T2Dx® instrument developed by T2 biosystems is an FDA-cleared test for rapid detection and identification of bacteria from whole blood based on magnetic molecular resonance technology. The time to result of the T2Dx® instrument is 3-4 hours and the time to result for QuickMIC™ is 2-4 hours. In this project, the possibilities and benefits of such a pipeline have been studied by comparison to a pipeline typically used today. Time, accuracy and practical aspects have been investigated during the project and the results are promising for future further studies.

This master thesis explores the launch process of new pharmaceuticals in Sweden. The path of a pharmaceutical from idea to innovation is a long and arduous process with only few new products actually reaching the patients in the end. Seeing as the drug development is also an expensive process, it is of importance that the products that get approval meet their expected revenue. New pharmaceuticals can also be life changing for the patient, and thus it is important that once approval is received the patients gain access to the new treatments. This study focuses on the post regulatory approval processes in Sweden, as well as activities carried out by the companies that affect the adoption of a new product. By utilizing a qualitative study, this thesis aims to describe the internal and external factors that affect the pharmaceutical launch process in Sweden. As well as exploring what future initiatives and possible changes that might affect it. Ten interviews with different company representatives as well as six interviews with governmental and regional stakeholders were analysed using grounded theory to answer what factors affect the adoption of new pharmaceuticals. Factors that were found to be important were: Utilisation of cross-functional teams, clear and simple strategy that includes the whole organisation, communicating with national and regional authorities, and get feedback from these, communicate with patient representatives and organisations as well as developing utility services for the product. From a couple of these factors a trend towards the servicification of the pharmaceutical industry was discovered.

