Dissertations / Theses on the topic 'DHA'

L'encapsulation peut affecter la digestion et la bioaccessibilité des composés encapsulés, ce qui peut ensuite affecter leur métabolisme. Le but de ce projet était d'étudier les effets de l'encapsulation sur la bioaccessibilité et le métabolisme du DHA, en étudiant une huile de DHA non-encapsulée ou encapsulée, apportée sous forme d'omelette comme matrice alimentaire.L'huile de DHA, composée de triacylglycérols riches en DHA, a été préparée sous forme d'émulsion de Pickering, stabilisée par des isolats de protéines de lactosérum dénaturés par la chaleur. De l'huile pure ou sous forme d’émulsion a ensuite été ajoutée à l’œuf liquide pour obtenir de l’omelette. Les effets de l'encapsulation sur la digestion ont été étudiés à l'aide d'un modèle de digestion statique in vitro pour adulte, puis l’impact sur le métabolisme du DHA a été mesuré sur un modèle rat pris au sevrage.Les résultats ont montré in vitro que l'encapsulation peut augmenter la surface de contact entre l'huile de DHA et les enzymes de digestion, favorisant l'hydrolyse lipasique de l’huile de DHA et améliorant ainsi la bioaccessibilité du DHA. In vivo, l'encapsulation n'a pas impacté le profil global des acides gras, et particulièrement l’accrétion du DHA dans le cerveau. En revanche, le profil des oxylipines, dérivés oxydatifs d’acides gras, a été fortement modifié dans le plasma, le cœur et même le cerveau. Les métabolites dérivés du DHA ont globalement été augmentés tandis que ceux issus des acides gras de la famille n-6 ont été essentiellement atténués.Par conséquent, l'encapsulation de l'huile de DHA pourrait non seulement améliorer la bioaccessibilité du DHA, mais constitue également un facteur clé dans le métabolisme du DHA pour produire des précurseurs de protectines et de maresines, améliorant ainsi le statut de santé global
Encapsulation may affect the digestion and bioaccessibility of the encapsulated bioactive compounds, which in turn affects their metabolism. The purpose of this project was to study the effects of encapsulation on DHA bioaccessibility and metabolism, based on omelet as a food matrix, which contains DHA oil as encapsulated or unencapsulated form.DHA oil composed of DHA-rich triacylglycerols was prepared as a Pickering emulsion, which is stabilized by heat-denatured whey protein isolates. Pure oil or emulsion was then incorporated into eggs and cooked in an omelet. The effects of encapsulation on the digestion and metabolism of DHA were studied by using INFOGEST static in vitro digestion model for adults and in a weanling rat model, respectively.The results showed that encapsulation can increase the contact surface between DHA oil and lipase during the in vitro digestion, thereby promoting the hydrolysis of DHA oil and improving DHA bioaccessibility. In vivo, encapsulation of DHA oil did not modulate the fatty acid profile in tissues, but remarkably modified the oxylipin pattern in plasma, heart and even brain. Specific oxidized metabolites derived from DHA were upgraded while those from n-6 fatty acids were essentially mitigated.Therefore, encapsulation of DHA oil could not only improve the bioaccessibility of DHA, but is also a key factor in the metabolism of DHA to produce protectins and maresins precursors, thereby improving global health status

