The desert ant Cataglyphis fortis is one of the hyperthermophilic species of Cataglyphis. It lives in the Sahara desert and forages during the hottest hours of the day when it can get up to 70˚C in the sand. The body temperature of the ant during the foraging runs can reach a maximum of 55˚C. Since C.fortis is one of few eukaryotic hyperthermophilic species, its proteins probably have a high thermostability. Investigating the thermostability can give valuable information about the principles of protein folding and stability in hyperthermophiles.Fatty acid binding proteins (FABPs) have an important role in the cell taking up and transporting fatty acids and regulating metabolic and inflammatory pathways. FABPs have been extensively studied and structures from several species have been determined. The determined structures of all FABPs are very similar why thermostability studies of FABP from C.fortis are highly relevant.Fluorescence spectroscopy is an easy and fast method to measure intrinsic protein fluorescence. Tryptophans were genetically introduced into three different positions in FABP to be used as environmental sensitive probes. Complementing the measurement results with a model of the 3D structure of FABP from C.fortis gave additional information about the ligand binding.The (local) thermostability of the mutants can be detected by shift in wavelength maximum during temperature ramping experiments. All mutants are stabilised in the presence of fatty acids. The mutant with tryptophan positioned closest to the supposed ligand binding residues (Y11W) is most affected. The mutant with tryptophan situated farthest from the supposed binding residues (Y52W) shows a stabilisation of Tm less evident than for Y11W. Thus, the structural changes following fatty acid binding are more obvious in the environment close to the binding site.However, the third mutant C87W shows no significant stabilisation although positioned closer to the fatty acid binding site than Y52. This is probably due to the size difference between the original and introduced amino acid in the mutation. Since the high value of the starting λmax for C87W implies that C87W is quite exposed to the aqueous solvent, the residue is likely to not have subsumed in the protein tertiary structure.Further, the myristic acid stabilise the melting temperature of all the mutants while octanoic acid only has a local effect of Y11W increasing the cooperativity. This implies different binding properties and that myristic acid stabilise the entire protein while octanoic acid only has a local stabilisation effect around the ligand binding site.

Resin and fatty acids (RFA) are predominantly components of coniferous trees having the natural function of protecting against microbial damage. These compounds are released from wood during the pulping process and a fraction reaches the wastewater treatment system. RFA are acutely toxic to aquatic organisms at concentrations on the order of parts per million, and their presence has been linked to toxicity outbreaks in receiving waters following process upsets. The chronic toxicity of resin and fatty acids in complex effluent matrices is poorly understood. Furthermore, the role of hydrophobic, pulp-derived solids as a removal pathway from wastewater streams has not been comprehensively studied. The objectives of this dissertation have been to quantify the relationship between resin and fatty acid concentration and chronic toxicity and to determine the role of partitioning in the removal of these compounds from pulp mill wastewater streams. Field and laboratory studies were conducted to measure toxicity using the Microtox™ whole effluent toxicity and Ceriodaphnia dubia 7-day, survival and reproduction bioassays. One resin acid in particular, dehydroabietic acid, was found to account for a significant fraction of final effluent chronic toxicity. Dissolved and sorbed RFA concentrations were quantified by solvent extraction, methyl ester derivatization, and GC-FID analysis. Partitioning to suspended solids was found to be a major removal pathway for the RFA from the effluent treatment system. A kinetic model for flotation was applied and compared to experimental data. Flotation was found to be effective at selectively removing RFA bound to pulp-derived solids from pulp mill and bleach plant sewers at moderate to high pH.

