Lai, Ka Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 114-121). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.x / Chapter Chapter1 --- General introduction --- p.1 / Chapter 1.1 --- Cell differentiation --- p.1 / Chapter 1.1.1 --- S tem cell treatments --- p.2 / Chapter 1.1.2 --- Differentiation therapy for cancer --- p.3 / Chapter 1.2 --- Cyclic adenosine monophosphate (cAMP) signaling involved in cell differentiation --- p.4 / Chapter 1.2.1 --- cAMP -signaling pathways leading to transcription activities --- p.4 / Chapter 1.2.1 --- Regulation of cell differentiation by cAMP/PKA signal --- p.5 / Chapter 1.3 --- Aim of thesis --- p.5 / Chapter Chapter2 --- "Materials, media, buffers and solutions" --- p.7 / Chapter 2.1 --- Mate rials --- p.7 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.12 / Chapter 2.2.1 --- General culture buffers --- p.12 / Chapter 2.2.2 --- Culture medium --- p.12 / Chapter 2.2.3 --- Assay buffers and solutions --- p.13 / Chapter 2.2.3.1 --- Buffers and solutions for RT-PCR --- p.13 / Chapter 2.2.3.2 --- Buffers and solutions for assay of [3H]cAMP production --- p.13 / Chapter 2.2.3.3 --- Buffers and solutions for Western blotting --- p.14 / Chapter 2.2.3.4 --- Buffers and solutions for histamine assay --- p.16 / Chapter 2.2.3.5 --- Buffers and solutions for flow cytometry --- p.17 / Chapter Chapter3 --- Methods --- p.18 / Chapter 3.1 --- Maintenance of rat pheochromocytoma (PC12) cells --- p.18 / Chapter 3.2 --- Dete rmination of AC isoforms expression in PC12 cells by RT-PCR analysis --- p.19 / Chapter 3.2.1 --- RNA isolation --- p.19 / Chapter 3.2.2 --- cDNA synthesis by reverse transcription (RT) --- p.20 / Chapter 3.2.3 --- Semi-quantitative PCR --- p.21 / Chapter 3.3 --- Maintenance of human erythroleukemia (HEL) cells --- p.23 / Chapter 3.4 --- Dete rmination of [3H]cAMP Production in HEL cells --- p.23 / Chapter 3.4.1 --- Principle of assay --- p.23 / Chapter 3.4.2 --- Column preparation --- p.24 / Chapter 3.4.3 --- Measurem ent of [3H]cAMP production in HEL cells --- p.24 / Chapter 3.4.4 --- Data analysis --- p.25 / Chapter 3.5 --- Im munodetection of STAT3 and pTyr705STAT3 by western blotting --- p.25 / Chapter 3.6 --- Harvesting of HE L cells after differentiation treatment --- p.27 / Chapter 3.7 --- Flow cyto metry analysis of HEL cells --- p.27 / Chapter 3.7.1 --- F ITC-conjugated CD41 -antibody staining --- p.28 / Chapter 3.7.2 --- P I staining --- p.28 / Chapter 3.8 --- Determination of extracellular and intracellular histamine of HEL cells --- p.29 / Chapter 3.8.1 --- Sample preparation --- p.29 / Chapter 3.8.2 --- Automated assay of histamine content --- p.30 / Chapter 3.9 --- siRNA mediated knockdown of STAT3 in HEK293 cells --- p.30 / Chapter 3.9.1 --- Culture human embryonic kidney (HEK293) cells --- p.30 / Chapter 3.9.1 --- siRNA transfection --- p.31 / Chapter Chapter4 --- mRNA expression of adenylyl cyclase isoforms during early stage of NGF-induced differentiation of PC12 cells --- p.33 / Chapter 4.1 --- Introduction --- p.33 / Chapter 4.1.1 --- Dif ferentiation of PC12 cells --- p.33 / Chapter 4.1.1.1 --- Induction of neurite outgrowth by NGF in PC12 cells --- p.33 / Chapter 4.1.1.2 --- Effect of cAMP on NGF-induced neurite outgrowth in PC12 cells --- p.34 / Chapter 4.1.1.3 --- Effect of cAMP on NGF-induced neurite outgrowth in PC12 cells --- p.35 / Chapter 4.1.2 --- Enhanced forskolin-stimulated [3H]cAMP productionin NGF-difFerentiated PC12 cells --- p.36 / Chapter 4.1.3 --- Classification of adenylyl cyclases --- p.38 / Chapter 4.1.4 --- Aims of study --- p.39 / Chapter 4.2 --- Results and discussion --- p.40 / Chapter Chapter5 --- Effect of cicaprost on PMA-mediated differentiation of human erythroleukemia (HEL) cells --- p.48 / Chapter 5.1 --- Introduction --- p.48 / Chapter 5.1.1 --- Differentiation of HEL cells --- p.48 / Chapter 5.1.2 --- Prostac yclin (PGI2) and human IP receptors --- p.49 / Chapter 5.1.3 --- Agonists and antagonists of IP receptors --- p.50 / Chapter 5.1.4 --- IP signaling in HEL cells --- p.52 / Chapter 5.1.5 --- Effect of cAMP on megakaryocytic differentiation --- p.52 / Chapter 5.1.6 --- Aims of study --- p.54 / Chapter 5.2 --- Results and discussion --- p.56 / Chapter 5.2.1 --- Preliminar y studies --- p.56 / Chapter 5.2.1.1 --- PMA induced cell adhesion and morphological change --- p.56 / Chapter 5.2.1.2 --- Cell proliferation and protein content --- p.57 / Chapter 5.2.1.3 --- IP signaling in HEL cells --- p.57 / Chapter 5.2.1.4 --- Presence of histaminase in FBS --- p.60 / Chapter 5.2.1.5 --- Summary of preliminary studies --- p.61 / Chapter 5.2.2 --- PMA -induced cell spreading of HEL cells --- p.63 / Chapter 5.2.3 --- PMA -induced DNA synthesis of HEL cells --- p.65 / Chapter 5.2.4 --- PMA -induced cell size and cell complexity of HEL cells --- p.67 / Chapter 5.2.5 --- PMA -induced CD41/CD61 expression of HEL cells --- p.69 / Chapter 5.2.6 --- PMA -induced histamine production of HEL cells --- p.72 / Chapter 5.2.7 --- IP receptor-dependent and IP receptor-independent actions of cicaprost --- p.74 / Chapter 5.2.8 --- STAT3 knockdown by siRNA --- p.75 / Chapter 5.3 --- Role of STAT3 in MK differentiation --- p.76 / Chapter 5.4 --- Summary --- p.78 / Chapter Chapter6 --- General discussions and future study --- p.105 / Chapter 6.1 --- General discussions --- p.105 / Chapter 6.2 --- Future study --- p.111 / References --- p.114

All organisms must sense and respond to their environment in order to survive. The processes that allow a living cell to sense changes in its environment, and respond appropriately are collectively referred to as ‘signal transduction’. Cyclic AMP is a ubiquitously used second messenger molecule that plays diverse roles from hormone signalling in mammalian cells to catabolite repression in enteric bacteria. In several bacterial pathogens such as Pseudomonas aeruginosa, cAMP has also been found to mediate pathogenesis, usually by regulating the production of several virulence factors aiding in colonisation of the host. Cyclic AMP signalling has been suggested to regulate the virulence of the obligate intracellular Mycobacterium tuberculosis. Mycobacteria, including M. tuberculosis, code for a large number of adenylyl cyclases, enzymes that synthesise cAMP. Of the 16 putative adenylyl cyclases encoded by M. tuberculosis H37Rv, 10 have received extensive biochemical attention. A knockout of one of these cyclases, Rv0386, resulted in compromised virulence of M. tuberculosis. Ten proteins predicted to bind cAMP and mediate its cellular roles have also been identified in M. tuberculosis. Among these are the cAMP-regulated transcription factor, CRPMt, and cAMP-regulated protein acetyl transferase, KATmt. Comparatively little information is available, however, regarding the roles of cAMP-degrading machinery in mycobacteria. Two phosphodiesterases, with modest activity against cAMP in vitro, have been identified from M. tuberculosis, and are encoded by the Rv0805 and Rv2795c loci. Of these, Rv2795c has orthologs in all sequenced mycobacterial genomes. However, Rv0805-like proteins are coded only by slow growing mycobacteria such as the M. tuberculosis-complex, M. marinum and M. leprae, several of which are human or animal pathogens. Rv0805 belongs to the metallophosphoesterase superfamily of proteins, consisting of metal-dependent phosphoesterases with substrates ranging from large polymers like nucleic acids to small molecules like cAMP and glycerophospholipids. Like other metallophosphoesterases, Rv0805 displays promiscuous substrate utilisation and efficient hydrolysis of 2’3’-cAMP in vitro. Rv0805 also hydrolyses 3’5’-cAMP in vitro. Overexpression of Rv0805 is reported to lead to reduction in intracellular cAMP levels in M. smegmatis and M. tuberculosis, suggesting that it is capable of hydrolysing cAMP in the bacterial cell as well. The structure