Dissertations / Theses on the topic 'Docosahexaenoic acid'

Neonatal lamb mortality is a major factor effecting profitability in the sheep industry. Lamb thermogenesis and immunocompetence are key elements in neonatal lamb survival. Research has shown an increase in lamb vigor, when ewes were supplemented during late gestation with algae-derived docosahexaenoic acid (DHA). However, the impact of DHA on lamb thermogenesis and immunocompetence has not been investigated. Eighty twin-bearing Targhee ewes were assigned randomly to 1 of 2 supplemental treatments to determine the effects of feeding (DHA) to ewes during late gestation and early lactation on lamb thermogenesis, immunocompetence, serum metabolites and hormones, and lamb growth. Treatments within supplements were: 1) 12 g/ewe daily of the product DHA Gold in the form of algal biomass (ALGAE), and 2) no DHA (CONTROL). Treatment supplements were individually fed daily during the last 30 d of gestation and pen fed (6 pens/treatment, and 6 or 7 ewes/pen) during the first 38 d of lactation. One h after lambing and before nursing, twin-born lambs were weighed, bled via jugular puncture, and placed in a dry cold chamber for 30 min (0°C). Lamb rectal temperatures were recorded every 1 min. After 30 min, lambs were removed from the cold chamber, bled, warmed for 15 min, and returned to their dam. Ewes were bled and colostrum samples collected 1 h postpartum. Ewe and lamb sera were assayed for glucose, non-esterified fatty acids (NEFA), cortisol, leptin and anti-Parainfluenza Type 3 (PI³) titers. Lamb rectal temperature, glucose, NEFA, cortisol, leptin, anti-PI³ titers, and birth weights did not differ between treatments. Thirty-eight-d BW was greater (P = 0.03) in lambs born to CONTROL-supplemented than lambs born to ALGAE-supplemented ewes; however, the colostrum of ALGAE-supplemented ewes had a greater specific gravity (P = 0.05), indicating greater IgG concentrations, than colostrum of CONTROL-supplemented ewes. Supplementation of DHA during late gestation and early lactation had a negative impact on lamb BW and did not affect indices of lamb thermogenesis, but may have improved IgG concentrations in ewe colostrum.

Physiology
Ph.D.
Dendritic cells (DC) are professional antigen presenting cells which link innate and adaptive immunity through recognition and processing of pathogens, migration to secondary lymph nodes, presentation of antigens to naïve T cells and contribution to T cell proliferation and differentiation into various classes of effector T cells. N-3 fatty acids including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported to have protective anti-inflammatory effects in clinical studies and animal models of colitis, sepsis, and stroke. However, the mechanisms involved in the anti-inflammatory function of EPA and DHA are not elucidated. Recent studies, including our own, investigated the effect of n-3 fatty acids on dendritic cells, and reported that DHA reduced the capacity of human monocyte-derived DC (hMo-DC) and of murine bone marrow derived conventional DC (BMDC) to produce IL-12 and to stimulate CD4+ T cell proliferation. The objectives of this thesis were to provide a comprehensive analysis of the in vitro effects of DHA on conventional myeloid DC, including CD4+ T cell activation and differentiation, and on the in vivo effects of dietary DHA on splenic DC and in a model of experimental autoimmune encephalomyelitis (EAE). First, I report on the effects of DHA on the phenotype and function of BMDC in terms of endocytosis, chemokine receptor expression and migration, and cytokine production. Next, I investigated the effects of DHA-treated DC on CD4+ T cell proliferation and differentiation in response to specifc antigens. Finally, I assessed the effects of dietary DHA in response to in vivo LPS stimulation and in a model of neuroautoimmune disease, i.e. EAE. My studies show that pretreatment with DHA prevents LPS-induced DC maturation, resulting in the maintenance of an immature DC phenotype characterized by low expression of MHCII and costimulatory molecules (CD40, CD80, CD86), maintenance of high CCR5 expression and lack of CCR7 upregulation. DHA also maintained a high level of endocytosis in DC. From a functional point of view, these phenotypic traits result in retention of fully phagocytic DC at the inflammatory site, lack of migration towards the lymph node, and poor stimulatory capacity for T cells. Exposure to DHA inhibited the production of proinflammatory cytokines and chemokines such as IL-6, TNF&alpha, CCL-4, and IL-12p70. I also found that DHA prevents IL-12p70 production in DC activated by ligands for toll like receptors (TLRs) located either on the plasma membrane or intracellular. In addition, I show for the first time that DHA has a similar inhibitory effect on IL-23 and IL-27, two other members of the IL-12 family. The effects of DHA on cytokine production were shown to be mediated through activation of PPAR&gamma and inhibition of NF&kappaBp65 nuclear translocation, associated with reduction in I&kappaB degradation. Dietary DHA inhibited production of IL-12 family cytokines in vivo. Regarding the effects on CD4+ T cells, I found that DC treated with DHA (DC-DHA) had poor stimulatory capacity for CD4+ T cells in terms of proliferation. This was associated with an increase in p27(kip1), a cell cycle arresting agent. A higher percentage of T cells were arrested in G0/G1 in co-cultures with DC-DHA compared to DC controls. Although DC-DHA reduced T cell proliferation, effector T cells were not anergic and did not have suppressive function. T cells activated by DC-DHA also showed lower levels of IFN&gamma and IL-17 secretion. The percentage of IFN&gamma producing cells was also lower in T cells activated by DC-DHA, suggesting that the lower levels of IFN&gamma were due not only to reduced proliferation but also to the inhibition of Th1 differentiation. Accordingly, there was decreased expression of Tbet, GATA-3 and ROR&gammat, master transcription factors for Th1, Th2, and Th17 in T cells co-cultured with DC-DHA. In contrast, T cells co-cultured with DC-DHA expressed higher levels of TGF&beta which might be the reason for reduced proliferation since TGF&beta plays an important role in the induction of p27(kip1). Dietary DHA had a preventive effect in EAE lowering the EAE clinical score and resulting in less weight loss. The beneficial effect of dietary DHA in EAE was associated with reduced numbers of IFN&ganna- and IL-17-producing CD4+ T cells in both spleen and central nervous system (CNS). In conclusion, DHA maintains DC in an immature stage characterized by lower levels of MHCII and co-stimulatory molecules (CD40, CD80 and CD86), and poor stimulatory capacity for T cells. The DHA inhibition of pro-inflammatory cytokines (IL-12p70, IL-23, and IL-6) production in DC results in decreased Th1 and Th17 differentiation, cells that play an important role in autoimmune diseases. The protective effect of dietary DHA in EAE is associated with a reduction in Th1 and Th17 cell differentiation and CNS infiltration.
Temple University--Theses

Der globale Prävalenzanstieg allergischer Erkrankungen wird mit der westlichen Ernährung und einem sich ändernden Fettsäurespektrum assoziiert. Die omega-3 Fettsäure Docosahexaensäure (DHA) wurde bereits bei verschiedenen chronisch-entzündlichen Erkrankungen erfolgreich therapeutisch eingesetzt. Die dabei zugrunde liegenden Wirkmechanismen sind jedoch nicht vollständig geklärt. Deshalb wurde hier der molekulare Mechanismus der DHA-vermittelten Hemmung der IgE-Produktion in humanen B-Zellen sowie der verminderten Differenzierung IgE-produzierender Plasmazellen in vitro untersucht. Analysen der beteiligten Signaltransduktionswege offenbarten eine Reduktion der IL-4-abhängigen STAT6-Phosphorylierung und der CD40-vermittelten NFkappaB-Translokation, was zu einer Inhibition des IgE-Klassenwechsels auf dem Niveau des epsilon-Keimbahntranskriptes sowie der Aktivierungsinduzierten Desaminase führte. Weiterhin wurde in einer randomisierten, kontrollierten Doppelblindstudie die Supplementierung mit hochdosierter DHA bei Patienten mit atopischem Ekzem hinsichtlich klinischer und immunologischer Parameter geprüft. Dabei führte DHA zu einer Reduktion des Schweregrades der Erkrankung und zu einer verminderten IgE-Produktion anti-CD40/IL 4-stimulierter Blutzellen ohne Beeinflussung des Serum-IgE-Spiegels. Schließlich wurden die lokalen Prozesse nach DHA-Verabreichung in einem Mausmodell für proteininduzierte Dermatitis analysiert. Dabei war die Reduktion der klinischen Ekzemausprägung mit der verminderten Zahl dermaler CD8+ T-Zellen verbunden. Andere histologische Parameter und das Serum-IgE blieben jedoch unbeeinflusst. Durch die Fähigkeit von DHA, in den IgE-Klassenwechsel in B-Zellen einzugreifen, stellt die Supplementierung mit DHA somit eine mögliche präventive Maßnahme gegen allergische Erkrankungen dar. Ebenso ist DHA in der Lage, den Schweregrad des atopischen Ekzems durch die positive Beeinflussung lokaler inflammatorischer Prozesse signifikant zu verbessern.
The prevalence of allergic diseases has increased worldwide. Westernised diet with its changing polyunsaturated fatty acid (PUFA) proportions is considered to contribute to this development. The omega-3 PUFA Docosahexaenoic acid (DHA) has been reported to be antiinflammatory, but its way of action is not completely understood. Initially, the molecular mechanisms of DHA impact on IgE production in human B cells were examined in vitro. Thereby, DHA inhibited IgE production and the differentiation of IgE secreting cells. This was mediated through direct inhibition of the immunoglobulin isotype switching process by decreasing epsilon germline transcript and activation induced desaminase transcription. Analysis of involved signalling pathways revealed an inhibition of IL-4 driven STAT6 phosphorylation and a reduced NFkappaB translocation into nucleus upon CD40 ligation. Next, in a randomised, double bind, controlled clinical study the efficacy of high-dose DHA supplementation in atopic eczema was determined by investigating the impact on clinical and immunological parameters. In the DHA, but not in control group a clinical improvement of atopic eczema and a reduction of CD40/IL-4 mediated IgE synthesis of peripheral blood cells were observed whereas serum IgE levels remained unchanged. Finally, in a mouse model the impact of oral DHA application on allergen induced dermatitis as well as the underlying local mechanisms were investigated. Thereby, the DHA mediated reduction of clinical skin score was associated with decreased dermal CD8+ T cell numbers, whereas other histological parameters or serum IgE values were not affected. In summary the results indicate that dietary DHA may be effective in prevention of allergic diseases by interference with the IgE switching process and improve the clinical outcome of atopic eczema by its positive impact on local inflammatory processes.