This study explored the effects of dietary unsaturated fatty acids on feline lipid metabolism. It was hypothesized that high dietary linoleic acid (18:2n-6, LA) would enhance conversion to arachidonic acid (20:4n-6), enrichment of dietary long chain n-3 FA (LCn-3FA) would affect lipid parameters, and n-3 FAs incorporation may blunt n-6 FA incorporation. Twenty-nine cats were randomized into groups (n = 9, 10, 10), and fed for 28 days with blood collections on days 0, 14, and 28. Experimental diets consisted of a commercial diet, supplemented with 8g oil/100g kibble. Oil supplements and subsequent diets were: high-oleic sunflower (H diet) with 82% oleic acid (18:1n-9), Menhaden fish (M diet) with LCn-3FA, and safflower (S diet) with 75% 18:2n-6. Dietary 20:4n-6 content was: 0.03 for H and S, and 0.09 for M (g FA/kg diet). Nonesterified fatty acid (NEFA), triacylglycerol (TG), total cholesterol (TC), lipoproteincholesterol (LP-C), plasma phospholipid (PL) FAs, red blood cell membrane (RBC) FAs, and ∆5 and ∆6 desaturase indices were measured. Statistical analyses were performed with SAS PROC MIXED with p < 0.05 determining significance. Neither TC nor NEFA showed significant effects. Diet M resulted in significant TG lowering, despite typically low feline TGs. Similarly, pre-β LP-C (i.e. TG-rich VLDL) was decreased in diet M. Plasma PL FAs revealed significant accumulations of the following: 18:1n-9 in diet H, 18:2n-6 in diet S, and LCn-3FA in diet M. Despite high dietary 18:2n-6, plasma PL 20:4n-6 was not increased in diet S over diets H or M. Increased docosadienoic acid (20:2n-6) in diet S demonstrated that 18:2n-6 chain elongation occurred in deference to its ∆6 desaturation further substantiating low feline ∆6 desaturase activity. Interestingly, no diet M blunting of 20:4n-6 incorporation occurred because fish oil supplementation provided additional 20:4n-6. Tissue 20:4n-6 content appears to be diet-dependent. Accumulation of eicosapentaenoic acid (20:5n-3), but low affinity for docosahexaenoic acid (22:6n-3) occurred in diet M RBC membranes. After 28 days, plasma PLs reflect dietary intake more readily than RBC membranes. Fish oil supplementation resulted in plasma PL LCn-3FA enrichment and lowered plasma TG concentrations, both of which may have physiological significance in cats.

Sixty-six percent of the ground beef consumed in the U.S. contains 16-30 percent fat by weight, and at the retail level, ground beef fat varies widely with regards to saturated, monounsaturated and trans-fatty acid content. Through two independent studies the effect of fatty acid composition of ground beef on selected cardiovascular disease risk indicators was evaluated. In the first study, 27 free-living normocholesterolemic men completed a three-way crossover dietary intervention. Subjects consumed five, 114-g ground beef patties per week for 5 wk with intervening 4-wk washout periods. Patties contained 24 percent total fat with monounsaturated fatty acid:saturated fatty acid (MUFA:SFA) of either 0.71 (low-MUFA, pasture-fed), 0.83 (mid-MUFA, short-term corn-fed), or 1.10 (high-MUFA, long-term corn-fed). Blood was collected from each subject before and at the end of each diet period. Overall, the ground beef interventions decreased plasma insulin, HDL2, and HDL3 particle diameter and α-linolenic acid (18:2 (n-3)), and increased plasma arachidonic (20:4(n-6)). The greatest increase in HDL cholesterol from baseline (0.07 mmol/L) was after the high-MUFA ground beef intervention. An increase from baseline in LDL particle diameter (0.5 nm) occurred after the mid- and high-MUFA interventions.We concluded that low-MUFA ground beef from pasture/hay-fed cattle was no more “heart healthy” than high-MUFA ground beef from corn-fed cattle as judged by common clinical criteria. In the second study, 19 of 29 post menopausal women completed a two-way crossover design. Subjects consumed five, 114-g ground beef patties per week for 6 wk periods separated by a 4 wk washout period. The low-MUFA patties contained 19.4 percent fat with MUFA:SFA of 0.9. The high-MUFA patties contained 22.5 percent fat with a MUFA:SFA ratio of 1.3. In addition to patty consumption, the subjects completed a bout of exercise during the last week of each phase. Blood was taken before, each diet phase (24 hr before exercise) and 24 hr post exercise. Total cholesterol was increased by the high-MUFA patties with the most significant increase seen in HDL cholesterol, mainly HDL2b subfraction. Lipid-rich lipoprotein fractions were increased with the low-MUFA diet, but not by the high-MUFA diet. Very long chain fatty acids were depressed by low MUFA patty consumption. When unadjusted for plasma volume shifts (raw), exercise decreased triglycerides in all three phases. Raw VLDL cholesterol was reduced after exercise during the intervention phases. Raw RLP and IDL cholesterol were reduced after exercise during the high-MUFA intervention. HDL2b was reduced after exercise during the high-MUFA phase. LDL mean size increased and LDL mean density decreased after exercise during the low-MUFA intervention. HDL mean density increased after exercise during both ground beef interventions. The data indicate that high-oleic ground beef can reduce some cardiovascular disease risk factors and can be a part of a healthful diet. Exercise can have a beneficial impact on cardiovascular disease risk factors independent and in conjunction with ground beef consumption.