L'acide docosahexaénoïque (DHA) est un acide gras polyinsaturé oméga-3 (AGPI [oméga]3) concentré dans le poisson. Il est l'AGPI [oméga]3 présent en plus grande quantité dans le cerveau. Une étude épidémiologique a démontré que la consommation de deux portions de poisson par semaine peut ralentir le rythme du déclin cognitif qui survient avec le vieillissement. Étude SUPPLÉMENT. Objectif : Évaluer le changement plasmatique en AGPI [oméga]3 chez des sujets jeunes et âgés lors d'une supplémentation en acide eicosapentaénoïque (EPA)/DHA. Résultats : Chez les sujets jeunes et âgés, le DHA (73 « 47% et 117 « 68 %) et l'EPA (133 « 67% et 97 « 52%) plasmatiques ont augmenté significativement (p < 0,05) avec la supplémentation. Le pourcentage de DHA était supérieur chez les sujets âgés par rapport aux sujets jeunes avec la supplémentation. Conclusion : Les différences observées entre les sujets jeunes et âgés suggèrent qu'il existe un changement dans le métabolisme du DHA avec le vieillissement. Étude TRACEUR. Objectif : Évaluer l'incorporation du DHA marqué au carbone 13 (indice supérieur 13]C-DHA) dans les lipides totaux plasmatiques et sa [bêta]-oxydation chez des participants jeunes et âgés en bonne santé. Résultats : Initialement, la concentration de DHA dans les lipides totaux plasmatiques était similaire entre les groupes. Le [indice supérieur 13]C-DHA avait tendance (p = 0,055) à s'incorporer davantage chez les participants âgés (0,80 « 0,35 nmol/ml) comparativement aux jeunes (0,35 « 0,12 nmol/ml) quatre heures après la prise du traceur. Conclusions : Ces résultats suggèrent que l'incorporation du DHA dans les lipides plasmatiques et sa [bêta]-oxydation dans les heures suivant son ingestion sont influencés par l'âge. La faible [bêta]-oxydation du DHA sur un mois montre qu'il est davantage conservé pour des rôles structuraux plutôt qu'à des fins énergétiques. Conclusion générale :L'âge est associé à une augmentation de l'incorporation du DHA dans les lipides plasmatiques qui se reflète par la plus grande augmentation du DHA dans le plasma lors d'une supplémentation chez des personnes âgées. À cause de sa plus grande incorporation, davantage de DHA est disponible pour la [bêta]-oxydation, ce qui explique la plus grande quantité de DHA [bêta]-oxydé chez les personnes âgées. Cependant la [bêta]-oxydation du DHA est trop faible pour entrer en contradiction avec les résultats obtenus lors de la supplémentation.--Résumé abrégé par UMI.

Fatty acid composition in the body displays a high level of heterogeneity and can rapidly respond to changes in diet regime or to starvation. Homeostasis of the level of certain fatty acids is an important factor for maintenance of structural integrity as well as for proper signaling within the organism. Hence, changes in fatty acid composition have been proposed as an important factor during the pathogenesis of many diseases.Concentration of polyunsaturated fatty acid (PUFA) within the body is modulated by the interplay between dietary intake, endogenous de novo synthesis or mobilization of fatty acids from tissue reservoirs. Endogenous synthesis of PUFA is regulated on different genetic levels as well as the level of substrate availability. Studies have reported a variation in PUFA biosynthesis between different developmental stages, age, gender, during pregnancy, lactation and under conditions of certain disorders. A member of the enzymatic machinery involved in PUFA synthesis is the elongase Elongation of very long-chain fatty acids 2 (ELOVL2) that controls the elongation of PUFA with 22 carbons to produce 24 carbons precursors for the production of the omega-3 PUFA, docosahexaenoic acid (DHA, 22:6n3) and the omega-6 PUFA, docosapentaenoic (DPAn6, 22:5n6). Deletion of Elovl2 in a mouse model (Elovl2KO) leads to systemic DHA deficiency at different physiological and early lifestages, and is related to certain metabolic dysfunctions. Mitochondria of Elovl2KO mice display structural and functional impairment. Compared to wild type littermates, Elovl2KO mice do not gain as much weight after high-fat diet treatment and do not develop hepatic steatosis, despite having a higher level of the positive regulator of denovo lipogenesis, nuclear transcription factor SREBP1c. Resistance to high fat diet induced-obesity in Elovl2KO mice is abolished by DHA supplementation together with high sucrose content in the background diet. In conclusion, deletion of Elovl2 in mice leads to systemic DHA deficiency that has pleiotropic effect on mouse energy metabolism.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.

Chez le lapin male ou femelle entre 40 et 150 jours, une methode par chromatographie liquide sur silice greffee permet de separer les 7 steroides. Au cours de la maturation sexuelle, il se produit d'importantes variations de concentrations plasmatiques d'androgenes dans les 2 sexes (episodes secretoires de quelques dizaines de jours). Il n'y a pas de dimorphisme sexuel pour les taux plasmatiques d'androstenedione, de dha et de son sulfate, on peut donc penser que la secretion en est surrenalienne. Chez les femelles, il existe une correlation entre les concentrations d'androgenes et celles de corticosterone ou de cortisol, ce qui suggere que les androgenes sont d'origine surrenalienne. Quelque soit l'origine des androgenes, le probleme de la signification de l'androgenisation plasmatique se pose, notamment entre 70 et 110 jours. Au cours de la maturation sexuelle, il se produit des modifications de synthese dans la surrenale, car chez le lapin adulte male ou femelle, la corticosterone est le principal steroide secrete, tandis que chez l'animal immature, elle est faible et meme inferieure a la corticolemie. Chez le foetus, les observations sont similaires

