Ribonucleoproteins (RNPs) are RNA-protein complexes that carry out a variety of key cellular functions and are essential for the regulation of gene expression. Small nuclear RNPs (snRNPs) are a class of RNPs that regulate gene expression at the level of RNA processing in the nucleus. These RNPs are subject to complex and highly regulated biogenesis pathways in order to ensure sufficient snRNP levels are present within the cell. snRNPs are required for viability of all eukaryotic cells and the importance of proper snRNP function in vivo is further highlighted by the fact that the fatal motor neuron disease spinal muscular atrophy (SMA) is caused by a genetic deficiency in the ubiquitously expressed survival motor neuron (SMN) protein, an essential component of the snRNP biogenesis machinery. The most well characterized targets of SMN for RNP assembly are the spliceosomal snRNPs, which are critical factors that carry out pre-mRNA splicing. However, SMN is not believed to be solely dedicated to spliceosomal snRNP biogenesis but rather is thought to be a general RNP assembly machine. Yet, no other RNP targets of the SMN complex had previously been characterized in a conclusive manner. Understanding the cellular targets of SMN-mediated RNP assembly is critical for elucidating basic mechanisms of RNA regulation. Furthermore, despite increased understanding of the molecular function of SMN in spliceosomal snRNP biogenesis and the cellular basis of SMA in animal models, the molecular mechanisms through which loss of SMN function leads to motor neuron disease remain poorly defined. Thus, identifying additional RNP pathways that are dependent on SMN is key to uncover the molecular mechanisms of SMA and may also help in the design of novel therapeutic approaches to this devastating childhood disorder that is currently untreatable. In an effort to expand on the established RNP targets of SMN for assembly, in this dissertation I explore the hypothesis that SMN is required for the biogenesis and function of U7 snRNP and that disruption of this pathway induced by SMN deficiency contributes to motor neuron pathology in SMA. While structurally analogous to spliceosomal snRNPs, U7 snRNP functions not in splicing but rather in the unique 3’-end processing mechanism of replication-dependent histone mRNAs. Here, I first provide detailed molecular characterization of the in vivo functional requirement of SMN for U7 snRNP biogenesis as well as histone mRNA 3’-end processing and proper histone gene expression. I go on to demonstrate that in a mouse model of SMA U7 snRNP biogenesis and function are severely impaired by SMN deficiency and these defects occur in disease-relevant SMA motor neurons. I then describe the development of a novel molecular strategy to restore U7 snRNP activity in a setting of SMN deficiency in order to investigate the functional consequences of U7 dysfunction in SMA. Finally, I apply this U7 restoration strategy to a mouse model of SMA using AAV9-mediated gene delivery and establish that disrupted U7 activity contributes to select aspects of motor neuron dysfunction in SMA mice. Collectively, my dissertation work provides a significant expansion in our understanding of RNP pathways controlled by SMN and, for the first time, establishes the contribution of an SMN-dependent RNA pathway to SMA pathology in a mouse model of the disease that best recapitulates the human condition both genetically and phenotypically. The continuation of this work in the future not only may lead to a detailed molecular understanding of the mechanisms of SMA but possibly also to the development of novel therapeutic approaches for this deadly disease that are complementary to SMN upregulation.

