Дисертації з теми "Omega-3 long-chain polyunsaturated fatty acid"

Alterations in cognitive function are characteristic of the aging process in humans and other animals. However, the nature of these age related changes in cognition is complex and is likely to be influenced by interactions between genetic predispositions and environmental factors resulting in dynamic fluctuations within and between individuals. These inter and intra-individual fluctuations are evident in both so-called normal cognitive aging and at the onset of cognitive pathology. Mild Cognitive Impairment (MCI), thought to be a prodromal phase of dementia, represents perhaps the final opportunity to mitigate cognitive declines that may lead to terminal conditions such as dementia. The prognosis for people with MCI is mixed with the evidence suggesting that many will remain stable within 10-years of diagnosis, many will improve, and many will transition to dementia. If the characteristics of people who do not progress to dementia from MCI can be identified and replicated in others it may be possible to reduce or delay dementia onset, thus reducing a growing personal and public health burden. Furthermore, if MCI onset can be prevented or delayed, the burden of cognitive decline in aging populations worldwide may be reduced. A cognitive domain that is sensitive to the effects of advancing age, and declines in which have been shown to presage the onset of dementia in MCI patients, is executive function. Moreover, environmental factors such as diet and physical activity have been shown to affect performance on tests of executive function. For example, improvements in executive function have been demonstrated as a result of increased aerobic and anaerobic physical activity and, although the evidence is not as strong, findings from dietary interventions suggest certain nutrients may preserve or improve executive functions in old age. These encouraging findings have been demonstrated in older adults with MCI and their non-impaired peers. However, there are some gaps in the literature that need to be addressed. For example, little is known about the effect on cognition of an interaction between diet and physical activity. Both are important contributors to health and wellbeing, and a growing body of evidence attests to their importance in mental and cognitive health in aging individuals. Yet physical activity and diet are rarely considered together in the context of cognitive function. There is also little known about potential underlying biological mechanisms that might explain the physical activity/diet/cognition relationship. The first aim of this program of research was to examine the individual and interactive role of physical activity and diet, specifically long chain polyunsaturated fatty acid consumption(LCn3) as predictors of MCI status. The second aim is to examine executive function in MCI in the context of the individual and interactive effects of physical activity and LCn3.. A third aim was to explore the role of immune and endocrine system biomarkers as possible mediators in the relationship between LCn3, physical activity and cognition. Study 1a was a cross-sectional analysis of MCI status as a function of erythrocyte proportions of an interaction between physical activity and LCn3. The marine based LCn3s eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have both received support in the literature as having cognitive benefits, although comparisons of the relative benefits of EPA or DHA, particularly in relation to the aetiology of MCI, are rare. Furthermore, a limited amount of research has examined the cognitive benefits of physical activity in terms of MCI onset. No studies have examined the potential interactive benefits of physical activity and either EPA or DHA. Eighty-four male and female adults aged 65 to 87 years, 50 with MCI and 34 without, participated in Study 1a. A logistic binary regression was conducted with MCI status as a dependent variable, and the individual and interactive relationships between physical activity and either EPA or DHA as predictors. Physical activity was measured using a questionnaire and specific physical activity categories were weighted according to the metabolic equivalents (METs) of each activity to create a physical activity intensity index (PAI). A significant relationship was identified between MCI outcome and the interaction between the PAI and EPA; participants with a higher PAI and higher erythrocyte proportions of EPA were more likely to be classified as non-MCI than their less active peers with less EPA. Study 1b was a randomised control trial using the participants from Study 1a who were identified with MCI. Given the importance of executive function as a determinant of progression to more severe forms of cognitive impairment and dementia, Study 1b aimed to examine the individual and interactive effect of physical activity and supplementation with either EPA or DHA on executive function in a sample of older adults with MCI. Fifty male and female participants were randomly allocated to supplementation groups to receive 6-months of supplementation with EPA, or DHA, or linoleic acid (LA), a long chain polyunsaturated omega-6 fatty acid not known for its cognitive enhancing properties. Physical activity was measured using the PAI from Study 1a at baseline and follow-up. Executive function was measured using five tests thought to measure different executive function domains. Erythrocyte proportions of EPA and DHA were higher at follow-up; however, PAI was not significantly different. There was also a significant improvement in three of the five executive function tests at follow-up. However, regression analyses revealed that none of the variance in executive function at follow-up was predicted by EPA, DHA, PAI, the EPA by PAI interaction, or the DHA by PAI interaction. The absence of an effect may be due to a small sample resulting in limited power to find an effect, the lack of change in physical activity over time in terms of volume and/or intensity, or a combination of both reduced power and no change in physical activity. Study 2a was a cross-sectional study using cognitively unimpaired older adults to examine the individual and interactive effects of LCn3 and PAI on executive function. Several possible explanations for the absence of an effect were identified. From this consideration of alternative explanations it was hypothesised that post-onset interventions with LCn3 either alone or in interation with self-reported physical activity may not be beneficial in MCI. Thus executive function responses to the individual and interactive effects of physical activity and LCn3 were examined in a sample of older male and female adults without cognitive impairment (n = 50). A further aim of study 2a was to operationalise executive function using principal components analysis (PCA) of several executive function tests. This approach was used firstly as a data reduction technique to overcome the task impurity problem, and secondly to examine the executive function structure of the sample for evidence of de-differentiation. Two executive function components were identified as a result of the PCA (EF 1 and EF 2). However, EPA, DHA, the PAI, or the EPA by PAI or DHA by PAI interactions did not account for any variance in the executive function components in subsequent hierarchical multiple regressions. Study 2b was an exploratory correlational study designed to explore the possibility that immune and endocrine system biomarkers may act as mediators of the relationship between LCn3, PAI, the interaction between LCn3 and PAI, and executive functions. Insulin-like growth factor-1 (IGF-1), an endocrine system growth hormone, and interleukin-6 (IL-6) an immune system cytokine involved in the acute inflammatory response, have both been shown to affect cognition including executive functions. Moreover, IGF-1 and IL-6 have been shown to be antithetical in so far as chronically increased IL-6 has been associated with reduced IGF-1 levels, a relationship that has been linked to age related morbidity. Further, physical activity and LCn3 have been shown to modulate levels of both IGF-1 and IL-6. Thus, it is possible that the cognitive enhancing effects of LCn3, physical activity or their interaction are mediated by changes in the balance between IL-6 and IGF-1. Partial and non-parametric correlations were conducted in a subsample of participants from Study 2a (n = 13) to explore these relationships. Correlations of interest did not reach significance; however, the coefficients were quite large for several relationships suggesting studies with larger samples may be warranted. In summary, the current program of research found some evidence supporting an interaction between EPA, not DHA, and higher energy expenditure via physical activity in differentiating between older adults with and without MCI. However, a RCT examining executive function in older adults with MCI found no support for increasing EPA or DHA while maintaining current levels of energy expenditure. Furthermore, a cross-sectional study examining executive function in older adults without MCI found no support for better executive function performance as a function of increased EPA or DHA consumption, greater energy expenditure via physical activity or an interaction between physical activity and either EPA or DHA. Finally, an examination of endocrine and immune system biomarkers revealed promising relationships in terms of executive function in non-MCI older adults particularly with respect to LCn3 and physical activity. Taken together, these findings demonstrate a potential benefit of increasing physical activity and LCn3 consumption, particularly EPA, in mitigating the risk of developing MCI. In contrast, no support was found for a benefit to executive function as a result of increased physical activity, LCn3 consumption or an interaction between physical activity and LCn3, in participants with and without MCI. These results are discussed with reference to previous findings in the literature including possible limitations and opportunities for future research.

Doctor of Philosophy
Department of Human Nutrition
Weiqun Wang
Many functional food related bioactive compounds have been discovered and draw the attention of scientists. This dissertation focused on sorghum phenolic compounds and omega-3 polyunsaturated fatty acids. Study 1: phenolic agents in plant foods have been associated with chronic disease prevention, especially cancer. However, a direct evidence and the underlying mechanisms are mostly unknown. This study selected 13 sorghum accessions and was aim to investigate: (1) the effect of extracted sorghum phenolics on inhibiting cancer cell growth using hepatocarcinoma HepG2 and colorectal adenocarcinoma Caco-2 cell lines; (2) and the underlying mechanisms regarding cytotoxicity, cell cycle interruption, and apoptosis induction. Treatment of HepG2 and Caco-2 cells with the extracted phenolics at 0-200 M GAE (Gallic acid equivalent) up to 72 hrs resulted in a dose- and time-dependent reduction in cell number. The underlying mechanism of cell growth inhibition was examined by flow cytometry, significant inverse correlations were observed between the decreased cell number and increased cell cycle arrest at G2/M or induced apoptosis cells in both HepG2 and Caco-2 cells. The cytotoxic assay showed that the sorghum phenolic extracts were non-toxic. Although it was less sensitive, a similar inhibitory impact and underlying mechanisms were found in Caco-2 cells. These results indicated for the 1st time that a direct inhibition of either HepG2 or Caco-2 cell growth by phenolic extracts from13 selected sorghum accessions was due to cytostatic and apoptotic but not cytotoxic mechanisms. In addition, these findings suggested that sorghum be a valuable functional food by providing sustainable phenolics for potential cancer prevention. Study 2: omega-3 polyunsaturated fatty acids (ω-3 PUFAs) especially long-chain ω-3 PUFAs, have been associated with potential health benefits in chronic disease prevention. However, the conversion rate from short- to long-chain ω-3 PUFAs is limited in human body. This study was aim to assess the modification of fatty acid profiles as well as investigate the conversion of short- to long-chain ω-3 PUFAs in the liver of Shan Partridge duck after feeding various dietary fats. The experimental diets substituted the basal diet by 2% of flaxseed oil, rapeseed oil, beef tallow, or fish oil, respectively. As expected, the total ω-3 fatty acids and the ratio of total ω-3/ ω-6 significantly increased in both flaxseed and fish oil groups when compared with the control diet. No significant change of total saturated fatty acids or ω-3 fatty acids was found in both rapeseed and beef tallow groups. Short-chain ω-3 α-linolenic acid (ALA) in flaxseed oil-fed group was efficiently converted to long-chain ω-3 docosahexaenoic acid (DHA) in the duck liver. This study showed the fatty acid profiling in the duck liver after various dietary fat consumption, provided insight into a dose response change of ω-3 fatty acids, indicated an efficient conversion of short- to long-chain ω-3 fatty acid, and suggested alternative long-chain ω-3 fatty acid-enriched duck products for human health benefits. In conclusion, the two studies in this dissertation provided a fundamental understanding of anti-cancer activity by sorghum phenolic extracts and the conversion of short- to long-chain ω-3 PUFAs in duck liver, contribute to a long term goal of promoting sorghum and duck as sustainable phenolic and ω-3 PUFAs sources as well as healthy food products for human beings.

La résistance des cellules tumorales à la chimiothérapie constitue une cause majeure d’échec des traitements anticancéreux. Des études précliniques montrent que les acides gras polyinsaturés oméga-3 à longues chaînes (AGPIn-3LC), apportés par l’alimentation, améliorent l’efficacité des chimiothérapies sans majorer les effets secondaires. Cette thèse a eu pour but d’identifier les mécanismes moléculaires impliqués dans l’augmentation de la sensibilité des cellules tumorales mammaires au docétaxel. Nous avons montré que le docétaxel induit un mécanisme de résistance via l’activation des voies de signalisation PKC/ERK et Akt impliquées dans la prolifération et la survie cellulaires. La modification de l’environnement lipidique membranaire par la supplémentation en AGPIn-3LC inhibe ces voies de signalisation et augmente l’efficacité du docétaxel dans des lignées tumorales mammaires et dans un modèle préclinique de tumeurs mammaires autochtones chez le rongeur. De plus, dans ce modèle in vivo, nous avons identifié une autre cible moléculaire régulée par les AGPIn-3LC : l’épiréguline, membre de la famille EGF. Les AGPIn-3LC bloquent l’induction de l’épiréguline par le VEGF dans les cellules endothéliales et induisent un remodelage de la vascularisation tumorale. Outre un effet direct des AGPIn-3LC sur les cellules tumorales, les AGPIn- 3LC agissent sur le microenvironnement tumoral. Ces travaux de thèse apportent des arguments supplémentaires pour l’utilisation des AGPIn-3LC comme molécules adjuvantes pour lutter contre la résistance des tumeurs mammaires aux agents anticancéreux
Chemotherapy-resistant tumor cells are a major cause of cancer treatment failure. Preclinical studies show that polyunsaturated omega-3 long chain fatty acids (AGPIn-3LC), provided by food, improve the efficacy of chemotherapy without increasing side effects. AGPIn-3LCs are incorporated in cancer and stromal cells. This thesis aimed to identify molecular mechanisms involved in the increased sensitivity of mammary tumor cells to docetaxel. We have shown that docetaxel induces a resistance mechanism via activation of PKC/ERK and Akt pathways involved in cell proliferation and survival. Modification of the membrane lipid environment by AGPIn-3LCs supplementation inhibits these signaling pathways and increases the efficacy of docetaxel in mammary tumor cell lines and in a preclinical rodent model of native mammary tumors. Moreover, in this mammary tumor model we have found another molecular target regulated by AGPIn-3LCs: epiregulin, a member of the EGF family. AGPIn-3LCs inhibit epiregulin-VEGF induced in endothelial cells and induce a remodeling of tumor vasculature. Furthermore, AGPIn-3LCs act on the tumor microenvironment directly. This thesis work provides additional arguments for the use of AGPIn-3LCs as adjuvant molecules to reduce the resistance of breast tumors to anticancer agents

