Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.

With crude oil demand rising and supplies being depleted, alternative energy, specifically biofuels, are of intense scientific interest. Current plant crop based biofuels suffer from several problems, most importantly the use of land needed for food. Cyanobacteria offer a solution to this problem as they do not compete with land for food and produce hydrocarbons that can be used as biofuels. Upon examination of metabolic pathways competing with hydrocarbon synthesis, it appeared that cyanobacteria lacked the major fatty acid degradative metabolic pathway β-oxidation, generally thought to be a universally occurring pathway. Lack of this pathway in cyanobacteria was confirmed by employing a range of analytical techniques. Bioinformatic analysis suggested that potential enzymes with β-oxidation activity were involved in other metabolic pathways. A sensitive assay was set up to detect acyl- CoAs, the substrates of β-oxidation, using liquid chromatography triple quadrupole mass spectrometry. None could be detected in cyanobacteria. No enzymatic activity from the rate-limiting acyl-CoA dehydrogenase/oxidase could be detected in cyanobacterial extracts. It was found that radiolabeled fatty acids fed to cyanobacteria were utilised for lipid membranes as opposed to being converted to CO2 by respiration or into other compounds by the TCA cycle. An element of the β-oxidation pathway, E. coli acyl-CoA synthetase was ectopically expressed in a strain of cyanobacteria and implications of the introduction of acyl-CoA synthesis were assessed. Finally, the regulation of the fatty acid biosynthetic pathway was investigated. It was determined that under conditions of excess fatty acid, the transcription of acetyl-CoA carboxylase and enoyl-ACP reductase was repressed and acyl-ACP synthetase involved in fatty acid recycling was induced. These results were discussed in relation to fatty acid oxidation and hydrocarbon biosynthesis in other organisms.

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Background: Endothelial dysfunction is a major factor in the development of atherosclerosis, thrombosis and heart disease. Evidence suggests dietary fat composition may modify cardiovascular risk, as well as surrogate markers of cardiovascular risk such as blood pressure, arterial stiffness and endothelium-dependent vasodilation. Aim: To investigate the impact of dietary fat composition on endothelial function and associated markers of vascular health. Methods: The effects of oils rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were separately investigated in a parallel-design, placebo-controlled randomised controlled trial (n=48, 6 weeks, 2.9 g/d), carried out in free-living healthy young men. Following a 2 week run-in period taking placebo capsules (olive oil), participants underwent baseline measurements of finger capillary density, endothelial progenitor cell numbers (EPC), platelet-monocyte aggregate numbers (PMA), ambulatory blood pressure (ABP), pulse wave analysis (PWA), digital volume pulse analysis (DVP), and gave blood samples for plasma lipid, glucose, insulin, nitric oxide metabolites (NOx) and isoprostanes. The same measurements were made at the study endpoint, 6 weeks. An in vitro investigation of the effects of physiologically-relevant fatty acid profiles on microvascular endothelial cell nitric oxide and prostacyclin production was also performed. Results: Neither EPA nor DHA supplementation influenced EPCs, capillary density, PMA, ABP, PWA, DVP or plasma cholesterol, triacylglycerol, glucose, insulin, NOx or isoprostanes compared to placebo. However, ambulatory night-time heart rate was increased following EPA supplementation compared to DHA. Furthermore, both EPA and DHA decreased plasma non-esterified fatty acids (NEFA) compared to placebo. The in vitro investigations suggested that the composition of circulating NEFA may differentially affect endothelial function in the microvasculature. Conclusion: Dietary EPA and DHA at relatively high doses do not improve a number of novel markers of vascular function, including microvascular function and a marker of endothelial repair in young healthy men. EPA and DHA have differing effects on heart rate during sleep, suggesting that further research is required into the possible adverse effects of higher doses of individual marine fatty acids in at-risk individuals. Further work is required to elucidate the role of physiological fatty acid profiles on endothelial function.

