Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear protein that catalyzes the cleavage of NAD⁺ into nicotinamide and ADP-ribose moiety, the latter of which may be covalently attached as a branched polymer of poly(ADP-ribose) to PARP-1 itself (automodification) or to other nuclear acceptor proteins (transmodification). PARP-1 plays pivotal roles in many fundamental biological processes, including DNA repair, gene expression, cell death and cell cycle regulation. The multiple functions of PARP-1 in various cellular events correlate well to its roles in carcinogenesis, inflammatory response, neural function, and aging. PARP-1 has a modular organization comprising six independent domains (domain A-F). Each domain has its own characteristic function in PARP-1 enzymatic catalysis. In this dissertation, the solution structure of domain C was determined by multi-dimensional NMR spectroscopy. To complement the structural results, the requirement of domain C for PARP-1 catalysis was demonstrated using activity assays. This structure-function relationship study will help to unveil the mechanism of the PARP-1 reaction, and should provide valuable information for the design of more potent and selective PARP-1 inhibitors. The determination of poly(ADP-ribosyl)ation sites is critical for understanding the biological roles of this modification. However, the identification of poly(ADPribosyl)ation sites has countered some daunting technical limitations due to the difficulties resulting from the heterogenous nature of this modification. In this dissertation, a methodology based on mass spectrometry is developed and used to identify ADP-ribosylation sites within the automodification domain (domain D) of PARP-1. Using this method, we were able to unambiguously localize three ADPribosylation sites on domain D. This method can be readily applied to study the transmodification of other substrates as well as PARP-1 automodification. As many as seventeen PARP homologues exist in the human proteome. The functional redundancy of the multiple PARP proteins has complicated the analysis of mammalian PARP-1 function in vivo. We have probed the biological roles of PARP-1 using an artificial PARP-1 pathway in yeast, an organism lacking the endogenous PARP-1. Our data suggest the heterologously expressed human PARP-1 in yeast retains some similar functions as it does in mammalian cells. Furthermore, a new function of PARP-1 in ribosome biogenesis was proposed. / text

Poly(ethylene oxide) (PEO) is known for facilitating the electrospinning of biopolymer solutions, that are otherwise not electrospinnable. The objective of this study was to investigate the mechanism by which PEO enables the formation of whey protein isolate (WPI) electrospun fibers under different pH conditions. This investigation revealed that the addition of PEO increased the viscosity of WPI/PEO (10% w/w WPI; 0.4% w/w PEO) solutions. Difference in pH levels of the polymer solutions affected electrospinnability and fiber morphology. Acidic solutions resulted in smooth fibers (700 ± 105 nm) while neutral solutions produced spheres (2.0 ± 1.0 um) linked with ultrafine fibers (138 ± 32 nm). In comparison, alkaline solutions produced fibers (191 ± 38 nm) that were embedded with spindle-like beads (1.0 ± 0.5 um). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses revealed that the native globular configuration of WPI was not altered under neutral conditions. By contrast, the electrophoresis and spectrometry data indicated that WPI was denatured and hydrolyzed under acidic conditions, which facilitated the formation of smooth fibers. C13 nuclear magnetic resonance (NMR) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopies showed that the increase random coil and a-helix secondary structures in WPI contributed to the formation of bead-less electrospun fibers. Also, C13 NMR analysis showed no evidence of chemical interaction between WPI and PEO. Scanning transmission electron microscopy coupled with energy dispersive X-rays (STEM-EDAX) revealed that WPI was uniformly distributed within WPI/PEO electrospun fibers. Observations by scanning electron microscopy (SEM) and field emission scanning electron microscopy (FESEM) indicated that fibers possessed a solid core. All these findings suggested that PEO enables the formation of WPI/PEO electrospun fibers by entanglement/entrapment/deposition. Preliminary studies were conducted on hydroxypropyl methyl cellulose (HPMC). In the absence of PEO, HPMC enabled the formation of WPI electrospun fibers under acidic conditions (124 ± 46 nm). FTIR analyses indicated that there was no interaction between HPMC and WPI, suggesting that HPMC aided in the electrospinning of WPI fibers, also by entanglement/entrapment/deposition. Hence, HPMC and PEO aid in the electrospinning of WPI fibers by entanglement/entrapment/deposition, which can be manipulated by alterations in the protein configuration and solution properties. / Natural Sciences and Engineering Research Council (NSERC) of Canada and the Dairy Farmers of Ontario (DFO)

