Hyaluronic acid (HA) is a high value biopolymer that has numerous biomedical and cosmetic applications. It is currently derived from two sources, namely animal tissues and bacterial fermentation (Fong Chong et al. 2005). The molecular weight (Mw) of HA can vary from several hundred thousand dalton (Da) to approximately 8 MDa (Widner et al. 2005). High Mw HA has surgical applications, and therefore constitutes a major component of the lucrative HA market. The current need is largely met by extraction from animal tissues, e.g., rooster comb and bovine vitreous humor (Shiedlin et al. 2004). However, the potential of contamination with adventitious agents (e.g., viruses) have raised regulatory concerns regarding the use of animal extracts in pharmaceutical products. Moreover, with recent reports of zoonotic diseases (e.g., bovine spongiform encephalitis and avian influenza virus), pharmaceutical companies are moving towards microbial HA sources. Although HA obtained from bacterial fermentation does not have the problem of viral contamination, this approach has not yet resulted in a process where HA of sufficiently high Mw for surgical applications can be derived. While attempts have been made to produce higher Mw HA through cross-linking, cross-linked HA is undesirable for certain medical procedures (e.g., ophthalmic applications) which requires a natural polymer with a short half-life. Nevertheless, due to its availability and the relative ease of purification, bacterial fermentation has the potential of replacing extraction from animal tissues as a preferred commercial source of HA. This thesis presents a good example of a metabolic engineering study where modern techniques (e.g., molecular biology, fermentation and omics technologies) are used to explain complex cell metabolism. The hypothesis for this study was that the precursors to HA, i.e., UDP-glucuronic acid and UDP-N-acetylglucosamine and consequently the genes involved in precursor generation, are important for HA Mw. However, environmental manipulation, e.g., anaerobic versus aerobic or glucose versus maltose, often results in large global changes in metabolite concentration and enzyme activities. This makes it impossible to resolve issues related to Mw control. Classical statistical methods do not provide a meaningful inference as the number of explanatory variables always exceeds the number of independent observations. Hence, it is difficult to distinguish between causative and accidental correlation. This work first examined the influence of manipulation of metabolite concentrations in the hyaluronan pathway to find an explanation for the mechanism of Mw control. To achieve this, the five essential genes of the hyaluronan synthesis (has) operon in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) were first examined. These genes are involved in two pathways which lead to the production of either UDP-glucuronic acid or UDP-N-acetylglucosamine. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased HA Mw, while overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased HA Mw. The Mw variation generated provided a stepping stone for further understanding of Mw control of HA. The highest Mw observed was achieved with combined overexpression of pgi and glmU. This study proved that there is a positive correlation between UDP-N-acetylglucosamine and Mw. The first model for HA Mw control based on the concentration of activated sugar precursors is described in this study (Chapter 3). This correlation observed led to the hypothesis that high Mw HA can be achieved when an appropriate balance of the two HA precursor is maintained. Three genes in the two precursor pathways are not found in the has operon of S. zooepidemicus. To obtain a complete overview of all genes in the HA pathway, these genes were also examined using overexpression studies. Individual overexpression of these genes had negligible effects on HA Mw and production. Despite the positive correlation previously observed between UDP-N-acetylglucosamine and Mw, sequential overexpression of genes involved in the UDP-N-acetylglucosamine precursor pathway did not increase Mw of HA produced. This is surprising since the highest pool of UDP-N-acetylglucosamine was achieved in this case. This suggests that a threshold effect is present in the correlation between UDP-N-acetylglucosamine and Mw. This threshold effect may be defined by a balance between the two precursors. To investigate this phenomenon further, the precursor ratio was also manipulated by co-metabolising glucose and N-acetylglucosamine. Similar to the previous experiment, a significant increase in UDP-N-acetylglucosamine levels was observed despite only a marginal increase in Mw (Chapter 4). Surprisingly, an increase in Mw was observed with the introduction of a plasmid in S. zooepidemicus. This plasmid effect was studied on a global scale using transcriptome and proteome analysis to understand the changes occurring in the system. The increase in Mw due to the plasmid effect is independent of the functions, i.e., nisin promoter or antibiotic resistance, encoded in the plasmid. A gene involved in UDP-N-acetylglucosamine production, UDP-N-acetylglucosamide 1-carboxivinyltransferase (murA), was significantly down-regulated in both the plasmid bearing strain and the high Mw strain (pgi). In addition, overexpression of murA decreased both the concentration of activated sugar precursors and HA Mw. There was however no evidence of down-regulation of murA in the plasmid containing strain from transcriptomics data. This suggests that control is exerted either at the translation level or by protein degradation (Chapter 5). This thesis contributes and represents an ongoing effort to understand the elusive mechanism of Mw control of HA.

Soluble, thermally stable phosphorus containing polyimides have been synthesized to controlled molecular weights and well-defined phthalimide endgroups via solution imidization. They were characterized by intrinsic viscosity, DSC, TGA, and thermomechanical measurements. Molecular weight control was successful and these polyimides were thermally stable via dynamic TGA up to 500°C. Flame resistance was suggested by the 10-20 % of char yield observed in air at 750°C. Good tensile modulus (500-550 ksi) via stress-strain measurements and excellent adhesion to Ti-6AI-4V substrates through single lap shear tests was demonstrated. Investigations of the effects of molecular weight and end group on thermal and mechanical properties of reactive amine terminated and non-reactive phthalic anhydride terminated PMDA-mBAPPO polyimides were pursued. Polyimides with non-reactive end groups do not crosslink and, hence, were essential for controlled processability. Rheological behavior was investigated with a parallel plate type viscometer and the results confirmed the thermoplastic nature of these systems. Phenyl ethynyl endcapped polyimides were synthesized for a broad based investigation of their processibility and mechanical properties. Thermally stable soluble polyimide oligomers were synthesized via the poly(amic acid) precursor route followed by subsequent solution imidization. These polyimides were successfully crosslinked upon heating, without the evolution of volatile products. The phenyl ethynyl endcapped polymers (T_cure ~ 350-400°C) exhibited a wider processing window above Tg, relative to the unsubstituted acetylene endcapped polymers (T_cure ~ 200°C). Studies of the chemorheological behavior of these polyimides as a function of varying temperatures and times were conducted. This research allows the conclusion that high Tg, thermostable, and solvent resistant new networks suitable for structural adhesives and matrix resins have been identified. This was proven by characterization of these novel networks. Ninety-nine percent gel fraction networks were observed after cure at 380°C for 60 minutes. Thermomechanical properties such as tensile properties, adhesion properties, creep behavior, and heat distortion temperatures have been generated which illustrate the excellent dimensional stability and mechanical properties achievable at optimum crosslink density. Polyarylene ether sulfones were also studied which demonstrated the desirable combination of excellent thermal stability and good mechanical properties. Biphenyl-based polysulfones were synthesized and endcapped with 3-phenyl ethyny] functional groups. High Tg, thermostable, ductile and solvent resistant networks were prepared after curing at 380°C. Thermo-mechanical behavior, including tensile properties and dynamic mechanical analyses have been determined and are reported in this thesis. / Ph. D.