Les stress oxydant et carbonylé interviennent dans le mécanisme physiopathologique de la dégénérescence maculaire liée à l'âge (DMLA) et de la maladie de Stargardt, constituant ainsi une cible thérapeutique intéressante. Une molécule lead de type lipophénol a préalablement été mise en évidence. Cette molécule est composée d’une partie phénolique de type alkylphloroglucinol, et lipidique de type oméga-3 (acide docosahexaènoïque, C22:6, ω3, DHA). Afin d’améliorer l’activité antioxydante de ce lipophénol, la synthèse totale de deux DHA deutérés en positions bis-allyliques a été réalisée. Leurs couplages au phloroglucinol alkylé a ensuite conduit aux dérivés lipophénoliques deutérés correspondants. L’impact des deutériums sur la diminution de la toxicité cellulaire et de la peroxydation lipidique a ensuite été mis en évidence lors d’une évaluation biologique in vitro réalisée en cellules de l’épithélium pigmentaire rétinien. / Age Related Macular Degeneration (AMD) and Genetic Stargardt’s disease share a common cytotoxic mechanism involving carbonyl and oxidative stress. Hence both stresses represent promising therapeutic targets. A lipophenol lead molecule has been previously highlighted. This molecule is composed by an alkylphloroglucinol moiety and a lipid part which is a polyunsaturated fatty acid, the docosahexaenoic acid (DHA, C22:6, ω3). In order to improve the antioxidant activity of this lipophenol, the total synthesis of two deuterated DHA on bis-allylic positions was performed. The cross-coupling between the deuterated DHA and the alkylphloroglucinol moiety led to the corresponding deuterated lipophenols. The impact of the deuterium to limit the cell toxicity and the lipid peroxidation was highlighted thanks to in vitro biological studies performed on retinal pigment epithelium cell line.

O experimento foi realizado no Biotério Experimental da USP em Pirassununga - SP. Na primeira fase foram utilizados 42 coelhos machos em crescimento da raça Nova Zelândia mantidos individualmente durante todo o experimento. O ensaio biológico totalizou 150 dias. Na Fase 1 os animais receberam dietas com indução de hipercolesterolemia (0,5% de colesterol), com desequilíbrio na proporção de Omega-6/Omega-3 (n-6:n-3) de 15:1 e vice versa e balanço na proporção de n-6:n-3 de 4:1 (controle), totalizando 6 ensaios, os quais foram divididos aleatoriamente em 7 grupos de 6 coelhos cada. Óleos de girassol e de peixe foram utilizados como fontes de ácidos graxos poli-insaturados n-6 e n-3, respectivamente. Na Fase 2, os animais remanescentes continuaram recebendo as mesmas dietas, exceto no grupo B (hipercolesterolemia) que passou a receber o tratamento controle para verificar o efeito. No final da Fase 1, três animais de cada grupo foram eutanasiados com retirada da artéria aorta para determinação de placas lipídicas, histopatológica, colesterol total e imunohistoquímica para verificação da expressão da enzima LDL-receptor e determinação histopatológica do tecido hepático. Em soro foram realizadas determinações de perfil de ácidos graxos, colesterol total, LDL e HDL-colesterol e triacilgliceróis. Células endoteliais aórticas de coelhos foram isoladas para a realização do teste dose resposta com óleo de peixe para identificar a melhor relação do consumo destes ácidos graxos comparando com o experimento in vivo. O objetivo deste trabalho foi monitorar os efeitos do fornecimento de cada dieta sobre o teor de colesterol total, LDLcolesterol, HDL-colesterol e triacilgliceróis em sangue de coelhos. No geral, o perfil de ácidos graxos no soro correspondeu diretamente com a dieta consumida. Os principais efeitos dos ácidos graxos poli-insaturados Omega 3 foi a redução dos lipídios séricos quando os coelhos remanescentes da dieta hipercolesterolêmica (0,5% de colesterol e n-6:n-3 de 2:1) passaram a receber o equilíbrio da relação de ácidos graxos 4:1 de n-6:n-3. O teor de colesterol total no soro, artérias e o crescimento dos ateromas foram influenciados pelo elevado consumo de ácidos graxos n-6 e n-3 associados à adição de colesterol nas dietas. Por outro lado