Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2009. / Includes bibliographical references (p. 131-147).

No description available.

O gene hrcA é encontrado em quase todos os ramos da árvore filogenética das eubactérias, e seu produto, a proteína HrcA, funciona como repressor da expressão dos operons de choque térmico groESL e dnaKJ, ligando-se à seqüência repetida invertida denominada CIRCE (controlling inverted repeat of chaperonin expression) presente na região regulatória destes operons. O sistema HrcA-CIRCE está, portanto, amplamente representado nas eubactérias. Particularmente, em Caulobacter crescentus, uma α-proteobactéria, este sistema está envolvido no controle da expressão do operon groESL durante o ciclo celular da bactéria. Conhecer a estrutura e as interações de HrcA é importante para entender este processo. Neste trabalho são apresentadas as análises de seqüência das HrcA\'s de C. crescentus e de Xylella fastidiosa, uma proteobactéria do grupo γ, as quais são muito similares. Este estudo levou à proposta de um modelo estrutural com a delimitação dos domínios da proteína, os dobramentos de cada domínio, com base nas interações da HrcA de C. crescentus com o elemento CIRCE e ATP, que estão sendo caracterizadas em nosso laboratório, assim como a atribuição de aminoácidos e motivos conservados funcionais. Adicionalmente, embora a expressão da HrcA recombinante de X. fastidiosa não tenha tido sucesso, a HrcA recombinante de C. crescentus purificada tem se prestado aos ensaios espectroscópicos, ainda que tenha sido detectada uma microagregação que está sendo enfrentada com um protocolo de purificação baseado no uso de α ciclodextrina. Os estudos espectroscópicos preliminares da HrcA C. crescentus dão suporte ao modelo estrutural proposto. / The hrcA gene is found in almost all branches of the filogenetic tree of eubacteria, and its product, the protein HrcA, functions as a repressor regulating the expression of the heat shock operons groESL and dnaKJ, by binding to the inverted repeat sequence called CIRCE (controlling inverted repeat of chaperonin expression). The system HrcA-CIRCE, therefore, is widely represented in eubacteria. Specifically in Caulobacter crescentus, an α-proteobacterium, this system is involved in the cell-cycle control of groESL expression (Baldini et al, 1998). Knowledge of the structure of HrcA and its interactions is important to understand this process. This work presents the analysis of the sequences of HrcA from C. crescentus and Xylella fastidiosa, a proteobacterium of the γ group, which are very similar. A structural model has been proposed, with protein domain delimitation, specific domain folding, based on known interactions of C. crescentus HrcA with the CIRCE element and ATP, obtained in our laboratory, as well as assignment of functional residues and conserved motifs. Additionally, even though no sucess was obtained the expression of recombinant HrcA from X. fastidiosa, purified recombinant HrcA from C. crescentus has been shown to be suitable for spectroscopic studies, in spite of microagregation observed, which is being faced with a purification protocol based on the use of α cyclodextrin. The preliminary spectroscopic studies of HrcA from C. crescentus support the proposed structural model.

Fong, Yu Hang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 102-107). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong, Ho Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 103-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