Master of Science
Department of Biological and Agricultural Engineering
Wenqiao Yuan
Many of the current industrial fermentation processes cannot use pentose as the carbon source. However, complete substrate utilization of sugars in lignocellulose is one of the prerequisites to render economic development of biofuels or bioproducts from biomass. In this study we proposed a new process for DHA production from renewable carbon sources by first using anaerobic bacteria, Clostridium tyrobutyricum to convert pentose into organic acids with butyric acid as the main product, and then using the organic acids to feed microalgae, Crypthecodinium cohnii to produce DHA. The effect of glucose and xylose on the yield of butyric acid produced by C. tyrobutyricum was investigated, separately. Cell growth of C. tyrobutyricum increased with increasing initial glucose or xylose concentration, but was not affected significantly when the concentration was above 55g/l for glucose or 35g/l for xylose. Butyric acid yield increased as the initial sugar concentration increased in both xylose and glucose, but the conversion rate from xylose or glucose to butyric acid decreased as the sugar concentration increased. The xylose to glucose ratio in their mixture did not significantly affect cell growth or butyric acid yield. The effect of arabinose on the yield of butyric acid produced by C. tyrobutyricum was also studied. As for butyric acid production, compared with glucose or xylose, the arabinose was in a low efficiency, with butyric acid output of 2.25g/l in 10g/l arabinose and a long lag period of about 3-4 d. However, a low concentration of arabinose could be used as a nutritional supplement to improve the fermentability of a mixture of xylose and glucose. The conversion rate of sugar to butyric acid increased as the supplement arabinose increased. In order to obtain low cost xylose, corncobs were hydrolyzed and this xylose-rich product was used to culture C. tyrobutyricum. The results showed that at end of the 9 d fermentation, the concentration of butyric acid from corncob hydrolysate reached 10.56 g/l, and the mimic medium reached 11.3 g/l. This suggests that corncob hydrolysate can be used as a carbon source for butyric acid production by C. tyrobutyricum, although some inhibitory effects were found on cell growth with corncob hydrolysate. The effect of butyric acid, lactic acid and acetic acid on the yield of DHA produced by C. cohnii was also investigated, separately. The DHA yield was highly related to both biomass and DHA content in the cell, whereas lower growth rate could bring higher DHA content. The best concentration for DHA yield seemed to be 1.2g/l in three single organic acid media. In two organic acids mixture media, acetic acid tended to be beneficial for biomass accumulation, regardless whether butyric acid or lactic acid was mixed with acetic acid, the OD could reach 1.3 or above. When butyric acid was mixed with lactic acid, the highest DHA yield was achieved, due to increased DHA content from mutual influence between butyric acid and lactic acid.

Thesis (M.S.)--West Virginia University, 2008.
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BACKGROUND: obesity may be associated with a later onset of chronic disorders and clinical complications. Insulin resistance, glucose intolerance and hyperinsulinemia are major components of the metabolic syndrome, which is highly prevalent among children and adolescents with severe obesity. Low levels of LCPUFAs, especially docosahexaenoic acid (C22:6 n-3, [DHA]) and a high n-6/n-3 LCPUFA ratio in skeletal muscle membrane phospholipids have been associated with insulin resistance in adults. Recent data suggest that the synthesis of DHA differs between obese children and normal weight children. In particular, in obese children, the highest quartile of BMI z-score was associated with higher plasma levels of the n-6/n-3 LCPUFA ratio. Given the high prevalence of insulin resistance in childhood obesity, we asked whether supplementation with DHA would be more effective than diet and physical activity alone in reducing this metabolic alteration. AIM: to determine whether DHA supplementation, in addition to adequate diet and lifestyle, may reduce insulin resistance compared with diet and physical activity only, in obese children. Secondary aims were to evaluate whether may exist an association of the change of steatosis degree after the intervention with DHA supplementation... SUBJECTS AND METHODS: this is a multicenter, longitudinal, double-blind, randomized, placebo-controlled trial that started on January 01, 2010. Up to September 30, 2012, thirty (14 boys, 16 girls, mean [SD] age, 11.4 [1.29] years, range 8-13), were recruited. All obese children consecutively admitted to the Department of Paediatrics, San Paolo Hospital, Milan, and to the Department of Paediatrics, Federico II Hospital, Naples, for routine examinations were assessed for eligibility. The study protocol scheduled daily oral supplementation of either an intervention “product”, that is two capsule of purified DHA (500 mg) or two capsule of wheat germ oil (500 mg). A nutritional-behavioural intervention was additionally recommended in all recruited children promoting a normocaloric balanced diet and an active lifestyle based on the Italian guidelines for treatment of childhood obesity. Additional recommendations were given to engage in a moderate daily exercise program (30-45 minutes/day aerobic physical exercise), tailored to individual preferences. Children were visited at the care centers within 3±1 days (baseline) after enrolment, and at 6 months after starting intervention. Evaluations included anthropometrical measurements, nutritional, metabolic assessment and liver ultrasonography. Children were randomly assigned to the intervention or control group based on a computer generated, blocked randomization list by each center. A block size of four was used, stratified according to gender. The investigator who generated the randomization sequence was independent of the research staff and unaware of children. RESULTS: at baseline there was no significant difference between groups for any anthropometrical (minimum P= 0.806) or dietary (minimum P= 0.318) or biochemical (minimum P=0.539) variable. No significant difference among groups occurred for daily intake of energy or any macronutrient and the end of the study (minimum P=0.111). At the end of the intervention a significant reduction of plasma fasting glucose (DHA group P=0.046; placebo group P=0.048), insulin (DHA group P=0.001; placebo group P=0.048) and HOMA (DHA group P=0.001; placebo group P=0.050) in both groups was observed. A higher percentage variation of plasma fasting insulin (P=0.0046) and HOMA (P=0.0045) in DHA than placebo group was showed. There was a difference between groups for percentage reduction of liver steatosis: in DHA group from 14 to 7 % (P=0.655), in placebo group from 20 to 13 % (P=0.275).

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

Docosahexaenoic acid (DHA; 22:6 n-3), a polyunsaturated fatty acid (PUFA), is linked to various health benefits in humans including cognitive and visual development of infants and reduced risk of cancer, cardiovascular diseases and mental illnesses of adults. Fish

oil, with an annual production of about 600,000 tons is at present the major source of DHA. However, the production of fish oils is expected to become inadequate for supplying an expanding market within few years. Thraustochytrids are marine heterotrophic producers of PUFA-rich triacylglycerols which represent an alternative source of DHA.

The focus of this thesis has been split between a basic study of the osmolyte system of traustochytrids and work towards an understanding of their growth kinetics, effects of nutrient depletion, and lipid and DHA accumulation. Three new osmotolerant thraustochytrid isolates (T65, T66, and T67) and the previously known Schizochytrium sp. strain S8 (ATCC 20889) were assigned to the genus Aurantiochytrium based on 18S ribosomal DNA phylogeny, morphology and PUFA profiles (approximately 80% DHA). Strains T66 and S8 displayed a nearly linear increase in cellular content of endogenously synthesized (-)-proto-quercitol and glycine betaine with increasing osmotic strength. This represented the first demonstration of accumulation of principal compatible solutes in thraustochytrids. A less osmotolerant isolate (Thraustochytriidae sp. strain T29), which was closely phylogenetically related to Thraustochytrium aureum (ATCC 34304) did not accumulate glycine betaine or (-)-proto-quercitol, illustrating a variation in osmolyte systems and osmotolerance levels among thraustochytrids.

To study the effects of nutrient limitations, Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate and glycerol containing medium with various medium limitations. N and P starvation and O₂ limitation initiated lipid accumulation. N starvation alone or in combination with O₂ limitation yielded the highest lipid contents obtained in this study, i.e., 54 to 63% of cell dry weight with a corresponding cell density of 90 to 100 g l^-1 dry biomass. The DHA-content of N starved cells was 29% of total fatty acids, while O₂ limitation increased the DHA-content up to 52%. Simultaneously, O₂ limitation abolished accumulation of monounsaturated fatty acids. We inferred that the biological explanation is that O₂ limitation hindered activity of the O₂-dependent desaturase(s) responsible for production of monounsaturated fatty acids, and favored the O₂-independent PUFA synthase. The highest DHA-productivity observed was 93 mg l^-1 h^-l, obtained during sequential N starvation and O₂ limitation. This

productivity approaches the highest values previously reported for thraustochytrids, and indicates that T66 may become a candidate organism for a future large-scale microbial PUFA production process.