verificamos que o excesso de Omega 3 associado ou não ao colesterol contribuiu para o agravamento das placas ateroscleróticas inclusive deposição de cálcio nas mesmas e paredes endoteliais. Provavelmente, o excesso de n-3 tenha ocasionado efeito contrário às suas funções anti-inflamatória, antiagregatória e anti-trombótica. Dietas hipercolesterolêmicas provocaram esteatose hepática e o elevado consumo de Omega-6 em detrimento do baixo consumo de Omega -3 induziram hepatite crônica. Já o consumo elevado de Omega-3, sem colesterol, regrediu a esteatose hepática nos animais. O consumo equilibrado de n-6:n-3 reduziu a expressão da enzima LDL-receptor no grupo que anteriormente recebeu dieta hipercolesterolêmica. Ressaltando-se a importância do consumo equilibrado destes ácidos graxos já que esta enzima é controlada pelo colesterol livre circulante. No teste dose-resposta em células endoteliais da aorta de coelhos foi evidenciado que a melhor proporção de n-6:n-3 seria de 9 vezes mais do que a concentração de Omega 3 recomendada de 5:1. / The experiment was conducted at the Experimental Biotery by USP in Pirassununga - SP. In the first phase were used 42 male rabbits growing New Zealand breed individually maintained throughout the experiment. The biological assay amounted to 150 days. In phase 1, the animals received diets induced hypercholesterolemia (0.5% cholesterol) with imbalance in the ratio of Omega-6 / Omega-3 (n-6 / n-3) 15:1 and vice versa, and balance the ratio of n-6 / n-3 of 4:1 (control), totaling six tests, which were randomly divided into 7 groups of six rabbits each. Sunflower and fish oils were used as sources of fatty acids polyunsaturated n-6 and n-3, respectively. In Phase 2, the remaining animals continued to receive the same diets except in group B (hypercholesterolemia) who went on to receive the control treatment to check the effect. At the end of Phase 1, three animals from each group were sacrificed with the aorta artery removed for determination of lipid plaques, histopathology, immunohistochemistry and total cholesterol to verify the expression of LDL-receptor enzyme and determination of histopathological liver tissue. In serum profile were made determinations of fatty acids, total cholesterol, LDL and HDL cholesterol and triglycerides. Aortic endothelial cells of rabbits were isolated to perform the dose response test with fish oil to identify the best value for the consumption of these fatty acids compared to the in vivo experiment. The objective of this work was to monitor the effects of provision for each diet on total cholesterol, LDL-cholesterol, HDLcholesterol and triglycerides in the blood of rabbits. In general, the profile of fatty acids in serum corresponded directly with the diet consumed. The main effects of polyunsaturated Omega 3 fatty acids was the reduction of serum lipids when the remaining rabbits with hypercholesterolemic diet (0.5% cholesterol and n-6: n-3 2:1) have received the balance of the relationship fatty acid 4:1 n-6: n-3. The total cholesterol content in serum, the growth of arteries and atheroma were influenced by a high intake of n-6 fatty acids and n-3 associated with the addition of cholesterol in diets. On the other hand we verified that the excess of Omega 3 with or without cholesterol contributed to the aggravation of the atherosclerotic plaques including deposition of calcium in them and endothelial walls. Probably the excess of n-3 has caused the opposite effect to their anti-inflammatory, anti-thrombotic and anti-aggregatory functions. Hypercholesterolemic diets caused hepatic steatosis and high intake of Omega-6 to the detriment of low consumption of Omega -3 induced chronic hepatitis. High consumption of Omega-3, no cholesterol, decreased hepatic steatosis in animals. Highlighting the importance of the balanced intake of this fatty acids, since this enzyme is controlled by circulating free cholesterol. In dose-response test in endothelial cells of the rabbits aorta was evident that the best ratio of n-6: n-3, nine times more than the recommended concentration of Omega 3 to 5:1.