Les acides gras polyinsaturés à longue chaîne (AGPI-LC) de la série n-3 jouent des rôles essentiels dans le fonctionnement du cerveau et notamment dans les processus de plasticité synaptique et de mémoire, altérés au cours du vieillissement. Il est maintenant bien admis que ces AGPI peuvent réguler la transcription génique en se liant à des récepteurs nucléaires spécifiques, les PPAR (peroxisome proliferator-activated receptors), mais aussi aux récepteurs de l’acide 9-cis rétinoïque, les RXR (retinoid X receptors). En tant que partenaires communs d’hétérodimérisation des PPAR et des RAR (récepteurs de l’acide tout-trans rétinoïque), les RXR sont des acteurs clés de la modulation de l’expression génique par les acides gras et les rétinoïdes. Dans ce contexte, l’objectif de ce travail de thèse était d’étudier, au cours du vieillissement chez le rat, les effets d’une supplémentation en AGPI-LC n-3 sur l’activité des voies de signalisation des acides gras et des rétinoïdes, les processus de plasticité cérébrale (plasticité synaptique et neurogenèse) et la mémoire spatiale. Nos principaux résultats montrent qu’une supplémentation en AGPI-LC n-3, pendant 21 semaines chez des rats à mi-vie, maintient dans l’hippocampe les niveaux d’expression des ARNm codant pour RXRγ et GAP-43 (protéine synaptique) altérés au cours du vieillissement. De plus les rats âgés supplémentés en AGPI-LC n-3 présentent une augmentation du nombre de néo-neurones hippocampiques et une amélioration de la mémoire spatiale de travail, comparés aux rats âgés contrôle. Les résultats de cette étude plaident en faveur d’un effet bénéfique des AGPI-LC n-3 sur la mémoire de travail au cours du vieillissement via notamment, une action sur la plasticité cérébrale. De plus, nos travaux suggèrent l’implication des RXR dans l’effet neuroprotecteur des AGPI-LC n-3, qui réguleraient l’expression de certains gènes cibles impliqués dans la plasticité synaptique et les processus de neurogenèse hippocampique
Long chain polyunsaturated fatty acids (LC-PUFA) of the n-3 series play essential roles in brain functions, including brain plasticity and memory processes which are altered during aging. It is now well accepted that these PUFA regulate gene transcription through binding and activating specific nuclear receptors such as PPAR (peroxisome proliferator-activated receptors) and RXR (retinoid X receptors, which also bind 9-cis retinoic acid). As a common heterodimeric partner of both PPAR and RAR (all-trans retinoic acid receptors), RXR is a key factor in the modulation of gene expression by fatty acids and retinoids. In this context, the purpose of this work was to study the effects of a n-3 LC-PUFA supplementation on fatty acid and retinoid signalling pathways and on cerebral plasticity and spatial memory processes. Our main results show that n-3 LC-PUFA supplementation for 21 weeks in mid-life rats, maintains the mRNA levels of RXRγ and GAP-43 (synaptic protein) which were altered in aged rat hippocampus. Besides, supplemented aged rats exhibited increased numbers of newly generated neurons and improved spatial working memory, when compared with control aged rats. To summarize, our results support the neuroprotective effects of n-3 LC-PUFA during aging, in particular on cerebral plasticity and working memory. Furthermore, our works suggest the implication of RXR in the set up of these effects through notably the regulation of some target genes involved in synaptic plasticity and hippocampal neurogenesis processes

L'infarctus du myocarde constitue la première cause de mortalité cardiovasculaire. Dans ce contexte, depuis plus de 40 ans et les premières études sur les populations du Groenland, il est connu qu'une consommation de poisson riche en acide gras polyinsaturés de type omégas 3 (AGPI n-3) a des effets cardioprotecteurs. De très nombreuses études cliniques, animales et cellulaires ont ensuite confirmé ces résultats cardioprotecteurs des AGPI n-3 qui semblent passer par une prévention des arythmies cardiaques post infarctus.Cependant, du fait de leurs nombreuses doubles liaisons carbone-carbone, les principaux AGPI n-3 que sont l'acide eicosapentaénoïque et l'acide docosahexaénoïque sont très sensibles à l'oxygénation à l'air et peuvent subir une peroxydation non enzymatique spontanée sous condition de stress oxydant qui accompagne notamment l'ischémie/reperfusion lors d'un infarctus du myocarde.Dans ce travail de thèse, nous posons la question de savoir quelle forme d'AGPI a des effets cardioprotecteur : la forme réduite ou oxydée. En effet, les effets des AGPI n-3 sur la fonction cardiaque sont très controversés, notamment due au manque d'information sur les mécanismes impliqués. Particulièrement, on ne sait pas quel lipide est actif : les AGPI n-3 ou un des leurs métabolites oxygénés.Durant l'ischémie reperfusion puis dans les mois qui suivent l'infarctus du myocarde, le stress oxydant est élevé et de nombreux métabolites non enzymatiques dérivés des AGPI n-3 comme les NeuroProstanes sont alors produits à tel point qu'ils sont reconnus comme biomarqueurs du stress oxydant. Ainsi, dans ce travail de recherche, nous spéculons que les NeuroProstanes ne sont pas seulement des biomarqueurs du stress oxydant mais auraient un rôle biologiquement actif qui expliqueraient les effets cardioprotecteurs connus de leurs précurseurs ; les AGPI n-3.Le but de cette thèse est dans un premier temps d'investiguer l'influence de la peroxydation lipidique du DHA sur ses propriétés antiarythmiques in cellulo sur des cellules ventriculaires cardiaques isolées puis in vivo sur des souris ayant subit un infarctus du myocarde par ligature de l'artère coronaire gauche. De la même manière, nous avons évalué les propriétés antiarythmiques des métabolites non enzymatique des AGPI n-3 et notamment le 4(RS)-4F4t-NeuroProstane. Dans un second temps et de manière plus précoce, nous avons observé si une infusion préventive de 4(RS)-4F4t-NeuroP chez le rat, 20 minutes avant un épisode d'ischémie reperfusion peut protéger le myocarde des dommages ischémiques (morts cellulaires), des arythmies et des altérations morpho-fonctionnelles.L'ensemble de ce travail de thèse a ainsi permis de mettre en évidence que un des médiateurs lipidiques des AGPI n-3 ; le 4(RS)-4-F4t-NeuroP peut exercer des effets biologiquement actifs qui passent par une prévention des arythmies dans les mois qui suivent l'infarctus du myocarde ; effets passant par une prévention des modifications post-translationnelles du RyR2 et in fine d'une régulation de l'homéostasie calcique. De manière plus précoce durant l'ischémie reperfusion, nos résultats montrent que le 4(RS)-4-F4t-NeuroP réduit les arythmies ventriculaires, la taille de la zone infarcie et la dysfonction cardiaque, effets cardioprotecteurs qui passent par des mécanismes mitochondriaux.Le travail de cette thèse démontre pour la première fois que le DHA n'exerce pas d'effets cardioprotecteurs mais que ce serait les produits issus de son oxydation non enzymatique tel le 4(RS)-4-F4t-NeuroP pouvant ainsi expliquer l'ensemble des effets connus des AGPI n-3. Cette découverte ouvre de nouvelles perspectives sur les produits oxydés non enzymatiques des AGPI n-3 comme des potentiels médiateurs dans les maladies comme durant l'infarctus du myocarde ou le stress oxydant qui est généré joue un rôle prépondérant dans les altérations physiopathologiques qui en découlent
Since 40 years, ω3 poly-unsaturated fatty acids (n-3 PUFA) are known to have cardioprotective properties in ischemic disease such as cardiac infarction following ischemia/reperfusion period. Many studies in isolated cells or in animals confirmed these effects and it has been suggested that n-3 PUFA have direct effects on targeted proteins such as ionic channels. However, due to the abundance of double carbone bounds, the main n-3 PUFA; eicosapentaenoic acid (C20: 5 n-3, EPA) and docosahexaenoic acid (C22: 6 n-3, DHA) are very sensitive to free radical oxidation and can undergo non-enzymatic spontaneous peroxidation under oxidative stress conditions as it occurs in ischemia/reperfusion. In the present work, we addressed the question of the form of DHA having cardioprotective properties: reduced or oxidized. Indeed, the effects of n-3 PUFA on cardiac function are controversial, notably due to the lack of information on the mechanisms involved. Particularly, it is not well understood which is the active lipid: the PUFA or one of its oxygenated metabolites. In the context of oxidative stress, during ischemia/reperfusion and in month following cardiac infarction, a lot of oxygenated metabolites of PUFA like Neuroprostane; 4(RS)-4F4t-NeuroP are produced and used as biomarkers of oxidative stress. This metabolite is associated to a lower atherosclerosis risk suggesting a beneficial role in cardiovascular diseases. In this context we speculate that Neuroprostane are not just a markers of stress conditions but have biological activities.The aim of this thesis was in first time to investigate the influence of DHA peroxidation on its potentially anti-arrhythmic properties in isolated ventricular cardiomyocytes and in vivo in post-myocardial infarcted (PMI) mice. In same way, we investigated in cellulo and in vivo anti-arrhythmic properties of oxygenated metabolites of n-3 PUFA such as 4(RS)-4F4t-NeuroP. In second time we investigated if the pericardial delivery 20 minutes before occlusion of 4F4t-NeuroP protects in prevention the myocardium from ischemic damages and arrhythmias during and following an I/R episode in rats.In this study, we challenged the paradigm that spontaneously formed oxygenated metabolites of lipids are undesirable as they are unconditionally toxic. This study reveals that the lipid mediator 4(RS)-4-F4t-NeuroP derived from non-enzymatic peroxidation of DHA, can counteract such deleterious effects through cardiac anti-arrhythmic properties in month following cardiac infarction by preventing deleterious post-translational modification of RyR2 and thus regulating calcium homeostasis. More early, during ischemia/reperfusion, our results show that pericardial delivery of 4(RS)-4-F4t-NeuroP reduced ischemia-induced ventricular arrhythmias, infarct sizes, and cardiac dysfonction ; cardioprotective effects involving mitchondria mecanisms.This thesis demonstrate for the first time that DHA per se has no anti-arrhythmic effects and 4(RS)-4-F4t-NeuroP as a mediator of the cardioprotection characteristics of DHA. This discovery opens new perspectives for products of non-enzymatic oxidized n-3 PUFA as potent mediators in oxidative stress diseases like during a cardiac infarction, where oxidative stress generated play fundamental role in pathophysiological alterations

The effects of LC n-3 PUFA, EPA and OHA, on aspects of early stage atherosclerosis including monocyte to endothelial cell adhesion, monocyte to macrophage differentiation, and macrophage cholesterol levels, lipoprotein receptor, and lipid droplet-associated protein expression were investigated. LC n-3 PUFA treatment increased monocyte adhesion to endothelial monolayers and adhesion molecule expression (E-selectin; ICAM-1) in unstimulated HAECs with an opposite effect found in TNF-a stimulated HAECs (ICAM-1; VCAM-l). Adhesion was also decreased upon THP-1 monocyte incubation with LC n-3 PUFA, with no effect on expression of monocyte adhesion molecules. In monocyte to macrophage differentiation immature macro phages were affected at a lower L( n-3 PUFA concentration, and to a greater extent than mature macrophages. Upon L( n-3 PUFA treatment cell-surface expression of the differentiation markers CD11c, CD11b and CD49d on immature and mature macrophages, and (014 mRNA expression on mature macrophages were down- regulated, as was cell-surface expression of the scavenger receptor (036 in immature and mature macrophages, as well as (036 mRNA expression in immature The extent of macrophage differentiation influenced LC n-3 PUFA effects on cholesterol levels and lipoprotein expression. LC n-3 PUFA decreased net cholesterol levels in untreated, immature macrophages, and free cholesterol levels in native LDL treated, immature macro phages; mature macrophages were unaffected. LOX-l and LDLr mRNA expression were also decreased in immature macrophages. DHA increased adipophilin mRNA expression regardless of cell treatment. LC n-3 PUFA treatment during differentiation decreased CD36 cell-surface and mRNA expression in immature macrophages, and CD36 cell-surface and LOX-l mRNA expression in mature macrophages, whereas, post-differentiation treatment reduced LOX-l mRNA expression in immature macrophages with no effect on CD36 expression. LDLr mRNA expression was reduced with treatment during and post- differentiation. Treatment with the LC n-3 PUFA, EPA and DHA, can decrease monocyte adhesion to the endothelium, the expression of monocyte to macrophage differentiation markers, and cholesterol levels within differentiated macrophages.

Tilapia are freshwater fish that have become important in aquaculture and as a stable global source of seafood due to their ability to thrive in different environments. However, tilapia are sometimes considered nutritionally undesirable due to their high n-6 to n-3 fatty acid ratios. A market study was conducted first to determine fatty acid compositions in tilapia fillets in different US markets. Then a research was conducted to enhance nutritional value of tilapia by improving the n-3 and oleic acid contents in fish fillets without compromising fish growth or feed conversion ratios. Feeds were formulated with combinations of high and low n-6, n-3, and oleic acid levels using soybean oil, fish oil, algae oil, and high-oleic sunflower oil. Then 12 diets, including a commercial diet, were assigned to 24 tanks, each with 25 tilapia per tank. A Recirculating Aquaculture System (RAS) was used to grow the fish for 8 weeks. Fatty acid compositions of tilapia fillets were determined and samples were vacuum packed and stored at -10oC and -20oC to test oxidative degradation and fatty acid compositional changes. The market survey data showed that there were significant differences in fatty acid composition, lipid content, and n-6:n-3 fatty acid ratios depending on the country of origin. Samples from USA had ideal n-6:n-3 ratios (1.3 ±0.85) while samples from Southeast Asia had higher n-6:n-3 fatty acids ratio (6.6 ±0.54). Algae oil incorporation significantly increased DHA level while fish oil incorporation significantly increased both EPA and DPA. High-oleic sunflower oil based diets improved oleic acid levels and reduced linoleic acid compared to the soybean oil based diets. Sensory evaluation indicated that lipid source did not significantly impact preference or overall fillet quality, including texture. Interestingly, a survey showed people were interested in value-added tilapia, and would pay up to 30% more for nutritionally enhanced fish compared to the $5.00/lb fresh fillet price currently available in supermarkets. There was no observable oxidation during long term frozen storage. The oxidation study proved that value-addition would not be compromised during the long term storage conditions, even under temperature abuse. It is possible to improve tilapia nutritional quality through diet to provide consumers with value-added products that maintain quality during frozen storage.
Ph. D.