of Rv0805 revealed a sandwich-like α/β fold, typical of metallophosphoesterases, along with a relatively flexible C-terminal domain of unknown function. Despite extensive biochemical and structural information on Rv0805 however, its roles in mycobacteria remain unknown. In this study, the cellular roles of Rv0805 are explored and using information from biochemical and structural analyses, novel activities and interactions of Rv0805 have been identified. Rv0805, when expressed in M. smegmatis, led to a reduction in intracellular cAMP, as previously reported. However, the extent of reduction was modest (~30 %) and limited to the exponential phase of growth when both Rv0805 and intracellular cAMP are at their highest levels. Overexpression of Rv0805 also resulted in hypersensitivity to cell wall perturbants like crystal violet and sodium dodecyl sulphate (SDS) indicative of a change in the properties of the cell envelope of M. smegmatis. Importantly, these effects were independent of cAMP-hydrolysis by Rv0805, as overexpression of catalytically inactive Rv0805N97A also elicited similar changes. Unexpectedly, Rv0805 was localised to the cell envelope, both in M. tuberculosis as well as in M. smegmatis. The ability of Rv0805 to localise to the cell envelope was dependent on it C-terminus, as truncation of Rv0805 in this region (Rv0805Δ10, Rv0805Δ20 and Rv0805Δ40) resulted in progressively greater enrichment in the cytosol of M. smegmatis. Overexpression of Rv0805Δ40, which was localised almost completely to the cytosol, did not result in hypersensitivity to SDS, suggesting that cell envelope localisation, rather than cAMP-hydrolysis, was crucial for the cell envelope modifying roles of Rv0805. A possible mechanism behind the cell envelope-related effects of Rv0805 overexpression was the ability of the protein to interact with the cell wall of mycobacteria in a C-terminus-dependent manner. Purified Rv0805, but not Rv0805Δ40, could associate with crude mycobacterial cell wall as well as purified cell wall core polymer (mycolyl-arabinogalactan-peptidoglycan complex) in vitro. In addition to the C-terminus, the architecture of the active site was also crucial for this interaction as mutations in the active site that compromised metal-binding also resulted in poor interaction with the cell wall. Most significant among these residues was His207, which when mutated to Ala almost completely abrogated interaction with the cell wall in vitro. Further, Rv0805H207A was unable to localise to the cell envelope when expressed in M. smegmatis, even in the presence of the C-terminus, highlighting the importance of this residue in maintaining the structural integrity of Rv0805, and demonstrating that the structure of the C-terminus, rather than its sequence alone, played a role in cell envelope localisation and interaction. In order to verify that the observed sensitivity of Rv0805-overexpressing M. smegmatis to cell wall perturbants was due to a change in cell envelope properties atomic force microscopy was employed. Two distinct modes of operation were used to analyse surface and bulk properties of the mycobacterial cell envelope. These were tapping mode phase imaging, and contact mode force spectroscopy. Using tapping mode phase imaging, it was found that the cell surface of M. smegmatis was inherently heterogeneous in its mechanical properties. Further, contact mode force-spectroscopy revealed that the cell envelope of M. smegmatis in cross-section had at least three layers of varying stiffness. Typically, a middle layer of high stiffness was observed, sandwiched between two lower stiffness layers. This organisation is reminiscent of the current model of the mycobacterial cell envelope, possessing a central polysaccharide rich layer and outer and inner lipid rich layers. Treatment of wild type M. smegmatis with cell wall-perturbing antibiotics isoniazid and ethambutol resulted in markedly altered