As shown by recent reports, number of obese people in recent years has been on the increase, there are about 4 million people in Taiwan who are considered to be overweight. World Health Organization (WHO) and United States Center for Disease Control and Prevention (CDC) publicly announced that: Obesity will be the greatest health killer of this century, its damage to personal health is comparable to that of cigarettes. Obesity can cause heart problems, diabetes, artery diseases, high blood pressure, increased chances of cancer occurrence, condition increase and deteriora- tion of Alzheimer¡¦s disease, gall bladder diseases, and shortening of life span. The cause of obesity is due to a fault in adipocytes metabolism functions, and because of this, research into adipocytes molecular regulation is becoming more popular and valued. The process of adipogenesis, the formation of adipose tissue, has become better understood by the studies of several cell types that can be induced to undergo differentiation into adipocytes. The first, and the best characterized, model of adipogenesis in vitro is the 3T3-L1 cell line, a substrain of Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets. Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer -binding protein £] (C/EBP£]). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels. At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBP£_. Immediately after exposure to exogenous inducers, the gene expression of C/EBP£] and C/EBP£_ significantly and transiently increases, C/EBP£] and C/EBP£_ may also regulate the expression of C/EBP£\ and PPAR£^. C/EBP£\ and PPAR£^ are considered to play a prominent role in regulating the gene expression of proteins necessary for the development fo the functional mature adipocyte. Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation. C/EBP£\ and PPAR£^ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin. The identification of regulators of adipogenesis raises the prospect of preventing or reversing obesity through pharmacological means. My research is aimed at investigating the adipocytes differentiation and regeneration adaptive mechanisms of mice 3T3L-1 preadipocytes and human processed lipoaspirate cells (PLA). By using adipocytes culture techniques in conjunction with adipocytes growth induction and gene delivery techniques to further study obesity related genes, POMC and PTEN, and downstream regulators , PPAR£^ and Adiponectin, in regards to their roles in the process of adipocytes differentiation.

