The current dietary recommendations for optimal health are designed to increase our intake of two bioactive omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), abundant naturally in fatty fish such as salmon. Health authorities recommend that the general population consume two to three fatty fish meals per week (1) for optimal health and for the prevention of cardiovascular disease. However, some modern Western societies consume only modest amounts of fish and seafood (2;3). Land based vegetable oils may provide an alternative to meet these needs. Linseed and canola oils are rich in alpha-linolenic acid (ALA, 18:3n-3) (4). ALA can be converted endogenously to EPA and DHA and suggests that increasing the dietary intake of ALA may increase the conversion and accumulation of DHA in tissues and plasma. However, elevated dietary intakes of ALA in animals and humans results in an increased level of EPA in tissues yet there is little or no change in the level of DHA (5-7). The current consensus is that the synthesis of DHA from ALA in humans is limited yet the mechanisms involved in regulating the accumulation of DHA in tissues are poorly understood. The reputed rate-limiting enzyme in the conversion of fatty acids is delta 6 desaturase (D6D). ALA is a substrate for D6D and undergoes a series of desaturation and elongation reactions to yield n-3 long chain polyunsaturated fatty acids (LCPUFA). The final step in the synthesis of DHA from ALA involves translocation of its immediate fatty acid precursor, 24:6n-3 from the endoplasmic reticulum to the peroxisome to be partially beta-oxidised to yield DHA. The involvement of multiple enzymes in the desaturation-elongation pathway, and the integration of other pathways, such as phospholipid biosynthesis, suggests there are various steps that may regulate the accumulation of DHA in cell membranes. This thesis aimed to examine the possible regulatory steps in the conversion of fatty acids to LCPUFA, particularly in the synthesis of DHA from n-3 fatty acid precursors. The human hepatoma cell line, HepG2, was used as an in vitro cell system to examine the accumulation of individual fatty acids and their metabolites in isolation from other competing fatty acid substrates. The accumulation of linoleic acid (LA, 18:2n-6) and ALA in HepG2 cell phospholipids following supplementation with increasing concentrations of each respective fatty acid correlated with that described in vivo, as was the accumulation of their conversion products. The accumulation of DHA in cells supplemented with ALA reached a plateau at concentrations above 5 micro g/ml and paralleled the accumulation of 24:6n-3 in cell phospholipids, suggesting that the delta 6 desaturation of 24:6n-3 was prevented by increasing concentrations of ALA, thereby limiting the accumulation of DHA. The accumulation of DHA in cells supplemented with eicosapentaenoic acid (EPA, 20:5n-3) or docosapentaenoic acid (DPA, 22:5n-3) was significantly greater than the level of DHA that accumulated in cells supplemented with ALA. However, regardless of substrate, the level of DHA in cell membranes reached a plateau at substrate concentrations above 5 micro g/ml. This thesis further aimed to examine the effect of fatty acid supplementation on the mRNA expression of D6D in HepG2 cells. The expression and activity of D6D mRNA is subject to nutritional and hormonal regulation. The mRNA expression of D6D in HepG2 cells following supplementation with oleic acid (OA, 18:1n-9), LA, ALA, arachidonic acid (AA, 20:4n-6) or EPA was examined by real time RT PCR. The expression of D6D mRNA was reduced by up to 50% in cells supplemented with OA, LA, ALA , AA or EPA compared with control cells and suggests that fatty acids modulate the expression of the key enzyme involved in the conversion of fatty acids. The effect of fatty acid co-supplementation on the fatty acid composition of HepG2 cell phospholipids was also examined in an attempt to gain insights into the role of D6D and the enzymes involved in peroxisomal beta-oxidation on the accumulation of DHA from n-3 fatty acid precursors. The reduction in the accumulation of DHA in cells co-supplemented with DPA and docosatetraenoic acid (DTA, 22:4n-6) was greater than in cells co-supplemented with DPA and LA, suggesting that peroxisomal beta-oxidation may have a greater role in determining the accumulation of DHA from DPA than the activity of D6D. Further investigation should be directed towards understanding the role that peroxisomal beta-oxidation may play in the synthesis of DHA from precursor fatty acids. The fatty acid composition of cell membranes in vivo is a result of several physiological processes including dietary intake, phospholipids biosynthesis and fatty acid conversion as well as catabolic processes. This thesis demonstrates that a greater understanding of the regulation of the conversion of fatty acids will help to define dietary approaches that enhance the synthesis of n-3 LCPUFA from n-3 fatty acid precursors to lead to improved outcomes for health.