phase images, as well as significantly lower stiffness of the bacterial cell envelopes, validating that the methodology employed could indeed be used to assess cell wall perturbation in mycobacteria. Further, M. smegmatis harbouring deletions in cell envelope biosynthesis related genes, MSMEG_4722 and aftC, showed significantly lower cell wall stiffness than wild type M. smegmatis, providing evidence that genetic perturbation of the cell wall of mycobacteria could also be studied using atomic force microscopy. While phase imaging revealed similar surface properties of Rv0805-overexpressing and control M. smegmatis, force spectroscopy revealed significantly lower cell envelope stiffness, particularly of the middle layer, of the former. Cell envelope stiffness was, however, unaffected by expression of Rv0805Δ40 in M. smegmatis, providing direct evidence for C-terminus-dependent cell envelope perturbation upon Rv0805 overexpression. Additionally, overexpression of Rv0805N97A, but not Rv0805H207A led to reduced stiffness of the cell envelope of M. smegmatis, demonstrating that the cell wall remodelling activity of Rv0805 was independent of cAMP-hydrolysis, but dependent on cellular localisation and cell wall interaction. Like in M. smegmatis, overexpression of Rv0805 also led to lower cAMP levels in M. tuberculosis. Using a microarray-based transcriptomics approach, pathways affected by Rv0805 overexpression were identified. Rv0805 overexpression elicited a transcriptional response, leading to the down-regulation of a number of virulence associated genes such as whiB7, eis, prpC and prpD. Importantly, Rv0805-overexpression associated gene expression changes did not include genes regulated by CRPMt, the primary cAMP-regulated transcription factor in M. tuberculosis. Further, Rv0805N97A overexpression in M. tuberculosis led to similar changes in gene expression as overexpression of the wild type protein. These observations reiterated that, at least upon overexpression, the effects of Rv0805 were largely independent of cAMP-hydrolysis. Using overexpression in M. smegmatis and M. tuberculosis, cAMP-hydrolysis independent roles of Rv0805 in mycobacteria were identified. To further validate these observations, a knockout strain of the Rv0805 gene was generated in M. bovis BCG, a well-established model to study M. tuberculosis. Curiously, deletion of Rv0805 did not lead to a change in intracellular cAMP levels, demonstrating that cAMP-hydrolysis by Rv0805 may not contribute to the modulation of mycobacterial cAMP levels under standard laboratory growth conditions. Rv0805 deletion led to altered colony morphology and possible reduction in cell wall thickness, reaffirming the roles of this phosphodiesterase in regulating cell envelope physiology of mycobacteria. Additionally, Rv0805 deletion also resulted in compromised growth of M. bovis BCG in fatty acid-deficient media, implicating Rv0805 as a possible regulator of carbon metabolism. In summary, this thesis explores novel links between Rv0805 and the mycobacterial cell wall and elucidates the critical importance of the C-terminus domain of this metallophosphodiesterase in modulating its cellular localisation to, and interaction with, the mycobacterial cell envelope. En route to understanding the effects of Rv0805 overexpression on the cell wall of M. smegmatis, an atomic force microscopy-based methodology to assess perturbation of the cell envelope of mycobacteria was also developed. Finally, using a combination of biochemical and genetic analyses, cellular roles of Rv0805, independent of cAMP-hydrolysis, were identified in slow-growing mycobacteria. This study therefore provides direct evidence against the sole role of this mycobacterial phosphodiesterase as a regulator of intracellular cAMP levels, and opens up new avenues to understanding the cellular functions of Rv0805 and indeed other members of the metallophosphoesterase superfamily.