Hepatic steatosis is common in the general population and is present in 13.25% of donor organs. It can affect graft survival and recovery of the donor after partial hepatectomy. Liver biopsy is the standard method to measure the degree of hepatic steatosis, but it¡¦s also an invasive procedure and may have sampling error. Non-invasive tools, such as computed tomography and magnetic resonance image, are generally utilized and developed. This study was designed to build a standard model for the quantification of the fat content in a fat-water mixed phantom model. Pork fat and pure water were mixed in different ratios by volume (from 0% fraction of fat to 100% fat in steps of 5%), and then measured for fat content in different concentrations of fat-water mixed phantom by using (1) CT number (Hounsfield unit; HU), (2) Dixon method (in-phase & opposed-phase), and (3) 1H spectroscopy (SVS30 & SVS136, without water suppression). The CT number decreased with increasing fat concentration. The Hounsfield units of pure fat were about -122 HU. At Dixon method, the fat image intensity increased to its maximum when the fat concentration reached 25% and then decreased. Fat concentration higher than 25% and lower than 25% both had the same value of the fat image intensity. Combined with SVS30 water/fat peak height ratio, the fat concentration could be estimated. Furthermore, the fat image could be utilized to observe the topographic distribution of hepatic steatosis. Then a rat fatty liver model fed with a choline deficient and iron supplemented L-amino acid defined (CDAA) diet was established. Fatty liver grade was evaluated by radiological and biochemical assessments. CT and MRS technique displayed the highest fat contents the same with histological examination in CDAA diet rats at 6 weeks. The results showed that MRS was a suitable method for quantifying fat to water concentration. As a result of this study, model of measurement scale can be established to measure fat concentration both in phanatoms and animal. Further study in human fatty liver was expected.

Obesity is a disorder of energy imbalance and the most prevalent nutritional diseases in developed countries. Besides, obesity is also strongly associated with health problems such as type 2 diabetes (NIDDM), hypertension, hyperlipidaemia, cardiovascular diseases and cancers. However, the defects in lipid metabolism underlying obesity-related disorders are extremely complicated. Thus, extensive studies on the mechanism of endogenous fatty acids synthesis would be one of the keys to elucidate molecular pathogenesis of obesity. In liver or adipose, fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA and NADPH to synthesize long-chain fatty acids (C16 or C18), which can be converted to triglycerides and stored as fat. During lactation, thioesterase II (TE II) expresses in mammary glands and interacts with FAS to produce medium-chain fatty acid (primarily C10) in milk, which provides immune protection and energy for the newborn. TE II causes premature termination of fatty acid synthesis catalyzed by FAS and releases medium-chain fatty acids. Unlike long-chain fatty acids, medium-chain fatty acids can enter mitochondria directly for beta-oxidation to generate ATP, thus provide energy more efficiently. Since TE II gene expression is under strict regulation, we utilized adenovirus gene transfer techniques to deliver and express TE II in hepatocytes. It was postulated that expression of TE II in hepatocytes might result in the increase of ATP and reduction of long-chain fatty acids, subsequently decrease the fat production. Recombinant adenovirus was used as gene delivery system for TE II because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant Ad-TE II by PCR, western blot analysis, and enzymatic assay, respectively. By using Ad-GFP, we have determined the optimal multiplicity of infection (MOI) for adenovirus to infect HepG2 cells is about 100-200. Adenovirus-mediated TE II expression in hepatocytes was demonstrated by western blot as well as TE II enzymatic assay. We have demonstrated that the adenovirus-mediated TE II expression was slightly cytotoxic to hepatocytes. Besides, an increase of free fatty acids, asparate transaminase, lactate dehydrogenase levels, as well as ATP synthesis was also noted in the TE II-expressed hepatocytes. The enhanced the release of asparate transaminase (AST/GOT) and lactate dehydrogenase (LDH) after TE II expression in the hepatocytes further supported its cytotoxcity to hepatocytes. In the future, we will carry out experiments to further characterize the effects of TE II expression on cellular lipid metabolism through adenovirus gene delivery. We hope that the present studies will not only provide further insights into mammalian lipid metabolism, but also enable us to evaluate the therapeutic potential of TE II on the treatment of obesity and its related disorders.