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-08T12:33:25Z No. of bitstreams: 1 Jeine Emanuele Santos da Silva.pdf: 2802282 bytes, checksum: 66fe306e48bd3ebef3e793500c49baea (MD5) / Made available in DSpace on 2016-06-08T12:33:25Z (GMT). No. of bitstreams: 1 Jeine Emanuele Santos da Silva.pdf: 2802282 bytes, checksum: 66fe306e48bd3ebef3e793500c49baea (MD5) Previous issue date: 2011-02-28 / The aim of the study was to investigate the effect of different fat dietary on serum lipid profile in Japanese quails and on vascular growth in the embryos vitelline membrane (VM). These birds were fed with supplemented feed with fish oil or soybean oil. The experiment was conducted with 80 quail, distributed in five treatments named Diet Control (DC), Fish Oil (FO 2%), Fish Oil 4% (FO 4%),Soybean Oil 2% (SO 2%) and Soybean Oil 4% (SO 4%), in a ratio of 1 male to 3 females. Fertilized eggs incubated at 37.5°C for 24 hours were opened and after removal of 1 mL of albumen the window was sealed with parafilm. From 72 hours incubation images of VM vascular network were captured at intervals of 24 hours over three days. DLA were generated in order to compare their fractal dimension (D) with the VM and test whether the method affects the value of D. To obtain the average value of D of the VM vascularization and DLA were used box-counting (Dbc) and information dimension (Di) methods. An enzymatic colorimetric method was used to estimate lipid profile. The total cholesterol (TC) levels of the groups showed no difference between itself. The total triglycerides (TG) of the FO 2% and FO 4% groups were higher than the groups DC, SO 2% and SO 4%. For HDL, the group FO 4% differ in relation to other groups. The embryos MV vascularization, evaluated by fractal dimension (D), of the group FO 4% was less than the MV vascularization of the group DC, SO 2% and FO 2%. The MV vascularization of the group SO 4% was lower only when compared with group DC. The PUFA supplementation in groups FO 2% and SO 2% did not interfered with MV vascularization. The DLA fractal dimension obtained by the Dbc and Di was higher than those observed for the MV fractal dimension for all experimental groups. Differences between the values of D obtained by these methods (Dbc e Di) were statistically significant for all groups. The vitelline membrane shows an experimental model suitable for vasculogenesis study. The fractal analysis shows to be a reliable mathematical method to quantify vascular growth in vivo such as the vitelline membrane. / O objetivo deste estudo foi investigar o efeito de diferentes dietas sobre o perfil lipídico sérico de codornas japonesas e sobre o crescimento vascular na membrana vitelina (MV) dos embriões das aves suplementadas com óleo de peixe e óleo de soja. Foram utilizadas 80 codornas, distribuídas em cinco tratamentos denominados Dieta Controle (DC), Óleo de Peixe 2% (OP 2%), Óleo de Peixe 4% (OP 4%), Óleo de Soja 2% (OS 2%) e Óleo de Soja 4% (OS 4%), numa relação de 1 macho para 3 fêmeas. Ovos fertilizados incubados a 37,5ºC durante 24 horas foram abertos e após a remoção de 1 mL de albúmen a abertura foi selada com parafilme. A partir de 72 horas de incubação imagens da rede vascular da MV foram capturadas em intervalos de 24 horas durante 3 dias. DLA foram gerados a fim de comparar a sua dimensão fractal com a da MV e testar se o método usado interfere no valor da dimensão. Para obtenção do valor médio da dimensão fractal da vascularização das MV e dos DLA foram utilizados os métodos de box-counting (Dbc) e dimensão de informação (Di). Método enzimático com reação colorimétrica foi usado para estimar o perfil lipídico das aves. Os níveis de colesterol total (CT) dos grupos não apresentaram diferença entre si. Os níveis de triglicerídeos totais (TG) das aves do grupo OP 2% e OP 4% foram superiores aos dos grupos DC, OS 2% e OS 4%. Para o HDL, apenas o grupo OP 4% apresentou