Yue Gar-Lee Grace. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of figures --- p.x / List of tables --- p.xiii / List of abbreviations --- p.xiv / Chapter Chapter I: --- Introduction --- p.1 / Chapter 1.1. --- Transepithelial electrolyte transport in colon --- p.1 / Chapter 1.1.1. --- Intestinal fluid secretion --- p.1 / Chapter 1.1.2. --- Cellular mechanism of chloride secretion --- p.3 / Chapter 1.2. --- Biological activities of flavonoids --- p.6 / Chapter 1.2.1. --- Classification and general activities of flavonoids --- p.6 / Chapter 1.2.2. --- Bioavailability and pharmacokinetic properties of flavonoids --- p.8 / Chapter 1.3. --- "What is Scutellariae radix""?" --- p.9 / Chapter 1.3.1. --- Usage in Traditional Chinese Medicine --- p.9 / Chapter 1.3.2. --- Relationship with Coptidis rhizoma --- p.9 / Chapter 1.4. --- Effect of flavonoids on gastrointestinal activities --- p.12 / Chapter 1.4.1. --- Genistein and quercetin --- p.12 / Chapter 1.4.2. --- Baicalein --- p.12 / Chapter 1.5. --- Possible intracellular signaling pathway involved in the secretory response by Scutellariae radix (SR) in T84 cells --- p.14 / Chapter 1.5.1. --- Human colonic T84 cell --- p.14 / Chapter 1.5.2. --- Intracellular signaling pathway --- p.14 / Chapter 1.6. --- Aim of study --- p.17 / Chapter Chapter II : --- Methods and Materials --- p.18 / Chapter II.1. --- Culture technique of the T84 cells --- p.18 / Chapter II.2. --- Simultaneous measurement of short-circuit current (Isc) and intracellular calcium ([Ca2+]i) --- p.21 / Chapter II.2.1. --- Experimental setup --- p.21 / Chapter II.2.2. --- Preparation of the permeable supports --- p.23 / Chapter II.2.3. --- Cell seeding --- p.27 / Chapter II.2.4. --- Dye loading --- p.27 / Chapter II.2.5. --- Simultaneous measurement of Isc and [Ca2+]i- --- p.30 / Chapter II.3. --- Conventional short-circuit current (Isc) measurement --- p.34 / Chapter II.3.1. --- Experimental setup --- p.34 / Chapter II.3.2. --- Preparation of the permeable supports --- p.36 / Chapter II.3.3. --- Cell seeding --- p.36 / Chapter II.3.4. --- Measurement --- p.38 / Chapter II.4. --- Measurement of cAMP --- p.39 / Chapter II.5. --- Solutions and chemicals --- p.40 / Chapter II.6. --- Statistical analysis --- p.42 / Chapter Chapter III : --- Results --- p.43 / Chapter III. 1. --- Effects of baicalein and its interaction with calcium and cAMP-dependent secretagogues --- p.43 / Chapter III. 1.1. --- Effects of baicalein on baseline Isc and [Ca2+]i --- p.43 / Chapter III. 1.2. --- Ionic basis of baicalein-evoked Isc --- p.43 / Chapter III. 1.3. --- Effect of baicalein on carbachol-evoked Isc --- p.47 / Chapter III. 1.4. --- "Effect of baicalein on Isc stimulated by another calcium mobilizing agonist, histamine" --- p.58 / Chapter III. 1.5. --- Effect of carbachol on Isc response stimulated by baicalein --- p.61 / Chapter III. 1.6. --- Chronic effect of baicalein on carbachol-evoked increase in Isc --- p.63 / Chapter III.1.7. --- Interaction of baicalein with forskolin --- p.65 / Chapter III.2. --- Effects of baicalein on cAMP generation in T84 cells --- p.69 / Chapter III.2.1. --- Effects of baicalein on cAMP production --- p.69 / Chapter III.2.2 --- Effects of baicalein on forskolin-induced cAMP production --- p.70 / Chapter III.3. --- Effects of Scutellariae radix extract on ion transport activities in T84 cells --- p.73 / Chapter III.3.1. --- Effects of Scutellariae radix extract (SRE) on baseline Isc --- p.73 / Chapter III.3.2. --- Ionic basis of SRE-evoked Isc --- p.77 / Chapter III.3.3. --- Effects of adenylate cyclase inhibitor and PKA inhibitor --- p.77 / Chapter III.3.4. --- PKC modulation --- p.86 / Chapter III.3.5. --- Involvement of intracellular calcium --- p.86 / Chapter III.3.6. --- Involvement of cAMP --- p.94 / Chapter Chapter IV : --- Discussion --- p.98 / Chapter IV. 1. --- Effects of baicalein on ion transport in human colonic T84 cells --- p.98 / Chapter IV. 1.1. --- Roles of baicalein in chloride secretion in intestinal epithelial cells --- p.98 / Chapter IV. 1.2. --- Potentiation effect of baicalein on calcium-mediated chloride secretion --- p.100 / Chapter IV. 1.3. --- Potentiation effect of carbachol on baicalein-stimulated chloride secretion --- p.102 / Chapter IV. 1.4. --- Interaction between baicalein