Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

Studies in asymmetric and heterocyclic synthesis: I. chiral ketones II. Quinolones III. Trifluoromethylated pyrones

This thesis is split into three sections based on three different areas of research. In the first section, investigations into the α-alkylation of ketones using a novel chiral auxiliary is reported. This chiral auxiliary was synthesised containing a pyrrolidine ring in the chiral arm and was applied in the preparation of α-alkylated ketones which were obtained in up to 92% ee and up to 63% yield over two steps. Both 3-pentanone and propiophenone based ketones were used in the investigation with a variety of both alkyl and benzyl based electrophiles. The novel chiral auxiliary was also successful when applied to Michael and aldol reactions. A diamine precursor en route to the chiral auxiliary was also applied as an organocatalyst in a Michael reaction, with the product obtained in excellent enantioselectivity. In the second section, investigations into potential anti-quorum sensing molecules are reported. The bacteria Pseudomonas aeruginosa is an antibiotic-resistant pathogen that demonstrates cooperative behaviours and communicates using small chemical molecules in a process termed quorum sensing. A variety of C-3 analogues of the quorum sensing molecules used by P. aeruginosa were synthesised. Expanding upon previous research within the group, investigations were carried out into alternative protecting group strategies of 2-heptyl-4-(1H)- quinolone with the aim of improving the yields of products of cross-coupling reactions. In the third section, investigations into fluorination and trifluoromethylation of 2-pyrones, pyridones and quinolones is reported. The incorporation of a fluorine atom or a trifluoromethyl group into a molecule is important in pharmaceutical drug discovery programmes as it can lead to increased lipophilicity and bioavailability, however late-stage incorporation is rarely reported. Both direct fluorination and trifluoromethylation were attempted. Eight trifluoromethylated 2-pyrones, five trifluoromethylated 2-pyridones and a trifluoromethylated 2-quinolone were obtained in a late-stage synthesis from their respective iodinated precursors using methyl fluorosulfonyldifluoroacetate as a trifluoromethylating reagent.

Factors relating to motivation to change behaviour in individuals who are overweight: application of the health belief model

Background: Obesity is the most important health challenge faced at a global level and represents a rapidly growing problem to the health of populations. Given the escalating global health problem of obesity and its co-morbidities, the need to re-appraise its management is more compelling than ever. The normalisation of obesity within our society and the acceptance of higher body weights have led to individuals being unaware of the reality of their weight status and gravity of this situation. Recognition of the problem is a key component of obesity management and it remains especially crucial to address this issue. A large amount of research has been undertaken on obesity however, limited research has been undertaken using the Health Belief Model. Aim: The aim of the research was to determine factors relating to motivation to change behaviour in individuals who perceive themselves to be overweight and investigate whether the constructs of the Health Belief Model help to explain motivation to change behaviour. Method: The research design was quantitative, correlational and cross-sectional. The design was guided by the Health Belief Model. Data Collection: Data were collected online using a multi-section and multi-item questionnaire, developed from a review of the theoretical and empirical research. Descriptive and inferential statistical analyses were employed to describe relationships between variables. Sample: A sample of 202 men and women who perceived themselves to be overweight participated in the research. Results: Following multivariate regression analysis, perceived barriers to weight loss and perceived benefits of weight loss were significant predictors of motivation to change behaviour. The perceived barriers to weight loss which were significant were psychological barriers to weight loss (p =<0.019) and environmental barriers to physical activity (p=<0.032).The greatest predictor of motivation to change behaviour was the perceived benefits of weight loss (p<0.001). Perceived susceptibility to obesity and perceived severity of obesity did not emerge as significant predictors in this model. Total variance explained by the model was 33.5%. Conclusion: Perceived barriers to weight loss and perceived benefits of weight loss are important determinants of motivation to change behaviour. The current study demonstrated the limited applicability of the Health Belief Model constructs to motivation to change behaviour, as not all core dimensions proved significant predictors of the dependant variable.