Forty-five Angus steers (358 kg BW) were utilized in a completely randomized block design with a 3 x 3 factorial arrangement of treatments to evaluate the hypothesis that differing dietary linolenic acid (from corn, flaxseed plus corn, or milo) and whole cottonseed (WCS) inclusion (0, 5, or 15% DM) would interact to alter fatty acid metabolism and deposition of conjugated linoleic acid (CLA) in subcutaneous (s.c.) and interfasicular (i.f.) adipose tissues, and thereby decrease carcass quality score. During the feeding period (135 d), steers receiving flaxseed or corn diets had a greater gain:feed ratio (0.119 and 0.108, respectively) than steers receiving the milo diet (0.093). Following transportation to a local abattoir and overnight starvation, there was less decrease in weight in flaxseed-fed steers (1.51%) than in steers fed the corn (2.89%) or milo diets (3.11%). Ribeye area of steers fed milo was less than that of steers fed the corn or flaxseed diets. Lipogenesis from acetate in s.c. adipose tissue was greater in steers fed flaxseed (5.42 nmol h-1 105 cells-1) than in the corn (3.10 nmol h-1 105 cells-1) or milo (1.92 nmol h-1 105 cells-1) groups. Stearoyl-CoA desaturase (SCD) activity in s.c. adipose tissue was unchanged between the 0% WCS group (88.1 nmol mg protein-1 7 min-1) and the 15% WCS group (20 nmol mg protein-1 7 min-1). The i.f. saturated fatty acid percentages increased with increasing levels of WCS. The i.f. cis-9, trans-11 CLA percentage increased with increasing WCS in the steers fed the corn diet, whereas it remained unchanged or even decreased slightly in the steers fed the flaxseed or milo-based diets. Steers fed flaxseed had a greater s.c. adipose concentration of vaccenic acid (18:1trans-11) than the steers fed milo. Steers fed flaxseed also had greater s.c. and i.f. percentages of linolenic acid (18:3, n-3) than steers fed either of the other grain sources. Increased dietary linolenic acid from flaxseed may have increased s.c. adipocyte volume by stimulating lipogenesis. These data indicate that rations formulated to provide increased levels of linolenic acid (i.e., flaxseed) will increase feed efficiency and lipogenesis from acetate without altering either the quality or composition of the beef carcasses.

Forty-five Angus steers (358 kg BW) were utilized in a completely randomized block design with a 3 x 3 factorial arrangement of treatments to evaluate the hypothesis that differing dietary linolenic acid (from corn, flaxseed plus corn, or milo) and whole cottonseed (WCS) inclusion (0, 5, or 15% DM) would interact to alter fatty acid metabolism and deposition of conjugated linoleic acid (CLA) in subcutaneous (s.c.) and interfasicular (i.f.) adipose tissues, and thereby decrease carcass quality score. During the feeding period (135 d), steers receiving flaxseed or corn diets had a greater gain:feed ratio (0.119 and 0.108, respectively) than steers receiving the milo diet (0.093). Following transportation to a local abattoir and overnight starvation, there was less decrease in weight in flaxseed-fed steers (1.51%) than in steers fed the corn (2.89%) or milo diets (3.11%). Ribeye area of steers fed milo was less than that of steers fed the corn or flaxseed diets. Lipogenesis from acetate in s.c. adipose tissue was greater in steers fed flaxseed (5.42 nmol h-1 105 cells-1) than in the corn (3.10 nmol h-1 105 cells-1) or milo (1.92 nmol h-1 105 cells-1) groups. Stearoyl-CoA desaturase (SCD) activity in s.c. adipose tissue was unchanged between the 0% WCS group (88.1 nmol mg protein-1 7 min-1) and the 15% WCS group (20 nmol mg protein-1 7 min-1). The i.f. saturated fatty acid percentages increased with increasing levels of WCS. The i.f. cis-9, trans-11 CLA percentage increased with increasing WCS in the steers fed the corn diet, whereas it remained unchanged or even decreased slightly in the steers fed the flaxseed or milo-based diets. Steers fed flaxseed had a greater s.c. adipose concentration of vaccenic acid (18:1trans-11) than the steers fed milo. Steers fed flaxseed also had greater s.c. and i.f. percentages of linolenic acid (18:3, n-3) than steers fed either of the other grain sources. Increased dietary linolenic acid from flaxseed may have increased s.c. adipocyte volume by stimulating lipogenesis. These data indicate that rations formulated to provide increased levels of linolenic acid (i.e., flaxseed) will increase feed efficiency and lipogenesis from acetate without altering either the quality or composition of the beef carcasses.