The potential of red meat, particularly that produced from grass-fed animals, to contribute to dietary intakes of long chain (LC) n-3 PUF A has become the focus of recent research. However, there remains little information on precise quantities of these fatty acids found in commercially produced red meat, the use of grass finishing diets by producers, or the ability of red meat produced from grass feeding regimes to contribute to LCn-3 PUF A intakes or status in consumers, especially in the UK and Ireland. Extensive fatty acid data were measured for beef and lamb produced in Northern Ireland over a 12 month period and the frequency of a grass diet being offered to animals before slaughter was determined by a survey carried out among a sample of beef and lamb producers. To investigate the effect of consumption of red meat produced from grass-fed animals on LCn-3 PUF A status, a human intervention study was carried out among healthy subjects. In addition, the potential for red meat produced from grass-fed animals to contribute to LCn-3 PUF A intakes in the Irish population was hypothetically assessed based on current intakes of meat. Beef and lamb were reported to contain concentrations ofLCn-3 PUFA similar to levels achieved with grass feeding by other studies. The season of slaughter and, to a lesser extent, the reported finishing diet of the animal were found to cause variation in concentrations ofLCn-3 PUFA and conjugated linoleic acids (CLA) found in beef and lamb. The survey revealed that grass finishing diets are commonly offered to animals by producers of beef and lamb, but the frequency of these diets also varied by season. In the human intervention study, consumption of red meat from grass-fed animals was associated with significantly increased plasma and platelet LCn-3 PUF A status among healthy subjects. Based on red meat consumed in the Irish diet being from grass-fed animals, the provision of red meat to LCn-3 PUF A intakes would be modestly increased than if meat consumed were from concentrate-fed animals. In conclusion, the production of red meat from grass-fed animals will contribute to increased LCn-3 PUF A intakes and status among Irish consumers, where red meat is habitually consumed. Further research is warranted to investigate the opportunity to further enhance concentrations ofLCn-3 PUF A within red meat by increased use of grass feeding regimes in beef and lamb production.

Dans une stratégie de diversification des apports en acides gras polyinsaturés très longue chaîne n-3 (AGPI TLC n-3), essentiels à la santé de l'Homme et majoritairement apportés par les produits de la mer, l'objectif de la thèse était de mieux comprendre les mécanismes cellulaires pouvant expliquer la faible teneur en AGPI TLC n-3 présente dans le muscle de bovin, ceci en explorant trois voies métaboliques de ces acides gras potentiellement limitantes (biosynthèse, captage facilité et peroxydation non-enzymatique) par des approches de qPCR ou de transcriptomique. Les principaux résultats montrent que le foie et les muscles de bovin possèdent tout le matériel génétique nécessaire à la synthèse des AGPI TLC n-3 et pourraient donc assurer leur synthèse, sauf chez le mâle entier où l'expression du gène de l'élongase 5 est réprimée par la présence d'hormones sexuelles mâles. D'autre part, les teneurs musculaires en AGPI TLC n-3 plus élevées avec un régime base d'herbe par rapport à de l'ensilage de maïs et dans un muscle glycolitique par rapport à un muscle oxydatif ne semblent pas s'expliquer par une modulation de l'expression des gènes impliqués dans leur biosynthèse, ni dans leur captage facilité et dans la régulation endogène de leur lipoperoxydation. En conclusion, ce travail de thèse a permis de considérablement faire progresser la compréhension des mécanismes impliqués dans la régulation des dépôts d'AGPITLC n-3 dans les muscles de bovin. Toutefois, ces régulations sont certainement plus complexes et probablement multifactorielles. De nombreuses pistes restent encore à explorer avant d'envisager augmenter la teneur en AGPI TLC n-3 dans la viande bovine
N-3 very long chain polyunsaturated fatty acids (n-3 VLC PUFA) are essential for human health but are almost exclusively present in seafood. Thus, in a strategy of diversification of n-3 VLC PUFA sources for human, the aim of this thesis was to better understand the cellular mechanisms that may explain the low n-3 VLC PUFA content in bovine muscle, by exploring three potentially limiting metabolic pathways of these fatty acids (biosynthesis, facilited uptake and non-enzymatic peroxidation) by qPCR or transcriptomic approaches. The main results indicate that the liver and muscles of cattle possess all the genetic material necessary for the synthesis of n-3 VLC PUFA and may therefore ensure their synthesis, except in entire males where gene expression of the elongase 5 is suppressed by the presence of male sex hormones. On the other hand, the higher n-3 VLC PUFA content in muscle of cattle with a grass based diet compard to corn silage and in glycolytic muscle compared to oxidative muscle do not seem to be explained by a modulation of gene expression of proteins involved in their biosynthesis or in their facilited uptake or in the endogenous regulation of their lipoperoxidation. In conclusion, this thesis has greatly advance our understanding of mechanisms involved in the regulation of n-3 VLC PUFA deposits in muscles of cattle. However, these regulations are certainly more complex and probably multifactorial. Many tracks remain to be explored before considering increasing n-3 VLC PUFA content in beef

Introdução: A doença hepática gordurosa não alcoólica é caracterizada pelo acúmulo hepático de lipídeos, principalmente na forma de triglicerídeos. Devido à atividade inflamatória progressiva pode evoluir para uma forma mais grave, a esteatohepatite não alcoólica. Os ácidos graxos poli-insaturados ômega-3 são associados a efeitos metabólicos positivos para redução da esteatose hepática, no entanto, são mais susceptíveis a peroxidação lipídica. Os triglicerídeos de cadeia média (TCMs) promovem a prevenção do bloqueio da beta-oxidação de ácidos graxos e redução da peroxidação lipídica, no entanto os efeitos na redução da esteatose ainda são controversos. Objetivo: O objetivo do estudo foi avaliar as implicações da dieta hiperlipídica (HL+) com óleo de peixe ou com óleo de TCM no desenvolvimento da esteatose hepática, no perfil de ácidos graxos hepáticos e no estresse oxidativo em ratos. Metodologia: Cinquenta ratos machos da linhagem wistar foram divididos em 5 grupos. Os animais receberam água e comida a vontade durante 45 dias. A adaptação a dieta HL+ foi realizada nos primeiros 15 dias. A composição da dieta do grupo que recebeu somente a gordura animal (HL+GA) era de 50% de gordura animal, e a dieta dos grupos HL+OS, HL+TCM e HL+OP era composta por 35% de gordura animal e 15% de óleo de soja, óleo de TCM e óleo de peixe, respectivamente. Resultados: Todos os grupos que receberam as dietas hiperlipídicas apresentaram maior acúmulo de gordura total e de triglicerídes hepaticos e somente os grupos HL+GA e HL+TCM apresentaram maior acúmulo de colesterol total hepático em relação ao controle. O grupo HL+TCM apresentou maior acúmulo percentual de gordura e um exacerbado acúmulo de triglicerídeos hepáticos em relação aos grupos alimentados com as dietas HL+. A redução do colesterol total sérico foi observada nos grupos HL+TCM e HL+OP, comparados ao controle. A maior incorporação hepática dos ácidos graxos EPA e DHA no grupo HL+OP contribuiu para o aumento do Índice de Peroxibilidade dos ácidos graxos e das substâncias reativas ao ácido tiobarbitúrico livres e totais e para a depleção da vitamina E no fígado. A maior razão AGS/AGPI hepática observada no grupo HL+TCM contribuiu para a preservação dos antioxidantes hepáticos. A alanina aminotransferase, um marcador de dano hepático, apresentou-se aumentada em todos os grupos que receberam as dietas HL+. Conclusões: A dieta hiperlipídica foi eficiente na indução do acúmulo de gordura hepática. O uso do óleo de TCM foi associado a uma maior concentração de lipídeos e preservação dos antioxidantes hepáticos. A dieta hiperlipídica com óleo de peixe foi associada ao aumento significativo na peroxidação lipídica, apesar do menor acúmulo de colesterol e triglicerídeos hepaticos.
Introduction: The Non-alcoholic Fatty liver disease is characterized by hepatic accumulation of lipids, mainly in the form of triglycerides. The disease may progress to a more severe form, the Non-alcoholic steatohepatitis, due to progressive inflammatory activity. Many authors have shown positive metabolic effects associated with the use of polyunsaturated omega-3 fatty acids and reduction in hepatic steatosis. However, these fatty acids are more susceptible to lipid peroxidation. The medium chain triglycerides (MCTs) are able to block beta-oxidation of fatty acids and reduce lipid peroxidation, but the MCT effects in steatosis are still controversial. Objective: The aim of this study was to assess the implications of high-fat diet (HF+) with fish oil or with MCT oil in the development of hepatic steatosis, liver fatty acid profile and oxidative stress markers in rats. Methodology: Fifty wistar male rats were divided into 5 groups. The animals had free access to food and water for 45 days. The first 15 days was dedicated for adaptation to high-fat diet. The HF+AF group received high-fat diet with 50% of animal fat and the other high-fat diets were made with 35% of animal fat plus 15% of other types of fat: soybean oil (HF+SO), MCT oil (HF+MCT) and fish oil (HF+FO). Results: The high-fat groups had higher hepatic total fat and triglycerides accumulation and only the groups HF+AF and HF+MCT had higher accumulation of hepatic cholesterol compared to control. The HF+MCT group had the highest percentage of hepatic fat accumulation and an exacerbated triglyceride accumulation in the liver among HF+ groups. The serum total cholesterol decreased in groups HF+MCT and HF+FO compared with the control group. The highest incorporation of hepatic fatty acids EPA and DHA in the HF+FO group contributed to the increased fatty acids peroxidizability index and total and free hepatic TBARS and depletion of hepatic vitamin E. The biggest ratio SFA/PUFA of liver fatty acids observed in the HF+MCT group contributed to the preservation of hepatic antioxidants. The alanine aminotransferase is a liver damage marker and was increased in all high-fat groups. Conclusions: The high-fat diet was effective to increase the hepatic fat concentration. The consumption of MCT oil can increase the hepatic lipid concentration and hepatic antioxidants. There was a significant increase in hepatic lipid peroxidation in the HF+FO group, although hepatic cholesterol and triglycerides were decreased.

Research Doctorate - Doctor of Philosophy (PhD)
Type 2 diabetes (T2D) is the fastest growing chronic health condition, driven by obesity-related insulin resistance (IR). Obesity accounts for 50% of the total diabetes burden in Australia and worldwide. As the global prevalence of obesity increases, diabetes-related health care costs will continue to rise. Interventions that prevent or delay the onset of diabetes can potentially reduce the overall diabetes burden and associated health care costs. Weight loss is clinically effective in ameliorating IR and reducing T2D risk, however long-term weight loss remains difficult to achieve and maintain, and research into adjunct therapies are warranted. Previous studies reported a sex-dependent association between long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) eicosapentaenoic acid (EPA; c20:5n-3) and docosahexaenoic acid (DHA; c22:6n-3) and T2D. Sex hormones are known to regulate synthesis of DHA from α-linolenic acid (ALA; c18:3n-3), though precise mechanisms through which they do so are not well understood. Sex hormones also affect T2D risk differentially in men and women. Interactions between sex hormones and LCn-3PUFA may in part explain some of the differential T2D risk in men and women. Therefore, this thesis aimed to investigate the interactions between sex hormones and LCn-3PUFA for the prevention of obesity-associated IR and subsequent development of T2D. A systematic review and meta-analysis was undertaken to review the existing evidence to determine whether currently available literature supported the prevailing hypothesis, that LCn-3PUFA reduce IR in a sex-dependent manner. The literature was searched to identify randomised controlled trials (RCT) which used a dietary or supplemental n3-PUFA intervention and included measures of IR or insulin sensitivity as an outcome. Thirty-two studies including n=1848 participants were included, with two studies conducted in men, seven in women and the rest in mixed population. Meta-analysis showed that interventions with LCn-3PUFA in females ≥6 weeks duration displayed a reduction in IR, with no change in IR in studies in male populations. However, few intervention studies were conducted in males and significant heterogeneity was present that could not be explained in our analysis, limiting conclusions that could be drawn. Furthermore, there were no studies in which men and women were exposed to an identical intervention protocol, necessitating development of a double-blind RCT. A cross-sectional study of n=2,092 older (55-85 years) Australians assessed relationships between LCn-3PUFA status and diabetes, with sub-group analyses used to investigate sex- and weight- related interactions. There was a significant three-way interaction between sex, weight status and LCn-3PUFA status in the prediction of T2D, therefore sub-group analysis was performed based on this. This demonstrated an inverse association between LCn-3PUFA and diabetes in overweight and obese women, which is consistent with previously reported findings. LCn-3PUFA was inversely correlated with HOMA-IR in men and women. However, this was attenuated when adjusted for BMI. A mediation analysis explored whether the observed association between LCn-3PUFA and T2D was mediated through inflammatory pathways, which demonstrated that interleukin-6, but not C-reactive protein, fibrinogen, or neutrophil-leukocyte ratio, was a partial mediator of the association in overweight and obese women. A double-blind RCT was designed and conducted to investigate the effects of a 12-week DHA-enriched fish oil supplementation on IR in men and women with abdominal obesity and no history of diabetes. This study found a significant reduction in IR in men and women with hyperinsulinaemia at baseline, with no significant interaction between sex and treatment. There was no change in fasting glucose or beta-cell function in any groups across the course of the study. Contrary to the hypothesis, there was a similar magnitude of change in both the males and females. Secondary analysis showed that there was a significant interaction between sex and treatment on testosterone across the course of the study, with fish oil increasing circulating testosterone levels in males compared with control, whilst no change was evident amongst females (p-interaction_{sex*treatment}=p<0.001). Changes in testosterone in males were inversely associated with changes in omega-6 PUFA dihomo-γ-linolenic acid content in erythrocyte membranes, and change in testosterone was a significant predictor of changes to insulin and HOMA-IR in men across the 12 week intervention. Finally, an observational study in n=20 transgender individuals undergoing cross-sex hormone treatment (feminising therapy: n=10; masculinising therapy: n=10) investigated effects of testosterone, androgen blockers and oestradiol on erythrocyte DHA levels and body composition. This suggested that exogenous testosterone administration in trans-men acutely downregulates DHA concentrations, but inhibiting the action of testosterone with androgen receptor antagonists does not increase erythrocyte DHA, suggesting an indirect effect; however, these did not reach statistical significance. Masculinising therapy was associated with significant increases in skeletal muscle mass at 3- and 6- months; feminising therapy was associated with decreases in skeletal muscle mass at 3- and 6- months, and increases in % body fat after 6 months treatment. Observational studies with larger sample sizes are required to determine whether change to erythrocyte DHA composition modulates any change in metabolic functioning. This thesis provides further evidence of an inverse association between LCn-3PUFA and T2D in overweight and obese women. However, the sex-dependency of the association appears to be independent of a direct effect on IR, and may represent differences in the pathogenesis of T2D in men and women. Investigations between DHA and testosterone in obese men is deserving of further research, and if substantiated, may have clinical applications for men with mild hypogonadism for whom testosterone is not clinically indicated. The inverse association between DHA and diabetes in women may have biological significance for the development of gestational diabetes. Future research should focus on associations between DHA, inflammation and IR in pregnancy.