Piu Wong. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 126-146). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xiii / List of figures --- p.xv / List of Tables --- p.xvii / Table of contents --- p.xviii / Chapter 1 --- Literature review / Chapter 1.1 --- Overviews --- p.1 / Chapter 1.1.1 --- Nutritional and economical significance of aspartate family amino acidsin human and animal nutrition --- p.1 / Chapter 1.1.2 --- Synthesis of aspartate family amino acids in plants --- p.2 / Chapter 1.2 --- Regulation of aspartate family amino acids between sink and source organs --- p.6 / Chapter 1.2.1 --- Co-ordination of genes/enzymes involved in amide amino acid metabolism to channel aspartate for aspartate family amino acid synthesis --- p.6 / Chapter 1.2.2 --- Sink-source regulation as a general mechanism in plants --- p.9 / Chapter 1.3 --- Source regulation at free amino acid level --- p.11 / Chapter 1.3.1 --- Regulation of free methionine synthesis --- p.11 / Chapter 1.3.1.1 --- Competition for OPHS between TS and CGS --- p.11 / Chapter 1.3.1.2 --- Turnover of CGS mRNA --- p.12 / Chapter 1.3.1.3 --- Post-translational regulation of CGS enzyme --- p.13 / Chapter 1.3.2 --- Regulation of lysine synthesis and catabolism --- p.15 / Chapter 1.3.2.1 --- Feedback regulation loop --- p.15 / Chapter 1.3.2.2 --- Possible intracellular compartmentalization of enzymes and metabolitesin regulating lysine level --- p.21 / Chapter 1.3.2.3 --- Co-ordination of gene/enzyme in aspartate kinase pathway in regulating flux to Lys --- p.21 / Chapter 1.3.3 --- Significance of lysine catabolism in mammals and plants --- p.24 / Chapter 1.3.3.1 --- Complex developmental regulation and stress response of LKR/SDH gene expression --- p.28 / Chapter 1.3.3.2 --- Regulation through a novel composite locus LKR-SDH --- p.28 / Chapter 1.3.3.3 --- Post-translational control of LKR-SDH activity --- p.31 / Chapter 1.3.3.4 --- Implication of two metabolic flux in Lys catabolism --- p.34 / Chapter 1.4 --- Source (free lysine) enhancement in transgenic plants --- p.36 / Chapter 1.4.1 --- Expression of feedback insensitive enzyme in transgenic plants to enhance free lysine supply in transgenic plant --- p.36 / Chapter 1.4.2 --- Reducing or eliminating lysine catabolism to enhance free lysine poolin transgenic plants --- p.40 / Chapter 1.5 --- Sink regulation --- p.41 / Chapter 1.5.1 --- Engineering transgenic plants through expression of seed storage protein (sink) --- p.41 / Chapter 1.5.2 --- "Dynamic relationship between sink protein, nitrogen metabolism and sulphur metabolism" --- p.45 / Chapter 1.6 --- Transgenic plants with improved source or enhanced sinks related to aspartate family amino acids available for our research --- p.47 / Chapter 1.6.1 --- Enhanced source: ASN1 over-expressers --- p.47 / Chapter 1.6.2 --- Enhanced source: metL transgenic plants --- p.47 / Chapter 1.6.3 --- Altered source: RNAi line --- p.47 / Chapter 1.6.4 --- Effective sink: LRP transgenic plants --- p.48 / Chapter 1.7 --- Overall concept of this study --- p.48 / Chapter 2 --- Materials and methods --- p.50 / Chapter 2.1 --- Materials and growth conditions --- p.50 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.50 / Chapter 2.1.2 --- Chemicals and reagents used --- p.53 / Chapter 2.1.3 --- Solutions used --- p.53 / Chapter 2.1.4 --- Commercial kits used --- p.53 / Chapter 2.1.5 --- Equipment and facilities used --- p.53 / Chapter 2.1.6 --- Growth condition --- p.53 / Chapter 2.1.7 --- Tagging of A. thaliana siliques of different developmental stage --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Expression pattern analysis --- p.55 / Chapter 2.2.1.1 --- RNA extraction --- p.55 / Chapter 2.2.1.2 --- Generation of single-stranded DIG-labelled ASN1 DNA probes --- p.55 / Chapter 2.2.1.3 --- Testing the concentration of DIG-labelled probes --- p.56 / Chapter 2.2.1.4 --- Northern blot --- p.57 / Chapter 2.2.1.5 --- Hybridization --- p.58 / Chapter 2.2.1.6 --- Stringency