Les acides gras polyinsatures (agp) sont presents dans la structure des membranes biologiques et jouent un role tres important d'un point de vue nutritionnel, car ils sont indispensables au bon fonctionnement de tous les tissus. Ce sont les precurseurs d'une grande variete de produits biologiquement actifs. L'acide arachidonique a ete l'agp le plus etudie, biologiquement et par le nombre de syntheses totales decrites dans la litterature. Actuellement, ce sont les acides gras a haut degre d'insaturation tels que l'acide eicosapentaenoique (epa) et l'acide docosahexaenoique (dha) qui font l'objet d'un grand nombre de travaux. L'acces a ces composes purs, naturels ou modifies, est d'un interet de premiere importance pour les etudes biologiques. Leur synthese est donc une etape indispensable pour mener a bien l'etude de leur metabolisme. Le but de ce travail repose sur la mise au point d'un agent d'homologation a six atomes de carbone permettant d'introduire en une seule etape deux doubles liaisons. Ce nouveau synthon est caracterise par une double liaison de stereochimie cis et a chaque extremite par des groupements fonctionnels suffisamment differencies pour pouvoir reagir, apres transformations, comme reactif ou substrat en reaction de wittig. Nous decrivons son utilisation dans les syntheses stereoselectives et convergentes de l'epa et du dha. Enfin, aucune etude systematique n'ayant ete realisee a ce jour, nous tenterons de definir un moyen simple pour permettre l'attribution des deplacements chimiques des atomes de carbone ethyleniques en rmn du carbone 13

Ce travail étudie l’obtention de phospholipides structurés enrichis en DHA et en acide caprylique (PC DHA-C8) par voie enzymatique. Deux voies de synthèse sont étudiées, l’acidolyse et l’estérification. Suite à un criblage enzymatique, la lipase retenue pour les deux voies de synthèse est TL-IM. Une optimisation des paramètres de la réaction d’acidolyse a été réalisée entre l’acide caprylique (C8:0) et la phosphatidylcholine de tournesol (PC) par le biais d’un plan d’expériences. Les conditions optimales déterminées sont une température de 38°C, une activité de l’eau de 0,7, une quantité d’enzyme de 15% de la masse en substrat ainsi qu’un rapport molaire C8:0/PC de 18. Ces conditions ont ensuite été utilisées pour l’acidolyse de phospholipides microalgaux riches en DHA issus de la microalgue Tisochrysis lutea afin d’obtenir de la PC DHA-C8. Les résultats n’ont pas été concluants. L’autre voie de synthèse étudiée est l’estérification par des lipases de la GPC, de l’acide caprylique et du DHA en milieu fondu. Cette réaction a été optimisée par la technique du pas par pas. Les paramètres étudiés sont la température, la quantité d’enzyme, le rapport molaire GPC/C8:0/DHA et l’application d’un vide. Pour l’obtention de PC DHA-C8, il faut fixer chacun de ces paramètres respectivement de la sorte : 45°C, 20% d’enzyme, un rapport molaire de 1/3/15 et un vide de 100 mbar. La production de PC DHA-C8, bien qu’optimisée ne dépasse pas 2% de rendement. Cependant, durant cette expérience, il a été constaté une forte production de LPC DHA, atteignant 16% sans optimisation des paramètres de synthèse
The enzymatic synthesis of structured phsopholipids enriched in DHA and caprylic acid (PC DHA-C8) is studied. Two different ways are studied, acidolysis and esterification. An enzymatic screening led to the choice of the immobilized lipase from Thermomyces lanuginosa (TL-IM) for the 2 reactions. Parameters of the acidolysis reaction between carpylic acid (C8:0) and sunflower phosphatidylcholine (PC) were optimized by means of an experimental design. The optimum conditions determined are a temperature of 38°C, an aw of 0.7, an amount of enzyme of 15% of the mass of substrate and a molar ratio of C8:0/PC of 18. These conditions were applied to the acidolysis of microalgal phospholipids from T. lutea, rich in DHA, in order to produce PC DHA-C8. The other studied reaction is the lipase catalyzed esterification of GPC with C8:0 and DHA in a solvent-free medium This reaction has been optimized by studying each factor independently. The parameters studied are the temperature, the amount of lipase, the molar ratio GPC/C8:0/DHA and the use of reduced pressure. In order to obtain PC DHA-C8, each of theses parameters are respectively set at: 45°C, 20% of enzyme, a molar ratio of 1/3/15 and a pressure of 100 mbar. The production of PC DHA-C8, although optimized, does not exceed a yield of 2%. However, during this experiment, a high production of LPC DHA is observed, up to 16% without optimization of the synthesis parameter