and forskolin --- p.104 / Chapter IV.2. --- Effects of Scutellariae radix extract on ion transport in human colonic T84 cells --- p.107 / Chapter IV.2.1 --- Characteristcs of Isc induced by Scutellariae radix extract --- p.107 / Chapter IV.2.2. --- Possible signaling mechanism involved in Isc induced by Scutellariae radix extract --- p.108 / Chapter IV.3. --- Comparison of the effects on ion transport in human colonic T84 cells produced by baicalein and Scutellariae radix extract --- p.110 / Chapter IV.3.1. --- Properties of baicalein- and Scutellariae radix extract- induced Isc response --- p.110 / Chapter IV.3.2. --- Summary --- p.111 / Chapter Chapter V : --- References --- p.113 / Publications --- p.120

A large number of evolutionarily conserved genes have been identified by comparative genomics approaches. However, a considerable fraction of these genes lack functional characterization despite the availability of several bioinformatics approaches for prediction of protein function. Moreover, with the advent of genome sequencing efforts, numerous disease associated genes have been identified. While high throughput approaches aid in identification of genes, studying individual genes is important to understand their cellular roles. During studies on cyclic AMP metabolism in mycobacteria conducted in the laboratory, a Class III cyclic nucleotide phosphodiesterase, Rv0805 was identified from Mycobacterium tuberculosis. Interestingly, additional bioinformatics analysis identified orthologs were in higher eukaryotes. These were members of the metallophosphoesterase-domain-containing protein 1 (MPPED1) and metallophosphoesterase-domain-containing protein 2 (MPPED2) family. Class III cyclic nucleotide phosphodiesterases were previously reported only in prokaryotes and are distinct from Class I cyclic nucleotide phosphodiesterases generally found in eukaryotes. Thus MPPED1 and MPPED2 proteins were the first identified eukaryotic Class III cyclic nucleotide phosphodiesterases. In humans, MPPED2 is located on chromosome 11 in the region p13-14 that has been associated with WAGR (Wilms’ tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome. Inspection of this region across sequenced mammalian genomes has revealed a shared synteny. Most interestingly, a stretch of 200 bp within the coding sequence of MPPED2 is identified to be one of 481 ultra conserved regions within the human genome. Furthermore, orthologs of MPPED2 can be traced all the way back to Drosophila melanogaster and Caenorhabditis elegans. All of these observations indicate that MPPED2 is highly conserved and hints at its likely importance in many organisms. MPPED1 and MPPED2 have been reported to be expressed in adult and fetal brain respectively and have been annotated as metallophosphoesterases. Metallophosphoesterases are a superfamily of proteins that show wide phyletic distribution and exhibit diversity in their substrate utilization and function. Previous studies from the laboratory have shown that MPPED1 and MPPED2 are indeed metallophosphoesterases and demonstrate cyclic nucleotide phosphodiesterase activity. The crystal structure of MPPED2 was obtained in collaboration with Dr. Marjetka Podobnik (National Institute of Chemistry, Slovenia). Interestingly, the crystal structure of MPPED2 revealed the presence of bound 5’GMP molecule at the active site, and this finding was investigated further in this thesis. MPPED2 bound 5’GMP and 5’AMP with high affinity (IC50 of ~70 nM) which inhibited the activity of MPPED2. Key residues involved in stabilising the 5’ nucleotide have been identified by structure guided mutational analysis. The MPPED2-G252H mutant, generated to mimic the active site of MPPED1, also bound 5’GMP or 5’AMP but with much lower affinity. Given the high affinity of MPPED2 towards 5’GMP/5’AMP, it can be speculated that MPPED2 may show poor phosphodiesterase activity in the cell, and could function in a catalytically-independent manner, perhaps as a scaffolding protein. MPPED1 on the other hand may have a catalytic role that could be regulated by intracellular levels of 5’AMP, 5’GMP and their respective cyclic nucleotides. In order to investigate the biological role of the MPPED1/MPPED2 family of proteins, Drosophila melanogaster was chosen as a