washes --- p.58 / Chapter 2.2.1.7 --- Chemiluminescent detection --- p.58 / Chapter 2.2.2 --- Amino acid analysis and nitrogen determination --- p.60 / Chapter 2.2.2.1 --- Free amino acids in A. thaliana --- p.60 / Chapter 2.2.2.2 --- Phloem exudates collection from A. thaliana --- p.60 / Chapter 2.2.2.3 --- Soluble Protein quantitation --- p.61 / Chapter 2.2.2.4 --- Extraction of salt and water soluble protein from A. thaliana seeds --- p.61 / Chapter 2.2.2.5 --- Purification and amino acid analysis of protein extracts from A. thaliana seeds --- p.62 / Chapter 2.2.2.6 --- Total amino acid determination in mature dry seeds --- p.63 / Chapter 2.2.3 --- Generation of crossing progenies --- p.64 / Chapter 2.2.3.1 --- Artificial crossing of A. thaliana --- p.64 / Chapter 2.2.3.2 --- CTAB extraction of genomic DNA --- p.64 / Chapter 2.2.3.3 --- PCR screening for successful crossing --- p.65 / Chapter 2.2.4 --- Generation of transgenic plants --- p.67 / Chapter 2.2.4.1 --- Cloning of E.coli dapA gene --- p.67 / Chapter 2.2.4.2 --- Preparation of recombinant plasmid --- p.68 / Chapter 2.2.4.3 --- Gene sequencing --- p.68 / Chapter 2.2.4.4 --- Homology search of differentially expressed genes --- p.69 / Chapter 2.2.4.5 --- Construction of chimeric dapA genes (TP-Phas-dapA) --- p.69 / Chapter 2.2.4.6 --- Transformation of electro-competent Agrobacterium cell --- p.73 / Chapter 2.2.4.7 --- Transformation of A. thaliana through vacuum infiltration --- p.73 / Chapter 2.2.4.8 --- Selection of hemizygous and homozygous transgenic plants --- p.74 / Chapter 2.2.4.9 --- Expression analysis of homozygous LRP/dapA transgenic plants --- p.75 / Chapter 3 --- Results --- p.77 / Chapter 3.1 --- Characterization of ASN1 over-expressers --- p.77 / Chapter 3.1.1 --- Overexpression of the ASN1 gene enhances the sink-source relationship of asparagine transport under regular daylight cycle --- p.88 / Chapter 3.1.2 --- Spatial distribution of total free amino acids under normal daylight cycle --- p.88 / Chapter 3.1.3 --- Over-expression of the ASN1 gene affects free amino acid level quantitatively under normal daylight cycle --- p.89 / Chapter 3.1.4 --- Over-expression of the ASN1 gene affects composition of total amino acid under normal daylight cycle --- p.89 / Chapter 3.2 --- Construction of dapA transgenic Arabidopsis --- p.91 / Chapter 3.2.1 --- Construction of chimeric gene for expression of the dapA gene --- p.91 / Chapter 3.2.2 --- Transformation of p1300/Phas-dapA into Arabidopsis and selection of homozygous progenies --- p.91 / Chapter 3.3 --- Generation of transgenic plants with altered sink-source relationship through crossing and in-planta transformation --- p.96 / Chapter 3.3.1 --- Rationale in methods for generating transgenic plants with different combination of sources and sinks --- p.96 / Chapter 3.3.2 --- Screening for double homozygous progenies through crossing --- p.98 / Chapter 3.3.3 --- Screening for F1 progenies of successful crossing --- p.100 / Chapter 3.3.4 --- Selection of homozygous crossing progenies --- p.102 / Chapter 3.3.5 --- Screening for homozygous dapA/LRP transgenic plants --- p.104 / Chapter 3.4 --- Amino acid composition analysis --- p.109 / Chapter 3.4.1 --- The change of aspartate family amino acids in mature seeds of transgenic plants with altered sources --- p.113 / Chapter 3.4.2 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.114 / Chapter 3.4.3 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.115 / Chapter 4. --- Discussion / Chapter 4.1 --- Characterization of ASN1 over-expressers --- p.116 / Chapter 4.1.1 --- Possible regulation of ASN1 mRNA stability through level of asparagine --- p.117 / Chapter 4.1.2 --- Over-expression of ASN1 gene may improve nitrogen remobilisation from source to sink tissues --- p.118 / Chapter 4.1.3 --- Over-expression of ASN1 gene has modified the composition of amino acidsin sink organs --- p.119 / Chapter 4.2 --- ASN1 RNAi transgenic plants increases the relative contents of lysine in the seeds --- p.122 / Chapter 4.2.1 --- Role of ASN1 in supplying or competing aspartate in developing seeds --- p.122 / Chapter 4.2.2 --- Possible role of glutamate receptor --- p.123 / Chapter 4.3 --- Lysine catabolism may strictly control the level of lysine --- p.123 / Chapter 4.3.1 --- Possible role of lysine-tRNA in protein synthesis --- p.124 / Chapter 5. --- Conclusion and prospective --- p.125 / References --- p.126 / Appendix --- p.147