The effects of conjugated linoleic acid (CLA) on lipogenesis and preadipocyte proliferation in young pigs were evaluated in two separate experiments. The first compared dietary effects of linoleic acid, beef tallow, and CLA on composition, lipogenesis, and DNA synthesis. Eighteen pigs weaned at 17 d of age were allotted randomly to corn-based diets supplemented with 1.5% corn oil, 1.5% tallow, or 1.5% CLA. The second experiment evaluated the effects of CLA included with diets high in polyunsaturated fat or beef tallow. Twenty-four pigs weaned at 17 d of age were allotted randomly to one of four corn-based diets supplemented with: 15% corn oil, 12% corn oil + 3% CLA, 15% tallow, and 12% tallow + 3% CLA. The piglets in both trials were fed a basal diet for 7 d and their respective diet for 35 d. [U-14C]Glucose incorporation into total lipids was (experiment 1): 10.64, 11.04, 13.64; (experiment 2): 21.15, 17.54, 21.34, and 19.52 nmol/(105 cells per h) for subcutaneous (s.c.) adipose tissue from corn oil, tallow, CLA; corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively. Tritiated thymidine incorporation into DNA was not different in s.c. adipocytes across treatment groups, but was 5,581, 2,794, 6,573, and 3,760 dpm/(105 cells per h) in s.c. stromal vascular cells from corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively (CLA main effect p<0.034). Additionally, there was a greater proportion of s.c. adipocytes in the smaller, 180-pL cell fraction from the corn oil + CLA-fed pigs (p<0.0074). CLA in the diet increased the s.c. adipose tissue concentration of 18:0 and decreased 16:1 and 18:1 (p<0.05), suggesting depression of stearoyl-coenzyme A desaturase (SCD) enzyme activity in the CLA-fed pigs. The concentration of CLA isomers was raised only slightly in s.c. adipose tissue with the addition of CLA to the diets even though the CLA oil contained 62% CLA isomers. No effects on the growth of young pigs were observed. However, CLA caused a more saturated fatty acid composition and may suppress preadipocyte proliferation, apparent SCD activity, and lipid filling of smaller cells.

This study was conducted to examine the antioxidative effect of conjugated linoleic acid (CLA) in irradiated, cooked ground beef patties. The hypothesis was that CLA would be retained during irradiation and would reduce lipid oxidation that is caused by irradiation. The objective was to evaluate the effects of CLA alone and in combination with irradiation on lipid oxidation, fatty acid composition, cooking loss, moisture and fat content, and trained panel sensory evaluations of beef patties. CLA was added at 0, 1, 2, or 4% level during the grinding process. Addition of CLA during the grinding process increased CLA cis-9,trans-11 and CLA trans-10,cis-12 isomers in both irradiated and non-irradiated cooked ground beef patties (irradiated at 1.6 kGy) (P = 0.0001). Weight loss during cooking was greater in irradiated beef patties than in non-irradiated patties (P = 0.004). Irradiation reduced the serumy/bloody aromatic attribute and increased browned aromatic attribute, browned aftertaste, and wet dog/hairy aromatic attribute (P < 0.05). There was no significant main effect of irradiation on the basic tastes. The linoleic acid, CLA cis-9,trans-11, and CLA trans-10,cis-12 were decreased by irradiation (P < 0.05). Although irradiation decreased the CLA isomers, higher percentages of CLA isomers were retained in irradiated patties containing a 4% free fatty acid preparation of CLA (FFA-CLA), reflecting the ability of the FFA preparation to reduce lipid oxidation that is caused by irradiation. The thiobarbituric acid reactive substances (TBARS) values were significantly higher in irradiated, cooked ground beef patties than in non-irradiated ground beef patties (P = 0.004). Although the FFA-CLA was effective in reducing lipid oxidation that is caused by irradiation, it increased painty aromatic attribute, bitter taste, and astringent aftertaste due to the soapy flavor of the free fatty acid (all P < 0.05). The FFA-CLA decreased cooked beef/brothy and serumy/bloody aromatic attribute and browned aftertaste (all P < 0.05). The 1% triacylglycerol (TAG) preparation of CLA reduced TBARS in irradiated, cooked patties to levels seen in control, non-irradiated patties. The 1% TAG concentration also provided good retention of CLA in the cooked ground beef.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Greek alphabet delta in title. Includes bibliographical references (leaves 136-155). Available also in a digital version from Dissertation Abstracts.