Research Doctorate - Doctor of Philosophy (PhD)
Despite an ever-growing body of research on obesity, investigating causes and possible solutions to address the problem, the prevalence of obesity continues to escalate. A major cause of obesity is attributed to poor eating behaviours driven by food advertising, lack of nutrition knowledge, lack of physical activity, lack of time and lack of will power to control food intake, and there is a plethora of research with a focus on changing dietary behaviour for weight management. In part, this research also addressed dietary change, employing a reduced energy intake for weight loss supported with nutrition education and counselling to enable maintenance of the weight lost. However, consideration was also given to the internal interactions and changes that occur in the body when energy intake exceeds energy output resulting in weight gain and obesity, and whether these mechanisms could be manipulated to reduce weight gain through the inclusion of long chain omega-3 polyunsaturated fatty acids (LCω-3PUFA) in the diet. Prospective studies in humans have reported that high levels of LCω-3PUFA were associated with low levels of obesity in males while higher intakes of LCω-3PUFA were associated with higher rates of obesity in females. The data on LCω-3PUFA concentrations in males and females had been sourced from dietary records with questionable reliability. Thus the first aim of this research was to investigate whether there was a relationship between plasma LCω-3PUFA and weight status in humans. The first research chapter (Chapter 3) reports on the relationship observed between plasma LCω-3PUFA composition and weight status in free living adults. Obese individuals, both males and females, had significantly lower levels of LCω-3PUFA compared to healthy-weight individuals. Thus the aim of the first clinical trial (Chapter 4) was to investigate whether LCω-3PUFA supplementation, combined with a healthful diet with portion control and energy restriction would facilitate weight loss, improve blood lipids and inflammatory mediators. This was a double-blinded randomised controlled trial with two parallel groups. Both groups were instructed to follow the same diet for 12 weeks, one group consumed fish oil capsules and the other group consumed placebo capsules (monounsaturated oil). Despite a two-fold increase of EPA and DHA in the treatment group, there were no significant differences in outcome measures between the two groups. Both the placebo and the fish oil supplemented groups lost similar amounts of weight, fat mass and fat free mass. Overall dietary compliance was poor representing a possible confounding factor on the outcomes. The aim of the next clinical trial (Chapter 5) was to investigate whether LCω-3PUFA supplementation would facilitate weight loss, but this time in combination with a very-low-energy-diet (VLED), using meal replacements (MRs), to improve dietary compliance. The protocol for this trial was the same as the previous one apart from the change to the diet and with a shortened intervention of 4 weeks. Also, because of the anticipated rapid weight loss, a 10 week weight maintenance phase with continued supplementation was included. Although there was a greater than two-fold increase of EPA and DHA in the treatment group, there were no significant differences in outcome measures between the two groups after 4 weeks of weight loss. However, after a further 10 weeks of supplementation during weight maintenance, there was a significant reduction in anthropometric measurements, apart from fat free mass, in the treatment group but not the placebo group. The differences between the two groups were not significant. The final clinical trial (Chapter 6) investigated potential benefits of loading the body cells/membranes with LCω-3PUFA prior to following a weight loss program. The protocol for the trial was similar to the previous two, but commenced with 4 weeks of prior-supplementation with fish oil or placebo in the treatment and placebo groups, respectively, while consuming their usual diet. This was followed by 4 weeks of dietary intervention where both groups again consumed a VLED with MRs plus continued supplementation. The same measurements were taken as for the previous trials. After 4 weeks of prior-supplementation there were no significant differences in outcome measures for either group. However, at 8 weeks a significant 3-way interaction between time, group and gender was observed for percentage reduction in weight and BMI, suggesting a significant effect of LCω- 3PUFA for the fish oil group. There was also a significant reduction in percentage weight loss for females in the fish oil group. These results suggest that priorsupplementation with LCω-3PUFA, followed by supplementation with LCω-3PUFA and a VLED regimen may assist weight loss.

Dietary omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), have beneficial health effects and hence increasing the consumption of these fatty acids is recommended by health authorities. The most common dietary source of EPA, DPA and DHA is seafood, but few Australians habitually consume fish and on average eat less than one meal of fish per week. Thus if Australians are to meet the dietary guidelines for n-3 fatty acid intake, there is a need to develop a source of n-3 rich foods that fit into a typical Australian diet. Feeding fish oils rich in n-3 LCPUFA to chickens has proven problematic due to alteration in organoleptic properties. The incorporation of vegetable oils rich in n-3 PUFA, alpha-linolenic acid (ALA, 18:3n-3) into the diet of chickens is potentially an alternative way to provide meat rich in n-3 LCPUFAs as ALA is the precursor of EPA and DHA. However, most vegetable oils also contain the n-6 (n-6) PUFA, linoleic acid (LA, 18:2n-6) which competes with ALA for the same enzymes in their metabolism to LCPUFA. This thesis addressed two crucial issues relating to the conversion of ALA into EPA, DPA and DHA of chicken tissues. The objectives of the first experiment were to examine the effects of increasing the ALA content of diets on the conversion of ALA into EPA, DPA and DHA by measuring their accumulation in chicken meat (breast and thigh) and to determine if there was an optimum level of ALA (at a fixed level of LA) in this process. The ratio of LA to ALA of the diets ranged from 10.5:1 to 0.6:1. The findings in this study demonstrated that there was no optimum level of dietary ALA and as indicated by the observation that EPA, DPA and DHA continued to increase in breast and thigh as the ratio of LA to ALA decreased in the diet. In general, DPA achieved higher levels than DHA. The experimental diets with the lowest LA to ALA ratio elevated the incorporation of EPA and DHA into breast and thigh meat to levels 5 and 4-fold, respectively relative to birds fed the highest LA to ALA ratio. In contrast, arachidonic acid (AA, 20:4n-6) in all groups reduced with decreasing LA to ALA ratio in the diets. The results indicated that the dietary treatments did not significantly change the growth performance of chickens. The objective of the second experiment was to assess the regulatory effect of dietary LA on the conversion of ALA into EPA, DPA and DHA. While in the first experiment the diets varied in the level of ALA but had a constant LA level, in this experiment the level of ALA in the diets was held constant and the level of LA was varied. The LA to ALA ratio of experimental diets ranged from 1.4:1 to 2.1:1. The results of this study indicated that the highest LA to ALA ratio (2.1:1) resulted in the lowest n-3 LCPUFAs, EPA, DPA and DHA in meat samples. For example, the total n-3 LCPUFA levels in the breast meat of birds fed with the lowest LA to ALA ratio was 16% higher than the n-3 LCPUFA in the breast of birds fed the highest LA to ALA ratio. This study indicated that the strongest influence on EPA, DPA and DHA accumulation in chicken tissues was the level of ALA in the diet. The experimental diets did not appear to affect the growth performance of chickens. In conclusion, increasing the ALA content of chicken diets may result in a meat source high in n-3 LCPUFAs that may reduce pressure on diminishing marine stocks as well as offering health benefits to Australians.
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Thesis (M.Ag.Sc.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

Dietary omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), have beneficial health effects and hence increasing the consumption of these fatty acids is recommended by health authorities. The most common dietary source of EPA, DPA and DHA is seafood, but few Australians habitually consume fish and on average eat less than one meal of fish per week. Thus if Australians are to meet the dietary guidelines for n-3 fatty acid intake, there is a need to develop a source of n-3 rich foods that fit into a typical Australian diet. Feeding fish oils rich in n-3 LCPUFA to chickens has proven problematic due to alteration in organoleptic properties. The incorporation of vegetable oils rich in n-3 PUFA, alpha-linolenic acid (ALA, 18:3n-3) into the diet of chickens is potentially an alternative way to provide meat rich in n-3 LCPUFAs as ALA is the precursor of EPA and DHA. However, most vegetable oils also contain the n-6 (n-6) PUFA, linoleic acid (LA, 18:2n-6) which competes with ALA for the same enzymes in their metabolism to LCPUFA. This thesis addressed two crucial issues relating to the conversion of ALA into EPA, DPA and DHA of chicken tissues. The objectives of the first experiment were to examine the effects of increasing the ALA content of diets on the conversion of ALA into EPA, DPA and DHA by measuring their accumulation in chicken meat (breast and thigh) and to determine if there was an optimum level of ALA (at a fixed level of LA) in this process. The ratio of LA to ALA of the diets ranged from 10.5:1 to 0.6:1. The findings in this study demonstrated that there was no optimum level of dietary ALA and as indicated by the observation that EPA, DPA and DHA continued to increase in breast and thigh as the ratio of LA to ALA decreased in the diet. In general, DPA achieved higher levels than DHA. The experimental diets with the lowest LA to ALA ratio elevated the incorporation of EPA and DHA into breast and thigh meat to levels 5 and 4-fold, respectively relative to birds fed the highest LA to ALA ratio. In contrast, arachidonic acid (AA, 20:4n-6) in all groups reduced with decreasing LA to ALA ratio in the diets. The results indicated that the dietary treatments did not significantly change the growth performance of chickens. The objective of the second experiment was to assess the regulatory effect of dietary LA on the conversion of ALA into EPA, DPA and DHA. While in the first experiment the diets varied in the level of ALA but had a constant LA level, in this experiment the level of ALA in the diets was held constant and the level of LA was varied. The LA to ALA ratio of experimental diets ranged from 1.4:1 to 2.1:1. The results of this study indicated that the highest LA to ALA ratio (2.1:1) resulted in the lowest n-3 LCPUFAs, EPA, DPA and DHA in meat samples. For example, the total n-3 LCPUFA levels in the breast meat of birds fed with the lowest LA to ALA ratio was 16% higher than the n-3 LCPUFA in the breast of birds fed the highest LA to ALA ratio. This study indicated that the strongest influence on EPA, DPA and DHA accumulation in chicken tissues was the level of ALA in the diet. The experimental diets did not appear to affect the growth performance of chickens. In conclusion, increasing the ALA content of chicken diets may result in a meat source high in n-3 LCPUFAs that may reduce pressure on diminishing marine stocks as well as offering health benefits to Australians.
Thesis (M.Ag.Sc.) - University of Adelaide, School of Agriculture, Food and Wine, 2009