Wong Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 73-85). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Abbreviations --- p.viii / Chapter 1 --- Introduction --- p.1 / Overview --- p.1 / Chapter a. --- Steroidogenesis --- p.1 / Chapter i --- Ectopic steroidogenesis --- p.2 / Chapter ii --- Steroidogenesis in the pituitary --- p.5 / Chapter iii --- Proteins and enzymes involved in steroidogenesis and their distribution in brain and other sites --- p.6 / Chapter iv --- Regulation of adrenal steroidogenesis --- p.11 / Chapter b. --- Regulation of pituitary ACTH secretion --- p.13 / Chapter i --- Hypothalamic regulation --- p.13 / Chapter ii --- Paracrine regulation --- p.14 / Chapter iii --- Feedback regulation --- p.15 / Chapter c. --- Aims of the study --- p.15 / Chapter 2 --- Materials and methods --- p.18 / Chapter a. --- Materials --- p.18 / Chapter b. --- In vivo experiments --- p.25 / Chapter i --- Steroidogenic enzyme mRNA expression in rat and mouse pituitary --- p.25 / Chapter ii --- "Effects of hormonal treatments on weights of body, adrenal glands and testis, and steroidogenic enzyme mRNA expression in rats" --- p.25 / Chapter iii --- Effects of adrenalectomy and/or gonadectomy on the expression of steroidogenic enzyme mRNAs in rat pituitary --- p.26 / Chapter c. --- In vitro experiments --- p.26 / Chapter i --- Effects of CRF and forskolin on cAMP production in mouse and rat pituitary cell-lines --- p.28 / Chapter ii --- Expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.28 / Chapter iii --- Effects of CRF on the expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.29 / Chapter d. --- Statistical analysis --- p.29 / Chapter 3 --- Results --- p.30 / Chapter a. --- In vivo experiments --- p.30 / Chapter i --- Steroidogenic enzyme mRNA expression in rat and mouse pituitary --- p.30 / Chapter ii --- "Effects of hormonal treatments on weights of body, adrenal glands and testis, and steroidogenic enzyme mRNA expression in rats" --- p.38 / Chapter iii --- Effects of adrenalectomy and/or gonadectomy on the expression of steroidogenic enzyme mRNAs in rat pituitary --- p.47 / Chapter b. --- In vitro experiments --- p.51 / Chapter i --- Effects of CRF and forskolin on cAMP production in mouse and rat pituitary cell-lines --- p.51 / Chapter ii --- Expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.55 / Chapter iii --- Effects of CRF on the expression of steroidogenic enzyme mRNAs in rat and mouse pituitary cell-lines --- p.57 / Chapter 4 --- Discussion --- p.58 / Further studies --- p.71 / References --- p.73 / Appendix 1 --- p.86

Xiao Ping. / "September 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 153-176) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

Abstract not available

Phosphate is an essential but often growth-limiting nutrient for bacteria. At low concentrations of phosphate in the growth medium, bacteria induce high-affinity uptake systems for phosphate, and this is usually the ABC-type phosphate specific transport system Pst. In the fully sequenced genomes of pathogenic species of mycobacteria, several copies of the genes encoding for the Pst system (pstSCAB) have been identified and some of these genes have been shown to be virulence factors. The reasons for the presence of multiple copies of pst genes in pathogenic mycobacteria are not understood, and phosphate transport by these bacteria, as well as the gene regulation involved, is poorly characterised. The fast-growing M. smegmatis contains only a single copy