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

Le maintien ou le renforcement de la masse et de la fonction musculaire, altérées chez les patients BPCO, est un objectif primordial pour préserver, voire améliorer leur tolérance à l'effort, leur qualité de vie et leur survie. Afin d'optimiser la prise en charge de cette dysfonction musculaire, la réhabilitation est complétée par des interventions nutritionnelles, encore appelées réhabilitations nutritionnelles. Dans ce contexte, l'apport d'acides gras polyinsaturés de la série n-3, et plus particulièrement d'acide docosahexaénoïque (DHA), pourrait s'avérer intéressant en raison de leurs effets bénéfiques démontrés dans plusieurs pathologies chroniques. L'objectif de ce travail était donc de caractériser les effets d'une supplémentation en DHA sur la tolérance à l'effort et sur le métabolisme énergétique des muscles squelettiques de rats adultes exposés à une hypoxie comme modèle de muscle de patient BPCO au stade de l'insuffisance respiratoire chronique. La tolérance à l'effort est améliorée par le DHA, que les rats soient conditionnés en normoxie ou en hypoxie. En normoxie, les mécanismes impliqués seraient liés à un effet du DHA mimétique de celui d'un exercice d'endurance, avec une activation de l'AMPK et une amélioration de la fonction mitochondriale étudiée sur fibres musculaires perméabilisées. En hypoxie, le DHA agirait différemment, réduisant les effets de l'hypoxie sur le muscle, sans que les mécanismes mimétiques de l'exercice d'endurance ne soient clairement retrouvés. La prise de DHA chez des rats entrainés et conditionnés en hypoxie permet également un gain d'endurance mais les mécanismes à l'origine de cet effet ne sont pas élucidés et nécessitent des travaux complémentaires. Au vu des résultats sur le muscle, la supplémentation en DHA pourrait donc être bénéfique dans la prise en charge de la dysfonction musculaire dans les maladies chroniques telles que la BPCO.

Orientador: Maria Cristina Thomaz
Coorientadora: Melissa Izabel Hannas
Banca: Pedro Henrique Watanabe
Banca: Fábio Enrique Lemos Budiño
Banca: Daniela Junqueira Rodrigues
Resumo: Objetivou-se avaliar os efeitos do ácido docosahexaenóico (DHA), utilizando como fonte a farinha de alga Schizochytrium sp., nas dietas de porcas gestantes, nos terços inicial e final, e na lactação, sobre escore corporal das porcas, número de leitões nascidos vivos e totais, natimortos, peso médio (PM), ganho de peso diário (GPD), mortalidade, coeficiente de variação dos pesos (CVP), conversão alimentar (CA), composição bromatológica do colostro e do leite, proteínas do colostro e do sangue das porcas, consumo de ração, intervalo desmame-cio (IDC) e viabilidade econômica. Foram utilizadas 51 porcas para o experimento I (0 a 38 dias de gestação), 45 para o experimento II (85 a 114 dias de gestação), e 45 porcas para o experimento III (22 dias de lactação). Em todos, os animais receberam as seguintes dietas experimentais: Controle: ração controle (sem aditivo); 2000: ração controle com adição de 15 g de farinha de algas Schizochytrium sp. (2333 mg de DHA por dia por porca); 4000: ração controle com adição de 30 g de algas Schizochytrium sp. (4666 mg de DHA por dia por porca). Os animais foram distribuídos em delineamento experimental em blocos casualizados, com 3 tratamentos. Conforme os níveis crescentes de inclusão de DHA nas dietas das porcas, houve efeito linear (P<0,05) para as seguintes variáveis: Experimento I - aumentaram o GPD dos leitões, melhorou a CA e reduziu a mortalidade dos leitões; no Experimento II - aumentou o DHA no colostro, e melhora o escore corporal das... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The aim of this study was to evaluate the effects of docosahexaenoic acid (DHA), using as source the Schizochytrium sp. algae meal, rich in docosahexaenoic acid (DHA), in diets of sows over the initial and final thirds of gestation, and lactation on body condition score, number of piglets born alive, stillborn, and total, average body weight (BW), daily weight gain (DWG), mortality, body weight coefficient of variation (BWCV), feed conversion (FC), chemical composition of colostrum and milk, proteins of colostrum and blood of sows, feed consumption, weaning-to-heat interval (WHI), and economical analysis. Fifty-one sows were assignet to experiment I (0 to 38 days of gestation), 45 sows were assigned to experiment II (85 to 114 days of gestation), and 45 sows were allocated to experiment III (22 d in lactation). In all experiments, animals received the following experimental diets: Control, control diet (with no algae meal); 2000, control diet supplemented with 15 g of Schizochytrium sp. algae meal (2333 mg DHA/d per sow); and 4000, control diet supplemented with 30 g of algae meal (4666 mg DHA/d per sow). Animals were sorted to the three treatments in a complete randomized block design. As the level of DHA in diets of sows increased, it was observed a linear effect (P < 0.05) for the following parameters: Experiment I - algae meal increased the ADG of piglets, and reduced the FC and mortality of piglets; in Experiment II - algae meal increased colostrum DHA concentration, and... (Complete abstract click electronic access below)
Doutor

