The creation of small molecule libraries for binding into the NS1A protein of influenza A viruses and the development of an indicator displacement assay for the differentiation of fatty acids are reported herein. Using Mitsunobu chemistry, a variety of structures based on hydroquinone, resorcinol and 2,7-dihydroxynaphthalene cores were synthesized. Both polar and non-polar functional groups were added to diversify the cores to help understand which molecule binds best to the protein. Because of poor protein binding, the focus of the project moved to a new lead compound, epigallocatechin-3-gallate (EGCG). EGCG showed promise in computational studies and efforts towards the synthesis of the epigallocatechin core were undertaken. Using a fluorescent indicator displacement assay (IDA), a sensing system for fatty acids was developed. The system consisted of bovine, rabbit, and human serum albumins as host molecules, while the fluorescent indicators were fluorescein, 2-anthracene carboxylic acid, and 1-anilino-8-naphthalene sulfonic acid. Fatty acids were able to be differentiated from one another based on their carbon chain length and the degree of unsaturation. The IDA was then subjected to a complex mixture of fatty acids, in the form of edible oils. The oils (extra virgin olive, hazelnut, peanut, sunflower and canola) with different fatty acid profiles were able to be differentiated from each other using principal component analysis. / text

Endothelial activation is considered to be an early and critical event in the pathology of atherogenesis which can be modified by environmental factors such as diet, pollutants, and lifestyle habits. Dietary andamp;ugrave;-6 and andamp;ugrave;-3 fatty acids have been reported to either amplify or diminish inflammatory responses related to atherosclerosis development. However, the interactions of andamp;ugrave;-6 and andamp;ugrave;-3 fatty acids with inflammatory cytokines or organic pollutants on endothelial cell activation are not well understood. The studies presented in this dissertation tested the hypothesis that andamp;ugrave;-6 and andamp;ugrave;-3 fatty acids alone, or in varying ratios can differently modulate pro-atherogenic mediators and inflammatory responses that are initiated by tumor necrosis factor- andamp;aacute; (TNF-andamp;aacute;) or polychlorinated biphenyls (PCBs) in endothelial cells. Exposure to TNF-andamp;aacute; induced oxidative stress, p38 MAPK, NF-andamp;ecirc;B, COX-2 and PGE2, which was amplified by pre-enrichment with linoleic acid but blocked or reduced by andamp;aacute;-linolenic acid. Furthermore, TNF-andamp;aacute;-induced caveolin-1 up-regulation and the co-localization of TNF receptor-1 with caveolin-1 was markedly increased in the presence of linoleic acid and diminished by andamp;aacute;-linolenic acid. Silencing of the caveolin-1 gene completely blocked TNF-andamp;aacute;-induced production of COX-2 and PGE2 and significantly reduced the amplified response of linoleic acid plus TNF-andamp;aacute;. These data suggest that omega-6 and omega-3 fatty acids can differentially modulate TNF-andamp;aacute;-induced inflammatory stimuli and that caveolae and its fatty acid composition play a regulatory role in these observed metabolic events. Besides cytokines, lipophilic environmental contaminants such as PCBs can also trigger inflammatory events in endothelial cells. Our data suggest that increasing the relative amount of andamp;aacute;-linolenic acid to linoleic acid can markedly decrease oxidative stress and NF-andamp;ecirc;B-responsive genes. The inhibitor study revealed that the modulation effect of andamp;ugrave;-6 and andamp;ugrave;-3 fatty acids on PCB toxicity was mainly through the oxidative stress sensitive transcription factor, NF-andamp;ecirc;B. In conclusion, our studies demonstrate that different dietary fats can selectively modulate vascular cytotoxicity caused by TNF-andamp;aacute; as well as by persistent organic pollutants such as PCBs. We also demonstrated the important relevance of substituting dietary andamp;ugrave;-3 fatty acids such as andamp;aacute;-linolenic acid for andamp;ugrave;-6 fatty acid such as linoleic acid in reducing cardiovascular diseases.

Although it is well-established that the fatty acid profile of breast milk will reflect the dietary fatty acids, the response time with which this occurs is not known. We hypothesized that fatty acids from a given meal would be transferred acutely from chylomicrons into breast milk. To test this hypothesis, the following experiment was performed. Fourteen lactating women drank 700 Calorie breakfast formulas containing six different test fats: 40 grams of cocoa butter, coconut, safflower or canola oil, 20 grams of menhaden oil or 7 grams of herring oil. Each fat contained a specific fatty acid whose appearance was tracked in the milk. After consuming the breakfast formula, subjects collected mid-feeding milk samples at 0, 6, 10, 14 and 24 hours, and one morning sample on days two through seven. Fatty acids specifically tracked in milk samples were: C12:0 (coconut oil), C18:0 (cocoa butter), C18:2n-6 (safflower oil), C18:3n-3 (canola oil), C22:ln-ll (herring oil), and C20:5n-3 and C22:6n-3 (menhaden oil). There was a significant increase in each of these fatty acids in human milk (p<0.001). Elevation of these fatty acids was first observed at 6 hours. Maximum increases of these fatty acids occurred 10 h after safflower oil (177% of baseline), 14 hours after cocoa butter (154%), coconut oil (216%), canola oil (206%) and menhaden oil (C20:5n-3 [1157%]), and 24 hours after the herring oil (2621%) and menhaden oil (C22:6n-3 [506%]). Compared to baseline, these fatty acids were significantly elevated (p<0.05) from 10 to 24 hours. However, after menhaden oil, C20:5n-3 was significantly elevated for 3 days and C22:6n-3 for 2 days. These data support the hypothesis that there is an acute transfer of dietary fatty acids from chylomicrons into human milk. / Graduation date: 1996