diferença em relação aos demais grupos. A vascularização da MV dos embriões, avaliada pela dimensão fractal D, do grupo OP 4% foi inferior a vascularização das MV dos grupos DC, OS 2% e OP 2%. A vascularização da MV do grupo OS 4% foi inferior apenas quando comparada ao grupo DC. A suplementação com PUFA nos grupos OP 2% e OS 2% não interferiu na vascularização das MV. A dimensão fractal do DLA obtida pelos métodos de Dbc e Di foi superior aquelas observadas para as dimensões das MV de todos os grupos experimentais. As diferenças entre as dimensões obtidas por estes métodos (Dbc e Di) foram estatisticamente significativas para todos os grupos avaliados. A membrana vitelina se mostra um modelo experimental adequado para o estudo da vasculogênese. A geometria fractal se mostra um método matemático confiável para a quantificação do crescimento vascular em modelos in vivo como a membrana vitelina.

Déterminer la composition en triacylglycérols des huiles végétales est un défi important à relever aussi bien en biologie végétale que dans le domaine médical ou celui de l’industrie agro-alimentaire. La chromatographie en phase liquide à polarité inversée de phases en milieu non aqueux (NARP-LC) est la méthode la plus utilisée, avec cependant une utilisation récurrente de solvants chlorés. Le premier objectif de ce travail a été de proposer des conditions analytiques alternatives n’utilisant pas de solvants chlorés. Pour ce faire, nous avons établi le diagramme de force éluante sur phases stationnaires en C18 des phases mobiles binaires constituées d’acétonitrile comme solvant faible et de divers solvants forts (acétone, iso-propanol, acétate d’éthyle, butanol) à quatre températures différentes (25, 43, 63 et 85°C). La comparaison dans des conditions iso-éluantes de l’analyse de 9 huiles de graines contenant une large gamme de TAG nous a permis de montrer que le mélange MeCN/BuOH 74/26 à 25°C est le meilleur choix, en terme de sélectivité pour l’analyse des TAG. Ce qui répond à notre premier objectif. Dans un second temps, nous avons comparé le potentiel séparatif des phases stationnaires de nouvelle génération à petit diamètre de particules, partiellement ou totalement poreuses. L’optimisation des conditions chromatographiques nous a permis de décrire deux systèmes chromatographiques, très performants, en termes d’efficacité et de rapidité. Le premier permet la séparation de TAG contenant des acides gras polyinsaturés isomères de position en C18 : 3 et C18 : 2. L’identification de ces isomères particuliers a été réalisée grâce à la synthèse d’informations complémentaires, obtenues en GC/MS, LC/MS ainsi que l’utilisation de lois de rétention chromatographiques. En outre, ce travail nous a permis de proposer un tableau récapitulatif regroupant un très grand nombre de TAG, qui n’ont jamais été décrits à notre connaissance. Le second permet une analyse rapide de l’huile d’olive, en moins de 5 min, tout en respectant les consignes qualitatives imposées par l’organisme «Conseil Oléique International (COI)». Comparée aux méthodes officielles couramment utilisées, elle mérite d’être proposée comme méthode de référence pour le contrôle de qualité de l’appellation de ces huiles. / The determination of triacylglycerols in vegetable oils is an important challenge in plant biology, in the medical field, and in food industry. Nowadays, non-Aqueous Reversed Phase Liquid Chromatography (NARP-LC) using chlorinated solvents is commonly used for this purpose. The first objective of this work was to develop alternative analytical conditions that can avoid using chlorinated solvents. In a first step, by using C18 stationary phases, we have established the eluotropic solvent strength scale as a function of temperature of several binary mobile phases consisting of acetonitrile as weak solvent and various strong solvents including acetone, isopropanol, ethyl acetate, and butanol. The