The industrial PhD project presented here is part of the R&D strategies of the Lipinutragen company. The innovation brought by the company concerns nutrilipidomics, i.e. the correlation between the lipid composition (in fatty acids) of the cell membrane and lipid-based nutraceuticals, especially starting from the well-known dependence of the lipid composition on the intake of essential fats, omega- 6 and omega-3 polyunsaturated fatty acids. Among the results obtained from the membrane lipidomic profiles, the case of autistic subjects is here highlighted, showing the significant deficiency of docosahexaenoic acid (DHA). The activity during the PhD was devoted to the nutrilipidomic approach. Part of the activities were devoted to scientific research in lipidomics: a) the study of lipidomic profiles in the frame of two collaboration projects: one with the group of Dr. I. Tueros at AZTI, Bilbao, regading obese population, and the other one regarding seed germination with the changes of the fatty acid profiles with the group of prof. A. Balestrazzi of the University of Parma; b) the liposome preparation for protection and lifetime prolongation of the peptide somatostatin, which was an important premise to the formulation of the DHA-containing microemulsion. The activities was also focused on the development of DHA-containing nutraceutical formulations in the form of emulsion, overcoming the difficulty of the capsule ingestion, to be administered orally. The work pointed to study the combination of active ingredients, based on the previous know-how regarding the bioavailability for the cell membrane incorporation. The ingredients of the formulation were studied and tested in vitro for the bioavailability of DHA to be incorporated in the cell membranes of different types of cultured cells. Part of this study is covered by non-disclosure agreement since it belongs to the know-how of Lipinutragen.

In the last century the amino alcohol serinol (2-amino-1,3-propanediol) has become a common intermediate for several chemical processes. Since the 40's serinol was used as precursor for synthesis of antibiotics (e.g. chloramphenicol). In the last years, new scopes of application were discovered. Serinol is used as building block in the synthesis of X-ray contrast agents, pharmaceuticals (anti-inflammatory or analgesic drugs) or in the cosmetics industry. It can either be obtained by chemical processes based on 2-nitro-1,3-propanediol, dihydroxiacetone (1,3-Dihydroxypropan-2-one or simply DHA) and ammonia [59]. One way to synthesize Serinol, in fact, is reacting DHA in a catalyzed reaction using Pt supported on carbon as catalyst. The DHA can be synthesized from Glycerol (1,2,3-propanetriol or glycerine) with a selective oxydation of the secondary carbon. The reaction from Glycerol to DHA must be catalyzed and nowadays only one method is well known. The method to obtain DHA uses gluconobacter oxydans as catalyst for the selective glycerol oxydation. Anyway the method presents many drawbacks cost not yet solved such as low productivity and high production. It is fundamental to underline how nowadays glycerol plants are closing and others are opening that use glycerol as a raw material as a result of the large surplus of glycerol that is formed as a byproduct in manufacturing biodiesel fuel by transesterification of seed oils with methanol. Over the last twenty years, indeed, biodiesel emerged as a viable fuel and as a fossil diesel additive to replace sulfur, whose content is being progressively lowered according to tighter environmental legislation. However, the increasing production of biodiesel is not artificially sustained and is predicted to spread and increase, as biodiesel provides sufficient advantages to merit subsidy. Besides the closure of production plants, industry reacted to this situation stimulating research to find new applications of glycerol as a low-cost feedstock for functional derivatives either for mass consumption, such as additives for concrete, or as a precursor of valued fine chemicals [60]. One investigated application of glycerol is exact its possible use as starting material for DHA synthesis (figure 1.4, chapter 1). The aim of this work was to investigate for new possibilities to transform glycerol into DHA, avoinding gluconobacter oxydans utilisation. In particular the attention was posed on new catalytic solutions investigating inorganic catalysts for the selective oxydation of glycerol. Two catalytic system were investigated. An heterogenous solution using noble metal nanoparticles supported on carbonious supports (mono- and bi-metallic systems) and an homogeneous one using an organometallic catalyst based on Pd. The study was approach from experimental and theoretical point of view becasue the reaction mechanism is not still clear. The research bring us to investigate deeply many weakness of the reaction and not still clear in literature, such as an efficient analytical method, problems linked to the catalysts aging, their deactivation and synthesis.