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

Wan Tai Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 102-111). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgements --- p.vii / General Abbreviations --- p.ix / Abbreviations of Chemicals --- p.xi / Table of Contents --- p.xii / List of Figures --- p.xvi / List of Tables --- p.xvi / Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Soybeans --- p.1 / Chapter 1.1.1 --- Nutrient composition of soybean --- p.1 / Chapter 1.1.2 --- Nitrogen fixation and assimilation in soybean --- p.3 / Chapter 1.1.3 --- The role in nitrogen allocation and controlling the nitrogen sink-source relationship of asparagine --- p.3 / Chapter 1.1.4 --- Characterization of asparagine synthetase --- p.8 / Chapter 1.1.4.1 --- Biochemistry and molecular background of plant asparagine synthetase --- p.8 / Chapter 1.1.4.2 --- Asparagine synthetase in Arabadopsis thaliana --- p.9 / Chapter 1.1.4.3 --- "Asparagine synthesis in soybean, Glycine max" --- p.10 / Chapter 1.1.4.4 --- "Asparagine synthetase in rice, Oryza sativa" --- p.11 / Chapter 1.2 --- Seed protein quality and quantity improvement --- p.13 / Chapter 1.2.1 --- Nutrition composition of rice --- p.13 / Chapter 1.2.2 --- Molecular approaches for improving seed storage protein quality --- p.14 / Chapter 1.2.2.1 --- Protein sequence modification --- p.14 / Chapter 1.2.2.2 --- Synthetic genes --- p.16 / Chapter 1.2.2.3 --- Overexpression of homologous genes --- p.17 / Chapter 1.2.2.4 --- Transfer and expression of heterologous genes --- p.18 / Chapter 1.2.2.5 --- "Manipulation of pathway synthesizing essential amino acids, aspartate family amino acid" --- p.19 / Chapter 1.2.3 --- Research in improving rice seed protein quality and quantity --- p.22 / Chapter 1.3 --- Hypothesis and objective of this study --- p.23 / Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Plant materials --- p.25 / Chapter 2.1.2 --- Bacterial strains and vectors --- p.26 / Chapter 2.1.3 --- Growth conditions for soybean --- p.26 / Chapter 2.1.4 --- Chemicals and reagents --- p.26 / Chapter 2.1.5 --- "Buffer, solution and gel" --- p.26 / Chapter 2.1.6 --- Commercial kits --- p.27 / Chapter 2.1.7 --- Equipments and facilities used --- p.27 / Chapter 2.1.8 --- Primers --- p.27 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Growth condition for plant materials --- p.28 / Chapter 2.2.1.1 --- General conditions for planting soybean --- p.28 / Chapter 2.2.1.2 --- Soybean seedlings for gene expression profile analysis --- p.28 / Chapter 2.2.1.3 --- Mature soybean for gene expression profile analysis --- p.29 / Chapter 2.2.1.4 --- Mature soybean for cloning of AS I and AS2 full length cDNA --- p.30 / Chapter 2.2.1.5 --- Mature soybean seed for amino acid profile analysis --- p.30 / Chapter 2.2.1.6 --- General conditions for planting transgenic rice in CUHK --- p.30 / Chapter 2.2.1.7 --- Transgenic rice seedling for PCR screening --- p.31 / Chapter 2.2.1.8 --- Transgenic rice for functional test and seed for biochemical analysis --- p.31 / Chapter 2.2.2 --- Molecular techniques --- p.32 / Chapter 2.2.2.1 --- Total RNA extraction --- p.32 / Chapter 2.2.2.2 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.2.2.3 --- Northern blot analysis --- p.33 / Chapter 2.2.2.3.1 --- Chemiluminescent detection --- p.33 / Chapter 2.2.2.3.2 --- Film development --- p.34 / Chapter 2.2.2.4 --- Preparation of single-stranded DIG-labeled PCR probes --- p.34 / Chapter 2.2.2.4.1 --- Primer design for the PCR probes of --- p.34 / Chapter 2.2.2.4.2 --- Amplification of AS1 and AS2 internal PCR fragments --- p.34 / Chapter 2.2.2.4.3 --- Quantitation of purified AS1 and AS2 PCR fragments --- p.35 / Chapter 2.2.2.4.4 --- Biased PCR to make single-stranded DNA probes --- p.35 / Chapter 2.2.2.4.5 --- Probe quantitation --- p.36 / Chapter 2.2.2.5 --- Probe specificity test --- p.37 / Chapter 2.2.2.6 --- Cloning of full length cDNA --- p.37 / Chapter 2.2.2.6.1 --- First strand cDNA synthesis from RNA of high protein content soybean leaf --- p.37 / Chapter 2.2.2.6.2 --- PCR for amplification of AS1 and AS2 full length cDNA --- p.38 / Chapter 2.2.2.6.3 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.38 / Chapter 2.2.2.6.4 --- Ligation of DNA inserts into pBluescript II KS(+) T-vector --- p.39 / Chapter 2.2.2.6.5 --- Preparation of E. coli DH5α CaCl2-mediaed competent cells --- p.39 / Chapter 2.2.2.6.6 --- Transformation of E. coli DH5α competent cell --- p.40 / Chapter 2.2.2.7 --- Screening of recombinant plasmids --- p.40 / Chapter 2.2.2.7.1 --- Isolation of recombinant plasimid DNA from bacterial cells --- p.41 / Chapter 2.2.2.7.2 --- PCR screening on recombinant plasmids --- p.41 / Chapter 2.2.2.7.3 --- DNA gel electrophoresis --- p.41 / Chapter 2.2.2.8 --- Sequencing and homology search --- p.42 / Chapter 2.2.2.9 --- Functional test using transgenic plant --- p.43 / Chapter 2.2.2.9.1 --- Preparation of chimeric gene constructs and recombinant plasmids --- p.43 / Chapter 2.2.2.9.2 --- Agrobacterium mediated transformation into rice calli to regenerate transgenic AS1/ AS2 rice --- p.44 / Chapter 2.2.2.10 --- PCR Screenig of homozygous and heterozygous transgenic plants --- p.44 / Chapter 2.2.2.10.1 --- Isolation of genomic DNA from transgenic plants --- p.45 / Chapter 2.2.2.10.2 --- PCR screening using genomic DNA --- p.46 / Chapter 2.2.2.11 --- Quantitative PCR analysis on transgenic plants --- p.48 / Chapter 2.2.3 --- Biochemical Analysis --- p.49 / Chapter 2.2.3.1 --- Quantitative amino acid analysis in mature soybean seeds --- p.49 / Chapter 2.2.3.2 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.49 / Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Amino acid analysis on mature soybean seeds --- p.50 / Chapter 3.2 --- Expression pattern analysis of AS genes by Northern Blot analysis --- p.54 / Chapter 3.2.1 --- Making of single strand digoxigenin (DIG)-labeled probe --- p.54 / Chapter 3.2.2 --- Probe specificity --- p.57 / Chapter 3.2.3 --- AS expression level under light/dark treatments by Northern Blot analysis --- p.58 / Chapter 3.2.4 --- AS expression level in young seedlings by Northern Blot analysis --- p.62 / Chapter 3.2.5 --- AS expression level in podding soybean by Northern Blot analysis --- p.64 / Chapter 3.3 --- Cloning of AS genes from high protein content soybeans --- p.66 / Chapter 3.3.1 --- "PCR amplification of AS1 and AS2 full length cDNA from the first-strand cDNA of high portein content cultivar soybean, YuDoul2" --- p.66 / Chapter 3.3.2 --- Nucleotide sequences analysis of AS1 and AS2 full-length cDNA clones --- p.68 / Chapter 3.4 --- Construction of AS1 and AS2 transgenic rice --- p.75 / Chapter 3.4.1 --- Construction of AS1 and AS2 constructs --- p.75 / Chapter 3.4.2 --- Transformation of chimeric gene constructs into Agrobacterium tumefaciens --- p.75 / Chapter 3.4.3 --- Agrobacterium mediated transformation into Oryza sativa calli to regenerate transgenic rice --- p.76 / Chapter 3.4.4 --- PCR screening of transgene from transgenic AS1 and AS2 rice --- p.76 / Chapter 3.4.5 --- Quantitative PCR analysis of the transgene expression --- p.81 / Chapter 3.4.6 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.83 / Chapter 4 --- Discussion --- p.89 / Chapter 4.1 --- The role of asparagine and asparagine synthetase in nitrogen assimilation and sink-source relationship in soybean --- p.89 / Chapter 4.2 --- Comparative study of AS between different high seed protein content crops --- p.92 / Chapter 4.3 --- The attempt to find out the reason for the strong AS1 expression detected in high protein soybean cultivars --- p.92 / Chapter 4.4 --- Other factors affecting seed protein contents --- p.93 / Chapter 4.5 --- Rice seed quality improvement by nitrogen assimilation enhancement --- p.94 / Chapter 4.6 --- Comparative study of amino acid profile and seed total protein in other transgenic rice --- p.95 / Chapter 4.7 --- Possible reason of higher seed protein content in AS2 transgenic rice --- p.96 / Chapter 4.8 --- Selectable marker --- p.97 / Chapter 5 --- Conclusion and Prespectives --- p.99 / Chapter 6 --- References --- p.102 / Chapter 7 --- Appendix --- p.112 / Appendix I: Major chemicals and reagents used in this research --- p.112 / "Appendix II: Major buffer, solution and gel used in this research" --- p.114 / Appendix III: Commercial kits used in this research --- p.117 / Appendix IV: Major equipments and facilities used in this research --- p.118 / Appendix V: Primer list --- p.119

