Orientador: Messias Borges Silva / Resumo: O cultivo de microalgas tem sido considerado como uma fonte promissora de lipídeos para a obtenção de ácidos graxos de alto valor agregado, como é o caso dos ômegas 3 (ω3) e ômegas 6 (ω6). O uso do planejamento de experimentos (DOE) permite estabelecer condições apropriadas de cultivo para as microalgas que favorece o acúmulo desses ácidos graxos. Desta forma, o presente trabalho teve como objetivo melhorar o desempenho dos cultivos das microalgas Nannochloropsis gaditana e Dunaliella salina, visando a produção de ω3 e ω6. Primeiramente, foram avaliadas, com o uso de uma matriz ortogonal Taguchi L4, as influências das concentrações de nitrato de sódio (de 25 a 75 mg L-1), de acetato de sódio e bicarbonato de sódio (ambas de 0 a 2 g L-1), sobre a produtividade de biomassa (Pb) e de lipídeos (Po). Como resultado, foi notado que tanto o acetato quanto o nitrato, influenciaram positivamente na Pb e na Po, para ambas as microalgas estudadas. O bicarbonato, por outro lado, não melhorou a Po, sendo excluído das etapas posteriores. Na etapa seguinte do trabalho foi realizado um planejamento fatorial completo 22 com ponto central, focando no estudo da concentração de nitrato (75 a 225 mg L-1) e acetato (2 a 6 gL-1) sobre a produtividade dos cultivos. As maiores Pb obtidas foram: 188,93 mg L-1 dia-1 para a microalga N. gaditana (225 mg L-1 de nitrato e 6 g L-1 de acetato) e 118,93 mg L-1 dia-1 para a D. salina (150 mg L-1 de nitrato e 4 g L-1 de acetato). Nestas condições de cultivo, a... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