The aim of this study was to gain important insights into the effects of peroxidized docosahexaenoic acid (pDHA) on the retinal pigment epithelium (RPE), and whether its effects can cause RPE cell dysfunction in a way similar to that observed in retinal ageing and the pathogenesis of age-related macular degeneration (AMD). Initially, the time-course of in vitro peroxidation of DHA was monitored, culminating in formation of products able to absorb light above 400 nm. Cultured RPE cells were then exposed to this pDHA, which was shown to be toxic, both in dark and light-exposed conditions. Various cell viability assays were carried out indicating RPE cell death after exposure to pDHA is likely to occur by apoptosis. The effects of pDHA were reduced in the presence of various agents - a-tocopherol, glutathione, N-acetylcysteine, and phosphatidylethanolamine (PE). Measurements of singlet oxygen, transient and superoxide production were carried out to determine the photosensitizing properties of pDHA. The ability of PE to reduce the production of these species was investigated but it was shown to have no effect on their yield, although singlet oxygen lifetime was reduced. Finally, lysosomal enzyme activity, lysosomal integrity and accumulation of fluorescent and undegraded material were monitored after exposure to pDHA, demonstrating that pDHA was able to disrupt the ability of the RPE to fully degrade phagocytosed material. In summary, pDHA is able to affect RPE cell viability directly when exposed extracellularly, and can also affect essential normal functions of the RPE intracellularly. In conjunction with published findings that pDHA is present in the retina - both within and around the RPE - the data presented here support the theory that pDHA can play an important role in causing RPE dysfunction, resulting in a loss of the protective role these cells play for photoreceptors, as occurs in retinal ageing and AMD.

Plasma membranes are essential to both the structure and function of mammalian cells. The first unifying paradigm of membrane structure, the Fluid Mosaic Model, is no longer considered adequate to describe the many non-homogeneous lipid structures that have been observed in both natural and model membranes over the past approximately thirty years. The field of membrane biophysics now appreciates that the complex mixture of different lipid species found in natural membranes produces a range of dynamic, laterally segregated, non-homogeneous structures which exist on time scales ranging from microseconds to minutes. When sphingomyelin (SM), POPC and cholesterol are all present in a bilayer there is wide range of compositional ratios where the bilayer consists of a coexistence between two fluid phases designated liquid ordered (lo) and liquid disordered (ld). The lo phase is cholesterol-rich phase characterized by relatively high molecular order and slow rotational and translational motion, while the ld phase generally has low molecular order and relatively rapid rotational and translational motion. The driving force for the formation of these two phases is the ability of cholesterol to form favorable van der Waals contacts with the two saturated acyl chains on PSM and the one saturated acyl chain on POPC. The ternary system is an important model system for examining the physical properties and functional implications of co-existing lo and ld phases. However, it does not include one of the most significant compositional variables found in many important mammalian membranes. Membranes in the nervous system contain high concentrations of the highly polyunsaturated fatty acid docosahexaenoic acid (DHA), which contains 22 carbons and 6 double bonds. A wide range of experimental evidence shows that DHA-containing phospholipids are important for optimal performance of a number of membrane signaling systems and membrane protein functions. The goal of this study is to determine how addition of a DHA-containing phospholipid, PDPC alters the biologically important lo and ld co-existence region.

Docosahexaenoic acid (DHA; 22:6n-3) is imperative for prenatal development and is found primarily in the flesh of marine life. Previous research has indicated that pregnant women do not meet current DHA recommendations of 200 mg per day. Much of the research has been conducted in coastal communities with greater access to marine sources and may not reflect non-coastal communities. PURPOSE: The purpose of this study was to describe maternal DHA intake in pregnant women from Southwest Montana and to determine changes in dietary DHA intake over time. METHODS: Thirty-nine participants were asked to complete a non-consecutive 3-day diet record and a DHA focused food frequency questionnaire (FFQ) at weeks 18, 28 and 35 (± 1 week). Maternal fasted plasma and red blood cell samples were obtained via venipuncture during the same data collection periods. Fetal cord blood samples were collected at delivery. Due to time restrictions, all blood lipid parameters were undetermined. After delivery, participants completed a post-partum survey. RESULTS: Participants' overall mean (MEAN ± SD (min. - max.)) DHA intake was 248 ± 321 mg/d (8 - 1,836 mg/d). According to those who reported using DHA supplements in their FFQ interviews at weeks 18 (n = 12), 28 (n = 13) and 35 (n = 11), the mean DHA intake was 272 ± 380 mg/d (147 - 1,490 mg/d). The mean DHA intake among non-supplement users at weeks 18 (n = 22), 28 (n = 20) and 35 (n = 21) was 147 ± 139 mg/d (8 - 555 mg/d). Dietary DHA intake from weeks 18, 28 and 35 did not differ significantly (F (2, 62) = 0.220, p = 0.803). CONCLUSION: Dietary DHA intake did not differ over time. The mean dietary DHA intake for non-supplement users was below the current prenatal recommendation. Therefore, pregnant women in Southwest Montana are not meeting current DHA recommendations through dietary means alone and should consider DHA supplementation as a method to meet fetal and maternal DHA needs during pregnancy.

Traumatic brain injury (TBI) is a devastating disease causing disability and death, and currently there are no effective treatments available. Therefore, there is an utmost need to improve our understanding of the pathophysiology of TBI and to identify potential therapies that can provide neuroprotection after injury. The aims of this thesis were to develop an in vivo and in vitro model of TBI, in which to assess the potential neuroprotective effects of an omega-3 polyunsaturated fatty acid (PUFAs), docosahexaenoic acid (DHA). Method The controlled cortical impact (CCI) in vivo model of TBI was optimized and performed in mice. Both a behavioural (Morris water maze (MWM) for cognitive deficits) and histological endpoints (astrogliosis, lesion size and activated microglia) were used to assess severity and neuroprotective effects of DHA. An in vitro model of mechanical TBI was also set up and optimized. This model employed 3D astrocyte cultures obtained from GFP positive rat pups. The CCI impactor from the in vivo studies was used to damage the cultures, and at 24 hours, 5 days and 10 days the astrogliosis and cell number was measured. Results The optimization of the in vivo studies demonstrated that at impaction depth of 2.2 mm produced an injury that was significantly different to the sham injury, in MWM performance and increased astrogliosis. Interestingly, there was an increase in the amount of astrogliosis on the contralateral side of the brain. A second study performed using the 2.2 mm injury parameters was performed, where an injection of DHA was administered via the tail vein 30 min after injury. The DHA-treated group did not demonstrate any neuroprotection compared to the injury-only group. However, there was an increase in the amount of astrogliosis in the contralateral hippocampus of the DHA-treat group. In the fat-1 studies it was shown that older male mice performed worse in the MWM, that the fat-1 gene did not confer neuroprotection but did lead to increased astrogliosis. The in vitro study revealed that astrocytes in the lesioned gels demonstrated an increase in astrogliosis, there was also an increase in the number of cell in the cultures following the lesion. Conclusion In conclusion, the in vivo model of CCI replicated components of the human TBI including a behavioural deficit and pathophysiological changes. Omega-3 PUFAs failed to demonstrate functional neuroprotection in this model, but histologically, promoted an increase in reactive astrogliosis. The development of a novel in vitro model of focal injury in a 3D culture system, that elicits reactive astrogliosis, could be used to support further studies of the astrocytic responses to mechanical injury.

Ovarian cancer is the fifth most lethal cancer in women (1) and the most lethal gynecological malignancy. In 2018, there were approximately 22,240 new diagnosed cases of ovarian cancer and 14,070 deaths in the United States alone (2). The lifetime risk for developing ovarian cancer in the United States is 1.3% or approximately 1 in 78 women. The five-year survival rate for women with ovarian cancer is a grim 47.6% (2) while the average five year survival rate for all cancers is about 68%. This dismal prognosis for ovarian cancer patients indicates the critical need for improved treatment options, efficient early detection methods and effective preventative measures for ovarian cancer (1). The objective of this study was to determine if DHA causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of NF-κB regulated transcription in the ovary. DHA is a 22 carbon long-chain omega-3 polyunsaturated fatty acid that is biologically derived from Alpha-linolenic acid (ALA) found in flaxseed. COX-2 is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. Prostaglandin E2 (PGE2) is a key regulator of inflammation which has been shown to be highly associated with ovarian cancer development and progression. Our laboratory studies ovarian cancer in the laying hen because it is the only known animal model to naturally develop ovarian cancer that both pathologically and histologically matches that of the human form of the disease. Dietary flaxseed is one of the richest vegetable sources of omega-3 polyunsaturated fatty acids. Our previous studies have shown that in laying hens, a long-term flaxseed supplemented diet reduces the incidence and severity of ovarian cancer and decreases COX-2 and PGE2. It was hypothesized that DHA, derived from ALA found in flaxseed, decreases inflammation in the ovaries by suppressing the activation of COX-2 and the production of PGE2 through inhibition of the NF-κB pathway. For this study, an NF-κB reporter plasmid was transfected into HEK293 cells. The reporter plasmid (“met-luc”) produces a secreted luciferase allowing sequential analysis of media from DHA and TNF-α treated cells to assess changes in NF-κB transcriptional activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB was used as a positive control. NF-κB activation was also assessed by measuring its nuclear translocation and cytoplasmic accumulation through immunocytochemistry (ICC) and western blot analysis. In a parallel study, immortalized ovarian surface epithelial (IOSE) cells were challenged with the same treatments of DHA and TNF-α. In these cells, COX-2 mRNA was assessed through RT-qPCR and COX-2 protein expression was analyzed through ICC and western blot.Our results indicate that DHA acts in a cell specific manner to reduce inflammation associated with cancer. We have found that in HEK293 cells DHA reduces TNFα induced NF-κB reporter activity. In contrast, ALA does not affect NF-κB reporter activity. HEK293 cells treated with TNFα alone indicated a dose-dependent increasing trend in nuclear translocation of the NF-κB p65 subunit and a decreasing trend in cytoplasmic p65, suggesting potential increased pathway activation. ICC suggests DHA treatment causes increased cytoplasmic sequestration of the NF-κB p65 subunits indicating inhibition of TNFα induced NF-κB activation. Western blot data also indicates a decreasing trend in nuclear NFκB p65 when cells are pretreated with DHA and subsequently challenged with TNF. The IOSE cells, were the only cells out of the cell lines tested (BG1, HEYC2, TOV112D, SKOV3, HEK293) to express COX-2. In these IOSE cells, TNFα alone showed a dose-dependent increasing trend in COX-2 protein (analyzed through ICC and western blot) and mRNA levels (analyzed through RT-qPCR). ICC analysis revealed that DHA reduces TNF induced COX-2 protein expression. However, the western blot did not further support this observation. Only a slight non-significant reduction with DHA treatment was observed. In addition, both DHA and TNFα, while also not significant, seemed to increase mRNA levels of COX-2 compared to control. This slight decreasing trend in COX-2 protein expression and increase in mRNA, could indicate a possible post-transcriptional mechanism of regulation of COX-2 by DHA independent of NF-κB in the IOSE cells. These data suggest that DHA could act via distinct mechanisms in a cell specific manner to potentially reduce COX-2 and subsequently PGE2 levels. DHA can act at the transcriptional level by reducing the nuclear translocation of NF-κB and transcriptional activation of NF-κB target genes such as COX-2 in some cell types. DHA also has the potential to work via a post-transcriptional mechanism to inhibit COX-2 and in turn reduce PGE2 levels. Both mechanisms ultimately have the potential to decrease the inflammation associated with ovarian cancer. This study describes the anti-inflammatory action of dietary flaxseed consumption, making flaxseed supplementation a promising preventive measure for reducing the risk of ovarian carcinogenesis.