Difficulties in controlling and forecasting avocado fruit ripening and the highly perishable nature of the crop once harvested, are the major causes of concern for avocado traders. In particular, the simultaneous presence of many suppliers may account for increased fruit variability during ripening. Avocado is a climacteric fruit with consistent ethylene production after harvest which is also related to high perishability. However, the mechanisms regulating ethylene biosynthesis and mesocarp softening are not completely understood. In order to study such effects, avocado fruit from different growing areas and harvested at various maturity stages, were investigated and the biochemical and physiological changes during ripening at both 18 and 23°C were studied. Mesocarp softening and fatty acid content discriminated fruit maturity and growing area, respectively, whereas C7 sugars (D-mannoheptulose and perseitol) discriminated length of fruit shelf life. For the first time, oleic acid content presents in the oil mesocarp was found to depend on fruit sources making of this a suitable indicator of avocado fruit growing area. In contrast, sugar content declined along fruit maturity and ripening. In particular the mannoheptulose presents in avocado mesocarp might be use to estimate avocado fruit shelf life. Indeed, fruit harvested late in season were found to have a lower C7 content than earlier harvest fruit and a faster softening, regardless fruit source. However, sugars content changed between growing area, thus a general C7 threshold defining fruit storability seems to be not definable. Furthermore, other possible indicators of fruit maturity and/or ripening stage have been searched in the cell wall constituents of avocado mesocarp. Thus, the structural carbohydrates profile of avocado mesocarp investigated with a new immunological method changed during ripening and harvest time (early and late season), suggesting a possible effect of cell wall composition on fruit ripening regulation. Also, the possible use of ethylene application in reducing the high heterogeneity noted on imported fruit from South Africa was also evaluated through different consignments. Results showed ethylene efficacy changed depending on harvest time and fruit dimension with less efficacy of the treatment on fruit harvested at the end of the season and characterised by smaller size.One of the most commercialized avocado cultivars, Hass, is peculiar in that its skin colour changes from green to deep purple as ripening progresses. The most common ripening indicator of avocado fruit is the mesocarp firmness and the destructive nature of this evaluation increases losses in the avocado industry. The availability of a non-destructive indicator of fruit ripening represents an important advantage for avocado consumers and importers. Thus, the possible relationship between mesocarp softening, skin colour were objectively evaluated (C*, L*, and H°), and the main pigment, cyanidin 3-O-glucoside, was investigated. Cyanidin 3-Oglucoside was confirmed to be the main anthocyanin present in avocado cv. Hass peel, regardless of preharvest factors. However, differences in its content were noted between shelf life temperatures. A higher relationship between hue angle and firmness was detected in late harvest fruit, whereas no correlation was found between anthocyanin content and firmness. Avocado skin is also involved in defence mechanisms due to the presence of antifungal and phenolic compounds. These phenolic compounds represent a natural protection against pathogenic infections and seem to be down regulated during ripening. The main phenolics were identified and quantified, using a new analytical method which was validated and optimised. Epicatechin, chlorogenic acid and procyanidin B2 were found to be present in the skin tissue and quantified using this assay and found to vary during shelf life and seasons. Although phenolics were present in minor amounts, in avocado pulp they are involved in mesocarp discoloration incidence, and therefore with fruit postharvest quality. Due to a lack of information, a new straightforward method for the identification and quantification of the main phenolics present in avocado mesocarp was developed. Finally, a commercial trial was undertaken to ensure that the results obtained in the laboratory can be reproduced in the market place. In conclusion, postharvest markers can define avocado fruit maturity and growing area and give guidelines in the control of avocado shelf life. Moreover, new methods for the investigation of the phenolic profiles (peel and mesocarp) and the characterisation of cell wall structures can be further tools in the management of avocado fruit postharvest quality.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Medical Sciences

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

<p>Obesity is today a problem that has reached epidemic proportions. One of the causes of obesity is the over-consumption of energy. Fat is the most energy-dense nutrient, where the quality seems to be more important for the development of the metabolic diseases than the quantity. The fatty acid composition in serum lipid fractions can be used to mirror the dietary fat quality.</p><p>Stearoyl-CoA-desaturase (SCD) is an enzyme that converts saturated to monounsaturated fatty acids. A surrogate measure of SCD activity can be estimated as a fatty acid ratio; 16:1/16:0 (palmitoleic acid/palmitic acid) and 18:1/18:0 (oleic acid/stearic acid). The aim of this project was to investigate the intra-day variation in the SCD-ratio in humans eating a standardized diet. The results showed that triacylglycerol and nonesterified fatty acid fractions in serum lipids had a significant variance in the 16:1/16:0 ratio during the day, whereas 18:1/18:0 ratio in the same fractions did not exhibit the same pattern. In this study 16:1/16:0 ratio also seems to be a better marker than 18:1/18:0 ratio for estimating SCD activity. For further evaluation of the intra-day variation there need to be a more long-term study of the SCD-activity for a larger group of subjects.</p>