The importance of dietary omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs) in human health has promoted interest in developing a range of n-3 rich foods. The inclusion of n-3 FA into chicken products can be achieved by feeding chickens marine n-3 LCPUFA sources such as fish oil. However, this dietary approach has proven problematic due to impaired sensory qualities in the chicken products. The inclusion of the plant n-3 PUFA source alpha-linolenic acid (ALA, 18:3n-3), the n-3 LCPUFA precursor, into the diet of chickens is potentially an alternative way to provide chicken products rich in n-3 LCPUFAs without these detrimental sensory effects. The objectives of the study were to investigate whether including ALA in the diets of two strains of laying hens and two strains of broilers would increase n-3 LCPUFA accumulation in eggs and meat, without altering production performance or the sensory characteristics of the products. The levels of dietary ALA tested in both laying hen and broiler experiment were 0.3 (low), 3 (moderate) and 6% energy (high; %en), while holding the level of LA constant at around 4%en in the moderate and high ALA diets. The findings in this study demonstrated that independent of strain, for both eggs and meat, the level of EPA was directly related to the level of ALA in the diet. On the other hand the longer chain fatty acids, DPA and DHA, tended to reach maximal levels when the level of dietary ALA reached 3%en. The level of total n-3 fatty acids in products from chickens fed ALA enriched diets (3 or 6%en for laying hens and 6%en for broilers) met the requirement needed for labelling as egg and meat n-3 PUFA sources (300mg/egg or 300mg/100g of meat). Fatty acid analysis of lipid fractions in breast meat showed that while ALA was mainly associated with triglyceride (TG) fraction, the n-3 LCPUFA were preferentially deposited in the phospholipids (PL). There was strain dependence in the ability of the chickens to convert ALA into n-3 LCPUFA. Among layers, brown hens were found to be more effective in converting ALA to n-3 LCPUFA than white hens, whereas in broilers, Cobb birds were more effective in the accumulation of n-3 LCPUFA than Ross birds. Dietary ALA enrichment up to a level of 6%en did not influence the sensory quality of boiled eggs whereas in scrambled eggs, high ALA diets tended to decrease egg aroma. Importantly, a diet enriched with 3%en ALA did not change the consumer acceptance of the eggs compared with eggs purchased from a local supermarket. In broilers, a diet containing ALA 3%en did not affect any of the sensory attributes tested. Importantly, the sensory quality of chicken breast meat from birds fed a dietary ALA of 3%en was comparable to that of commercial breast meat purchased from a local supermarket. There were strain effects on the sensory attributes of the eggs, with boiled brown eggs having a significantly (P < 0.05) stronger after-taste than boiled white eggs whereas white eggs had a stronger (P < 0.05) sulphur flavour than brown eggs. In scrambled eggs, stronger egg aroma, sulphur flavour, and butter flavour were detected in brown eggs (P < 0.05) than white eggs. In conclusion, a dietary ALA level of approximately 3%en could be recommended as a good ALA level for producing eggs and chicken meat n-3 LCPUFA by industry. The findings of this study demonstrated that incorporating n-3 rich vegetable oils into chicken diets could be an alternative to marine sources to produce eggs and meat higher in n-3 LCPUFA, without influencing either production performance of birds or sensory qualities of the chicken products. This strategy would help to provide consumers with a variety of foods rich in n-3 LCPUFA, and help to achieve recommended intakes for human health.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013

The potential for supplemented omega-3 polyunsaturated fatty acids (PUFA) to increase sow reproductive performance when supplied from isocaloric diets containing low levels (3 – 6 g/kg of diet) of fish oil as a partial replacement for tallow was investigated. In the first experiment, there was an increase of 1 piglet live born (P < 0.05) to sows at the subsequent parity fed a supplemented diet before farrowing and during lactation. In contrast, litter size was unaffected when gilts were fed a supplemented diet with fish oil during puberty and early pregnancy. Furthermore in gilts, increasing the duration or level of supplementation did not improve litter size or embryo survival, possibly due to their inherently high level of fertility (82% embryo survival). In subsequent experiments, the effect of omega-3 supplementation on reproduction was evaluated in older parity sows known to have an inherently lower level of fertility compared with gilts. In parity 4 – 7 sows fed a supplemented diet prefarrowing and during lactation continuing to mating, embryo survival at 23 d was increased (Omega-3 70% vs 61% in Controls; P =0.054), without affecting ovulation rate. Subsequent experiments examined the response when supplemented diets were fed either during lactation continuing to mating; or after mating and during early gestation; or across both periods from lactation through to early gestation. Litter size born was maximised in the subsequent parity in sows fed fish oil diets from lactation to early gestation for 28 d, with the response being greatest in higher parity sows (+0.7 live born; and +0.9 total born, P < 0.05). In the following experiment this increases was associated with a 19% increase in embryo survival with omega-3 supplementation (P = 0.061). There was no effect on live weight or backfat during lactation; litter weight gain; piglet wean weight; and sow intake when gilts or sows were fed supplemented diets. The increase in embryo survival and litter size consistently observed in the sow studies was associated with increases in the omega- 3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and were independent of energy intake or energy metabolism. Partially replacing tallow (wt/wt) with 3 – 6 kg levels of fish oil did not change plasma levels of the essential omega-6 fatty acids, linoleic acid (LIN) and arachidonic acid (ARA). Using in-vitro cultures of granulosa cells it was demonstrated that progesterone production is increased with prostaglandin E3 and there was evidence for PGE₃ to enhance the steroidogenic response to PGE₂. It is proposed that specific long-chain omega-3 fatty acids increases embryo survival in older sows due to improved oocyte quality and/or embryo development, possibly through synergistic activities of PGE₂ and PGE₃ on progesterone levels in the local ovarian- uterine circulation. Supplementation of diets with EPA and DHA from fish oil offers pig producers a nutritional approach to improve sow litter size in older parities thereby increasing longevity and lifetime performance.
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2012

Research Doctorate - Doctor of Philosophy (PhD)
Type 2 diabetes (T2D) is the most common chronic metabolic disorder resulting from either deficit of insulin secretion and/or action. The transition of normal glucose tolerance to T2D is usually accompanied by a cluster of metabolic risk factors such as low-grade inflammation, oxidative stress, insulin resistance (IR) and dyslipidaemia. IR is one of the marked independent predictors among these cluster of metabolic abnormalities that mediates the transition in high risk states such as obesity, impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) to overt T2D. IR also is often associated with decreased clearance of lipids and lipoprotein abnormalities, together representing a greater risk of cardiovascular disease (CVD) in both high risk and individuals with T2D. Several studies have employed lipid ratios, homeostatic models, and anthropometric measures as surrogate markers for predicting IR. However, none of these accounted for both insulin and lipid availability in a single model to predict IR or metabolic syndrome (MetS). Therefore, the first aim of my PhD project, presented in the chapter 3, was to develop a novel marker for IR and MetS that accounts for both insulin and lipid availability in a single model. We proposed and evaluated a novel physiologically relevant marker, InsuTAG (product of fasting insulin and fasting triglycerides) as a predictor of IR and MetS. Cross-sectional analysis of data from the Retirement Health and Life-style Study (RHLS, n=618) showed that InsuTAG is a strong predictor of IR over existing lipid based surrogate markers and anthropometric measures. Receiver operating curve analysis indicated InsuTAG (93%) as the favourable marker for IR over other lipid based surrogate markers and anthropometry measures. Prevalence of MetS was significantly higher in individuals with InsuTAG values above the optimal cut-off value of 11.2. InsuTAG exhibited a greater area under than curve than HOMA-IR for identifying MetS. Together these observations indicate the potential of InsuTAG for predicting IR and MetS. Despite effective lifestyle and pharmacological interventions, the prevalence of T2D is growing at an alarming rate in Australia, in line with global prevalence. Failure of long term compliance to these interventions is a major barrier for their effectiveness in halting the transition to T2D in high risk state individuals, indicating a necessity for alternative effective approach. Given the fact that pathogenesis of T2D is chronic, complex and often involving multiple pathological pathways, use of well tolerated dietary bio-active compounds appears to be a potential strategy for delaying the onset of T2D. Several pre-clinical and in-vitro studies have reported the ability of dietary bio-actives to down regulate multiple pathological mechanisms (chronic low-grade inflammation, IR, oxidative stress and β-cell dysfunction) that are involved in the pathogenesis of T2D. We hypothesised that a combination of two lipid-lowering and anti-inflammatory dietary bio-active compounds, curcumin and long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA), could potentially act in multiple pathways to improve the glycaemic control in individuals at high risk of developing T2D. My second aim, presented in chapter 4, was to evaluate the acute effects of curcumin and/or LCn-3PUFA on glycaemic responses. Therefore, in a randomised, cross over trial we investigated the postprandial glucose and insulin response to a single dose of curcumin and/or LCn-3PUFA in healthy individuals. The glucose levels were reduced by curcumin at as early as 30 min, and the maximum effect was observed at 60 min post meal consumption. Curcumin was found to be effective for lowering the insulin demand to control postprandial glucose levels. Similar results were observed following dietary supplementation with curcumin plus LCn-3PUFA. It was apparent that the postprandial effects on glycaemic control were primarily due to curcumin even in the combined treatment group. Thus, providing basis for long-term supplementation study with curcumin for glycaemic control. In chapter 5, a detailed study protocol for 2x2 factorial placebo controlled, double blinded randomised trial with long term (12 weeks) curcumin and LCn-3PUFA supplementation (COP-D trial) was presented. In chapter 6, we examined the effects of curcumin with or without LCn-3PUFA on glycaemic control and blood lipid levels in people at high risk of T2D. 12 weeks of supplementation with curcumin has effectively reduced the fasting insulin levels and IR in individuals with high risk of T2D. Parallel to these results, both curcumin and LCn-3PUFA were able to reduce the fasting triglycerides and atherogenic index of plasma, however the magnitude of reduction was greater with LCn-3PUFA supplementation. InsuTAG levels were also reduced with curcumin and LCn-3PUFA supplementation. However, this study failed to show any complimentary effects with concurrent administration of curcumin and LCn-3PUFA. Though IR and fasting triglycerides, were effectively reduced by these two bio-actives, we did not find any beneficial effects of curcumin and LCn-3PUFA supplementation on fasting glucose and glycosylated haemoglobin levels. In chapter 7, we designed a study to target commonly prevalent dyslipidaemia with curcumin and/or LCn-3PUFA in individuals with T2D (CALFOR-CVD trial). Participants were randomised to either placebo or curcumin or LCn-3PUFA, or curcumin plus LCn-3PUFA for six weeks. This pilot study has demonstrated that supplementation of curcumin can effectively reduce the TG. Contrasting to the results from chapter 6, magnitude of reduction in triglycerides in this study was higher with curcumin than LCn-3PUFA. Preliminary observations also presented a non-significant, but a noteworthy reduction of 0.5 mmol/L in total cholesterol and LDL-Cholesterol with curcumin supplementation. In line with observations from the COP-D trial, curcumin and LCn-3PUFA did not have any complimentary and/or added benefits. In conclusion, the results presented in this thesis demonstrate that InsuTAG has the potential to predict IR and MetS. This provides a basis for further research to validate InsuTAG with gold standard technique for IR and a longitudinal data analysis to determine the ability of InsuTAG to predict T2D in general population. With regards to the intervention trials, our hypothesis of targeting multiple pathways (IR and dyslipidaemia) in high risk and T2D patients with curcumin and LCn-3PUFA supplementation was successful. However, this thesis failed to provide any evidence on beneficial effects of combining curcumin and LCn-3PUFA for better glycaemic control to delay the onset of T2D. This could partly be due to presence of any unknown interactions between the two bio-actives or may be due to uncertainties in co-administration of curcumin and LCn-3PUFA. Thus, paving a way for further research to investigate beneficial effects with single formulation (curcumin and LCn-3PUFA) for achieving glycaemic control. This thesis constitutes a noted contribution to the research area of bio-markers and novel intervention strategies for T2D, and also presents a set of riddles that provides an extensive scope for future research.

Research Doctorate - Doctor of Philosophy (PhD)
The consumption of foods rich in saturated fats has been associated with elevated blood lipid levels and consequently with risk for numerous chronic diseases, such as coronary heart disease. However, understanding the effects of replacing saturated fat in the diet is complex, and the health effects of reducing saturated fat consumption clearly depend on what substitutions are made. Furthermore, studies using animal models have demonstrated that dietary saturated fats raise triglyceride levels only when the diet is deficient in omega-3 polyunsaturated fatty acids (n-3PUFA). The n-3PUFA are known for their potential to help in managing hyperlipidaemia for the prevention of coronary heart disease, as well as for their anti-inflammatory, anti-arrhythmic and anti-aggregatory potential. In addition, research in human and animal models has shown competition for important enzymes in the metabolism of omega-6 polyunsaturated fatty acids (n-6PUFA) and n-3PUFA, with high consumption of n-6PUFA leading to an increase in their metabolism at the expense of n-3PUFA. This leads to an increased production of n-6PUFA derived eicosanoids, which are pro-inflammatory and pro-aggregatory, in contrast with those derived from n-3PUFA, which are less inflammatory and aggregatory. Therefore, we hypothesised that consumption of saturated fats, would not adversely influence coronary heart disease risk factors (blood lipid levels, lipoprotein profiles, platelet aggregation and inflammation) when the diet was balanced with an adequate intake of n-3PUFA. Moreover, we hypothesized that the health benefits obtained with the consumption of n-3PUFA would be maximised by including foods rich in saturated fat and reducing the consumption of vegetable oils (rich in n-6PUFA) in the diet. Our first aim, addressed in chapter 3, was to establish the basis for our hypothesis, analysing the literature on saturated fatty acids to identify the contradictions in the literature to date and to highlight the gaps in knowledge gained from previous interventional and epidemiological studies. We have observed that although many studies have associated saturated fatty acids with hyperlipidaemia and cardiovascular disease risk factors, there is still much contradiction on the subject with not all studies finding the same association. The key studies relating saturated fat consumption and heart health made no mention about the presence or absence of n-3PUFA as a possible confounding factor. This may have been due to the lack of knowledge about the existence of n-3PUFA or an inability to determine n-3PUFA concentration in most of the early studies. Therefore the missing link in the research on cardiovascular disease risk and dietary fats could be an ignorance of the interactions between different dietary fats and the effect of this interaction. In Chapters 4a and 4b we aimed to determine if LCn-3PUFA and the other dietary fats interact during digestion, absorption, re-esterification into triglycerides and assembly into chylomicrons to modulate circulating lipid levels postprandially. In a randomised cross-over design, we investigated the effect of feeding meals rich in either saturated fatty acids or n-6PUFA in conjunction with LCn-3PUFA on plasma lipid (triglycerides and total, low density lipoprotein and high density lipoprotein cholesterol) and fatty acid levels. The postprandial lipemic response and fatty acid kinetics were similar after the consumption of both meals and suggest that the competition between n-3 and n-6PUFA may be a longer term phenomenon, not just a postprandial effect. The aim of Chapter 5 was then to determine if there were interactions between LCn-3PUFA and other dietary fats in the longer term (6 weeks). Therefore, in a randomized parallel design intervention we investigated the longer-term effects of LCn-3PUFA supplementation in subjects consuming diets enriched in either saturated fatty acids or n-6PUFA, on blood lipid profiles and on the incorporation of fatty acids into plasma and erythrocyte lipids. Long chain omega-3 polyunsaturated fatty acids were incorporated to a greater extent into the plasma and erythrocyte lipids of subjects consuming the saturated fat rich diet compared to the n-6PUFA rich diet, although total and low density lipoprotein (LDL) cholesterol were also increased. Plasma samples of the subjects who completed the intervention in chapter 5 were then further analysed in chapter 6 for lipoprotein profiles, with the aim of determining if the increase in plasma cholesterol levels was due to changes in the lipoprotein particle concentration or size. The increase in LDL cholesterol was due to an increase in the less atherogenic, large, buoyant LDL particles rather than the small, dense LDL particles. In chapter 7, the aim was to determine if pre-supplementation rather than co-supplementation with LCn-3PUFA would improve the effect of the major dietary fat groups on plasma lipids and lipoprotein profiles. Therefore, in a randomized parallel design clinical intervention, we examined the effect of increasing the omega-3 index of subjects before randomizing them to a diet rich in either saturated fatty acids or n-6PUFA. The diet rich in saturated fatty acids increased, while the diet rich in n-6PUFA decreased, total and LDL cholesterol, independently of LCn-3PUFA supplementation. However, the saturated fatty acid rich diet caused a further increase in plasma and erythrocyte LCn-3PUFA compared to the n-6PUFA rich diet. In conclusion, the results presented in this thesis demonstrate that the background dietary fat is a determinant of the degree of incorporation of LCn-3PUFA into plasma and tissue lipids. The consumption of a saturated fat rich diet did indeed cause an increase in plasma cholesterol levels. However, the rise in circulating cholesterol levels following saturated fat consumption is accompanied by an increase in the less atherogenic LDL particle size, when the LCn-3PUFA status is adequate, which is likely to reduce the detrimental effects. In addition, there was a concurrent increase in incorporation of LCn-3PUFA into plasma and erythrocytes, which may have benefits, independent of cholesterol or blood lipids. Hence, this thesis paves the way for further research to examine the impact of increased plasma and tissue LCn-3PUFA levels as a result of saturated fat consumption with adequate LCn-3PUFA intakes, on cardiovascular health risk indicators, such as inflammation, hypertension, platelet aggregation and endothelial function.