Maternal dietary intake during pregnancy needs to meet increased nutritional demands to maintain metabolism and support fetal development. Docosahexaenoic acid (DHA) is essential for fetal neuro-/visual development and in immunomodulation, accumulating rapidly within the developing brain and central nervous system. Levels available to the fetus are governed by the maternal diet. In this multicenter, parallel, randomized controlled trial, we evaluated once-daily supplementation with multiple micronutrients and DHA (MMS) on maternal biomarkers and infant anthropometric parameters during the second and third trimesters of pregnancy compared with no supplementation. Primary efficacy endpoint: change in maternal red blood cell (RBC) DHA (wt% total fatty acids) during the study. Secondary variables: other biomarkers of fatty acid and oxidative status, vitamin D, and infant anthropometric parameters at delivery. Supplementation significantly increased RBC DHA levels, the omega-3 index, and vitamin D levels. Subscapular skinfold thickness was significantly greater with MMS in infants. Safety outcomes were comparable between groups. This first randomized controlled trial of supplementation with multiple micronutrients and DHA in pregnant women indicated that MMS significantly improved maternal DHA and vitamin D status in an industrialized setting – an important finding considering the essential roles of DHA and vitamin D.

The American Heart Association recommends consuming fish (particularly oily fish) at least two times per week, which would provide ae 0.5 g/day of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) for cardiovascular disease risk reduction. Previous analyses indicate that this recommendation is not being met; however, few studies have assessed different ethnicities, subpopulations requiring additional n-3 fatty acid intake (i.e., children and pregnant and/or lactating women), or deciles of intake. Data from the National Health and Nutrition Examination Survey 2003-2008 was used to assess n-3 fatty acid intake from foods and supplements in the US population, according to age, sex, and ethnicity. A unique "EPA equivalents" factor, which accounts for potential conversion of shorter-chain n-3 fatty acids, was used to calculate total long-chain n-3 fatty acid intake. Data are reported for 24,621 individuals. More than 90% consumed less than the recommended 0.5 g/day from food sources (median = 0.11 g/day; mean = 0.17 g/day). Among the top 15% of n-3 fatty acid consumers, fish was the largest dietary contributor (71.2%). Intake was highest in men aged 20 years or more, and lowest in children and women who are or may become pregnant and/or are lactating. Among ethnicities, intake was lowest in Mexican-Americans. Only 6.2% of the total population reported n-3 fatty acid supplement use, and this did not alter median daily intake. Additional strategies are needed to increase awareness of health benefits (particularly among Mexican-Americans and women of childbearing age) and promote consumption of oily fish or alternative dietary sources to meet current recommendations.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Introduction: The ability of different polyunsaturated fatty acids (PUFAs), especially n-3 PUFAs, to prevent the development of cancer has been under intense investigation the past three decades. Numerous studies have shown that these fatty acids can kill cancer cells in vitro as well as in vivo whilst normal cells remain unaffected. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood. This study investigated the signalling pathways modulated by docosahexaenoic acid (DHA) in an adenocarcinoma cell line, in order to shed some light on these unknown mechanisms. Materials & Methods: NCM460 (normal colon epithelial) and CaCo2 (colon adenocarcinoma) cells were cultured and treated with low doses of palmitic acid (PMA), oleic acid (OA), arachidonic acid (AA), and DHA. The effects of these fatty acids on the proliferation of the cells were measured with the MTT assay. The composition of membrane phospholipids of CaCo2 cells was determined after 48h supplementation with different fatty acids by gas chromatography. Also, CaCo2 cells were treated with DHA (10 μM) only and proteins were harvested at fixed time points ranging from 2 minutes to 48 hours. The protein inhibitors wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB 203580 (p38 inhibitor) and also RNA interference (RNAi) of the p38 MAPK protein were used to investigate cross-talk between signalling pathways. ERK, p38 MAP kinase, Akt, and p53 were then analysed by Western blotting using phospho-specific and total antibodies. The cleavage of the apoptotic proteins, caspase-3 and PARP were also analysed. Results and discussion: MTT assays revealed that none of the fatty acids were toxic to normal cells. In addition, DHA was shown to be most effective to kill CaCo2 cells whilst protecting NCM460 cells and a subsequent dose response experiment revealed that lower concentrations are most suitable for this purpose. DHA was also shown to be readily incorporated into phospholipids, along with AA. This is associated with increased membrane fluidity, which could affect the localisation, and downstream effects, of various signalling proteins within the membrane. Western blot analysis revealed a rapid increase in activity in most proteins under investigation, especially ERK and Akt (Ser473). Long-term DHA supplementation suppressed the full activation of Akt. This down regulation of survival signalling could lead to cell death in CaCo2 cells. In addition, it was shown that after 48h, DHA induced the cleavage of caspase-3 and PARP, which is indicative of apoptosis. RNAi experiments suggested a possible role for p38 MAPK in the phosphorylation of p53 at Ser15, a site which is associated with DNA damage. Conclusion: DHA exerts its effects by means of cellular signal transduction pathways, particularly by suppression of the important survival-related kinase, Akt. This could have implications for future therapeutic interventions in cancer patients, as fatty acids are safe to use and do not interfere with the functionality of normal tissue.
AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van verskillende poli-onversadigde vetsure (POVSe), veral n-3 POVSe, om die ontstaan van kanker te voorkom, is intens nagevors die afgelope drie dekades. Menigte studies het aangevoer dat hierdie vetsure kankerselle in vitro asook in vivo kan doodmaak, terwyl normale selle nie daardeur beïnvloed word nie. Ongelukkig word die sellulêre and molekulêre meganismes onderliggend tot hierdie verskynsel nie goed begryp nie. Hierdie studie het verskeie seintransduksie-paaie wat deur dokosaheksaenoësuur (DHS) in ‘n adenokarsinoom sellyn gemoduleer word, ondersoek. Materiale & Metodes: NCM460 (normale kolonepiteel) en CaCo2 (kolon adenokarsinoom) selle is onderhou in ‘n selkultuur-laboratorium en behandel met lae dosisse palmitiensuur (PMS), oleïensuur (OS), aragidoonsuur (AS), en DHS. Die invloed van hierdie vetsure op die proliferasie van die selle is d.m.v. die MTT toets bepaal. The samestelling van membraan-fosfolipiede van CaCo2 selle is na 48h behandeling met die verskillende vetsure bepaal deur middel van gaschromatografie. Die CaCo2 selle is ook met DHA (10 μM) alleenlik behandel en teen vaste tydpunte wat wissel van 2 minute tot 48h, waarna proteïene geëkstraeer is. Die proteïen-inhibitore wortmannin (PI3 kinase inhibitor), PD 98059 (MEK inhibitor), en SB 203580 (p38 inhibitor) asook RNAinterferensie (RNAi) teen die p38 MAPK proteïen is ingespan om oorvleueling tussen seintransduksie–weë te ondersoek. ERK, p38 MAPK, Akt, en p53 is geanaliseer deur middel van die Western–klad metode met fosfo–spesifieke en totale antiliggame. Die kliewing van die apoptotiese proteïene caspase-3 en PARP is ook bepaal. Resultate en bespreking: MTT toetse het ontul dat geen vetsure toksies was vir die normale selle nie. Daar is ook gevind dat DHS die mees effektiewe vetsuur was om CaCo2 selle te dood, terwyl NCM460 selle beskerm word. Gevolglik het ‘n dosis-respons eksperiment getoon dat laer konsentrasies die beste geskik is vir hierdie doel. Daar is ook gevind dat DHA maklik in fosfolipiede geïnkorporeer word, tesame met AS. Dit word geassosieer met verhoogde membraan-vloeibaarheid, wat die ligging, en ook stroom-af werking, van verskeie seintransduksie proteïene in die membraan, kan beïnvloed. Westernklad analises het ‘n vinnige verhoging in die aktiwiteite van die meeste proteïene onder die soeklig, getoon, veral ERK en Akt (Ser473). Langdurige DHS behandeling het die maksimale aktiwiteit van Akt onderdruk. Hierdie afname van oorlewing-gerigte seine kan lei tot seldood in CaCo2 selle. Daar is boonop geving dat DHS die kliewing van caspase-3 en PARP geïnduseer het na 48, wat dui op apoptose. Uit die RNAi eksperiment kon daar ook ‘n moontlike rol vir p38 MAPK in die fosforilering van p53 by Ser15, wat geassosieer word met DNS-skade, getoon word. Gevolgtrekking: DHS beoefen sy effekte deur middel van seintransduksie paaie, veral deur die oorlewing-geassosieerde kinase, Akt, te onderdruk. Dit kan implikasies hê vir toekomende terapeutiese ingrypings in kankerpasiënte, aangesien vetsure veilig is om te gebruik en nie skadelik is vir normale weefsel nie.

