Vibrio vulnificus is a foodborne bacterial pathogen associated with raw oyster consumption. Shellfish depuration for 48 hours is a dynamic process where coliform bacteria are purged; however, this process is ineffective against V. vulnificus. The current study investigated the use of prolonged two-week depuration on V. vulnificus populations in Gulf Coast oysters. The study evaluated the impact of prolonged depuration on V. vulnificus fatty acid profile change and the ability to survive in simulated gastric fluid. Oyster depuration in seawater (10 or 22oC, 14 days) reduced V. vulnificus counts, but not to non-detectable level, indicating close ecological relationship between the pathogen and mollusk. Greatest V. vulnificus count reductions were seen in 12 ppt 10°C seawater (2.7 log10 CFU/g) and in 20 ppt 22°C seawater (2.8 logs). Mesophilic vibrios dominated the overall microflora of freshly harvested oysters, while refrigeration selected for psychrotrophic bacteria. Depuration at 22°C retained dominance of mesophilic vibrios, including pathogenic species. Although aerobic plate counts were lower in 22°C depurated oysters (5.0 logs vs 6.0 logs), depuration at 10°C had little to no advantage over 22°C in terms of vibrio population reduction. Use of prolonged depuration remains economically questionable since this method failed to completely eliminate V. vulnificus. Starved V. vulnificus behavior in artificial seawater showed that low temperature (4oC) and high seawater salinity (35 ppt) contributed to pathogen population reduction. Starved V. vulnificus did not adjust membrane fluidity to storage temperature within the investigated time frame. However, a significant fatty acid switch from C18:1w7c to C18:1w6c by double bond relocation was observed. The relocation was faster at ambient temperatures compared to refrigerated temperatures. The majority of V. vulnificus foodborne infections occur during warm summer months. Vibrio vulnificus ATCC 27562 was significantly less resistant (3.7 min D-value) to simulated gastric fluid (pH 4.0) after 7-day storage at 4oC compared to the control (7.8 min D-value). Therefore, greater gastric fluid sensitivity of the pathogen may occur in winter-harvested oysters and may partially explain the low number of winter outbreaks.

The growth of Rhodotorula glutinis in sweet sorghum juice in three levels of three factors of temperature, carbon to nitrogen ratio and pH was evaluated. Accompanying of this growth was the generation of lipids converted to fatty acid methyl ester (FAMEs) and carotenoids. The optimized condition for maximum biomass and carotenoid accumulation was determined to be at 25C, pH of 5.5 and carbon to nitrogen ratio of 10. This condition yielded 22.7 g/L biomass with specific growth rate of 0.213 hr-1. At this condition the carotenoids generation was also maximum with 2.6 mg/gram biomass, comprising of torularhodin, beta-carotene and torulene. The accumulation of lipids following generation of biodiesel was highest at same temperature and pH but carbon to nitrogen ratio of 70, generating 96.3 mg of FAMEs/gram of biomass containing methyl ester of oleic acid, linoleic acid, palmitic acid, stearic acid and linolenic acid.

Fatty acid amide hydrolase (FAAH) is an enzyme that terminates the signaling role played by the lipid mediators, N- acylethanolamines (NAEs), present both in plants and animals. FAAH is responsible for NAE hydrolysis and has been extensively studied in mammalian systems and the model plant Arabidopsis thaliana; it has been reported in various organisms as well as some crop plants such as rice and Medicago truncatula. To understand the role of FAAH in diverse organisms, here we report the identification and biochemical characterization of a FAAH homolog in tomato. Previously identified and cloned candidate FAAH from tomato was expressed in Escherichia coli as a fused protein with 6X his-tag for identification. Supernatant containing recombinant FAAH showed the ability to hydrolyze NAE substrates. The optimal reaction conditions for enzyme assay and kinetic parameters for tomato FAAH were determined and effect of inhibitor on enzyme was determined. Characterization of FAAH in tomato will contribute to further understanding of NAE metabolic pathway and its implications.