comparison of the results obtained under iso-eluotropic conditions for nine seed oils containing a wide range of TAG enabled us to show that the MeCN/BuOH (74/26, v/V) mixture operating at 25 °C are the best mobile phase conditions for TAG analysis, in terms of selectivity, thus avoiding the use of chlorinated solvents. In a second step, we compared the separation of TAGs on new generation of fully or partially porous stationary phase particles of small diameter. After optimizing the separating conditions, the obtained data allowed us to propose two highly efficient chromatographic systems. The first system enables the efficient separation of C18:3 and C18:2 positional isomers of C18 polyunsaturated fatty acids containing TAG. For the identification of these TAG isomers, it was necessary to combine the data obtained by GC-MS and LC -MS as well as the data obtained by the application of some chromatographic retention laws. Taken together, these results allowed us to provide a list containing a large number of TAG unknown to date. The second system allows rapid analysis of olive oil in less than 5 min. This system obeying the guidelines of the International Olive Council can be proposed as a candidate reference method for rapid quality control of olive oils.

Obesity and associated metabolic disorders, e. g. metabolic syndrome, represent a considerable health threat for modern society. Due to sedentary lifestyle, high caloric intake and changes in composition of diet, prevalence of obesity is increasing worldwide. One of the possible causes contributing to higher prevalence of obesity in recent population could be the change of fatty acids (FA) composition of dietary lipids, with the shift in the content of n-6 and n-3 FA toward n-6 FA. In contrast to n-6 FA, n-3 FA are known for their anti-atherogenic, anti-obesogenic and anti-inflammatory properties. In our experiments in mice, the capability of naturally occurred and chemically modified n- 3 long chain polyunsaturated fatty acids (LC-PUFA) in prevention and reversal of specific parts of metabolic syndrome was demonstrated. A specific chemical derivative of docosahexaenoic acid was proven to be very effective in preventing and improving metabolic conditions of animals exposed to high-fat (HF) diet challenge. Further, the involvement of AMP-activated protein kinase (AMPK), a master regulator of lipid metabolism, in skeletal muscle thermogenesis induced by HF-feeding was investigated. Activation of AMPK in the HF-fed mice is most possibly caused by increased leptin levels and represents an important link...

Obesity and associated metabolic disorders, e. g. metabolic syndrome, represent a considerable health threat for modern society. Due to sedentary lifestyle, high caloric intake and changes in composition of diet, prevalence of obesity is increasing worldwide. One of the possible causes contributing to higher prevalence of obesity in recent population could be the change of fatty acids (FA) composition of dietary lipids, with the shift in the content of n-6 and n-3 FA toward n-6 FA. In contrast to n-6 FA, n-3 FA are known for their anti-atherogenic, anti-obesogenic and anti-inflammatory properties. In our experiments in mice, the capability of naturally occurred and chemically modified n- 3 long chain polyunsaturated fatty acids (LC-PUFA) in prevention and reversal of specific parts of metabolic syndrome was demonstrated. A specific chemical derivative of docosahexaenoic acid was proven to be very effective in preventing and improving metabolic conditions of animals exposed to high-fat (HF) diet challenge. Further, the involvement of AMP-activated protein kinase (AMPK), a master regulator of lipid metabolism, in skeletal muscle thermogenesis induced by HF-feeding was investigated. Activation of AMPK in the HF-fed mice is most possibly caused by increased leptin levels and represents an important link...