Parkinson disease (PD) is the main neurodegenerative disease that involves motor symptoms. About 1% of population above 65 years is affected by PD. Main symptoms are bradikinesia, resting tremor, postural instability, muscle rigidity, and sometimes, cognitive problems and personality. Neuropathological features of PD are neuronal death in the substantia nigra pars compacta and formation of cytoplasmatic inclusion, named Lewy bodies, constituted by fibrillar form of α-synuclein (aS). aS is a 140 amino acid protein, whose structure and function is yet not well defined. As a consequence of specific genetic mutations or environmental factors, it undergoes aggregation and forms amyloid fibrils. It is highly expressed in neuronal pre-synaptic nerve terminals. Its sequence is characterized by an amphipathic lysine-rich amino terminus, which governs binding to lipids and interactions with membranes and contains seven imperfect repetition of the sequence KTKEGV; by a hydrophobic central region (NAC, non-amyloid component), responsible for protein aggregation and α-sheet formation and a highly acidic C-terminal, rich in Pro and acidic residues. Overexpression of aS and mutations in its gene are associated with a premature development of PD. Mechanism that make aS a toxic protein has not yet been well clarified, but it seems clear that oligomeric forms, and not the final fibrillar forms, are the main responsible for the pathogenesis of PD. The project of this thesis focuses on the characterization of oligomers of aS that form in the presence of docosahexaenoic acid (DHA), and their interaction with membrane, to understand the mechanism of toxicity. DHA is one of the most abundant fatty acids in neuronal membrane and it has been correlated to PD. It has been demonstrated that dopaminergic cell cultures exposed to PUFAs accumulate soluble cytotoxic aS oligomers (Assayag et al., 2007). Indeed, aS seems to be involved in fatty acids metabolism (Golovko et al., 2007). Moreover, it was reported that, in PD patients, DHA concentration is enhanced in those area affected by aS inclusions. In vivo studies demonstrated that a DHA enriched diet enhances formation of aS oligomers (Sharon et al., 2003). In previous studies in this laboratory, it was analyzed the aggregation process of aS in the presence of DHA using different protein to DHA molar ratios (De Franceschi et al., 2009; 2011). Oligomers obtained in these conditions were characterized from a morphological point of view (De Franceschi et al., 2011). The presence of DHA (50:1 lipid:protein molar ratio) leads to the formation of stable oligomers, off-pathway in the aggregation process of aS, that have significant toxic activity on cells, suggesting that they are potentially relevant in the pathogenesis of PD (De Franceschi et al., 2011). In the first part of this thesis a characterization of oligomers have been conducted using several biophysical methods, since these oligomers are sufficiently stable to allow the use of these techniques. In particular, transmission electron (TEM) and atomic force (AFM) microscopy were used to study oligomers morphology and dimension. The secondary structure was evaluated by circular dichroism (CD). This spectroscopic analysis reveals that oligomers have a partial α-helix structure, in contrast with the majority of oligomeric species described in literature. It was analyzed also the ability of oligomers to interact with membrane, using liposomes of different size and composition and cell cultures. Interaction of oligomers with membrane, analyzed by CD measurements and leakage assays, causes the leakage of small molecule, demonstrating their ability to destabilize membranes. Oligomers activity was tested also on dopaminergic cell culture that showed an altered permeabilization after treatment. To determine the mechanism by which oligomers cause membrane permeabilization, different tests were performed. Initially, dynamic light scattering (DLS) and TEM allow to exclude a detergent-like effect. Moreover, aggregation studies and planar lipid membrane (PLM) measurements lead to hypothesize a toxicity mechanism that depends on the formation of a transient aperture or on the enhancement of flip-flop. This part of the thesis is object of a publication (Fecchio et al., 2013). Another aspect faced in this thesis is the study of chemical modification occurring on oligomers after exposition to DHA. Different chemical modification were evidenced by mass spectrometry: carbonylation and the formation of adduct with DHA at the level of His50. To deepen the role of this residue in the interaction with FA, it was used an aS variant, H50Q, that has recently been linked to familiar form of PD. Finally, also the interaction with DHA of other pathological variants of aS (A30P, E46K, A53T) was studied. In particular their secondary structure and oligomerization in the presence of DHA were analyzed, in comparison with results obtained with aS. In conclusion, this study supplied further information about structure and activity of oligomeric species that are potentially relevant in PD pathogenesis. These data can be compared to oligomers produced in different conditions or formed by different amyloidogenic proteins: this knowledge would be fundamental to the development of therapeutic agent that would prevent or defeat these kind of debilitative diseases
Il morbo di Parkinson (PD) è la principale malattia neurodegenerativa riguardante la funzionalità motoria. L'1% della popolazione sopra i 65 anni è affetto da questa malattia. I sintomi principali sono bradichinesia, tremore a riposo, instabilità posturale, rigidità muscolare e, talvolta, problemi cognitivi e della personalità . Le principali caratteristiche neuropatologiche del PD sono la morte dei neuroni dopaminergici a livello della substantia nigra pars compacta e la formazione di corpi d'inclusione citoplasmatici composti da aggregati proteici fibrillari di tipo amiloide, Lewy bodies (LBs), il cui costituente principale è α-sinucleina (aS) (Spillantini et al., 1998). aS è proteina di 140 amminoacidi, natively unfolded, la cui funzione, nonostante il suo ruolo chiave nel PD, non è ancora completamente chiarita. E' espressa in livelli alti nel sistema nervoso centrale ed è abbondante nei terminali presinaptici neuronali. Strutturalmente è caratterizzata dalla presenza di sette ripetizioni imperfette di sequenza aminoacidica (KTKEGV) nella regione N-terminale, da una regione idrofobica centrale (NAC, non-amyloid component) e da una coda C-terminale con numerosi residui acidi. La sovraespressione di aS e mutazioni nel suo gene sono associati a forme precoci della sindrome di Parkinson. Il meccanismo con cui un cambiamento nella struttura e nell'espressione della proteina possa portare allo sviluppo della malattia non è ancora stato chiarito, ma è sempre pi๠accreditata l'ipotesi che siano le forme oligomeriche e non gli aggregati finali fibrillari ad essere responsabili della malattia. Il progetto di questa Tesi riguarda la caratterizzazione di oligomeri tossici di α-sinucleina (aS) ottenuti in presenza di acido docosaesaenoico (DHA) e lo studio della loro interazione con membrane lipidiche, allo scopo di comprendere il meccanismo con cui esercitano la loro tossicità . Il DHA è uno dei principali acido grassi cerebrali, strettamente correlato al PD e ad aS. L'esposizione di colture cellulari dopaminergiche a PUFAs porta all'accumulo di oligomeri solubili di aS, responsabili della citotossicità associata alla neurodegenerazione (Assayag et al., 2007). Esistono poi evidenze dell'implicazione di aS nella regolazione del metabolismo degli acidi grassi (Golovko et al., 2007). Inoltre è stato osservato che, nei pazienti affetti da PD, la presenza di DHA è maggiore nelle aree cerebrali contenenti inclusioni di aS. Studi in vivo, infine, hanno dimostrato che il DHA induce la formazione di oligomeri di aS (Sharon et al., 2003). In precedenti studi condotti nel laboratorio dove è stata svolta questa Tesi è stato analizzato il processo di aggregazione di aS in presenza di DHA, utilizzando due diversi rapporti molari proteina/acido grasso) (De Franceschi et al., 2009) e gli aggregati proteici ottenuti in queste condizioni sono stati caratterizzati da un punto di vista morfologico e strutturale (De Franceschi et al., 2011). E' stato osservato che la presenza di DHA in rapporto molare 50:1 rispetto alla proteina, porta alla formazione di oligomeri stabili, off-pathway nel processo di fibrillazione, che presentano una significativa attività tossica sulle cellule rispetto al monomero di aS. Nella prima parte di questa ricerca è stata condotta una caratterizzazione di queste specie oligomeriche che sono sufficientemente stabili nel tempo da consentire l'uso di diverse tecniche biofisiche. In particolare gli oligomeri sono stati analizzati mediante microscopia elettronica a trasmissione (TEM) e a forza atomica (AFM), per studiare le dimensioni e la morfologia. Il tipo di struttura secondaria è stata valutata mediante dicroismo circolare che ha dimostrato un'altra peculiarità di questi oligomeri, ovvero la presenza di struttura parzialmente in α-elica, diversamente dalla maggior parte degli oligomeri descritti in letteratura. E' stata analizzata anche la capacità degli oligomeri di interagire con le membrane, utilizzando liposomi di diversa dimensione e diversa composizione e colture cellulari. L'interazione tra gli oligomeri e i liposomi, studiata mediante CD e saggi di leakage, causa il rilascio di piccole molecole interne alle vescicole, dimostrando così un loro effetto destabilizzante sulle membrane. L'attività degli oligomeri è stata anche testata su cellule in coltura che mostrano un'alterata permeabilità in loro presenza. Per determinare quale sia il meccanismo di destabilizzazione degli oligomeri, sono stati eseguiti vari saggi. Si è dimostrato tramite dynamic light scattering (DLS) e TEM che le vescicole, in seguito al legame con gli oligomeri, non vengono distrutte. Inoltre mediante studi di aggregazione e analisi su planar lipid membrane portano a ipotizzare un meccanismo di tossicità dovuto alla formazione di un'apertura transiente o un aumento di flip-flop a livello delle membrane. I risultati di questa parte di tesi sono oggetto di una pubblicazione (Fecchio et al., 2013). Un altro aspetto che è stato approfondito in questo lavoro di Tesi è lo studio degli oligomeri da un punto di vista chimico, allo scopo di caratterizzare le modifiche chimiche presenti sulla sequenza della proteina in seguito all'esposizione al DHA. Sono state evidenziate diverse modifiche mediante spettrometria di massa tra cui carbonilazioni e presenza di addotti. Quest'ultimo tipo di modifica in particolare avviene a livello dell'istidina in posizione 50. Per approfondire il ruolo di questo aminoacido nell'interazione con gli acidi grassi è stato studiato il mutante H50Q di aS. Questa proteina modificata è tra l'altro responsabile di forme familiari del PD. Infine è stata studiata anche l'interazione di altre varianti patologiche di aS associate a PD, A30P, E46K e A53T con DHA. In particolare, è stata analizzata la loro struttura e la loro tendenza ad aggregare in presenza di DHA, nonchè la loro capacità di formare oligomeri, in confronto ai risultati ottenuti con aS. In conclusione questo studio ha permesso di fornire maggiori informazioni sulla struttura e di studiare l'attività di specie oligomeriche che sono potenzialmente molto rilevanti per la patogenesi del PD. La struttura e l'attività di questi oligomeri potrà essere confrontata con quelle di oligomeri prodotti in diverse condizioni sperimentali o di oligomeri prodotti da altre proteine amiloidogeniche. Questa conoscenza è fondamentale per sviluppare agenti terapeutici che prevengano o debellino queste malattie debilitanti ed in continuo aumento