Orientador: Eliana Pereira Araújo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Enfermagem
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Resumo: O processo cicatricial é um fenômeno complexo e altamente especializado, onde é necessário a integração de vários tipos de células, fatores de crescimento e enzimas. O processo ocorre através de várias fases: formação do coágulo; inflamação; reepitelização; angiogênse; formação do tecido de granulação; contração da ferida; formação da cicatriz; e, remodelamento do tecido. Estudos recentes revelaram que o receptor acoplado a proteína G, GPR120, é ativado por ácidos graxos insaturados de cadeia longa e é importante sinalizador relacionado com vários aspectos da função celular, incluindo o efeito anti-inflamatório exercido através da ligação do GPR120 com estes ácidos graxos. Nosso objetivo foi avaliar a expressão e atividade do GPR120 em pele íntegra e em feridas cutâneas de ratos Wistar tratados com o ácido docosahexaenóico (DHA). Tal estudo se justifica pela importância do melhor entendimento dos mecanismos imunoreguladores e inflamatórios da cicatrização de feridas que levem a abordagens terapêuticas mais efetivas. Para tal, pele íntegra e feridas cutâneas de ratos Wistar foram avaliadas por imunohistoquímica, imunoblot e PCR em tempo real. Nós encontramos que o GPR120 é expresso na pele e em tecido cicatricial de Wistar, fato ainda nunca relatado na literatura. Além disso, o GPR120 foi co-localizado em queratinócitos importante células nativas da epiderme. O tratamento tópico das feridas com DHA acelerou a cicatrização. Os efeitos moleculares deste tratamento na ferida foram: a associação de GPR120 e 'beta'-arrestina; diminuição de Interleucina-1 Beta (IL-1'beta'); aumento do Fator Transformador Beta (TGF-'beta') e Ivolucrin (IVL), um marcador de queratinócitos
Abstract: Wound healing is a complex process and highly specialized, where it is necessary to integrate various types of cells, growth factors and enzymes. The healing process involves several phases: clot formation, inflammation, reepithelialization, angiogenesis, formation of granulation tissue, wound contraction, scar formation, and tissue remodeling. Recent studies have shown that the G protein-coupled receptor, GPR120 is activated unsaturated long chain, such as docohexaenoic acid, and are important signaling relating to various aspects of cellular function, including inflammatory effect exerted by connecting the GPR120 with these fatty acids. Our objective was to evaluate the expression and activity of the GPR120 in intact skin and in skin wounds in rats treated with docosahexaenoic acid (DHA). This study is justified by the importance of better understanding of inflammatory and immunoregulatory mechanisms of wound healing leading to more effective therapeutic approaches. For this purpose, intact skin and wounds of rats were evaluated by immunohistochemistry, immunoblotting and real-time PCR. We found that GPR120 is expressed in the skin and scar tissue of Wistar fact still never been reported in the literature. Additionally, the GPR120 co-located important native keratinocyte cells of the epidermis. The topical treatment of wounds with DHA accelerated healing. The molecular effects of this treatment on the wound were: the association of 'beta'-arrestin and GPR120; decreased interleucin - 1 beta ( IL - 1'beta'), increasing the Transformer Factor Beta (TGF - 'beta') and Ivolucrin (IVL), a marker of keratinocyte
Mestrado
Enfermagem e Trabalho
Mestra em Enfermagem