Muscle wasting is a significant complication of many diseases including diabetes mellitus, renal and liver failure, HIV/AIDS, and cancer. Sustained loss of skeletal muscle can severely impair a patient's quality of life and often results in poor tolerance and responsiveness to disease treatments. The increased protein breakdown observed during muscle atrophy has been attributed to accelerated activity of the ubiquitin-proteasome pathway, but the precise mechanisms by which this activation stimulates muscle protein loss are poorly understood. Previous work showed that the deubiquitinating enzyme USP19 is upregulated in rat skeletal muscle in various forms of muscle wasting, including streptozotocin induced diabetes, cancer, and dexamethasone treatment. 1 To further explore the role of USP19 in muscle wasting, siRNA-mediated depletion of the enzyme was carried out in L6 myotubes. Knockdown of USP19 resulted in more rapid differentiation of myoblasts into myotubes, with a greater extent of myoblast fusion. It also produced tubes that were visibly larger than those formed by myoblasts transfected with a control siRNA. At the molecular level, silencing of USP19 increased the amount of myosin heavy chain (MHC) and tropomyosin proteins. It also increased levels of MHC transcript, suggesting that USP19 acts at the level of gene transcription or mRNA stability rather than protein degradation. USP19 may mediate its effects on muscle-specific gene expression through the myogenic transcription factor myogenin, since depletion of USP19 increased protein and mRNA levels myogenin but did not affect protein levels of the related transcription factor Myf5. Moreover, the increased tropomyosin and MHC observed upon USP19 knockdown could be abolished when myogenin was simultaneously depleted using siRNA. Collectively, these results suggest that USP19 functions to inhibit the synthesis of key muscle proteins and may therefore be a promising target for the treatment of muscle atrophy.