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Chronic wounds often result from prolonged inflammation involving excessive polymorphonuclear leukocyte activity. Studies show that the omega-3 polyunsaturated fatty acids eicosapentaenoic and docosahexaenoic acids found in fish oils generate bioactive lipid mediators that reduce inflammation and polymorphonuclear leukocyte recruitment in numerous inflammatory disease models. The purpose of this study was to test the hypotheses that boosting plasma levels of eicosapentaenoic and docosahexaenoic acids with oral supplementation would alter lipid mediator levels in acute wound microenvironments and reduce polymorphonuclear leukocyte levels. Eighteen individuals were randomized to 28 days of either eicosapentaenoic + docosahexaenoic acid supplementation (Active Group) or placebo. After 28 days the Active Group had significantly higher plasma levels of eicosapentaenoic (p<0.001) and docosahexaenoic acid (p<0.001) than the Placebo Group and significantly lower wound fluid levels of two 15-lipoxygenase products of omega-6 polyunsaturated fatty acids, [9- hydroxyoctadecadienoic (HODE) acid (p = 0.033) and15-hydroxyeicosatrienoic acid (HETrE) (p = 0.006)], at 24 hours post wounding. The Active Group also had lower mean levels of myeloperoxidase, a leukocyte marker, at 12 hours and significantly more re-epithelialization on Day 5 post wounding. We suggest that lipid mediator profiles can be manipulated by altering polyunsaturated fatty acid intake to create a wound microenvironment more conducive to healing.

Crude glycerol is the major byproduct of the biodiesel industry. In general, for every 100 pounds of biodiesel produced, approximately 10 pounds of crude glycerol are created. Because this glycerol is expensive to purify for use in food, pharmaceutical, or cosmetics industries, biodiesel producers must seek alternative methods for its disposal. Using crude glycerol as a carbon source for fermentation is a promising alternative use for this waste material. In this project, we propose to use crude glycerol in the fermentation of the microalga Schizochytrium limacinum, which is a prolific producer of docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid with proven beneficial effects on treating human diseases such as cardiovascular diseases, cancers, and Alzheimerâ s. The first part of this study provided â proof-of-conceptâ that Schizochytrium limacinum was capable of utilizing crude glycerol while producing DHA. The results show that pretreated crude glycerol was a viable carbon source for algal growth and DHA production, with 75 g/L to 100 g/L of glycerol being the optimum concentration range. In addition to glycerol concentration, temperature, trace metal levels, ammonium acetate concentration, and NH4Cl concentration also had significant effects (P < 0.1) on algal DHA production. Optimizing these factors using response surface methodology led to a DHA yield of 4.91 g/L and a DHA productivity of 0.82 g/L-day. The second part of this study investigated the effects of impurities within the glycerol on DHA production and algal biomass composition, with a goal of ensuring that the algal biomass produced from crude glycerol can be safely utilized as an ingredient in omega-3 fortified foods or animal feed additives. Crude glycerol samples obtained from different biodiesel producers were tested. All the samples contained methanol, soaps, and various elements including calcium, phosphorus, potassium, silicon, sodium, and zinc. Both methanol and soaps in the glycerol negatively influence algal DHA production. The two impurities can be removed from the culture medium by evaporation through autoclaving (for methanol) or by precipitation through pH adjustment (for soap). The results showed that the crude glycerol-derived algal biomass contained 45-50% lipid, 14-20% protein, and 25% carbohydrate, with 8-13% ash content. Palmitic acid (C16:0) and DHA (C22:6) were the two major fatty acids in the algal lipid. ICP analysis showed that that boron, calcium, copper, iron, magnesium, phosphorus, potassium, silicon, sodium, and sulfur were present in the algal biomass, but no heavy metals (such as mercury) were detected. The crude glycerol-derived algal biomass had a high level of DHA and a nutritional profile similar to glucose-derived commercial algal biomass, suggesting a great potential for using crude-glycerol derived algae in omega-3 fortified food or feed. Overall, this study shows that crude glycerol can be used as a safe and effective substrate for algal culture to produce high levels of omega-3 fatty acids. With the rapid expansion of the biodiesel industry, there is an urgent need to develop new markets for large quantities of crude glycerol. This research provides an exciting opportunity to utilize a large quantity of this low grade glycerol.
Master of Science

Crude glycerol is a major byproduct if the biodiesel industry. Biodiesel manufacturers are currently facing the challenges of appropriate disposal of this waste material. Crude glycerol is expensive to purify for use in food, cosmetic, and pharmaceutical industries and therefore, alternative methods for use of this crude glycerol are needed. A promising alternative is to use this crude glycerol as a carbon source for microalgae fermentation. In this project, we investigated the use of crude glycerol as a less expensive substrate for the fermentation of the microalgae Schizochytrium limacinum and Pythium irregulare which are prolific producers of omega-3 polyunsaturated fatty acids. Omega-3 fatty acids have many beneficially effects on treating human diseases such as cardiovascular diseases, cancers, and neurological disorders. In addition, the omega-3 fatty acids docosahexaenoic acid (DHA) has been shown to be an important factor in infant brain and eye development. The first part of this study focused on the continuous fermentation of S. limacinum, a prolific producer of DHA. The objective of this study was to examine the algal cellular physiology and maximize its DHA productivity. Two important parameters used in continuous fermentation were studied: dilution rate (D) and feed glycerol concentration (S₀). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day^-1 and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day^-1 and S₀ = 90 g/L. The cells had a true growth yield of 0.283 g/g, a maximum specific growth rate of 0.692 day^-1, and a maintenance coefficient of 0.2216 day^-1. The second part of this study focused on morphology issues with P. irregulare, a prolific producer of eicosapentaenoic acid (EPA). P. irregulare has a filamentous morphology, which can make fermentation difficult. The mycelium can stick to the agitation blades resulting in mechanical problems. In addition, this filamentous morphology prevents adequate amounts of oxygen from reaching some cells resulting in decreased productivities. The focus of this research was to control the fermentation conditions to make the algae grow in small pellets, a morphology more suitable for fermentation. In flask culture studies, pellets were formed at an agitation speed of 110 rpm in both regular and baffled flasks. Baffled flasks resulted in pellet formation at 90 and 130 rpm as well. Fermentation studies resulted in pellet formation at agitation speeds of 150 and 300 rpm. Pellets were better able to form when a baffle was not in place. In addition, agitation speed influenced pellet size, with smaller pellets forming at the higher agitation speed. Overall, this study showed that crude glycerol can be used as a carbon source for the continuous fermentation of S. limacinum with high DHA productivity and the morphology of P. irregulare could be controlled by manipulating culture conditions, mainly agitation speed. These results show the potential for scale-up studies for both algal species.
Master of Science