L’obésité est un état physiopathologique d’origine multifactorielle caractérisé par une accumulation excessive de tissu adipeux (TA). Elle est associée à une augmentation du risque de développer une insulino-résistance (IR), un syndrome métabolique et, à terme, un diabète de type 2. L’altération des fonctions du TA au cours de l’obésité joue un rôle central dans l’apparition des troubles métaboliques, tels qu’une accumulation ectopique de graisse et une IR périphérique, notamment dans le muscle. Dans ce contexte, la qualité des apports énergétiques et plus précisément en lipides pourrait jouer un rôle important dans l’adaptation des tissus au cours de l’obésité. Ainsi le palmitate (PAL), un acide gras saturé (AGS) est pro-lipogénique, pro-inflammatoire et lipotoxique, ce qui favorise l’apparition d’une IR. Les acides gras polyinsaturés oméga-3 (3) auraient des effets antagonistes au PAL et donc potentiellement protecteurs vis-à-vis des perturbations métaboliques associées à l’obésité. Parmi les 3, les effets spécifiques des trois principaux acides gras alimentaires, les acides alpha-linolénique (ALA), éicosapentaénoïque (EPA) et docosahexaénoïque (DHA), ont été très peu décrits.L’objectif principal de ce travail de thèse a été d’étudier les effets propres de l’ALA, de l’EPA et du DHA sur les altérations métaboliques induites en situation d’obésité. Des explorations mécanistiques ont été réalisées sur les cellules musculaires C2C12 dans lesquelles l’IR a été induite par le PAL et sur des adipocytes 3T3-L1 pour étudier l’impact des AGPI 3 sur la différenciation adipocytaire. Les effets des AGPI 3 ont ensuite été étudiés in vivo, en supplémentant des souris C57BL/6 sauvages ou déficientes en leptine (ob/ob) lors de la consommation d’un régime obésogène riche en lipides et en sucrose (mimant un régime occidental).Dans les cellules musculaires C2C12, les trois 3 co-incubés avec le PAL ont induit de façon comparable une diminution du contenu en composés lipotoxiques et une amélioration de la captation du glucose, mais seuls l’EPA et le DHA ont restauré la -oxydation du PAL et l’activation de la voie de signalisation de l’insuline. De plus, l’EPA et le DHA ont eu un effet protecteur supérieur à l’ALA vis-à-vis de l’inflammation induite par le PAL. Dans le modèle in vivo, seul la supplémentation en EPA a amélioré l’homéostasie du glucose en comparaison avec les supplémentations en ALA et en DHA. Alors que l’EPA a réduit la prise de masse grasse, le DHA a induit une hypertrophie des cellules adipeuses associée à une augmentation de la sécrétion de leptine et une baisse de la sécrétion d’adiponectine. Dans un modèle d’adipocytes 3T3-L1 en culture, le DHA a accéléré la différenciation des préadipocytes en comparaison avec l’ALA et l’EPA, pouvant expliquer son effet hypertrophique in vivo.En conclusion et dans nos conditions expérimentales, les 3 ALA, EPA et DHA ont bien des effets communs sur le métabolisme lipidique et glucidique in vitro mais également des effets propres qui ont permis de montrer qu’une supplémentation nutritionnelle en EPA serait plus intéressante pour limiter l’IR in vivo par rapport au DHA ou à l’ALA. Le DHA a quant à lui favorisé l’hypertrophie du TA, perturbant ainsi la sécrétion des adipokines participant à la régulation de la sensibilité à l’insuline des tissus périphériques, comme le muscle squelettique. / Obesity is characterized by an excess of adipose tissue (AT) mass and may be caused by multiple factors. It is associated with an increased risk of the development of insulin-resistance (IR) and metabolic syndrome, leading to type 2 diabetes. The impairment of lipid storage in the AT play a central role in obesity-associated disorders, as it leads to ectopic lipid accumulation and peripheral IR notably in muscles. In this context, the quality of dietary lipids may play a role in the regulation of AT and muscle metabolisms. In fact, palmitic acid (PAL), a saturated fatty acid (SFA) induces lipogenesis, inflammation and lipotoxicity favoring IR in many tissues. On the contrary, omega-3 polyunsaturated fatty acids (3) have protective effect against obesity-associated disorders. Among them, linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) specific effects remained partially described.This work aimed at exploring the specific effects of 3 on metabolic disorders and the development of obesity. Mechanisms were studied in C2C12 muscle cells during PAL-induced IR and in 3T3-L1 adipocytes to determine the impact of 3 on adipocyte differentiation. In vivo, the effects of 3 were investigated by supplementating C57BL/6 wild-type or leptin-deficient (ob/ob) mice with ALA, EPA or DHA during a high fat / high sucrose diet (mimicking a western diet).In C2C12 muscle cells, co-incubation of 3 with PAL induced a similar decrease in the content of lipotoxic compound and improved glucose uptake, whereas only EPA and DHA restored -oxidation and insulin signaling activation. Furthermore, EPA and DHA were more potent to reduce PAL-induced inflammation compared to ALA. In mice, only EPA improved whole body glucose homeostasis compared to ALA and DHA. While EPA reduced body fat gain, DHA induced hypertrophy in AT, increased leptin secretion and decreased those of adiponectine. In cultured 3T3-L1 adipocytes, preadipocyte differentiation was also induced by DHA compared to ALA and EPA and might explain the hypertrophy observed in mice.In conclusion and in our experimental conditions, ALA, EPA and DHA have common effects on in vitro lipid and glucose metabolism but also specific effects, demonstrating that EPA would be more interesting to limit IR in vivo compared to DHA or ALA. DHA favored hypertrophy of AT and disturbance of adipokine secretion involved in peripheral regulation of insulin sensitivity, notably in muscle.

Errata slip inserted / Bibliography: leaves 178-199 / xxi, 199, [55] leaves, [3] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, and Rheumatology Unit, Royal Adelaide Hospital, 1994

