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Triacylglycerol (TAG) is the main storage lipid in plants, found both in seed and non-seed tissues (e.g. root, leaves, mesocarp etc.). Plants use TAGs as a carbon and energy source during seed germination while humans use plant lipids for biofuel production, industrial feedstocks and nutrition. It is expected that by 2030 the demand for biodiesel will be doubled. To meet this demand it is important to understand the regulation of rate limiting reactions involved in TAG accumulation in nonseed tissues because of their higher biomass relative to the seed tissues. In this study, avocado (Persea americana) is used as a model organism because it is a basal angiosperm and can store up to 70% oil content in the form of TAG in its mesocarp, a nonseed tissue. Typically, the last acylation of diacylglycerol (DAG) to form TAG in seed tissues is catalyzed by diacylglycerol acyltransferases (DGAT) and/or phospholipid:diacylglycerol acyltransferases (PDAT). Based on the transcriptome of avocado, it is hypothesized that both DGAT and PDAT are responsible for catalyzing the terminal step of TAG biosynthesis in mesocarp of avocado. Fulllength coding sequences for DGAT1 and PDAT1 were identified based on the avocado transcriptome data and expressed in TAG-deficient yeast strain (SCY-1998) for complementation. Total lipid extracts from complemented yeast lines will be analyzed for presence of TAG. Furthermore, the enzyme activity and substrate specificity for PaDGAT1 and PaPDAT1 will be determined from microsomal preparations of avocado and eukaryotic expression systems containing the avocado transgenes. This study is expected to identify the enzymes responsible for the terminal acylation step in TAG synthesis in avocado, thereby contributing to the basic understanding of TAG accumulation in nonseed tissues.

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No description available.

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A variety of plants are natural resources for oil and are capable of synthesizing and storing up to 90% oil (dry weight) in the form of triacylglycerols (TAGs). TAGs are commonly used as vegetable oils of which, >35% is derived from fleshy part of the fruits, such as mesocarp of oil palm, avocado, and olive. Studies on TAG synthesis in seed tissues mostly implicated an acyl CoA-dependent enzyme, diacylglycerol (DAG) acyltransferase (DGAT) to catalyze the conversion of DAG to TAG. However, recent studies on Arabidopsis and oil palm suggested participation of a phospholipid:diacylglycerol acyltransferase (PDAT), which is an acyl-CoA-independent enzyme. In avocados, which store up to 70% oil in mesocarp, I hypothesize that both DGAT and PDAT are likely involved in TAG synthesis. To test the hypothesis, I determined TAG content and composition by gas chromatography (GC) and expression levels of DGAT and PDAT genes by real-time PCR, in developing mesocarp. These data will be compared to that of seed tissues of avocado to associate gene expression levels with changes in oil accumulation. Future studies on cloning and characterization of these potential acyltransferase genes involved in TAG synthesis will allow us to develop genetic tools that may increase oil yield; a step towards meeting the consumption demand for oil that is expected to almost double by 2030.

Aluminium is an element of increasing clinical importance. It not only has uses as a medicinal substance but also in recent years it has been shown to be the cause of considerable toxicity, particularly in the setting of chronic renal failure. Diseases that have been shown to be associated with aluminium, or in which it has been implicated, include dialysis dementia, renal osteodystrophy and Alzheimer's disease. This thesis has studied aspects of the interaction between aluminium and the kidney. The work has addressed two major issues. Firstly, a study is described where Malvin's stop-flow technique was used to determine any excretory/absorptive tubular site for Al in the pig kidney. Al was found to be excreted in the distal nephron of the pig kidney. Secondly, the toxic effects of Al in vitro on the DNA of pig kidney cell line LLC-PKl were investigated, in an attempt to elucidate some of the mechanisms of toxic action. DNA synthesis was measured using ³H-TdR incorporation. Over increases of both time (9-72 h) and Al concentration (0.01-8.0 mM), ³H-TdR incorporation was diminished. Effects were evident at concentrations as low as 0.05 mM Al. The production of DNA strand breaks was assessed by the increase in size of cell nucleoids (ie DNA in supercoiled form). Nucleoid size was analyzed in a Epics 753 Fluorescence Activated Cell Sorter interfaced with an MDADSII data acquisition and analysis system. After 90 min incubation with Al (over the concentration range 0.001-32 mM), an increase in nucleoid size was noted at concentrations above 0.05 mM. The data demonstrate that Al exerts an effect on kidney cells in vitro which is expressed as diminished DNA synthesis and production of DNA strand breaks. These effects on DNA may have important long-term implications on various disease states associated with Al toxicity.

Oil palm can accumulate up to 90% oil in its mesocarp, the highest level observed in the plant kingdom. In contrast, the closely related date palm accumulates almost exclusively sugars. To gain insight into the mechanisms that lead to such an extreme difference in carbon partitioning, the transcriptome and metabolite content of oil palm and date palm were compared during mesocarp development. Compared with date palm, the high oil content in oil palm was associated with much higher transcript levels for all fatty acid synthesis enzymes, specific plastid transporters, and key enzymes of plastidial carbon metabolism, including phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. Transcripts representing an ortholog of the WRI1 transcription factor were 57-fold higher in oil palm relative to date palm and displayed a temporal pattern similar to its target genes. Unexpectedly, despite more than a 100-fold difference in flux to lipids, most enzymes of triacylglycerol assembly were expressed at similar levels in oil palm and date palm. Similarly, transcript levels for all but one cytosolic enzyme of glycolysis were comparable in both species. Together, these data point to synthesis of fatty acids and supply of pyruvate in the plastid, rather than acyl assembly into triacylglycerol, as a major control over the storage of oil in the mesocarp of oil palm. In addition to greatly increasing molecular resources devoted to oil palm and date palm, the combination of temporal and comparative studies illustrates how deep sequencing can provide insights into gene expression patterns of two species that lack genome sequence information.