O gene hrcA é encontrado em quase todos os ramos da árvore filogenética das eubactérias, e seu produto, a proteína HrcA, funciona como repressor da expressão dos operons de choque térmico groESL e dnaKJ, ligando-se à seqüência repetida invertida denominada CIRCE (controlling inverted repeat of chaperonin expression) presente na região regulatória destes operons. O sistema HrcA-CIRCE está, portanto, amplamente representado nas eubactérias. Particularmente, em Caulobacter crescentus, uma α-proteobactéria, este sistema está envolvido no controle da expressão do operon groESL durante o ciclo celular da bactéria. Conhecer a estrutura e as interações de HrcA é importante para entender este processo. Neste trabalho são apresentadas as análises de seqüência das HrcA\'s de C. crescentus e de Xylella fastidiosa, uma proteobactéria do grupo γ, as quais são muito similares. Este estudo levou à proposta de um modelo estrutural com a delimitação dos domínios da proteína, os dobramentos de cada domínio, com base nas interações da HrcA de C. crescentus com o elemento CIRCE e ATP, que estão sendo caracterizadas em nosso laboratório, assim como a atribuição de aminoácidos e motivos conservados funcionais. Adicionalmente, embora a expressão da HrcA recombinante de X. fastidiosa não tenha tido sucesso, a HrcA recombinante de C. crescentus purificada tem se prestado aos ensaios espectroscópicos, ainda que tenha sido detectada uma microagregação que está sendo enfrentada com um protocolo de purificação baseado no uso de α ciclodextrina. Os estudos espectroscópicos preliminares da HrcA C. crescentus dão suporte ao modelo estrutural proposto. / The hrcA gene is found in almost all branches of the filogenetic tree of eubacteria, and its product, the protein HrcA, functions as a repressor regulating the expression of the heat shock operons groESL and dnaKJ, by binding to the inverted repeat sequence called CIRCE (controlling inverted repeat of chaperonin expression). The system HrcA-CIRCE, therefore, is widely represented in eubacteria. Specifically in Caulobacter crescentus, an α-proteobacterium, this system is involved in the cell-cycle control of groESL expression (Baldini et al, 1998). Knowledge of the structure of HrcA and its interactions is important to understand this process. This work presents the analysis of the sequences of HrcA from C. crescentus and Xylella fastidiosa, a proteobacterium of the γ group, which are very similar. A structural model has been proposed, with protein domain delimitation, specific domain folding, based on known interactions of C. crescentus HrcA with the CIRCE element and ATP, obtained in our laboratory, as well as assignment of functional residues and conserved motifs. Additionally, even though no sucess was obtained the expression of recombinant HrcA from X. fastidiosa, purified recombinant HrcA from C. crescentus has been shown to be suitable for spectroscopic studies, in spite of microagregation observed, which is being faced with a purification protocol based on the use of α cyclodextrin. The preliminary spectroscopic studies of HrcA from C. crescentus support the proposed structural model.

No description available.