model organism owing to the presence of a single ortholog, CG16717, in its genome. Biochemical characterization of CG16717 revealed that the protein was in fact a metallophosphodiesterase capable of hydrolysing cyclic AMP and cyclic GMP, albeit poorly. CG16717 could be inhibited by 5’ nucleotides at high concentrations that may seldom be achieved in-vivo, suggesting that CG16717 may have roles in the organism that depend on its catalytic activity. CG16717 has not been functionally characterized previously. In this thesis, a detailed analysis of CG16717 expression pattern has been performed. CG16717 was found to be expressed in all stages of the fly lifecycle. In adult female flies, levels of CG16717 increased across age. Moreover, CG16717 was not differentially regulated under conditions of starvation, paraquat-induced oxidative stress or in the presence of heavy metals. Spatial expression analysis revealed that CG16717 was expressed in all adult tissues tested, with maximal expression in the brain, suggesting that neuronal expression of CG16717 may be important for its function. Attempts to identify specific cells expressing CG16717 using an enhancer-promoter analysis were not successful. In order to elucidate the physiological role of CG16717, and after having ruled out options of using a P-element insertion mutant and RNA interference approaches, a targeted knock-out of CG16717 was generated using homologous recombination based genomic engineering. CG16717KO flies generated were homozygous viable suggesting that CG16717 was dispensable for fly survival at least under normal laboratory conditions. In line with high expression of CG16717 in the brain and in-vitro ability of CG16717 to hydrolyse cAMP and cGMP, CG16717KO flies showed two to three-fold higher levels of cyclic nucleotides in the head fraction than wild-type flies. C25E10.12, one of the three C. elegans orthologs of CG16717 has been identified to be a target of the transcription factor daf-16 (FOXO) that is inhibited by active insulin signalling. Moreover, knock-down of C25E10.12 reduced the lifespan of age-1 (PI3K) mutant worms. In contrast to this, CG16717 was not found to be differentially regulated in dFOXO null flies. CG16717KO flies however, showed median lifespan that was shorter than control wild-type flies even in the presence of functional PI3K. Various genetic approaches were employed to verify if reduced lifespan was indeed a consequence of loss of CG16717. In the first approach, a wild-type copy of CG16717 was re-introduced at the genomic locus of CG16717 in the CG16717KO flies using attP-attB recombination. However, this approach could not rescue the reduced lifespan of CG16717KO flies, probably due to very low expression of CG16717. In the second approach, CG16717 was reconstituted using genomic constructs containing a copy of CG16717. Finally, CG16717 was expressed ubiquitously using the bipartite Gal4/UAS system. Both the genomic construct and the expression of CG16717 using the Gal4/UAS approach were able to restore the lifespan of CG16717KO flies. More importantly, overexpression of CG16717 in an otherwise wild-type fly led to enhanced lifespan over and above that of control flies. All of these together suggested that CG16717 plays a critical role in regulating lifespan. Mutants of the insulin and target of rapamycin (TOR) signalling pathways have previously been reported to show lifespan extension. Moreover, these mutants have also been associated with reduced growth, increased stress resistance and reduced fecundity. Given the reduction in lifespan of CG16717KO flies, the other insulin/TOR signalling associated phenotypes were tested. While CG16717KO flies showed no difference in terms of developmental growth, and resistance to starvation or paraquat induced oxidative stress, CG16717KO flies were less fecund compared to wild-type controls. Multiple approaches were adopted even in the case of reduced fecundity to verify if the observed phenotype was a consequence of loss of CG16717. However, neither reconstitution of CG16717 using the genomic construct nor ubiquitous expression of CG16717 using the bipartite Gal4/UAS system were able to rescue the reduced fecundity phenotype of CG16717KO flies. This suggested that reduced fecundity in CG16717KO flies