INTRODUCTION: Specialized pro-resolving lipid mediators (SPM) such as resolvin-D1 (RvD1) act to resolve vascular inflammation and may guard against the progression of restenosis following cardiovascular interventions. Stimulating synthesis of these mediators directly in vascular cells may increase their local availability, and thus, protect against restenotic injury. However, the ability of endothelial (EC) and vascular smooth muscle cells (VSMC) to produce SPMs from their polyunsaturated fatty acid precursor decosahexaenoic acid (DHA) via lipoxygenase (LO) enzymatic transformation remains unknown. We sought to determine whether vascular cells produce SPMs from DHA and, if they do, how inflammation and mechanical injury of the vasculature alter biosynthesis. METHODS: Primary cultures of human saphenous vein endothelial and smooth muscle cells were treated with DHA in cell culture media (+ 10% serum) for 4h-24h. Freshly dissected rabbit aorta was incubated intact or following gentle endothelial denudation in cell culture media (+10% serum) with or without DHA for 48h. SPM levels in media were quantified by LC-MS/MS and ELISA and lipoxygenase expression and localization were assessed by western blotting and immunofluorescence staining, respectively. RESULTS: EC and SMC receiving media without DHA did not synthesize SPMs within the detection limits of the assay, whereas DHA treatment produced 17-HDHA, 14-HDHA, Mar1, RvD5, RvD2, and a dose and time-dependent increase in RvD1 production in EC (10.1 ±1.0 pg for 1000nM at 24h) and SMC (7.4 ± 0.2 pg for 1000nM at 24h). Intact rabbit aorta incubated in DHA+ media produced 0.24 ± 0.05 pg RvD1/mg tissue whereas aorta incubated in DHA− media produced 0.13 ± 0.007 pg RvD1/mg tissue. Moreover, EC-denuded aortas produced less RvD1/mg tissue than intact aortas. 5-LO was expressed in both cell types, however DHA induced 5-LO expression in EC (1.3 fold -DHA) but not in SMC. DHA promoted a nuclear to cytoplasmic shift of 5-LO in both EC and SMC. Finally, TNF-α stimulated an increase in RvD1 production in EC. CONCLUSIONS: Human vascular cells and rabbit vascular tissue can biosynthesize SPMs de novo from their precursor DHA, signifying a new source of SPMs in the vasculature.

This dissertation is focused on exploring the potential of bacteria for the biosynthesis of natural products with the purposes of generating novel natural product derivatives and of improving the titer of pharmaceutically important natural products. A wide variety of compounds from various sources have been historically used in the treatment and prevention of diseases. Natural products as a major source of new drugs are extensively explored due to their huge structural diversity and promising biological activities such as antimicrobial, anticancer, antifungal, antiviral and antioxidant properties. For instance, penicillin as an early-discovered antimicrobial agent has saved millions of lives, indicating the historical importance of natural products. However, the alarming rise in the prevalence of drug resistance is a serious threat to public health and it has coincided with the decreasing supply of new antibiotics. Bacteria with a tremendous undiscovered potential have still been one of the richest sources of bioactive compounds to tackle the growing threat of antibiotic-resistant pathogens. Nevertheless, the production level of those important compounds is often quite low, and often undetectable using current analytical techniques. To expand the chemical repertoire of nature and to increase the titer of the natural products, researchers have developed various strategies, such as heterologous expression, co-cultivation of different bacteria, optimization of fermentation conditions, discovery of new species, engineering of biosynthetic enzymes, and manipulating regulatory elements. Thus, in my dissertation research, I have exploited a few of these strategies. First, I heterologously expressed some of the biosynthetic genes from the sch biosynthetic gene cluster, resulted in the production of a novel glycosylated angucycline. I was also able to generate another new glycosylated derivative of angucycline through gene disruption of tailoring enzymes. In this research, I isolated two novel angucycline derivatives and gained new insights into the glycosylation steps in the biosynthesis of Sch47554 and Sch47555. Next, I engineered the regulatory elements in Streptomyces sp. SCC-2136 through the overexpression and targeted gene disruption approaches for enhanced production of pharmaceutically important angucyclines. The highest titer of Sch47554 was achieved in Streptomyces sp. SCC-2136/ΔschA4 (27.94 mg/L), which is significantly higher than the wild type. This work thus provides an initial understanding of functional roles of regulatory elements in the biosynthesis of Sch47554 and Sch47555 and several engineered strains with enhanced production of Sch47554. Last, I isolated a carotenoid-producing endophytic bacterium from the leaves of the yew tree and optimized the fermentation conditions for an improved yield of zeaxanthin diglucoside up to 206 ± 6 mg/L. With the introduction of an additional copy of the Pscrt gene cluster through an expression plasmid, the engineered strain Pseudomonas sp. 102515/pOKF192 produced zeaxanthin diglucoside at 380 ± 12 mg/L, which is 85% higher than the parent strain. This strain holds a great potential for the production of pharmaceutically important antioxidant agent, zeaxanthin diglucoside.