Mouse models of human disease are valuable tools for studying pathogenesis and for evaluating novel therapies. T-cell lymphoma is a relatively rare disease in humans, affecting 100-150 persons yearly in Sweden. It exists in both aggressive and more indolent forms. We have established a mouse model for an aggressive T-cell lymphoma, the T-cell lymphoma/leukemia (TLL) mouse. In the present thesis, the TLL mouse model was characterized and used for experimental therapeutic and primary prevention studies. The TLL mouse was established unintentionally in our laboratory during work on VH-gene replacement in a “knock-in” mouse experimental setting. The generated chimeras all developed aggressive T-cell lymphomas affecting the lymphoid organs, lungs, kidneys and liver. The lymphoma phenotype segregated from the targeted locus and we could demonstrate the presence of Moloney murine leukemia virus (MMLV) in the germline of the affected mice. MMLV is a retrovirus known to induce T-cell lymphomas when inoculated in newborn mice.  We further characterized two TLL substrains; TLL-2 and TLL-14 carrying the proviral integrations on chromosomes 2 and 14 respectively. Significant differences were found between the substrains regarding lymphoma frequency and immunophenotype, the TLL-14 substrain developing tumors with higher frequency than TLL-2 and with a more mature immunophenotype. A transfer model was developed in which TLL cells could be readily transferred intravenously to syngenic recipients causing aggressive lymphomas. The transfer model was used in a therapeutic study where the selective COX-2 inhibitor celecoxib was evaluated as a single agent and in combination with the established anti-tumor agent cyclophosphamide. The study was based on results from other tumor types that have indicated celecoxib, originally an anti-inflammatory and analgetic drug, to have possible anti-tumor effects. In our TLL model, however, we could not demonstrate any benefit of celecoxib monotherapy or any additive effect to cyclophosphamide. Dietary fatty acids, in particular omega-3 fatty acids, have been a focus of public and scientific interest due to observed effects on the prevention of cardiovascular disease, cancer and inflammatory conditions. In addition, omega-3 fatty acids inhibit T-cell proliferation in vitro. We supplemented the diet of TLL mice with omega-3 and omega-6 fatty acids respectively and could demonstrate a significant delay in lymphoma onset between 5-8 months of age in the group receiving an omega-3 rich diet.

In this study, pharmaceutical-grade and food-grade linker-based lecithin self-emulsifying delivery systems (SEDS) were developed with a combination of lipophilic and hydrophilic linkers. These additives at suggested concentrations are safe for pharmaceutical and food applications. The ratio of surfactant lecithin and linkers in these systems was optimized to develop surfactant in oil preconcentrates. The preconcentrates containing different surfactant concentrations and oil were diluted with fed state simulated intestinal fluid to produce pseudo-ternary phase diagrams and to identify the formulations that produced self-emulsifying or self-microemulsifying delivery systems. Optimal SEDS preconcentrates were evaluated using a dialyzer model to simulate intestinal uptake. An uptake of 39.6 mg/cm2 for the pharmaceutical-grade SEDS was obtained within 72 minutes, which promises substantial improvement in the bioavailability of hydrophobic actives. The optimal uptake of 12.2 mg/cm2 for food-grade SEDS suggests enhancement in the bioavailability of omega-3 fatty acids.

Omega-3 fatty acids, particularly docosahexaenoic (DHA) and eicospentaenoic acid (EPA), are important mediators for cardiovascular disease, fetal/infant development, neurological disorders and inflammatory diseases. Supplementation and washout studies are important for future research on the physiological effects of omega-3 fatty acids and for determination of the proper washout period for future cross-over studies. In this study, omega-3 fatty acid blood biomarker comparisons are made for the n-3 HUFA score (% of n-3 HUFAs in total HUFAs) and omega-3 index (sum of EPA + DHA) in plasma, erythrocytes, whole blood and a novel finger-tip prick blood method (FTPB) of analysis. This FTPB method of fatty acid analysis is further tested to determine the potential for its use in fatty acid analysis. In addition, gender differences in response to omega-3 fish oil supplementation are analyzed in all four blood fractions. Nine males and seven females were supplemented with 8 fish-oil capsules per day (providing 3.2 g/day EPA and 1.6 g/day DHA) for four weeks, followed by an eight-week omega-3 washout period. Venous plasma, erythrocyte and whole blood samples were collected during weeks 0, 4, 8 and 12 and FTPB samples were collected weekly during supplementation and washout fatty acid analysis was performed. EPA and DHA incorporation is lowest in magnitude in erythrocytes relative to all other blood fractions. Omega-3 blood biomarker comparisons demonstrate that the n-3 HUFA score is a more reliable measure across all blood fractions compared to the omega-3 index. In addition, the n-3 HUFA score demonstrates no differences (p > 0.05) between FTPB and whole blood analysis, providing evidence to support its usefulness as a tool for fatty acid analysis. However, differences (p < 0.05) do exist between these methods for saturated fatty acid, monounsaturated fatty acids, omega-6 polyunsaturated fatty acids (PUFAs) and omega-3 PUFAs. Baseline fatty acid levels for DHA, and the DHA:EPA and DHA:DPA ratios tend to be higher (p < 0.05) in females, and docosapentaenoic acid n-3 (DPAn-3) is higher (p > 0.05) in males across all blood fractions. Furthermore, a gender effect (p < 0.05) is seen for the DHA:EPA ratio across all blood fractions. At baseline, female DHA:EPA is higher (p < 0.05) than males with supplementation lowering both male and female values and removing any differences (p > 0.05) between genders. Washout results in a return of levels towards baseline, however, baseline levels are not fully reached. Furthermore, while gender differences do begin to reform during washout, these differences are not significant (p > 0.05). In conclusion, omega-3 fatty acid responses, particularly DHA:EPA ratio, demonstrate significant gender differences that may be related to differences in long-chain PUFA synthesis pathways between males and females. In addition, the n-3 HUFA score may be a more valuable omega-3 blood biomarker than the omega-3 index, as the n-3 HUFA score displays more consistent levels across all blood fractions. Finally, the FTPB method of analysis may be a useful tool in the measurement of fatty acid composition, however, some microwave methylation problems do exist, specifically in the phospholipid class of lipids.