was probably not linked to CG16717 and was a consequence of a second mutation at a site distinct from CG16717. Two other approaches were employed to confirm these observations. When CG16717KO/Deficiency lines were tested, these showed fecundity comparable to wild-type control flies despite the lack of CG16717. CG16717KO flies were extensively out-crossed in an attempt to segregate the second site mutation from the CG16717 locus and their fecundity was tested. However, these flies which retained the deletion of CG16717, showed fecundity comparable to wild-type control flies, reiterating that reduced fecundity was not linked to loss of CG16717. In an attempt to find possible links between reduced longevity of CG16717KO flies and the well-established insulin/TOR pathways, transcript levels of key players of these pathways were measured by qRT-PCR. The translational repressor 4EBP was found to be upregulated in CG16717KO flies compared to wild-type control flies. Interestingly, increased 4EBP levels have been associated with enhanced lifespan but in this case despite higher levels of 4EBP, CG16717KO flies showed reduced lifespan. Phosphorylation status of 4EBP and other players involved in the insulin/TOR phosphokinase signalling cascade would shed light on the activity of these pathways. In summary, this thesis has attempted to understand the biochemistry and physiological functions of an evolutionarily conserved metallophosphoesterase. Its apparent role in regulating life span in the fly suggests that the functions of this protein are likely to impinge on a number of diverse and important pathways involved in basic physiological processes in the organism. Further investigation would shed light on the molecular basis by which CG16717 affects lifespan, and opens up new avenues to understanding the contributions of CG16717 in regulating lifespan and diverse neurological functions.

Wong Cheuk Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 101-124). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vi / List of Abbreviations --- p.vii / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Methods --- p.4 / Chapter 2.1 --- Measurement of cAMP and cGMP in VSMCs --- p.4 / Chapter 2.1.1 --- Cell culture --- p.4 / Chapter 2.1.2 --- Enzyme-immunoassay colorimetric measurement for cAMP and cGMP --- p.5 / Chapter 2.1.3 --- Statistical analysis --- p.6 / Chapter 2.2 --- Measurement of apoptosis in VSMCs and bone marrow-derived stem cells --- p.6 / Chapter 2.2.1 --- Cell culture --- p.6 / Chapter 2.2.2 --- Hoechst33258 --- p.7 / Chapter 2.2.3 --- Cell Death ELISA plus --- p.7 / Chapter 2.2.4 --- Protein extraction and Western blot analysis of PKG expression --- p.8 / Chapter 2.2.5 --- Statistical analysis --- p.9 / Chapter 2.3 --- Measurement of cell proliferation in VSMCs and bone marrow-derived stem cells --- p.9 / Chapter 2.3.1 --- Cell culture --- p.9 / Chapter 2.3.2 --- Cell count --- p.10 / Chapter 2.3.3 --- MTT assay --- p.11 / Chapter 2.3.4 --- BrdU-(5`Bromo-2-deoxyuridine) ELISA colorimetric assay --- p.11 / Chapter 2.3.5 --- Statistical analysis --- p.12 / Chapter Chapter 3. --- Effects of hyperglycemia on CGRP-induced cAMP response in VSMCs / Chapter 3.1 --- Introduction --- p.13 / Chapter 3.2 --- Results --- p.18 / Chapter 3.3 --- Discussion --- p.22 / Chapter Chapter 4. --- Role of cGMP and protein kinase G in regulation of apoptosis in VSMCs / Chapter 4.1 --- Introduction --- p.26 / Chapter 4.2 --- Results --- p.30 / Chapter 4.3 --- Discussion --- p.44 / Chapter Chapter 5. --- Role of protein kinase G in regulation of proliferation in VSMCs / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Results --- p.58 / Chapter 5.3 --- Discussion --- p.67 / Chapter Chapter 6. --- Effects of aging and eNOS- and iNOS-gene deletion (using eNOS- and iNOS-knockout mice) on apoptosis of VSMCs / Chapter 6.1 --- Introduction --- p.73 / Chapter 6.2 --- Results --- p.76 / Chapter 6.3 --- Discussion --- p.79 / Chapter Chapter 7. --- Role of protein kinase G in regulation of apoptosis and proliferation of bone marrow stromal stem cells / Chapter 7.1 --- Introduction --- p.81 / Chapter 7.2 --- Results --- p.84 / Chapter 7.3 --- Discussion --- p.92 / Chapter Chapter 8. --- Overall discussion --- p.95 / Chapter Chapter 9. --- References --- p.101