WRINKLED 1(WRI1), a member of AP2/EREBP class of transcription factors regulates carbon allocation between glycolytic and fatty acid biosynthetic pathway. Additionally, among the four WRI1 paralogs in arabidopsis, WRI3 and 4 but not WRI2, are also able to increase fatty acid content in different tissues. While the role of WRI1 is well established in seeds, the potential or WRI1 or its paralogs as master regulators in oil-rich nonseed tissues is poorly understood. Recent transcriptome studies of avocado (Persea americana) mesocarp revealed that the ortholog of WRI2, along with WRI1 and WRI3 was highly expressed during oil accumulation. Through transient expression assays, we further demonstrated that both PaWRI1 and PaWRI2 can accumulate oil in tobacco leaves. We conducted a comprehensive and comparative in silico analysis of WRI paralogs from a dicot, monocot and a basal angiosperm to identify distinct features associated with function. These data provide insights into the possible evolutionary changes in WRI1 homologs and allow for identification of new targets to enhance oil biosynthesis in diverse tissues.

Over the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the susceptibility of serum lipoproteins to undergo oxidation, thereby contributing to the progression of atherosclerosis. The intestinal cells are responsible for the absorption of dietary fatty acid peroxides (FAOOH) which has been reported to enhance anti-atherosclerotic effects by inducing apolipoprotein A1 (apoA1) gene and protein levels. Therefore, there is a void in the knowledge of when to expect “harmful” or “beneficial” effects of dietary lipid peroxides. The formation of toxic products like aldehydes from the decomposition of FAOOH is well documented. On the other hand, carboxylic acids particularly azelaic acid, formed as an end product of FAOOH decomposition has been reported to have anti-atherosclerotic effects. Hence, we hypothesize that intestinal cells may decompose FAOOH to aldehydes, which might get converted to carboxylic acids that can be transported across the intestine. Linoleic acid is the most abundant polyunsaturated fatty acid (PUFA) present in the diet. So, we will use peroxidized linoleic acid (13- HPODE) and incubate with intestine derived cells or Caco-2 cells as an in-vitro model for determining its decomposition to aldehydes and carboxylic acids. We propose that the decomposition products of FAOOH in the presence of intestinal cells might be iv responsible for causing an increase in apoA1 levels, which might suggest that lipid peroxidation derived products might actually be beneficial for reducing the progression of atherosclerosis as compared to the absorption of intact FAOOH.

The glycoprotein CD36 is expressed in the plasma membrane (PM) of many cell types that surround or contact arteries, including macrophages, myocytes, and endothelial cells. CD36 binds oxidized low density lipoprotein (oxLDL), which promotes atherosclerosis, and fatty acids (FA), which promotes their cellular uptake. To gain insights into the molecular mechanisms of uptake, HEK293 cells expressing CD36 were studied by cell biological and fluorescence methods. To test our hypothesis that the PM is not an impermeable barrier to FA and that FA move into cells by diffusion via their uncharged form, we first applied biophysical fluorescence spectroscopy to directly measure transmembrane FA movement and membrane fluidity. Expression of CD36 in HEK293 cells did not increase either transport across the PM or the fluidity of the PM compared to HEK293 cells without CD36; however, CD36 enhanced intracellular FA esterification. Furthermore, the widely used “inhibitors” of FA transport did not alter either the rapid FA transmembrane diffusion in HEK293 cells or diffusion in control experiments with protein-free phospholipid bilayers. To gain new insights into the physiological relevance of FA binding to CD36, we applied surface plasmon resonance (SPR) to quantify FA and oxLDL binding to the ectodomain of CD36. Structurally distinct FA [saturated, monounsaturated (cis and trans), polyunsaturated, ω-3, ω-6, and oxidized FA] were pulsed in a solubilized form (bound to methyl-β-cyclodextrin) across SPR channels, generating real-time association and dissociation binding curves. With the exception of the oxidized FA hydroxyoctadecadienoic acid (HODE), all FA tested bound to CD36 with rapid association and dissociation kinetics similar to human serum albumin. In addition, FA increased oxLDL binding to CD36. To investigate whether FA affect CD36-mediated oxLDL uptake in live cells, we monitored fluorescent oxLDL (Dii-oxLDL) uptake using confocal microscopy. Addition of exogenous FA to serum-free media enhanced dose-dependent oxLDL uptake. Exceptions were ω-3 FA, which bound to CD36, and HODE, which did not bind to CD36, demonstrating FA structure-specific effects on a major function of CD36 and a new mechanistic link between atherosclerosis and high levels of FA in obese and Type-II diabetic individuals.