Omega-3 fatty acids, particularly docosahexaenoic (DHA) and eicospentaenoic acid (EPA), are important mediators for cardiovascular disease, fetal/infant development, neurological disorders and inflammatory diseases. Supplementation and washout studies are important for future research on the physiological effects of omega-3 fatty acids and for determination of the proper washout period for future cross-over studies. In this study, omega-3 fatty acid blood biomarker comparisons are made for the n-3 HUFA score (% of n-3 HUFAs in total HUFAs) and omega-3 index (sum of EPA + DHA) in plasma, erythrocytes, whole blood and a novel finger-tip prick blood method (FTPB) of analysis. This FTPB method of fatty acid analysis is further tested to determine the potential for its use in fatty acid analysis. In addition, gender differences in response to omega-3 fish oil supplementation are analyzed in all four blood fractions. Nine males and seven females were supplemented with 8 fish-oil capsules per day (providing 3.2 g/day EPA and 1.6 g/day DHA) for four weeks, followed by an eight-week omega-3 washout period. Venous plasma, erythrocyte and whole blood samples were collected during weeks 0, 4, 8 and 12 and FTPB samples were collected weekly during supplementation and washout fatty acid analysis was performed. EPA and DHA incorporation is lowest in magnitude in erythrocytes relative to all other blood fractions. Omega-3 blood biomarker comparisons demonstrate that the n-3 HUFA score is a more reliable measure across all blood fractions compared to the omega-3 index. In addition, the n-3 HUFA score demonstrates no differences (p > 0.05) between FTPB and whole blood analysis, providing evidence to support its usefulness as a tool for fatty acid analysis. However, differences (p < 0.05) do exist between these methods for saturated fatty acid, monounsaturated fatty acids, omega-6 polyunsaturated fatty acids (PUFAs) and omega-3 PUFAs. Baseline fatty acid levels for DHA, and the DHA:EPA and DHA:DPA ratios tend to be higher (p < 0.05) in females, and docosapentaenoic acid n-3 (DPAn-3) is higher (p > 0.05) in males across all blood fractions. Furthermore, a gender effect (p < 0.05) is seen for the DHA:EPA ratio across all blood fractions. At baseline, female DHA:EPA is higher (p < 0.05) than males with supplementation lowering both male and female values and removing any differences (p > 0.05) between genders. Washout results in a return of levels towards baseline, however, baseline levels are not fully reached. Furthermore, while gender differences do begin to reform during washout, these differences are not significant (p > 0.05). In conclusion, omega-3 fatty acid responses, particularly DHA:EPA ratio, demonstrate significant gender differences that may be related to differences in long-chain PUFA synthesis pathways between males and females. In addition, the n-3 HUFA score may be a more valuable omega-3 blood biomarker than the omega-3 index, as the n-3 HUFA score displays more consistent levels across all blood fractions. Finally, the FTPB method of analysis may be a useful tool in the measurement of fatty acid composition, however, some microwave methylation problems do exist, specifically in the phospholipid class of lipids.