L'acide docosahexaénoïque (DHA, 22:6n-3) est un acide gras polyinsaturé (AGPI) oméga-3. Il est particulièrement abondant dans le cerveau et la rétine et est nécessaire pour le bon développement et fonctionnement du cerveau. Tandis qu'une déficience en DHA a été montrée être liée à l'émergence de maladies cérébrales (i.e. maladie d'Alzheimer ou maladie de Parkinson), des études ont également montré qu'un apport alimentaire en AGPI oméga-3 pouvait empêcher ou atténuer les perturbations neurologiques liées au vieillissement ou aux maladies neurodégénératives. Il est alors primordial de transporter efficacement le DHA au cerveau. Le laboratoire français a synthétisé auparavant une forme stabilisée de la lysophosphatidylcholine-DHA, qui est le vecteur principal d'apport de DHA au cerveau, de structure 1-acétyl,2-docosahexaénoyl-glycérophosphocholine, brevetée et nommée AceDoPC®. L'injection d'AceDoPC ou de DHA après un accident vasculaire cérébral ischémique provoqué expérimentalement a montré que ces deux molécules étaient neuroprotectrices. Ces effets sont supposés être dus en partie à la conversion du DHA en métabolites oxygénés. Notre étude vise à examiner les effets du DHA et de ses métabolites dérivés, estérifiés ou non dans des phospholipides structurés sur un modèle de neurogenèse in vitro en conditions physiologiques ou pathologiques. Le premier objectif de ce travail a été de synthétiser le phospholipide structuré contenant du DHA, l’AceDoPC®, la protectine DX (métabolite oxygéné du DHA), et un nouveau phospholipide structuré contenant la protectine: 1-acétyl,2-protectine DX-glycérophosphocholine (AceDoxyPC). Le second objectif était d’étudier les effets du DHA, de l'AceDoPC et de la PDX sur la neurogenèse en utilisant un modèle in vitro de neurogenèse, constitué de cultures de cellules souches progénitrices neurales (NSPCs) dérivées de cerveaux de souris adultes, dans des conditions physiologiques ou pathologiques (ischémiques ici). Enfin, le troisième objectif de cette thèse a été d'identifier les mécanismes impliqués dans la réponse des cellules aux conditions ischémiques. La synthèse du phospholipide structuré AceDoxyPC a été réalisée avec succès par une double lipoxygénation enzymatique de l'AceDoPC, et l'identification du produit a été possible grâce à l'utilisation de techniques avancées de chromatographie liquide couplée à la spectrométrie de masse (LC/ESI/MS). De futures études sur ce transporteur de molécule neuroprotectrice potentielle doivent être réalisées prochainement. Les cellules incubées en présence d’AceDoPC présentent une augmentation de neurogenèse comparativement à celles cultivées avec addition de DHA non estérifié ou du véhicule contrôle, notamment sous conditions pathologiques. Les études préliminaires des mécanismes potentiellement impliqués dans la neuroprotection indiquent que les effets neuroprotecteurs et régénératifs de l'AceDoPC pourraient être en partie dus à des effets anti-oxidants
Docosahexaenoic acid (DHA, 22:6n-3) is an essential omega-3 polyunsaturated fatty acid (PUFA). It is specifically enriched in the brain and the retina and it is required for visual acuity, proper brain development and cerebral functions. While DHA deficiency in the brain was shown to be linked to the emergence of cerebral diseases (i.e. Alzheimer’s disease or Parkinson’s disease), studies showed that a dietary intake of omega-3 PUFA could prevent or attenuate neurologic disturbances linked with ageing or neurodegenerative diseases. In this context, it is primary to deliver DHA efficiently to the brain. Targeting the brain with DHA might offer great promise in developing new therapeutics for neurodegenerative diseases. The French host laboratory previously synthesized a stabilized form of lysophosphatidylcholine-DHA, which is main vector of DHA transportation to the brain, of structure 1-acetyl,2-docoshexaenoyl-glycerophosphocholine, patented and named AceDoPC®. Injection of AceDoPC or DHA after experimental ischemic stroke showed that both molecules also had neuroprotective effects. These potential neuroprotective effects are expected to be due, in part, to DHA conversion into oxygenated metabolites. This study aims to investigate the beneficial effects of DHA and its derived metabolites either unesterified or esterified within structured phospholipids on a model of neurogenesis in vitro under physiological or pathological conditions. The first objective of this work was then to synthesize the DHA-containing structured phospholipid AceDoPC®, DHA oxygenated derivative protectin DX (PDX) and a novel structured phospholipid of protectin: 1-acetyl,2-protectinDX-glycerophosphocholine (AceDoxyPC). The second objective was to investigate the effects of DHA, AceDoPC and PDX on neurogenesis using an in vitro model of neurogenesis, namely cultures of neural stem progenitor cells (NSPCs) derived from the adult mouse brain under physiological or pathological conditions (ischemic conditions). Following this, the third objective of this work was to identify the mechanisms involved in such response to stress induced under pathological conditions. Synthesis of the novel structured phospholipid AceDoxyPC was successfully performed by double enzymatic lipoxygenation of AceDoPC and identification of the product was possible using advanced techniques of liquid chromatography (LC)/electrospray ionization (/ESI)/mass spectrometry (/MS). Future studies on this potential neuroprotective molecule transporter are to be investigated in the near future. Neurogenesis study of cell cultures with AceDoPC showed enhanced neurogenesis compared to addition of unesterified DHA or vehicle control, especially under pathological conditions. Preliminary studies of the potential mechanisms involved in neuroprotection hinted that AceDoPC neuroprotective and regenerative effects might be due in part to its anti-oxidative effects

Colon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously.

Correct localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 µM DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras, N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
This work aimed to develop refining processes, chemical alcoholysis followed by separation of fatty acids using the complexation with urea technique for the synthesis of poly-unsaturated esters from waste of fish oils. The special crude fish oil was purchased from Company Campestre - SÃo Paulo. Initially this oil has undergone a process of physical and chemical refining. From the refined oil, an alcoholysis process was carried out to obtain the mixture of free fatty acids. From the hydrolyzed material were obtained 32.78% p/p of PUFAs against 19.73% p/p of ω-3 concentrates. The free fatty acids were separated using the complexation with urea technique. The best operating conditions for separation of the fatty acids was: ratio 7:1 (urea / oil) and the crystallization temperature at -23ÂC for a time of 20 hours. After treatment of the material, the total PUFAs production was 47.87%, a ω-3 concentration of 27.59% with a saturated fraction of 4.48%. When the temperature was raised to -10ÂC, the PUFAs production was halved, reaching the value of 28.08% and 25.49% of ω-3 which was slightly altered and a saturated fraction of 42.44%. For the ester synthesis was mounted a statistical factor of two levels in order to determine the parameters which optimized the process. In the synthesis phase, the combination of temperature, glycerin concentration and catalyst was significant, and it was observed a greater influence of the glycerin concentration due to the excessive use of glycerin to favor the formation of the ester. After the analysis of the kinetic results was observed that the interactions temperature-glycerin and temperature-glycerin-catalyst were not significant (below 95%). The response interaction graphic showed the least free fatty acids index after one hour of reaction, and that the greater interaction was glycerin (5%)-catalyst (3%). It was concluded that the yields to obtain the polyunsaturated ω-3 and ω -6 from the waste of fish oil were satisfactory (85,3%). Therefore, it is concluded that it is feasible the synthesis of polyunsaturated esters of marine oils from fish waste, because this technology provides important results to avoid environmental impacts, reduce imports of fish oils and, consequently, reduce improper fishing. The aquaculture industry may be stocked with diets enriched with EPA and DHA for shrimp and fish farming, besides contributing to supply ω-3 for nutraceutical purposes.
Este trabalho teve o objetivo de desenvolver os processos de refino, alcoÃlise quÃmica seguida da separaÃÃo dos Ãcidos graxos utilizando a tÃcnica da complexaÃÃo com urÃia para a sÃntese de Ãsteres poli-insaturados a partir de resÃduos de Ãleos de pescado. O Ãleo bruto especial de peixe foi adquirido da Empresa Campestre â SÃo Paulo. Inicialmente este Ãleo sofreu um processo de refino fÃsico e quÃmico. A partir do Ãleo refinado, foi realizado um processo de alcoÃlise para se obter a mistura de Ãcidos graxos livres. Do material hidrolisado, foram obtidos 32,78% p/p de PUFAs contra 19,73% p/p de concentrados de ω-3. Os Ãcidos graxos livres foram separados utilizando-se a tÃcnica da complexaÃÃo com urÃia. As melhores condiÃÃes operacionais para separaÃÃo dos Ãcidos graxos foram: a relaÃÃo 7:1 (urÃia/Ãleo) e a temperatura de cristalizaÃÃo a -23ÂC por um tempo de 20 horas. ApÃs o tratamento do material, a produÃÃo total de PUFAs foi de 47,87%, uma concentraÃÃo de ω-3 de 27,59% com uma fraÃÃo saturada de 4,48%. Quando se elevou a temperatura para -10ÂC, a produÃÃo de PUFAs reduziu pela metade, atingindo o valor de 28,08% e 25,49% de ω-3, que pouco foi alterada e uma fraÃÃo de saturados de 42,44%. Para a sÃntese do Ãster de glicerina foi montado um fatorial estatÃstico de dois nÃveis a fim de se determinar os parÃmetros que otimizaram o processo. Na fase de sÃntese, a conjugaÃÃo de temperatura, concentraÃÃo de glicerina e catalisador foram significantes, tendo sido observado uma maior influÃncia da concentraÃÃo de glicerina, em virtude do uso excessivo de glicerina para favorecer a formaÃÃo do Ãster. ApÃs as anÃlises dos resultados cinÃticos, foi observado que as interaÃÃes temperatura-glicerina e temperatura-glicerina-catalisador nÃo foram significantes (abaixo de 95%). O grÃfico da interaÃÃo para resposta mostrou o menor Ãndice de Ãcidos graxos livres apÃs uma hora de reaÃÃo, e que a maior interaÃÃo foi glicerina (5%)-catalisador (3%). Foi concluÃdo que os rendimentos para obtenÃÃo dos poli-insaturados ω-3 e ω -6 dos resÃduos de Ãleo de pescado foram satisfatÃrios (85,3%). Conclui-se, portanto, que à viÃvel a sÃntese de Ãsteres poli-insaturados de Ãleos marinhos a partir de rejeitos de pescados, pois esta tecnologia proporciona resultados importantes para evitar impactos ambientais, diminuir as importaÃÃes de Ãleos de peixe e, consequentemente, reduzir a pesca indevida. O setor aquÃcola poderà ser abastecido com raÃÃes enriquecidas com EPA e DHA para camarÃes e peixes de cultivo, alÃm de contribuir para oferta de ω-3 para fins nutracÃuticos.