by Chow Fung-cheung, Judy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 106-117). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF ABBREVIATIONS --- p.ix / LIST OF FIGURES AND TABLES --- p.xii / Chapter SECTION I- --- ISOLATION OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1 --- Functions of Actin --- p.1 / Chapter 1.1.1 --- Functions of Actin in Animals --- p.1 / Chapter 1.1.2 --- Functions of Actin in Plants --- p.2 / Chapter 1.1.2.1 --- In Lower Plants --- p.2 / Chapter 1.1.2.2 --- In Higher Plants --- p.2 / Chapter 1.2 --- Molecular Studies of Actin Gene Families in Plants --- p.4 / Chapter 1.2.1 --- Multigene Family --- p.4 / Chapter 1.2.2 --- Homologies Across Kingdom --- p.4 / Chapter 1.2.3 --- Homologies Within Kingdom --- p.5 / Chapter 1.2.4 --- Position of Intron --- p.5 / Chapter 1.2.5 --- Differential Expression of Actin Genes --- p.7 / Chapter 1.3 --- Objectives of Present Studies --- p.7 / Chapter CHAPTER 2: --- GENERAL TECHNIQUES / Chapter 2.1 --- Growth of Algal Strain --- p.9 / Chapter 2.2 --- Growth of Bacterial Strains --- p.10 / Chapter 2.3 --- Agarose Gel Electrophoresis --- p.10 / Chapter 2.4 --- Restriction Enzyme Digestion --- p.10 / Chapter 2.5 --- Recovery of DNA Fragments from Agarose Gel --- p.11 / Chapter 2.5.1 --- Glass Powder Elution of DNA --- p.11 / Chapter 2.5.2 --- Sephaglas´ёØ BandPrep Kit --- p.11 / Chapter 2.6 --- Large Scale Preparation of Plasmid by Using Magic´ёØ Maxipreps DNA Purification System --- p.12 / Chapter 2.7 --- Ligation --- p.13 / Chapter 2.8 --- Preparation of Competent Cells --- p.13 / Chapter 2.9 --- Transformation of Competent Cells --- p.14 / Chapter 2.10 --- Screening of Recombinant Plasmids --- p.14 / Chapter 2.11 --- Spun Column Techniques --- p.15 / Chapter CHAPTER 3: --- PCR-CLONING OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter 3.1 --- Introduction --- p.16 / Chapter 3.2 --- Materials and Methods --- p.16 / Chapter 3.2.1 --- Preparation of Genomic DNA from C. vulgaris --- p.16 / Chapter 3.2.2 --- Amplification of Actin Genomic Fragments by PCR --- p.17 / Chapter 3.2.3 --- Cloning of PCR Products --- p.17 / Chapter 3.2.4 --- Southern Blotting --- p.18 / Chapter 3.2.5 --- Radiolabeling of DNA Probe --- p.19 / Chapter 3.2.6 --- Prehybridization and Hybridization --- p.19 / Chapter 3.2.7 --- Sequencing Strategies --- p.20 / Chapter 3.2.7.1 --- Isolation of Template DNA --- p.20 / Chapter 3.2.7.2 --- Template Denaturation and Primer Annealing --- p.21 / Chapter 3.2.7.3 --- Labeling and Termination Reaction --- p.21 / Chapter 3.2.7.4 --- DNA Sequencing Electrophoresis --- p.21 / Chapter 3.3 --- Results and Discussion --- p.22 / Chapter CHAPTER 4: --- CLONING OF ACTIN COMPLEMENTARY DNA FRAGMENT FROM CHLORELLA VULGARIS / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Materials and Methods --- p.31 / Chapter 4.2.1 --- Preparation of RNA --- p.31 / Chapter 4.2.2 --- RT-PCR --- p.32 / Chapter 4.2.3 --- Southern Blotting and Hybridization --- p.32 / Chapter 4.2.4 --- Radiolabeling of DNA Probe --- p.32 / Chapter 4.2.5 --- Cloning of RT-PCR Product --- p.33 / Chapter 4.2.6 --- DNA Sequencing --- p.33 / Chapter 4.2.7 --- Sequence Analysis --- p.33 / Chapter 4.3 --- Results and Discussion --- p.34 / Chapter CHAPTER 5: --- SEQUENCE COMPARISON OF ACTIN GENES / Chapter 5.1 --- Introduction --- p.44 / Chapter 5.2 --- Materials and Methods --- p.44 / Chapter 5.3 --- Results and Discussion --- p.44 / Chapter 5.3.1 --- Nucleotide Sequence Analysis --- p.44 / Chapter 5.3.2 --- Analysis of the Predicted Amino Acid Sequence --- p.49 / Chapter 5.3.3 --- Codon Usage --- p.49 / Chapter 5.3.4 --- Intron-Exon Structure in Plant Actin Genes --- p.51 / Chapter 5.3.5 --- General Discussion --- p.53 / Chapter CHAPTER 6: --- ISOLATION OF FURTHER UPSTREAM SEQUENCE FOR ACTIN GENE (CAc18G) FROM CHLORELLA VULGARIS / Chapter 6.1 --- Introduction --- p.54 / Chapter 6.2 --- Materials and Methods --- p.54 / Chapter 6.2.1 --- Genomic Southern Analysis --- p.54 / Chapter 6.2.2 --- Preparation of Actin-Enriched DNA Fraction --- p.55 / Chapter 6.2.3 --- Ligation of Actin-Enriched Fragments with Specific DNA Cassette --- p.55 / Chapter 6.2.4 --- Amplification of Upstream Sequence by Nested PCR --- p.55 / Chapter 6.2.5 --- DNA Sequencing --- p.56 / Chapter 6.3 --- Results and Discussion --- p.57 / Chapter SECTION II - --- CONSTRUCTION OF TRANSGENIC CASSETTES FOR THE PRODUCTION OF BACILLUS TOXIN IN CHLORELLA VULGARIS / Chapter CHAPTER 1： --- INTRODUCTION / Chapter 1.1 --- Characteristics of Algae --- p.64 / Chapter 1.2 --- Biotechnology Potential of Algae --- p.66 / Chapter 1.3 --- Transgenic Algae --- p.68 / Chapter 1.3.1 --- Genes of Selection for Transformant --- p.70 / Chapter 1.3.1.1 --- Homologous Genes --- p.70 / Chapter 1.3.1.2 --- Heterologous Genes --- p.70 / Chapter 1.3.2 --- Transformation Technologies Used in Algae --- p.71 / Chapter 1.3.3 --- Expression of Transgenes in Algae --- p.73 / Chapter 1.4 --- Bacillus Toxin --- p.73 / Chapter 1.4.1 --- Bacillus thuringiensis --- p.73 / Chapter 1.4.2 --- Classification of Bacillus Toxin Genes (Cry Genes) --- p.74 / Chapter 1.4.2.1 --- Type I (CtyI Genes) --- p.74 / Chapter 1.4.2.2 --- Type II (CryII Genes) --- p.75 / Chapter 1.4.2.3 --- Type III (CryIII Genes) --- p.76 / Chapter 1.4.2.4 --- Type IV (CryIV Genes) --- p.76 / Chapter 1.4.3 --- Mode of Action of Insecticidal Effects --- p.77 / Chapter 1.5 --- Insect-Resistance Transgenic Plants --- p.78 / Chapter 1.5.1 --- Transgenic Plants Expressing Crystal Protein Gene --- p.79 / Chapter 1.5.2 --- Problems Encountered --- p.80 / Chapter 1.6 --- Aims of Present Studies --- p.80 / Chapter CHAPTER 2: --- CONSTRUCTION OF TRANSGENIC CASSETTES / Chapter 2.1 --- Introduction --- p.82 / Chapter 2.2 --- Materials and Methods --- p.82 / Chapter 2.2.1 --- Preparation of Plasmids Involved in the Construction of Master Cassette --- p.82 / Chapter 2.2.2 --- Construction of Master Cassette --- p.83 / Chapter 2.2.3 --- Multiple Cloning Site (MCS) of Master Cassette --- p.83 / Chapter 2.2.4 --- Preparation of Plating Cells --- p.85 / Chapter 2.2.5 --- Titering --- p.85 / Chapter 2.2.6 --- Preparation of Plate Lysate --- p.86 / Chapter 2.2.7 --- Amplification of Coding Region of CryIVC Gene --- p.86 / Chapter 2.2.8 --- Cloning of PCR Products --- p.87 / Chapter 2.2.9 --- Construction of Transgenic Cassette --- p.87 / Chapter 2.2.10 --- Confirmation of the Junction Sites --- p.89 / Chapter 2.2.11 --- Testing for the Sensitivity of Algae Towards Kanamycin --- p.90 / Chapter 2.3 --- Results and Discussion --- p.91 / Chapter CHAPTER 3: --- TRANSFORMATION OF ALGAE BY ELECTROPORATION / Chapter 3.1 --- Introduction --- p.97 / Chapter 3.2 --- Materials and Methods --- p.97 / Chapter 3.2.1 --- Harvesting of Algae --- p.97 / Chapter 3.2.2 --- Electroporation at Different Field Strength --- p.98 / Chapter 3.2.3 --- Plating Culture of Algal Cells --- p.98 / Chapter 3.2.4 --- Preparation of Plasmids for Electrop oration --- p.98 / Chapter 3.2.5 --- Transformation of Algae --- p.100 / Chapter 3.2.6 --- Study on the Uptake of DNA after Electrop oration --- p.100 / Chapter 3.2.6.1 --- Genomic DNA Preparation --- p.100 / Chapter 3.2.6.2 --- Analysis of DNA Uptake --- p.101 / Chapter 3.3 --- Results and Discussion --- p.101 / REFERENCES --- p.106 / APPENDIX --- p.118