Research Doctorate - Doctor of Philosophy (PhD)
In Western society, increased asthma prevalence over recent years has coincided with changes in dietary patterns, leading to the hypothesis that a Western diet increases susceptibility to asthma. Components of the diet that may be important are antioxidants (e.g. lycopene) and fatty acids. Lycopene and long chain n-3 polyunsaturated fatty acids (LCn-3PUFA) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory effects. As asthma is a disease linked to oxidative stress and inflammation, it was hypothesised that these nutrients may have a beneficial effect individually, and may have a synergistic anti-inflammatory effect when used in combination. The aim was to examine the ability of lycopene and/or LCn-3PUFAs to protect against virus-induced inflammation, as rhinovirus infection is the primary cause of asthma exacerbation. The results presented demonstrate that both lycopene and DHA (but not EPA) individually decreased the inflammatory response of airway epithelial cells infected with rhinovirus. The results also showed that DHA supplementation increased the utilization of lycopene by cells. Furthermore, lycopene reduced rhinovirus replication. A combination of lycopene and DHA also reduced the inflammatory response of cells to rhinovirus infection, however, no synergistic anti-inflammatory effect was apparent. It is concluded that consumption of foods containing lycopene and DHA may exhibit a beneficial effect on the inflammatory response to rhinovirus infection. This may have important clinical implications, as increased dietary intake of foods rich in these nutrients may lead to a reduction in the frequency and severity of asthma exacerbations.

Research Doctorate - Doctor of Philosophy (PhD)
In Western society, increased asthma prevalence over recent years has coincided with changes in dietary patterns, leading to the hypothesis that a Western diet increases susceptibility to asthma. Components of the diet that may be important are antioxidants (e.g. lycopene) and fatty acids. Lycopene and long chain n-3 polyunsaturated fatty acids (LCn-3PUFA) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory effects. As asthma is a disease linked to oxidative stress and inflammation, it was hypothesised that these nutrients may have a beneficial effect individually, and may have a synergistic anti-inflammatory effect when used in combination. The aim was to examine the ability of lycopene and/or LCn-3PUFAs to protect against virus-induced inflammation, as rhinovirus infection is the primary cause of asthma exacerbation. The results presented demonstrate that both lycopene and DHA (but not EPA) individually decreased the inflammatory response of airway epithelial cells infected with rhinovirus. The results also showed that DHA supplementation increased the utilization of lycopene by cells. Furthermore, lycopene reduced rhinovirus replication. A combination of lycopene and DHA also reduced the inflammatory response of cells to rhinovirus infection, however, no synergistic anti-inflammatory effect was apparent. It is concluded that consumption of foods containing lycopene and DHA may exhibit a beneficial effect on the inflammatory response to rhinovirus infection. This may have important clinical implications, as increased dietary intake of foods rich in these nutrients may lead to a reduction in the frequency and severity of asthma exacerbations.

Preterm infants have an underdeveloped immune system and as such they are predisposed to developing unregulated inflammatory responses that are associated with disease in the postnatal period. Docosahexaenoic acid (DHA) is an omega-3 long-chain polyunsaturated fatty acid (LCPUFA) with known immunomodulatory properties, however the effect of dietary DHA on the regulation of immune responses in preterm infants is largely unknown. This thesis employs a multi-system approach to address questions related to the efficacy of omega-3 DHA to regulate inflammation in preterm infants and in human type II alveolar epithelial cells (AEC). The N3RO randomised controlled trial (RCT) provided the opportunity to carry out a single-centre nested study to examine the effect of supplemental DHA in preterm infants on pro-inflammatory and regulatory biomarkers in blood and levels of a common bacterial pathogen in the gastrointestinal tract. The aim of the N3RO RCT was to assess the efficacy of an enteral DHA emulsion to reduce bronchopulmonary dysplasia (BPD) in preterm infants < 29 weeks gestation compared to a standard soy emulsion without DHA. Prior to analysis of biological samples from preterm infants, the immune response to enteral DHA and soy emulsions in human type II AECs, one of the primary cell types affected in respiratory disorders, was assessed in vitro. The enteral emulsions assessed in the N3RO RCT were tested in conjunction with other commercially available parenteral lipid emulsions. Omega-3 DHA in both enteral and parenteral emulsions significantly reduced pro-inflammatory cytokines (IL-1β, IL-8 and IFNγ) when compared to soy-based emulsions. There are very few studies that have assessed what, if any, targets DHA interacts with to exert an immunomodulatory effect in preterm infants. Inflammatory cytokines are known to play a crucial role in the progression of airway inflammation, epithelial and vascular damage and subsequent development of BPD. Such inflammatory mediators are also involved in the development of other neonatal inflammatory disorders such as sepsis, necrotising enterocolitis and retinopathy of prematurity. A total of 144 blood samples were collected from 51 preterm infants enrolled in the nested study. Supplemental DHA did not reduce pro-inflammatory cytokine levels in plasma or whole blood culture supernatants (after a 24 hour incubation with E. coli lipopolysaccharide). Inflammatory mediators in the gut environment can influence initial colonisation and resulting abundance of both commensal and pathogenic bacteria. Staphylococcus is among the first colonisers of the respiratory and gastrointestinal tracts and it is one of the most important pathogens in the neonatal intensive care unit. Colonisation by methicillin-resistant bacteria including Staphylococcus in preterm infants also causes significant morbidity and mortality in the neonatal intensive care unit. In the neonatal period, diet has a significant effect on microbial colonisation of the gut, however the effect of supplemental omega-3 LCPUFA on Staphylococcus colonisation in preterm infants is unknown. A total of 220 stool samples were collected from 41 preterm infants enrolled in the nested study. Levels of Staphylococcus and bacteria carrying the gene coding for methicillin-resistance (mecA) decreased significantly over time in both groups, but DHA did not have an effect on abundance. The original contribution this thesis makes to the knowledge base is that supplementing preterm infants < 29 weeks gestation enterally with 60 mg/kg/day of DHA does not affect circulating levels of pro-inflammatory or regulatory cytokines, the immune response to an infectious stimuli nor does it influence Staphylococcus and mecA+ bacteria in the gut. This thesis contributes important information regarding the use of DHA at supplemental levels in nutrition regimens for preterm infants.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, Adelaide Medical School, 2017.

Omega-3 (n-3) long chain polyunsaturated fatty acids (LCPUFA), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are important for normal health as well as growth and brain development in humans. These fatty acids can be consumed in the diet directly, or synthesised from short chain PUFA consumed in the diet. Fish, particularly in species with a high fat content like salmon, are a major source of these beneficial fatty acids in the human diet. Fish production from aquaculture continues to expand due to a growing human population and demand for fish. Currently there is a reliance on fish oil and fish meal derived exclusively from wild fish as the primary lipid and protein source in fish feeds. Depleted wild fish stocks have made this source of n-3 LCPUFA unsustainable and alternative sources of n-3 LCPUFA are required to fill the void. Most animal species can convert the plant derived 18 carbon (C18) n-6 linoleic acid (LA, 18:2 n-6) and n-3 α-linolenic acid (ALA, 18:3 n-3) to 20 and 22 carbon (C20-C22) LCPUFA by using a series of enzymes to extend and alter the saturation level. There are two types of enzymes responsible for desaturating and elongating fatty acids are desaturases and elongases. The genes associated with these processes appear to be regulated by the extremes of dietary PUFA intake but the extent is currently unclear. This thesis is aimed to examine the effect of dietary PUFA on tissue n-3 LCPUFA levels in animals (rat and fish) after the consumption of diets with increasing levels of ALA, and to investigate whether the expression of desaturases and elongases is involved in the regulation of lipid metabolism and therefore LCPUFA biosynthesis. Furthermore, this thesis also investigated the potential enzyme functions of barramundi Δ6 desaturase and elongase using a yeast heterologous system. Experiments showed that while high ALA diets consistently produced higher levels of n-3 LCPUFA in rat tissues than low ALA diets, mRNA abundance of the Δ6 desaturase (FADS2) and elongase 2 (ELOVL2) genes were increased only in animals fed the low PUFA reference diet compared to those fed diets with adequate to high PUFA levels. There was no correlation between the gene expression of desaturases, elongases or transcription factors and the levels of EPA, docosapentaenoic acid (DPA, 22:5 n-3) or DHA in rat blood, liver and other tissues as a result of feeding increasing levels of ALA. In barramundi however, while vegetable oils induced significant increases in mRNA abundance of FADS2 and ELOVL genes compared with those fed the fish oil-based commercial diet, the tissue EPA, DPA and DHA levels were not increased. It is therefore hypothesised that the enzyme activity of barramundi Δ6 desaturase was low and therefore limited the effectiveness of the enzymes in the LCPUFA pathway to produce EPA and DHA. Furthermore, a large amount of variation between individual fish in DHA levels among those fed the vegetable oil-based diets was found, and this may provide a possibility for a future breeding program of barramundi for better DHA production. Barramundi FADS2 and ELOVL genes were also cloned into yeast cells and performed functional expression of the two enzymes. Results revealed that the barramundi Δ6 desaturase also showed Δ8 desaturase activity and the elongase showed a broad range of fatty acid specificity with the greatest activity with EPA. In addition, a significant amount of the desaturation and elongation fatty acid products could be detected in the culture medium at various time points after the addition of fatty acid substrates, and that it was important to take the levels of fatty acids in the medium into account when it came to calculating enzyme activity.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2011