of the pst operon, but we recently identified a gene locus containing three genes, phnDCE, which encode for a putative ABC-type phosphate/phosphonate transport system, and a gene, phnF, which encodes for a putative transcriptional regulator of the HutC subfamily of GntR like regulators. To identify a function for the PhnDCE transport system and to characterise high-affinity phosphate transport in M. smegmatis, we created allelic exchange mutants in phnD and pstS, as well as a phnD pstS double deletion mutant. All three mutants failed to grow in minimal medium containing 10 mM phosphate, while the wildtype was able to grow in the presence of micromolar phosphate concentrations. No differences were observed in complex growth medium. Steady-state levels of [��P]-phosphate uptake were approximately 25% lower in all mutant strains as compared to the wildtype. Kinetics of phosphate uptake in the wildtype strain when grown at low phosphate concentrations (50 [mu]M P[i]) were biphasic, suggesting the presence of two inducible transport systems with apparent K[m] values of 16 [mu]M P[i] and 64 [mu]M P[i], respectively. Analysis of the kinetics of phosphate transport in the mutant strains led us to the proposition that the Pst system has an apparent Km value of ca. 16 [mu]M P[i], and the Phn system has an apparent Km of ca. 60 [mu]M P[i]. A third inducible phosphate transport system, which was active in the double mutant strain, had an apparent K[m] of ca. 90 [mu]M P[i]. Uptake of phosphate in all strains was not inhibited by the presence of excess phosphonates or phosphite, suggesting that all three transport systems were specific for phosphate. The study of phosphate transport in the presence of various metabolic inhibitors revealed that uptake by the Phn and Pst systems is driven by ATP-hydrolysis, consistent with ABC-type transport, while the third, unidentified transport system may be driven by the proton motive force. We showed that phnDCE formed an operon, and that the promoter area of the operon lies within 200 bp of the start of phnD. To investigate the regulation of the phn and pst genes, β-galacosidase activities of strains carrying transcriptional lacZ-fusions of the pstSCAB, phnDCE and phnF promoter areas, and levels of mRNA of the phn and pst genes were studied. All genes were induced when phosphate concentrations fell below a threshold value of 30 [mu]M, which coincided with a shift in the growth characteristics of M. smegmatis. Expression of the pst operon appeared to be controlled directly by the PhoPR two-component regulatory system, while the phn operon may be under direct or indirect control by PhoPR. To identify a role for PhnF in the regulation of phn gene expression, we created a phnF deletion mutant. PhnF appeared to repress transcription of phnDCE and phnF under phosphate-replete conditions. We identified two putative binding sequences for PhnF in the intergenic region between phnD and phnF with the sequence TGGTATAGACCA, which is similar to the proposed recognition consensus for HutC-like transcriptional regulators. Using site-directed mutagenesis of these sequences, we demonstrated that they are required for the repression of phnDCE and phnF. To prove PhnF binding to these potential binding sites, we attempted to express the M. smegmatis PhnF protein in E. coli, but could not obtain soluble recombinant protein. Electrophoretic mobility shift assays of the phnDCE promoter fragment using cell-free crude extracts of M. smegmatis were not successful. We propose that Pst and Phn both constitute high-affinity phosphate specific transport systems of M. smegmatis, and that a third inducible phosphate transport system is present in this bacterium. PhnF is required for repression of phnDCE and phnF transcription under phosphate-replete conditions, while induction of the pst operon, and possibly the phn operon, under phosphate-limited conditions involves the PhoPR system.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.