The biological activity of α-linolenic acid (ALA) is poorly understood and primarily associated with its conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This study used the Δ6 desaturase knockout (D6KO) mouse, which lacks Δ6 desaturase and therefore cannot convert ALA, to evaluate the independent effects of ALA on preventing non-alcoholic fatty liver disease (NAFLD). First, the capacity of very long chain fatty acids to rescue the D6KO lipid profile was established. Next, to evaluate the independent effects of ALA, D6KO or wild-type mice were fed diets containing lard, canola, flaxseed, or fish oil. Following treatment, liver phospholipid fatty acid composition was evaluated and livers were scored for steatosis and inflammation. Glucose tolerance was also evaluated. D6KO mice fed ALA-rich diets had lower liver lipid accumulation, lower hepatic inflammation (8 weeks) and improved glucose tolerance (20 weeks) relative to lard-fed D6KO mice. Overall, this thesis supports an independent biological role for ALA. / D.W.L. Ma is funded by the Canola Council of Canada, Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation Leaders Opportunity Fund with matching from the Ontario Research Fund; Jessica Monteiro is funded Ontario Graduate Scholarship.

Stearoyl-coA desaturase-1 (SCD1) activity has been linked to the development of obesity and the metabolic syndrome (MetS) through its central role in lipid metabolism. Understanding how changes in SCD1 activity affect obesity and MetS risk biomarkers and investigating how these changes in activity affect nutrient handling in non-hepatic tissues is also important. This study investigated how acute SCD1 inhibition effected plasma lipids, skeletal muscle nutrient handling and insulin signaling in mice fed a high-carbohydrate very-low fat diet for 10 weeks. This study demonstrated that SCD1 inhibition created acute dyslipidemia, altered nutrient handling protein activity and increased the percentage of saturated fatty acids (SFA) in hepatic and muscle tissue, independent of dietary oleic acid content. However, the molecular controls of protein synthesis in the mTOR pathway were not affected by the loss of SCD1 activity. In conclusion, we observed that inhibiting hepatic SCD1 activity and subsequently changing the monounsaturated fatty acid (MUFA) to SFA ratios in tissues alters normal nutrient handling in skeletal muscle.

Oleic acid (18:1) is one of the important monounsaturated fatty acids, which is synthesized upon desaturation of stearic acid and this reaction is catalyzed by the SSI2 encoded stearoyl-acyl-carrier-protein-desaturase. A mutation in SSI2 leads to constitutive activation of salicylic acid (SA)-mediated defense responses. Consequently, these plants accumulate high levels of SA and show enhanced resistance to bacterial and oomycete pathogens. Replenishing 18:1 levels in ssi2 plants, via a second site mutation in GLY1 encoded glycerol-3-phosphate (G3P) dehydrogenase, suppresses all the ssi2-triggered phenotypes. Study of mechanism(s) underlying gly1-mediated suppression of ssi2 phenotypes showed that 18:1 levels are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of defense signaling pathways. To establish a role for 18:1 and G3P during host defense, interaction between Colletotrichum higginsianum and Arabidopsis was studied. Resistance to C. higginsianum correlated with host G3P levels. The gly1 plants showed increased susceptibility while act1 plants, defective in utilization of G3P, showed enhanced resistance. Plant overexpessing GLY1 showed enhanced resistance in both wild type as well as camalexin deficient backgrounds. Together, these results suggested that G3P conferred resistance acted downstream or independent of camalexin. Exogenous application of glycerol lowered 18:1 levels and produced ssi2-like phenotypes in wild-type plants. Furthermore, glycerol application or the ssi2 mutation produced similar phenotypes in fatty acid desaturation mutants and mutants defective in SA/resistance gene signaling. Expression studies showed that ssi2 phenotypes were likely due to increased expression of resistance genes. Epistatic analysis suggested that certain components of SA pathway had redundant function and were required for 18:1-regulated signaling.

Stearoyl-coA desaturase-1 (SCD1) activity has been linked to the development of obesity and the metabolic syndrome (MetS) through its central role in lipid metabolism. Understanding how changes in SCD1 activity affect obesity and MetS risk biomarkers and investigating how these changes in activity affect nutrient handling in non-hepatic tissues is also important. This study investigated how acute SCD1 inhibition effected plasma lipids, skeletal muscle nutrient handling and insulin signaling in mice fed a high-carbohydrate very-low fat diet for 10 weeks. This study demonstrated that SCD1 inhibition created acute dyslipidemia, altered nutrient handling protein activity and increased the percentage of saturated fatty acids (SFA) in hepatic and muscle tissue, independent of dietary oleic acid content. However, the molecular controls of protein synthesis in the mTOR pathway were not affected by the loss of SCD1 activity. In conclusion, we observed that inhibiting hepatic SCD1 activity and subsequently changing the monounsaturated fatty acid (MUFA) to SFA ratios in tissues alters normal nutrient handling in skeletal muscle.