Research Doctorate - Doctor of Philosophy (PhD)
Thrombosis is a critical event that accounts for considerable morbidity and mortality in the Western world. Thrombosis is associated with arterial diseases including, myocardial infarction, stroke, and peripheral occlusive disease as well as with venous thromboembolic disorders. Consequently, the primary goal for the prevention of arterial and venous thrombosis to combat disease progression is to limit thrombus extension. Platelet activation and aggregation is considered to be central to thrombus production; thus anti-thrombotic treatments to inhibit platelet activity have been a major drug target to retard the thrombotic and atherosclerotic processes. Despite extensive resource investment in cardiovascular research and treatment, the current pharmacological strategies for the inhibition of platelet aggregation, although effective, may present limitations and adverse health effects have been reported. Given the toll taken by thrombotic complications, a safe and efficacious non-pharmacological approach may be paramount for the prevention and management of thrombotic disease. While a wealth of evidence supports that fish oil provides preventative or ameliorative effects against thrombotic disease, the mechanisms responsible for this association are not understood and are further complicated by contrasting reports. Fish oils are a rich source long chain omega-3 polyunsaturated fatty acids including eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), however it is not clear whether the anti-thrombotic effects are due to EPA or DHA or whether both are equally effective. In the available literature relating fish oil and platelet aggregation, wide variability in terms of dosage, concentration ratios, study design, subject characteristics and gender inequality are apparent, hence there is discrepancy regarding the effect of fish oils on platelet activity. Consequently, the anti-thrombotic potential of fish oil supplementation is controversial and largely disregarded by the medical community. This dissertation investigated the independent effects of EPA and DHA on platelet and coagulant activity. A series of three controlled studies were undertaken to elucidate the mechanisms by which EPA and DHA influence hemostatic parameters with the hope to resolve the existing controversy. The ultimate and unifying theme of these studies was to provide a safe and efficacious approach to optimise cardio-protection via anti-thrombotic potential of EPA versus DHA. Firstly, an in vitro investigation was carried out that compared the effects of EPA with DHA on platelet aggregation in healthy male and female subjects. The inhibition of platelet aggregation by EPA/DPA/DHA was equally effective and correlated with lag time; however most strikingly the results were influenced in a gender-specific manner. These observations suggest that interactions between sex hormones and fish oils exist to influence platelet response differentially. With a new perspective of gender bias effects, an acute supplementation study monitored the platelet responses up to 24 hours after consumption of a single dose of an EPA versus DHA-rich oil capsule in thirty male and female subjects. The kinetics of the EPA and DHA supplement on platelet activity was examined according to gender stratified treatment. Subgroup gender analysis showed that the anti-aggregatory effects of EPA were predominately evident in males while female platelets were more responsive to DHA. The marked decrease in platelet aggregation with EPA supplementation was paralleled with a reduction in platelet microparticle activity in the male subjects only, and an inverse relationship between testosterone levels and platelet responses were observed. Findings from this study reflected the in vitro observations and suggest that EPA and DHA inhibit platelet aggregation via independent pathways compounded by sex hormonal influences. Confirmation of gender-specific platelet responses with omega-3 fatty acid supplementation was achieved in a chronic supplementation study involving ninety-four healthy male and female subjects. Subsequently, this four week dietary intervention trial demonstrated that the anti-thrombotic potential is apparent with longer term exposure to EPA/DHA and explored the mechanistic pathways. Significant interactions between gender and treatment were observed; the effects of EPA were specific in reducing platelet aggregation and specific coagulation factors in males, whereas no effects were observed in the female cohort. Conversely, the effects of DHA were unique to females with a similar decrease in platelet aggregability. Interactions between sex hormones with coagulation factors and retention of EPA and DHA in plasma were also observed.In conclusion, the study findings presented in this thesis provide evidence that the effects of EPA and DHA on platelet aggregation are apparent; the effects are neither shared nor complementary, rather they are gender-specific. Furthermore, the results herein may explain the existing controversy between fish oils, platelets and thrombosis that have intrigued clinical investigators for several decades. With respect to thrombotic disease risk, males would likely benefit more from supplementation with EPA while females are more responsive to DHA. The significance of these findings allows optimal cardio-protection tailored for both gender groups offering a safe and efficacious non-pharmacological approach.

Circulating lipids play an important role in human physiology and pathophysiology. Lipids, as the major components in various cellular membranes, are involved in homeostatic regulation, particularly in relation to immune function and inflammatory mechanisms. With the growing global prevalence of lifestyle-related diseases, including obesity, diabetes, cardiovascular disorders and cancers, dietary lipids have received a great attention. Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) have been associated with a broad range of health benefits. The three main LC n-3 PUFA are eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and docosapentaenoic acids (DPA, 22:5n-3). Fish oil and krill oil are currently the most available sources of EPA and DHA as over-the-counter supplements, although other marine sources such as algae oil are also rich in EPA and DHA. Krill oil, derived from Antarctic krill (Euphausia Superba), is rich in EPA and DHA found in phospholipids (predominantly phosphatidylcholine) rather than triacylglycerol (TAG), in which EPA and DHA in fish oil are found. Krill oil also contains astaxanthin, a carotenoid contributing to its red colour which may also have beneficial health effects (Barros et al. 2014a, Pashkow et al. 2008). Despite a number of studies examining the effects of krill oil compared with fish oil on the incorporation of LC n-3 PUFA into different tissues, the outcomes have been conflicting, which might be associated with the different study designs using different chemical forms of fish oil and/or different doses of LC n-3 PUFA, and focusing at different target tissues. The research presented in this thesis consists of nine chapters covering a literature review (Chapter two) and two intervention studies in humans (Chapters four, five, six and seven) which have examined the effect of krill oil compared with fish oil on the incorporation of LC n-3 PUFA into plasma lipid fractions. There were a postprandial and a longer-term (30 days) intervention studies, and both clinical studies were randomised crossover designs involved healthy women (n = 10 and n = 11, respectively). All participants were instructed to maintain the habitual dietary intake and habitual physical activity throughout the interventions. The aim of the postprandial study was to compare the incorporation of LC n-3 PUFA into the plasma and circulating lipids in plasma and chylomicron fractions from five capsules (1 g each) of krill oil compared with five capsules (1g each) of fish oil and 5 g of the olive oil (control) over a 5-hour postprandial period. The second study aimed to investigate the longer-term effect of krill oil supplementation (containing 1,269 mg/d of LC n-3 PUFA including EPA, DHA and DPA) on the plasma LC n-3 PUFA, plasma circulating TAG and inflammatory biomarkers compared with fish oil supplementation (containing the closest possible match to these fatty acids from the capsules, 1,441 mg/d) over a 30-day intervention period. In both studies, lipidomics, was applied to identify the differences in plasma lipid molecular responses between krill oil and fish oil supplementation. Using this technique, a number of plasma lipid classes, and lipid molecular species containing EPA and DHA were identified and quantified. In the 5-hour postprandial study (Chapters four and five), there were no significant differences in the levels of TAG or cholesterol in plasma or chylomicron between the three study oil interventions, although the expected increases in chylomicron TAG were observed in all groups. In comparison to the olive oil, both krill oil (containing 907 mg of LC n-3 PUFA) and fish oil (containing 1,441 mg of LC n-3 PUFA) supplementation significantly increased the level of plasma EPA, which plateaued after three hours; there were no significant differences in the plasma EPA levels between krill oil and fish oil supplementation groups. There were no significant changes in either DHA or DPA between the three groups. Krill oil, with a lower dose of EPA in this study, showed a similar incorporation outcome of EPA into plasma lipids as fish oil. Given that there were 31% less EPA from krill oil, these results indicate a differential extent of incorporation of EPA between krill oil and fish oil, suggesting that EPA from krill oil may be more efficiently incorporated into the plasma than fish oil. The advanced technique for lipidomics was performed by high-performance liquid chromatography-mass spectrometer analysis (HPLC MS/MS), which was able to identify and quantify changes in various lipid molecular species containing LC n-3 PUFA in both the postprandial and the longer-term studies. Therefore, the HPLC MS/MS facilitated a comparison between differences in the individual lipid molecular species between krill oil and fish oil supplementation. A more sensitive setting of HPLC MS/MS was applied to the postprandial data than the longer-term data, based on the settings applied by the research laboratory at Baker Heart and Diabetes Institute where these analyses were conducted. In Chapter five, the postprandial plasma lipidomic changes are reported at hours zero (baseline), 3 and 5. A total of 29 lipid classes (≥ 500 pmol/mL) (for example: TAG, diacylglycerol (DAG), phosphatidylcholine (PC), cholesterol esther (CE)) were identified; six of these including O-linked phosphatidylethanolamine classes had significantly greater the incremental area under the curve from baseline (net iAUC 0-5 h) after krill oil supplementation compared with fish oil supplementation. Over the postprandial period, 56 EPA-containing and 76 DHA-containing molecular species (for example 16:0-20:5-PC, 16:0-18:1-20:5-TAG, 16:0-22:6-PC, 16:0-18:1-22:6-TAG) were significantly increased after both krill oil and fish oil supplementation. There were 33 phospholipid molecular species containing EPA, and 16 of these molecular species, including six ether-phospholipid molecular species had significantly greater increased net iAUC 0-5 h after krill oil than fish oil supplementation. In contrast, for TAG and DAG molecular species containing EPA, seven out of a total of 21 showed significantly increased net iAUC 0-5 h for fish oil compared with krill oil. Put simply, the EPA from krill oil was associated with increases in phospholipid EPA-molecular species, while the EPA from fish oil was associated with increased TAG and DAG EPA-molecular species. There were 49 phospholipid molecular species containing DHA, and 11 of these including six ether-phospholipid molecular species, had significantly greater increased net iAUC 0-5 h after krill oil supplementatin than fish oil supplementation. In a total of 61 AA-containing molecular species (for example 16:0-20:4-PC, 16:1-20:4-DAG) identified, there were 51 phospholipid molecular species containing AA, and seven of these including six ether-phospholipid molecular species, had significantly greater increased net iAUC 0-5 h after krill oil supplementation than fish oil. A novel finding from this postprandial study was that there was a consistent trend that ether-phospholipid classes (O-linked (containing an alkyl bond) or P-linked (containing an alkenyl bond) phosphatidylcholine and phosphatidylethanolamine) were significantly increased after krill oil supplementation, but decreased after fish oil supplementation. Consistently, it was found that EPA- and DHA-containing ether-phosphatidylethanolamines were significantly increased after the krill oil supplementation, but decreased after the fish oil supplementation. While the significance of this finding is not clear, it is worth noting that plasma levels of O- and P-linked phosphatidylethanolamine have been reported to be decreased in a number of disease states including Alzheimer’s disease. Little is known about the origin of these ether-phospholipids in plasma, but the fact that krill oil increased their post-prandial levels and fish oil decreased them is a clear differentiation between these two omega-3 oils. In the longer-term study (Chapters six and seven), EPA, DHA and DPA were significantly increased after both krill oil and fish oil supplementation over the 30-day period (p < 0.001). The main response to the 30-day krill oil supplementation was that the increase of plasma EPA level was significantly greater in the net iAUC 0-30 d than that of fish oil supplementation (p < 0.05). Both krill oil and fish oil significantly reduced plasma TAG over the intervention period (p < 0.05 and p < 0.01, respectively), but no significant differences were observed between the two groups. Over the 30-day intervention period, some plasma pro-inflammatory cytokines including IL-1β, IL-10, IL-4 and IL-5 (p ≤ 0.05) were significantly reduced after krill oil supplementation, while no such changes were found after fish oil supplementation. In Chapter seven, the long-term lipidomic changes (≥ 500 pmol/mL), at days zero (baseline), 15 and 30, are reported. Twenty three EPA-containing and 46 DHA-containing molecular species were significantly increased after both krill oil and fish oil supplementation over the 30-day supplementation period. Among EPA-, DHA-, and DPA-containing molecular species, there were 20 cases of net iAUC 0-30 d significant differences between the two supplementation. Fourteen of these molecular species in phospholipid species, including 12 ether-phospholipid species, had significantly greater increased net iAUC 0-30 d after krill oil than fish oil (p ≤ 0.05) supplementation. Consistently, it was found that EPA- and DHA-containing ether-phospholipid species, including six ether-phosphatidylethanolamines, were significantly increased after the krill oil supplementation, and decreased after the fish oil supplementation. The changes in the ether-phospholipids in the long-term trial were consistent with the changes described in the postprandial trial (Chapter five). These results support strongly the differentiation between krill oil and fish oil although there are still many unanswered questions flowing from this novel finding. What is known about plasmalogens is that they play a role in anti-inflammatory response, which might be linked to the significant decrease in pro-inflammatory cytokines observed in the present study. Overall, both postprandial and longer-term studies demonstrated that EPA from krill oil is efficiently incorporated into plasma, has a similar effect on the plasma TAG-lowering and a greater efficacy on the plasma inflammatory biomarkers when compared with fish oil. No previous studies have investigated plasma lipidomic responses to krill oil and fish oil supplementation in humans. There were significant increases in molecular species containing EPA and DHA following supplementation with krill oil and fish oil over both the postprandial and the longer-term periods. The plasma lipidomic changes of net iAUC over both intervention periods were significantly different between krill oil and fish oil supplementation, particularly for phospholipids (krill oil resulted in a greater increase than fish oil) and TAG (fish oil resulted in a greater increase than krill oil, as described in Chapter five). A novel aspect identified in this study was that krill oil increased ether-phospholipids, particularly ether-linked phosphatidylethanolamine, whereas fish oil decreased ether-phospholipids. The biological relevance of this novel lipidomic finding has yet to be fully explored.

Yellowtail Kingfish (Seriola lalandi) (YTK) are carnivorous marine finfishes that are commercially farmed in Australia. YTK present the greatest opportunity for expansion of the Australian aquaculture industry, but improved diet formulations and feed conversion ratios are essential for production gains and economic upscaling. Lipids constitute a major cost component of aquafeeds but lipid composition has not been optimised for YTK. The purpose of this research was to increase understanding of how YTK utilise dietary lipids, and to improve feed conversion efficiency and product quality for human consumers. Fish oil (FO) as a dietary lipid source is central to this research as YTK require dietary omega 3 (n-3) long chain polyunsaturated fatty acids (LC PUFA) from FO for healthy development and growth, but FO is limited and less economically sustainable than other types of oil/lipid. The first study presented in this thesis sought to benchmark the fatty acid composition of wild YTK compared to aquacultured YTK. Tissue total lipid content was on average 4-times higher in aquacultured than wild YTK, with significantly higher concentrations of total saturated, omega 9, omega 7 and omega 6 fatty acids in tissues, but n-3 LC PUFA concentrations were not significantly different in the white muscle of wild and aquacultured YTK. The second and third studies were carried out with YTK grown in tanks using aquafeeds with varying lipid formulations. Generally, the fatty acid composition of aquacultured fish is reflective of the composition of aquafeeds, however this was not always the case for YTK in the following experiments. The key findings were that YTK have the capacity to spare in full Docosahaexonoic Acid (DHA) in white muscle at the expense of oleic acid (18:1n-9) when dietary levels of n-3 LC PUFA were <1.6 g 100 g-1 feed and that the digestibility of saturated fatty acids decreased with increasing chain length. Both of these findings could be used to manipulate dietary formulations and improve utilisation of n-3 LC PUFA. The fourth study investigated the potential for finishing diets to be utilised to modify the tissue fatty acid composition of YTK prior to harvest. Results showed significant changes in white muscle n-3 LC PUFA over 33 days at warm water temperatures, however further research was recommended to optimise the duration of finishing periods under a range of conditions. It was also recommended that the n-3 LC PUFA content of YTK feeds be closely monitored with strict lower limits set to ensure optimal product quality. The fifth and final study validated a method for the quantification of bioactive free fatty acid and oxylipin levels in YTK blood plasma. The approach was then used to determine the effects of dietary levels of n-3 LC PUFA on plasma free fatty acids and oxylipin bioactives. This method provides a new tool for aquaculture nutritionists to assess the impact of changes to YTK aquafeed formulations. In summary, this thesis has provided insight into the factors that affect fatty acid utilisation in YTK which have the potential to positively influence future aquafeed formulations, while also providing new methods to investigate lipid metabolism in the future.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2019