The homeoviscous adaptation hypothesis states that the relative abundance of polyunsaturated fatty acids (PUFAs) in membrane phospholipids of ectothermic organisms decreases with increasing temperatures to maintainvital membrane properties. We reared Daphnia magna at 15°, 20°, and 25°C and increasing dietary concentrations of the long-chain PUFA eicosapentaenoic acid (EPA) to test the hypothesis that the well-documented increase in heat tolerance of high-temperature-reared Daphnia is due to a reduction in body PUFA concentrations. Heat tolerance was assessed by measuring the time to immobility at a lethally high temperature (Timm at 37°C), and whole body lipid fluorescence polarization (FP) was used as an estimate of membrane fluidity. At all rearing temperatures, EPA supplementation resulted in an increase in the relative abundance of EPA in body tissues, but only at 15° and 25°C did this result in a decrease in heat tolerance, and only at 20°C was this associated with an increase in membrane fluidity (i.e., decrease in FP). Overall, however, the degree of tissue fatty acid unsaturation correlated well with heat tolerance and FP. Our results support the homeoviscous adaptation hypothesis by showing that cold-reared Daphnia accumulate PUFAs within their body tissues and thus are more susceptible to heat than hot-reared Daphnia accumulating fewer PUFAs. However, our data also point out that further studies are required that elucidate the complex relationships between PUFA supply, membrane fluidity, and heat tolerance in ectotherms.

Indiana University-Purdue University Indianapolis (IUPUI) / Omega-3 polyunsaturated fatty acids (n-3 PUFAs) relieve the symptoms of a wide variety of chronic inflammatory disorders. Typically, they must be obtained in the diet from sources such as fish oils. Docosahexaenoic acid (DHA) is one of these n-3 PUFAs. As yet the structural mechanism responsible for the health benefits within the body is not completely understood. One model that has emerged from biochemical and imaging studies of cells suggests that n-3 PUFAs are taken up into phospholipids in the plasma membrane. Thus the focus here is on the plasma membrane as a site of potential structural modification by DHA. Within cellular membranes, the huge variety of molecules (called lipids) which constitute the membrane suggest inhomogeneous mixing, thus domain formation. One potential domain of interest is called the lipid raft, which is primarily composed of sphingomyelin (SM) and cholesterol (chol). Here the molecular organization of [2H31]-N-palmitoylsphingomyelin (PSM-d31) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane was examined by solid state 2H NMR spectroscopy. Solid state 2H NMR spectroscopy extracts details of molecular orientation and anisotropy of molecular reorientation by analysis of the lineshape. This essentially non-invasive technique allows for a direct measurement of dynamics in bulk materials which has been extensively applied to biological materials. It is a niche area of NMR for which standard software often lack necessary features. Two software programs, “EchoNMR processor” and “EchoNMR simulator”, collectively known as “EchoNMR tools”, that were developed to quickly process and analyze one-dimensional solid-state NMR data, will be described along with some theoretical background of the techniques used. EchoNMR tools has been designed with a focus on usability and the open-source mindset. This is achieved in the in the MATLAB® programming environment which allows for the development of the graphical user interfaces and runs as an interpreter which allows the code to be open-source. The research described here on model membranes demonstrates the utility of the software. The NMR spectra for PSM-d31 in mixtures with PDPC or POPC with cholesterol were interpreted in terms of the presence of nano-sized SM-rich/chol-rich (raft-like) and PC-rich/chol-poor (non-raft) domains that become larger when POPC was replaced by PDPC. An increase in the differential in order and/or thickness between the two types of domains is responsible. The observation of separate signals from PSM-d31, and correspondingly from [3α-2H1]cholesterol (chol-d1) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31), attributed to the raft-like and non-raft domains enabled the determination of the composition of the domains. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain. There was also a substantial amount of PDPC (70%) in the raft-like domain that appears to have minimal effect on the order of SM. PDPC molecules sequestering into small groups to minimize the contact of DHA chains with cholesterol is one possible explanation that would also have implications on raft continuity. These results refine the understanding of how DHA may modulate the structure of raft domains in membranes.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19047号 / 農博第2125号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4929(農学部図書室) / 31998 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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