abstract: The most abundantly studied societies, with the exception of humans, are those of the eusocial insects, which include all ants. Eusocial insect societies are typically composed of many dozens to millions of individuals, referred to as nestmates, which require some form of communication to maintain colony cohesion and coordinate the activities within them. Nestmate recognition is the process of distinguishing between nestmates and non-nestmates, and embodies the first line of defense for social insect colonies. In ants, nestmate recognition is widely thought to occur through olfactory cues found on the exterior surfaces of individuals. These cues, called cuticular hydrocarbons (CHCs), comprise the overwhelming majority of ant nestmate profiles and help maintain colony identity. In this dissertation, I investigate how nestmate recognition is influenced by evolutionary, ontogenetic, and environmental factors. First, I contributed to the sequencing and description of three ant genomes including the red harvester ant, Pogonomyrmex barbatus, presented in detail here. Next, I studied how variation in nestmate cues may be shaped through evolution by comparatively studying a family of genes involved in fatty acid and hydrocarbon biosynthesis, i.e., the acyl-CoA desaturases, across seven ant species in comparison with other social and solitary insects. Then, I tested how genetic, developmental, and social factors influence CHC profile variation in P. barbatus, through a three-part study. (1) I conducted a descriptive, correlative study of desaturase gene expression and CHC variation in P. barbatus workers and queens; (2) I explored how larger-scale genetic variation in the P. barbatus species complex influences CHC variation across two genetically isolated lineages (J1/J2 genetic caste determining lineages); and (3) I experimentally examined how CHC development is influenced by an individual’s social environment. In the final part of my work, I resolved discrepancies between previous findings of nestmate recognition behavior in P. barbatus by studying how factors of territorial experience, i.e., spatiotemporal relationships, affect aggressive behaviors among red harvester ant colonies. Through this research, I was able to identify promising methodological approaches and candidate genes, which both broadens our understanding of P. barbatus nestmate recognition systems and supports future functional genetic studies of CHCs in ants. / Dissertation/Thesis / Doctoral Dissertation Biology 2016

Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild type Arabidopsis plants regarding their sensitivity to low temperature, and the size and height of the plants.

Studies were conducted to investigate the tissue distribution of stearoyl-CoA desaturase-1 (SCD) and the regulation of SCD1 protein expression by dietary fat, insulin, polyunsaturated fatty acids (PUFA), and linoleic acid (cis-9, cis-12 18:2). The first study examined tissue distribution of SCD1 protein in Holstein calves (n=6/diet) fed one of four milk replacer diets for a nine wk period after which they were sacrificed. Milk replacer diets varied in fat content and were formulated and administered as follows: 0.4 kg/d 20% protein, 20% fat (20:20; CON), 0.97 kg/d (28:20; HPLF), 0.97 kg/d (28:28; HPHF), or 1.46 kg/d (28:28; HPHF+). Samples of subcutaneous adipose tissue (AT), perirenal AT, omental AT, duodenum, proximal jejunum, distal jejunum, ileum, and liver were collected from calves fed the HPHF+ diet to determine SCD1 tissue distribution. Tissue homogenates were prepared and used for Western blotting. Additionally, dietary effects were analyzed on tissues expressing SCD1 protein for all 24 calves. The second study investigated the regulation of SCD1 protein expression by insulin, fatty acids increasing in degree of unsaturation, and increasing concentrations of linoleic (18:2) acid. Subcutaneous AT was collected from Smith Valley Meats in Rich Creek, VA and used to prepare explants cultured in treatment media for 24 h. Treatments consisted of insulin at 0, 7, 14, and 21 nM; stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids at 100 μM; and linoleic (18:2) acid at concentrations of 0, 25, 50, 75, and 100 μM. Tissue explant homogenates were used for Western blotting to detect SCD1. In the first study, we found that SCD1 protein was detectable in subcutaneous AT, perirenal AT, and omental AT; however, it was not detectable in liver or small intestine samples. Also, the HPHF+ diet increased SCD1 protein expression in subcutaneous AT and perireanl AT. In the second study, SCD1 protein expression increased linearly with insulin concentration. There was no fatty acid treatment effect, but there was a negative linear effect with increase in degree of unsaturation. Finally, there was no effect on SCD1 protein expression with linoleic acid increasing in concentration. In conclusion, results indicate that SCD1 protein expression was detected in bovine AT depots, regulated by dietary fat, insulin, and by PUFA . / Master of Science