In a number of mammalian species, susceptibility to enteric coronavirus infection has been shown to be age-related. This is thought to be associated with enterocyte maturation and receptor protein expression. One of the factors that can influence differentiation and maturation of enterocytes is the availability of short-chain fatty acids (SCFA) in the intestinal lumen. These compounds are by-products of the bacterial fermentation of dietary fiber and serve as the primary energy source for enterocyte metabolism. The overall objective of this dissertation was to evaluate the effects of dietary fiber and short-chain fatty acids on enterocyte differentiation, maturation, and susceptibility to coronavirus infection in turkeys. Initial work involved the development of an indirect immunoperoxidase assay (IPA) for the identification and localization of turkey coronavirus (TCV) in paraffin-embedded, acid-ethanol fixed tissue. IPA was found to be superior to indirect immunofluorescent antibody test (IFA) for this and other diagnostic purposes. To evaluate cellular differentiation and maturation, an SDS-PAGE/immunoblot technique was developed to determine relative levels of villin expression in turkey embryos. Villin is an actin-bound cytoskeletal protein known to be expressed in increasing quantities at the apical surfaces of maturing enterocytes. Villin expression level was found to increase linearly as a function of embryo age. Villin localization was performed by IPA on paraffin-embedded, acid-alcohol fixed tissue. As enterocytes (embryos) matured, villin was found to concentrate at the apical surfaces and eventually at the basolateral membranes. Experiments were also conducted to see what effect in ovo butyrate administration would have on developing embryonic enterocytes. Butyrate has been shown to enhance differentiation of non-neoplastic and neoplastic cells in culture as well as promote healing of damaged intestinal epithelium in human. Villin expression was significantly enhanced in embryos receiving 0.2 and 0.3 M butyrate 36 hours post-administration. Butyrate appeared to enhance villin expression and therefore enterocyte maturation in a dose-dependent manner. Susceptibility of turkey embryos to TCV infection as a function of age and butyrate treatment was investigated as well as epithelial localization of TCV infection in poults. The level of TCV infection of epithelium was found to increase with embryo age between 17 and 23 days. Poults showed higher levels of infection on the distal 2/3 of villi and no evidence of infection in the intestinal crypts. Butyrate administration in 21-day-old embryos followed by TCV inoculation caused a significant increase of the number of infected cells per villus. This data suggested that butyrate might be used as a means to manipulate enterocyte susceptibility to TCV infection. In the final set of experiments, the effects of fiber-fortified poult diets containing 5% cellulose or 5% guar gum on luminal SCFA levels, enterocyte maturation, and TCV infection were investigated. SCFA levels in cecal contents were determined by gas chromatography. Enterocyte maturation was assessed by the determination of villin expression on immunoblot and the severity of TCV infection was determined by IPA and lesion score. Fiber-fortified diets enhanced SCFA production and villin expression, but contrary to embryo studies, TCV infection appeared to be reduced. In general, poults performed better on the diet containing cellulose. Mechanisms regarding the roles of dietary fiber and SCFA in enterocyte differentiation, maturation, and TCV susceptibility are proposed as well as future directions for research. The in ovo and poults system used in this research may serve as models for further investigation of the influences of host and dietary factors on enteric viral infection and recovery. / Ph. D.

Producing biodiesel from triacylglycerol (TAG) generates glycerol as a byproduct which could be recycled and used to grow the oleaginous yeast Rhodotorula glutinis. R. glutinis has the ability to produce up to 70% of its weight in the form of TAG. This study is designed to determine the effects of glycerol on the TAG and fatty acids produced by R. glutinis. After 24 hrs, R. glutinis cultured on medium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or dextrose and glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry weight basis, respectively. The fatty acids derived from R. glutinis were mostly saturated, however, cells cultivated on glycerol alone had the highest degree of unsaturated fatty acids (53%). Growth on dextrose may be enhanced by the addition of glycerol, but it cannot be determined if using glycerol as a secondary carbon substrate enhances lipid production.

No description available.

No description available.