In block-selective solvent, the rod-coil block copolymers can form various micellar structures. With block copolymers that contain a conjugated polymer block, the conformation of the conjugated polymer can be reflected by spectral changes in the solution. Therefore, it is of interest to study the relationship between the spectral changes and the nature of the conjugated polymer. The fundamental physical properties of poly(3-hexyl thiophene) (P3HT) were studied. Five P3HT samples with different molecular weights were used. We have determined the relationship between physical and spectral properties of this polymer and its molecular weight. In particular, we have found that the refractive index increments, the maximum absorbance wavelength, extinction coefficients, and the emission wavelengths, increase with molecular weight. Diblock copolymers of poly(3-hexyl thiophene)-block-poly(2-hydroxyethyl methacrylate) (P3HT-b-PHEMA) were also studied. The morphological and spectral changes of these block copolymers were studied at various stages of micelle formation in block selective solvents. The relationship between the volume fraction of the P3HT block and their physical and spectral properties were also discussed. / Thesis (Master, Chemistry) -- Queen's University, 2010-03-31 11:30:44.539

A series of medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHAs) with enriched 3-hydroxynonanoate (HN) content (up to 95.8 mol% compared to 68.6 mol% without acrylic acid) or 3-hydroxyoctanoate (HO) content (up to 97.5 mol% compared to 88.0 mol% without acrylic acid) was produced in continuous culture by co-feeding fatty acid and glucose plus inhibiting fatty acid β-oxidation using acrylic acid. Using a similar feeding strategy in fed-batch fermentation, similar monomeric compositions but a higher biomass concentration and PHA content could be obtained. However, at a lower growth rate (0.15 h-1 vs. 0.25 h-1), the biomass concentration and PHA content could be greatly enhanced from 17.1 to 71.4 g L-1 and from 64.4 to 75.5%, respectively, while the HN content decreased slightly from 92.2 to 88.9 mol%. PHAs produced under acrylic acid inhibition possessed improved physical properties including a higher melting point, faster crystallization rate, and greater tensile strength at break and Young’s modulus. Two recovery methods were developed for the recovery of MCL-PHA from Pseudomonas putida KT2440. One applied acetone extraction which was capable of recovering all the PHA from the cells with a purity of 91.6% and no detectable polymer molecular weight loss using Soxhlet extraction. Further purification was achieved by redissolving in acetone and reprecipitating in cold methanol. The other method used sodium hydroxide to solubilize the non-PHA cellular material. PHA purity of about 85% was obtained from a biomass containing 65.6% PHA after treatment with 0.2 N NaOH at 22 ± 1oC for 2 h or with 0.1 N at 80 ± 1oC for 15 min. However, a treatment at 22 ± 1oC followed by a second NaOH treatment at 80 ± 1oC resulted in higher PHA purity (94.7%) with a recovery efficiency of 88%. Under these conditions, NaOH digestion had a negligible effect on PHA molecular weight. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2010-08-30 22:44:44.501

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Aliphatic polyesters prepared by the ring opening polymerization of lactones and lactides, are versatile polymers having good hydrolyzability, mechanical properties and biocompatibility. These characteristics make them a leading material in biomedical and pharmaceutical industries as a resorbable implant and a vehicle for controlled drug delivery. An extensive research effort has been made to develop new initiators, catalysts for the ring opening polymerization of cyclic esters. Many effective initiators based on alkali metals, metal oxides have been developed for anionic polymerization of lactones. The main objectives of this project were to develop a novel catalyst by utilizing fully biocompatible and non-toxic reagents for the synthesis of polycaprolactone (PCL) by ring opening polymerization of cyclic esters at reasonably low temperature and a synthesis of hybrid silica nano-composite for biomedical applications and its characterization. Silica and dry calcium hydride reagents were used to successfully prepare heterogeneous catalysts for the ring opening polymerization of cyclic ester monomer å-caprolactone at reasonably low temperature of 100 oC. Two kinds of catalyst were prepared with non-functionalized and silane functionalized silica. The GP silane functionalized silica catalyst showed higher activity and higher product yield as compared to non-functionalized catalyst during polymerization at the same temperature. The in-situ polymerization kinetics of both reactions was studied using Raman spectroscopy. A silica based nano-composite was also synthesized which has a potential application in bone tissue engineering and possible drug delivery. The synthesized polyester and hybrid silica nano-composite were characterized with different analytical techniques to confirm required product formation.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.