The purpose of this investigation was to modify the procedure of Bills, Khatri and Day for use in the development of a suitable method for determining the quantitative distribution of the free fatty acids (FFA) in normal, heated, and rancid milk and milk fractions The method consists of extracting the FFA from lyophilized milk, separating the FFA from neutral fat, converting the FFA to methyl esters, and analyzing the methyl esters by gas-liquid chromatography (GLC). The salts of FFA present in lyophilized milk or milk fractions were released by lowering the pH of the sample with H₂SO₄, and subsequently extracted with ethyl ether. The extracted FFA were simultaneously isolated from the extract and methylated by using the one-step procedure of Bills, Khatri and Day. A specially designed concentration flask was employed with a reflux system to concentrate the methyl esters. The esters were then separated by GLC. Quantitative calculations were made from the GLC peak areas using internal standards. The major esterified fatty acids of milk are n-saturated, evennumbered 4:0-18:0 and unsaturated 18:1 and 18:2 acids. The distribution of major FFA in whole milks was found to be essentially the same as that of the esterified fatty acids of milk fat. Heat treatments of milk, whether pasturization or extended holding at 100°C, effect a progressive reduction in total FFA. Decreases in long chain fatty acids are also characteristic of extended heating. Milks determined to be rancid by acid degree value and organoleptic analysis showed high levels of FFA, 1.5-3.6% of the fat content of the sample. The increases in 6:0-12:0 resulting from lipase hydrolysis approximate the amounts shown by Al-Shabibi and co-authors to produce rancid flavor when added to good quality milk. Milk triglycerides, fat globule membrane, skim milk, and buttermilk show characteristic FFA patterns which appear to be related to the solubility properties of the individual acids. Seventy-one percent of the total FFA in a given portion of 40% cream was found in the triglyceride fraction, 26% in the crudie fat globule membrane preparation and 3% in the skim milk fraction. The total concentrations of FFA were found to be: 1% of the fat in whole milk or 0.04% of the fluid milk weight; 0.16% of the total weight of 40% cream; 0.28% of centrifuged triglycerides; 1.7% of the fat globule membrane; and 0.008% of the fluid weight of skim milk. / Graduation date: 1965

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In order to achieve a better understanding of the impact of exercise on the concentration of serum free fatty acids (FFA) during post-exercise recovery, the purposes of this study were: (1) to determine the relationships between exercise intensity, total exercise energy expenditure, and the concentration of serum FFA during post-exercise recovery; (2) to examine the effects of exoge- nous glucose on post-exercise serum FFA and hormones controlling the FFA response; and (3) to examine the impact of acute exercise on hunger. Untrained, 12-h fasted, college-age males performed cycle ergometer exercise at exercise intensities ranging from 29 to 59% peak ‘VO₂ for total energy expenditures ranging from 162 to 320 kcal. Blood samples, hunger ratings, and metabolic indices were collected or measured before, during, and for 3 h post-exercise. In response to exercise of approximately 300 kcal, FFA was elevated for 3 h post-exercise. The FFA response was a function of total exercise energy expenditure, rather than exercise intensity, or combined effects of these factors. The response was associated with low insulin concentration but no changes were observed in blood glucose, glucagon, growth hormone, or cortisol. Glucose ingestion and infusion studies demonstrated that possible mechanisms con- tributing to the post-exercise FFA response included decreases in FFA re-esterification, increases in triglyceride hydrolysis, and decreases in sympathetic input to adipose tissue. Exercise caused a suppression of hunger for 2 h post-exercise which was a function of the combined effects of exercise intensity and total energy expenditure. An increase in core temperature may have contributed to the anorexigenic effect of exercise. In conclusion, exercise, performed in and followed by a period of fasting caused an elevation of FFA for 3 h during post-exercise recovery. The post-exercise recovery period should be considered an important phase in the physiological impact of exercise on the storage and utilization of fat.

The objectives of this study were to investigate the different characteristics of the newly introduced breed, Waguli (Wagyu x Tuli) when comparing it with the Brahman breed. Twenty-four animals were used. Six steers of each breed were fed 94% concentrate diet (94C) and the other six were fed 86% concentrate diet (86C). Eight steers, two from each group, each were harvested at 128 days, 142 days, and 156 days on feed. Feedlot performance data indicated that Waguli steers were highly efficient (P < 0.05) and gained more than Brahman steers on a daily basis (P < 0.05). Carcass characteristic data showed that Waguli steers have larger ribeye area with more 12th rib fat thickness, marbling score and higher quality grade (P < 0.05). It is well known that Wagyu is a highly marbled and tender Japanese breed. It was found that the reason for the Waguli tenderness and low shear force values to be the low level of calpastatin activity (P < 0.05), the inhibitor of the postmortem proteolytic enzyme-calpain. While the toughness of the Brahman meat was due to the high level of calpastatin activity. The calpain activity did not differ between the two breeds. Shear force values agreed with the calpain and calpastatin activities data, in which the Waguli steaks showed less shear force values at day 7 and 10 postmortem than the Brahman steaks (P < 0.05). However, at day 14 postmortem there was no difference in shear force values between the two breeds (P < 0.05). Fatty acid data analysis indicated that Waguli steers had a profile with less saturated fatty acids (SFA) and more unsaturated fatty acids (UFA) content than those in Brahman steers fat. Looking at the ratio of unsaturated fatty acids (UFA) to saturated fatty acids (SFA), it is observed that Waguli steers had a greater ratio than Brahman steers. In conclusion, Waguli steers produce tender meat with good marbling ability, which likely to satisfy the consumers demand. In addition, they have a desirable average daily gain and feed efficiency with high polyunsaturated fatty acids comparatively with Brahman steers.