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016
Os ácidos gordos polinsaturados, conhecidos por PUFA (do inglês, polyunsaturated fatty acids), contêm duas ou mais ligações duplas de carbono e incluem os ácidos gordos essenciais, ómega-6 (n-6) e ómega-3 (n-3). O ómega-6 deriva do ácido linoleico (LA, 18:2n-6) e origina o ácido araquidónico (AA) como metabolito final. O ómega-3 deriva do ácido linolénico (ALA, 18:3n-3) e tem como metabolitos principais o ácido eicosapentaenóico (EPA, 20:5n-3) e ácido docosahexaenóico (DHA, 22:6n-3), que são ácidos gordos de cadeia longa (LC-PUFA, do inglês, long-chain polyunsaturated fatty acids). Os metabolitos do n-6 e do n-3 são originados através de uma cascata de reacções de dessaturação, alongamento e oxidação, com enzimas específicas. O n-6 produz também alguns eicosanóides com propriedades pró-inflamatórias e pró-trombóticas: lipoxinas (LXs), prostaglandinas (PGs), thromboxanos (TXs) e leucotrienos (LTs), que são contrabalançados pelos eicosanóides anti-inflamatórios do n-3. Os PUFA são considerados ácidos gordos essenciais porque não conseguem ser sintetizados de novo pelo nosso organismo e precisam de ser obtidos através da dieta. As melhores fontes de ácidos gordos são o peixe gordo e os seus óleos, os óleos vegetais, como óleo nozes, chia, canola e linhaça e também óleo ou extracto de algas marinhas. Os PUFA podem também ser encontrados em suplementos alimentares e no leite materno. O rácio de ingestão n-6/n-3 é considerado um factor de promoção da saúde humana, sendo os níveis baixos deste rácio recomendados para se obterem os efeitos protectores destes ácidos gordos, nomeadamente ao nível das suas propriedades anti-inflamatórias, cardiovasculares e neurobiológicas. Na dieta ocidental moderna existe um consumo excessivo de n-6 relativamente ao n-3, o que origina uma desregulação do metabolismo normal destes ácidos gordos, onde o n-6 compete com o n-3 pelas mesmas enzimas e leva ao aumento dos eicosanóides pró-inflamatórias do n-6. Há, no entanto, estudos recentes que colocam em causa o papel do rácio n-6/n-3 e reforçam a ideia do consumo de EPA e DHA em maior quantidade, ao invés de n-3 sob a forma de ALA. Actualmente é recomendado o consumo de 1g/ dia de n-3 PUFa, sob a forma de EPA+DHA, para se obterem efeitos benéficos no sistema cardiovascular. O consumo de óleo de peixe, rico em EPA e DHA, tem sido associado a efeitos protectores no sistema nervoso central, promovendo o desenvolvimento dos circuitos corticais e afectando o funcionamento de neurotransmissores (serotonina, adrenalina, noradrenalina e dopamina), tendo consequentemente um impacto positivo na progressão de patologias neurológicas do foro inflamatório e também comportamental, como a depressão, ansiedade, stress e perturbações de humor. Condições como a depressão, a ansiedade e o stress têm um impacto negativo na sociedade, podendo levar a situações fatais. Assim, é necessário avaliar o impacto dos ácidos gordos de cadeia longa na prevenção destes distúrbios. A maior parte dos estudos sobre EPA e DHA foca-se na toma conjunta destes ácidos gordos e, por isso, não clarificam o papel individual de cada um destes compostos sobre a saúde. O objectivo principal deste trabalho é, portanto, explorar os efeitos benéficos da toma de EPA e DHA, comparando a sua acção isolada com a sua acção conjunta, na promoção de comportamentos activos, opostos aos encontrados em situações de depressão e outros distúrbios comportamentais. Para este trabalho foram usados 32 ratos Wistar como modelo de estudo, distribuídos aleatoriamente por 4 dietas diferentes (com 8 animais por grupo) e ricas em ácidos gordos de diferentes origens, de forma a avaliar qual destes compostos tem um efeito benéfico maior sobre o comportamento: óleo de peixe, rico em EPA+DHA (grupo Fish Oil), óleo de Nannochloropsis, uma microalga marinha rica em EPA (grupo Nanno) e óleo de Schizochytrium, uma alga marinha rica em DHA (grupo Schyzo). Uma dieta pobre em EPA e DHA (grupo Milk Fat) foi usada como controlo negativo. Os animais foram pesados duas vezes por semana durante dois meses, registando-se igualmente a quantidade de alimento ingerido nesse período. Para avaliar estado de actividade/passividade dos animais recorreu-se a um teste de natação forçada (Forced Swimming Test, FST, em inglês), em que os animais são colocados numa piscina com 30 cm água, num ambiente controlado e do qual não podem escapar. O teste foi realizado em duas fases, em dois dias consecutivos (pré-teste de 15 minutos + teste de 5 minutos), sendo o segundo teste gravado para análise dos movimentos natatórios, frequência de movimentos, tempo de latência e tempo de imobilidade dos animais. O maior tempo de imobilidade está associado a um estado menos activo e pode ser interpretado como uma maior tendência para um comportamento depressivo. Posteriormente, os animais foram sacrificados e procedeu-se à recolha dos seus órgãos e sangue, usados para análise do perfil de ácidos gordos, quantificação de parâmetros bioquímicos e análise dos níveis de serotonina e catecolaminas. As fezes (previamente recolhidas) foram também analisadas para determinar o perfil de ácidos gordos e a eventual absorção destes pelo organismo. Os resultados do teste comportamental revelam um maior poder benéfico no consumo conjunto de EPA+DHA, uma vez que o grupo Fish Oil revelou tempos de imobilidade menores e uma maior latência de imobilidade. O grupo Schyzo, rico em DHA, teve valores próximos, embora inferiores, aos encontrados no grupo Fish Oil, tendo os grupos Milk Fat e Nanno uma pior prestação global no teste comportamental. Os resultados nas fezes revelam um maior poder de absorção para o grupo Fish Oil e menor para o grupo Nanno. A análise ao plasma revelou valores mais baixos de lípidos totais, colesterol total, triglicéridos e glucose para o grupo rico em EPA+DHA, bem como níveis mais altos de dopamina e adrenalina, associados a um maior índice de actividade e motivação. O grupo Nanno, rico em EPA, apresentou bons resultados nos parâmetros ligados à saúde cardiovascular, o que pode indicar um papel mais benéfico deste ácido gordo, relativamente à toma de DHA. Os eritrócitos e o cérebro apresentaram também níveis elevados de EPA e DHA para o grupo Fish Oil, em comparação com os níveis encontrados nos outros grupos, revelando uma maior incorporação de ácidos gordos por parte da dieta rica em óleo de peixe. Pode concluir-se que a toma conjunta de EPA+DHA é mais benéfica para a saúde cardiovascular geral e para melhorar os níveis de actividade nos indivíduos do que a toma isolada destes compostos, uma vez que o grupo alimentado com EPA+DHA apresentou melhores resultados em todos os parâmetros analisados, comparativamente aos que apenas tomavam EPA ou DHA.
Polyunsaturated fatty acids include the essential omega-6 (n-6) and omega-3 (n-3) fatty acids, which are not synthesised by our body and must be obtained through diet. The most abundant sources of PUFA are fish, plant and algae oils. Omega-3 has an important anti-inflammatory power and is known for its benefit effect on the prevention of cardiovascular diseases. The main n-3 metabolites are eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6,n-3), the long-chain PUFA (LC-PUFA), mainly found in fish oil and known to have a protective role regulating brain development and neurotransmitter functioning. Therefore, LC-PUFA are implied for the prevention of neurodegenerative and neurological conditions, as well as behavioural disturbances like depression and anxiety-related disorders. However, there is a lack of information about the individual role of these fatty acids on these stated conditions. The purpose of this work was to test and compare the effects of EPA and DHA, in form of isolated and combined diet, on the promotion of active behaviours, favourable in neurologic disorders. An experimental design was made using 32 Wistar rats, divided into 4 different diets to assess the specific effects of each fatty acid: Milk Fat, the negative control diet without EPA or DHA added; Fish Oil, the positive control diet, rich in EPA+DHA; Nanno group, rich in EPA; Schyzo group, rich in DHA. A behavioural Forced Swimming Test (FST) was performed to evaluate the active/passive state in rats. The animals were later euthanized, with their blood and organs removed for biochemical analysis. Fatty acid profile in faeces, erythrocytes and brain, as well as biochemical markers, serotonin and catecholamines levels were determined. Behavioural FST revealed benefit effects of the EPA+DHA intake, rather than individual fatty acid intake, since Fish Oil group presented a better overall performance. Both Milk Fat and Nanno groups presented the worse results in FST, with higher immobile levels, low latency times and higher frequencies. Schyzo group has more similar results to Fish Oil group than Nanno group, which might indicate a better role of individual DHA, contrarily to individual EPA, on promoting active behaviours. Plasma metabolites, as well as dopamine and epinephrine levels, also presented better results in Fish Oil group, with Nanno group having similar results as Fish Oil regarding plasma metabolites related with cardiovascular health. It can be concluded that an EPA+DHA diet is more adequate for the promotion of global health, as well as increasing active behaviours, which can be benefit for neurologic conditions.

Parkinson's disease is one of the most common neurodegenerative disorder that is slowly progressive and manifested by muscle rigidity, tremor, decreased mobility and postural instability. The disease is caused by a combination of genetic and environmental factors. The most prominent pathological features are the severe loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of cytoplasmic protein inclusions called Lewy bodies, primarily composed of fibrillar α-synuclein and ubiquitinated proteins within some remaining nigral neurons. Autophagy is a catabolic process that maintain cellular homeostasis, through the selection of misfolded proteins, damaged organelles, and even pathogenic organisms to be degraded by lysosomes. Autophagy can mediate cytoprotection (for instance neuroprotection and cardioprotection in the context of ischemic preconditioning) and delay the pathogenic manifestations of aging. Dysregulation of autophagy has been observed in the brain tissues from Parkinson’s disease patients and animal models. In recent years, some reports have shown a new relationship between macroautophagy and lipid metabolism. In this work, we used the most consumed polyunsaturated fatty acid in our diet, linoleic acid, to evaluate if it induces autophagy and if there is a possible relationship between linoleic acid-induced autophagy and the neuroprotective/toxic mechanisms triggered by this compound. We found that linoleic acid induces autophagy at concentrations equal or higher than 200 μM, and we describe its activation pathway, using Western blotting and immunofluorescence assays. Our results suggest that linoleic acid-activated autophagy process is mammalian target of rapamycin-independent, class III phosphatidylinositol 3-kinase/Beclin1-independent and AMP-activated protein kinase-dependent. As for the neuroprotective capacity of linoleic acid, we observed that alone it shows some toxicity. However, if co-administered with an inducer of reactive oxygen species (such as paraquat), linoleic acid does not increase paraquat toxicity. On the other hand, when linoleic acid is co-administered with puromycin (protein aggregates generator) it has a neuroprotective effect.

Salmon is a good source of long chain (LC) omega 3 fatty acids and selenium; these are well recognised for their health benefits. Recommendations for LC omega 3 fatty acid intakes presume equivalence between fish and fish oil. The aim of this research was to compare the effects of consuming salmon with salmon oil capsules on LC omega 3 fatty acid and selenium status. Forty four healthy subjects were randomly assigned to consume either two servings of 120 g farmed New Zealand King (FNZK) salmon/week or 2, 4 or 6 capsules of salmon oil/day for 8 weeks. Fasting blood samples, anthropometric measures, food consumption habits information and blood pressure (BP) measurements were obtained at the study commencement and ending. Each subject’s intake of LC omega 3 fatty acids and selenium was determined by analysing the fatty acid and selenium content of duplicate portions of cooked salmon and capsules. The amount of salmon consumed was then calculated by subtracting unconsumed amounts of salmon and then calculating the intake of LC omega 3 fatty acids as grams of LC omega 3 fatty acids consumed per day. Percentage of compliance to capsule intake, based on counts of unconsumed capsules, was calculated to determine the amount of LC omega 3 fatty acids consumed per day from capsules. Change in red blood cells (RBC) LC omega 3 fatty acid levels from equivalent amounts of LC omega 3 fatty acids consumed from capsules and salmon were compared using linear regression analysis predictive models fitted to the capsule data. Omega 3 index was calculated. LC omega 3 fatty acid intakes from salmon and 2, 4 and 6 capsules were 0.82, 0.24, 0.47 and 0.68 g/day, respectively. Equal amounts of LC omega 3 fatty acids consumed from salmon and capsules resulted in similar increases in RBC LC omega 3 fatty acids and omega 3 index (RBC eicosapentaenoic acid (EPA): 0.80 [0.58 – 1.02] vs. 1.00 [0.71 – 1.27] %; RBC docosahexaenoic acid (DHA): 0.93 [0.58 – 1.29] vs. 0.99 [0.68 – 1.31] %; omega 3 index: 1.92 [1.46 – 2.38] vs. 2.25 [1.65 – 2.83] %). The capsules did not contain selenium, but the salmon provided 6.84 µg selenium/day. Plasma selenium concentrations increased significantly in the salmon group compared to the capsule