Eating disorders (ED) usually develop during adolescence, and intervention to stop further weight loss is believed to improve outcome and long-term prognosis. Adolescents with ED who do not receive effective treatment risk poor outcome and even untimely death as adults. The first aim of this thesis was to investigate long-term mortality and causes of death in a series of female adults with chronic ED. The second aim was to study the one-year outcome of an unselected series of adolescent girls with anorexia nervosa (AN) and “other restrictive eating disorders” who had been treated within a specialist ED out-patient service focused on nutritional rehabilitation based on family therapy and without planned hospitalization. The third aim was to investigate the possible metabolic and hormonal side effects of olanzapine when used as an adjunct to facilitate nutritional rehabilitation. The fourth aim was to investigate the relationship between polyunsaturated fatty acid (PUFA) status and depression. In adult women with chronic ED, a very low body mass index and psychiatric co-morbidity confer a substantially increased risk of premature death. A treatment programme for adolescent ED with rapid access to assessment and prompt start of treatment with initial emphasis on nutritional rehabilitation proved efficient. The outcome was encouraging, as 43% of all patients with ED and 19% of those with AN did not have an ED at one-year follow-up. Of the remaining patients the vast majority had gained weight and regained menstruation, and were back in school on a full-time basis. Olanzapine was used to reduce anxiety, excessive exercise and rumination over weight and shape. Side effects were similar to those observed in normal-weight individuals, and do not preclude its use in underweight adolescents with ED. Low ω3 PUFA were associated with depression. The ω3 PUFA status improved during nutritional rehabilitation with ordinary foods and without supplementation. The investigations indicate that adolescent ED can be successfully treated in an out-/day-patient setting. An essential feature of the service is rapid handling and weight gain. Further weight loss can be avoided, and chronic disease hopefully prevented.

Objective: The objective of this thesis was to facilitate clinical trials of the optimal lipid based approach (e.g.: omega-3 containing lipid emulsions or minimization of conventional lipid) for the prevention of Intestinal Failure Associated Liver Disease (IFALD). This was achieved through 3 related projects. Project 1: The first project examined the risk of advanced IFALD associated with exposure to conventional intravenous lipid in a logistic regression model. The study demonstrated that each day of conventional lipid (> 2.5 g/kg/day) was associated with a significant increase in the risk of advanced IFALD [Odds Ratio: 1.04 95% CI: 1.003 – 1.06]. Project 2: The second project surveyed experts in Intestinal Failure regarding their beliefs of the efficacy of lipid minimization and lipid emulsions containing omega-3 fatty acids relative to conventional emulsions. The goal of the project was to develop prior distributions of the treatment response for these therapies that can be used in Bayesian analyses of clinical trials. Our results demonstrated consistent expert opinion that the novel lipid based approaches are superior to conventional therapy. Estimates of the treatment effect were similar for the two approaches (median elicited treatment response, relative to conventional lipid, was a relative risk of 0.53 for omega-3 lipid and 0.45 for lipid minimization). Project 3: The final project was a pilot randomized controlled trial of an omega-3 emulsion. The study demonstrated that the randomized design is a feasible strategy for evaluating lipid based approaches for the prevention of IFALD. A Bayesian preliminary assessment of the results of the trial, suggests a high likelihood that the trial will demonstrate a difference between the conventional and omega-3 emulsion evaluated in the trial. However, since the analysis was blinded, the direction of the difference is not known. Conclusion: This thesis will contribute to the design and analysis of high quality and feasible randomized trials that will allow investigators to address the optimal lipid based approach to the management of IFALD.

This purpose of this thesis was to develop a pilot study to determine if omega-3 fatty acids and vitamin D3 will improve body weight loss and improve risk factors for Metabolic Syndrome within a weight loss program. Risk factors include obesity, hypertension, hyperglycemia, and dyslipidemia. Thirty-five men and women between 18 and 65 years of age with risk factors for Metabolic Syndrome were recruited for this study. All participants followed an energy-restricted, low glycemic-index based diet and exercise program for 16 weeks. Half of these participants received omega-3 fatty acid and vitamin D3 supplements. In those that received these supplements, it was seen that their serum 25-hydroxyvitamin D2/D3 levels and incorporation of docosahexaenoic acid and eicosapentaenoic acid into red blood cell phospholipids improved. The effect of supplementation on changes to body weight and risk factors for Metabolic Syndrome did not reach significance (p<0.05). It was however demonstrated, that an energy-restricted, low glycemic index diet with exercise was effective in inducing weight loss and improving Metabolic Syndrome risk factors with a 50% reduction in participants who had the criteria for diagnosis of Metabolic Syndrome by week 16.