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

No description available.

This thesis addresses the need for environmentally and socially responsible sources of energy. Biofuels, made from organic matter, have recently become a viable alternative to petroleum-based fossil fuel. Sugar and starch make up the majority of feedstock used in biofuel production as it is easily digested. However, the use of these feedstocks is problematic as they consume resources with negative implications. By using a bacterium able to utilise five and six carbon sugars, such as the thermophile Geobacillus thermoglucosidans, organic lignocellulosic waste material can be used as a feedstock. The aim of this project was to investigate and utilise key genetic regulators of fermentation in G. thermoglucosidans and to construct genetic engineering tools that enable strain development for second generation biofuel production. We have focused on the redox-sensing transcriptional regulator Rex, widespread in Grampositive bacteria, which controls the major fermentation pathways in response to changes in cellular NAD+/NADH ratio. Following the identification of several members of the Rex regulon via bioinformatics analysis, ChIP-seq and qRT-PCR experiments were performed to locate genome-wide binding sites and controlled genes in G. thermoglucosidans. Initial electromobility shift assay experiments were performed to demonstrate the potential for use of Rex from Clostridium thermocellum as an orthogonal regulator. To further this research, novel in vivo synthetic regulatory switches were designed and tested with the aim of controlling gene expression in response to changes in cellular redox state. In addition, new tools for the efficient genetic engineering of G. thermoglucosidans were produced and optimised, including an E. coli-G. thermoglucosidans conjugation method for plasmid transfer and gene disruption.