Doctor of Philosophy
Department of Human Nutrition
Mark D. Haub
Mark D. Haub
Psychological problems and human error are leading causes of death and disability among military service members. Strategies to improve the psychological health and cognitive performance of those in the military are much needed. Recent advances in neuroscience suggest that omega-3 fatty acids may play an important role in the psychological well-being of those in the military. The purpose of this research was to explore the relationship between omega-3 status and psychological outcome variables among soldiers deploying to combat. Data collection was preceded by the development and reliability testing of a novel food frequency questionnaire (FFQ) designed to capture intake from contemporary sources of omega-3 fatty acids including functional foods and supplements. Based on the instrument assessment study (Chapter 2) conducted among university students (n = 165), this FFQ appears to be a comprehensive and reliable (n = 54, ρ = 0.86, p < 0.001) instrument for measuring docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intakes in young adults. As described in Chapter 3, intake of EPA + DHA as estimated by the FFQ was positively correlated (r = 0.39, p < 0.001) with biomarker measurements of omega-3 status. Primary data were obtained from a volunteer sample of soldiers (n = 272) scheduled for deployment to Iraq. Preliminarily analyses revealed relationships between attention deficit hyperactivity disorder (ADHD) screening scores and psychological outcome variables (Chapter 4). Primary analyses (Chapter 5) indicated intake of EPA + DHA was not significantly correlated with mood, nor were omega-3 exposure variables correlated with cognitive performance based on the required p value (< 0.001) calculated using the Bonferroni correction for multiple tests. Among participants with EPA + DHA intakes at or below the median, omega-3 HUFA was related (p < 0.002) to happiness (β = -0.46), depression (β = 0.44), and fatigue (β = 0.43). Although exploratory in nature, the results of this study suggest a relationship between omega-3 fatty acids and mood. Given the current concerns regarding the psychological health of those in the military, additional research is warranted.

Alzheimer's disease (AD) is the most common form of dementia in the elderly. Despite over 100 years of research, there is no cure for the disease. Thus, ways of preventing its onset and/or slowing its progression are of particular interest. Evidence from epidemiological and animal studies has suggested that dietary docosahexaenoic acid (DHA) may reduce the incidence of AD and, more specifically, attenuate P-amyloid (Ap) pathology and improve cognitive symptoms associated with the disease. However, the efficacy of such an intervention remains controversial. Some clinical trials and animal studies have shown limited or no effect of DHA supplementation on behaviour or pathology. Therefore, further research is required to test the hypothesis that dietary DHA supplementation improves cognition and alleviates Ap pathology. The strategy adopted in this thesis was to evaluate dietary DHA supplementation on cognition and pathology in a mouse model of p-amyloid pathology. Tg2576 transgenic mice (Tg), which overexpress the human APPswe mutation, and wild type littermates were fed a diet containing approximately 1.8% DHA or a control diet from the age of 4 months. The mice were tested at different times (8, 12 and 16 months of age) using two different spatial memory tasks. Lipid analyses were carried out on plasma and specific brain regions and the distribution of Ap was analysed using immunohistochemistry and enzyme-linked immunosorbent assay. The results showed that the levels of DHA were increased in plasma and in cortex, hippocampus and cerebellum of DHA-fed mice. In addition, the brain lipid analysis showed that phosphatidylethanolamine (PE), a major phospholipid in brain, was one of the main DHA-containing phospholipids and was the phospholipid that was most clearly affected by dietary DHA and Ap pathology. However, long-term DHA supplementation had only a mild positive effect on learning and memory in the Tg mice. There was no statistically significant effect of DHA supplementation on the accumulation of soluble and insoluble Apl-40 and Apl-42 in the cortex and the hippocampus of Tg mice. These findings suggest that DHA may improve cognitive functions in Tg2576 mice, perhaps by reducing the inflammatory and oxidative effects caused by Ap, rather than reducing the accumulation of the Ap peptide per se and that PE may have a key role in this process.

碩士
輔仁大學
營養科學系
96
Docosahexaenoic acid (DHA) is a long-chain polyunsaturated fatty acid, essential for the growth and development of brain in infants. Main dietary source of DHA is fish oil. The purpose of this study is to compare plasma and brain tissues’ DHA levels between pups by breast-fed and tube-fed. The first part of this study was to feed pregnant rats with different levels of DHA in the diets, including control (without DHA added), DHA-adequate ( 0.01 g DHA/kg B.W. added) and DHA-high group ( 0.1 g DHA/kg B.W. added) diets. Rat’s milk was collected at the 14th day of lactation, followed by determination of DHA concentration. Pups were sacrificed at the 21th day of lactation. Results showed that DHA-high group had higher DHA content in milk (254 ± 2.5 mg DHA/ 100 ml rat milk) than rat in the control group (82 ± 0.1 mg DHA/ 100 ml rat milk, p＜0.05). However, compared with the control group, higher DHA concentration was discovered in plasma, liver, cerebellum and medulla oblongata of pups in the DHA-high group. Higher recovery rate of dietary DHA to organs or tissues were found in rat control group than that in other two groups. The second part of this study was to feed neonatal pups with different levels of DHA in the artificial rat milk by gastrostomy. DHA contents in the formula (control, adequate and high group) were designed to be 0, 5 and 300 mg DHA/ 100 ml milk, respectively. After 14 days of feeding, pups were sacrificed. Significantly higher DHA concentration was found in plasma, liver and brain tissues of the pups in the DHA-high group than pups in control group. The recovery rate of pups by gastrostomy, and there dietary DHA to plasma, liver or brain tissues in DHA-adequate pups were higher than that in pups of DHA-high group. Except liver and thalamus and hypothalamus, DHA contents in the rest of the tissues of breast-fed group were higher than that of tube-fed group. Compared of DHA concentration between DHA adequate (92.70 ± 54.35μg/ml) and control (64.57 ± 36.81μg/ml) group by tube-fed, the results suggested that the amount of DHA added in the commercial infant formula, i.e. up to 5 mg DHA/ 100 ml milk, might not be high enough to influence level of body DHA in the neonatal pups. The DHA added in the tube-fed formula over 300 mg DHA/100 ml might have the similar effect on elevation of DHA levels in plasma, liver and brain tissues as breast milk (254 mg DHA/100 ml rat milk).

Introduction: One potential new therapy for epilepsy involves the omega-3 polyunsaturated fatty acids (PUFAs), and more specifically docosahexaenoic acid (DHA).   Methods: The anticonvulsant properties of the n-3 PUFAs were assessed in a series of different experiments. Subjects received chronic dietary supplementation, sub-chronic and acute injections of either fish oil (chronic) or DHA (sub-chronic, acute). Animals were tested in the electrical afterdischarge thresholds (ADTs) model in the amygdale and the maximal pentylenetetrazol (PTZ) model.   Results: Chronic, sub-chronic, and acute administrations of n-3 PUFAs were anticonvulsant in both the electrical stimulation and maximal PTZ models. In chronic experiments, amygala ADTs increased following 3 months of fish oil administration. Fourteen days of DHA i.p. injections increased latencies to maximal PTZ seizures. Acute injection of DHA s.c. and i.v. increased unesterified serum DHA and seizure latency. Conclusions: The present research suggests that n-3 PUFAs, and more specifically DHA, have anticonvulsant effects in vivo.

碩士
大葉大學
生物產業科技學系碩士在職專班
96
In the last few years , everyone started paying attention to personal health, and disease prevention due to the improvement of the society. Docosahexaenoic acid (DHA) has physiological effects in heart and circulatory system, inflammatory and dermatitis disease. The study was investigated on batch production of DHA by Thraustochytrium sp. using 20 L fermentor. During the batch cultivation, temperature and pH were controlled at 25 ℃ and 6.5. Agitation and airflow were adjusted to keep the dissolved oxygen above 30 ％ of Saturation. Samples were taken for measurements of biomass, lipid and DHA contents. The medium contained per liter:20.0 g glucose, 1.0 g ammonium sulfate. The results of experiment show that after 30 hrs, the nitrogenous source was exhausted, but there was still 11.54 g/L of carbonic source in the broth. However, the biomass and lipid were keeping raising. After 33 hrs, the biomass and lipid increased greatly. Until glucose was exhausted, biomass and lipid was decreasing. Under no carbonic source, microbe would consume the lipid which made by itself so that lipid was just decreasing. At 37 hrs, biomass was 3.48 g/L and lipid was 1.58 g/L. It was close to the stationary phase of microbial growth and lipid. At this time, docosahexaenoic acid was 251.07 mg/L. The lipid was 48 ﹪of biomass and the doc- osahexaenoic acid was 7.61 ﹪of biomass, respectively. Key Words：Thraustochytrium sp. docosahexaenoic acid (DHA)