L’ostéoporose est une pathologie osseuse chronique dont la prévalence augmente avec l’âge. Elle résulte d’une altération de la quantité et de la qualité du squelette (micro-architecture et densité minérale osseuse) avec pour conséquence un risque accru de fracture et de comorbidités. Selon des études épidémiologiques récentes, on estime en Europe que cette pathologie touche environ 30% des personnes de plus de 50 ans, entraine une surmortalité de près de 30%, pour un coût socio-économique annuel proche de 30Mds d’euros. La prise en charge actuelle est limitée par une prophylaxie non systématique et les effets secondaires des traitements curatifs dont la compliance ne dépasse à 20% à 1 an. C’est pour répondre à ces challenges que de nouvelles alternatives innovantes de prévention nutritionnelles reposant sur l'utilisation combinée d’actifs alimentaires est plus que jamais nécessaire. La fisétine est un polyphénol de la classe des flavonoïdes décrit pour son activité anti-inflammatoire. On la trouve principalement dans les fruits rouges comme la fraise. Nous avons précédemment démontré comment et dans quelle mesure la fisétine inhibe la différenciation des ostéoclastes, stimule l’activité des ostéoblastes, et prévient la perte osseuse sur des modèles ostéoporotiques murins. De façon à optimiser cet effet bénéfique nous avons recherché des partenaires nutritionnels susceptibles d’agir en synergie avec la fisétine. Nous avons ainsi montré que le DHA, un acide gras polyinsaturé de type oméga 3, pouvait potentialiser les effets biologiques de la fisétine au niveau osseux. Nos travaux ont permis de mettre en évidence la forme le DHA et les ratios de combinaisons les plus pertinents. Des travaux collaboratifs partenariaux ont permis de développer des formulations et extraits sur la base des résultats de recherche. L’objectif est à terme la transposition de ces travaux vers les patients, afin de fournir des outils de prévention commercialisables et scientifiquement étayés
Osteoporosis is a chronic bone disease whose prevalence increases with age. It results from an alteration in the quantity and quality of the skeleton (micro-architecture and bone mineral density) with an increased risk of fracture and co-morbidities. According to recent epidemiological studies, it is estimated that this pathology affects around 30% of people over 50 years of age in Europe, resulting in an excess mortality rate of almost 30%, and in an annual socio-economic cost of nearly 30 billion euros. Current treatment is limited by non-systematic prophylaxis and the side effects of curative treatments, with compliance rates of no more than 20% within one year. To meet these challenges, new innovative preventive nutrition alternatives based on the combined use of active food ingredients are more necessary than ever. Fisetin is a polyphenol of the flavonoid class described for its anti-inflammatory activity. It is mainly found in red fruits such as strawberries. We have previously demonstrated how and to what extent fisetin inhibits osteoclast differentiation, stimulates osteoblast activity, and prevents bone loss in murine osteoporotic models. In order to optimize this beneficial effect, we have sought nutritional partners who could act in synergy with fistin. We have shown that DHA, an omega-3 polyunsaturated fatty acid, can potentiate the biological effects of fisetin at the bone level. Our work has made it possible to highlight the form of DHA and the most relevant combination ratios. Collaborative work in partnership has made it possible to develop formulations and extracts on the basis of the research results. The long-term objective is to transpose this work to patients, in order to provide marketable and scientifically supported prevention tools

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.

Tesis de grado para obtener el grado de Maestra en Ciencias de la Salud
El glioblastoma es el tipo de tumor cerebral más común y agresivo, ya que tiene una mortalidad superior al 90% en 5 años, aún con tratamientos combinados agresivos, los cuales tienen efectos secundarios graves. Debido a ello, se buscan nuevas formas de tratarlo de manera más segura y con menos efectos secundarios. La manipulación del sistema endocanabinoide es una alternativa atractiva, ya que este sistema ha demostrado funciones anticancerígenas, inhibiendo la proliferación, supervivencia e invasividad de las células tumorales. Los endocanabinoides son compuestos derivados de ácidos grasos y su producción se relaciona con el consumo de estos en la dieta, particularmente, con los ácidos grasos esenciales omega-3, como el ácido docosahexaenoico (DHA), que también ha mostrado tener propiedades anticancerígenas. Debido a su relación metabólica y sus efectos antitumorales, es relevante comprobar la posible sinergia entre endocanabinoides y DHA para potenciar sus efectos anti-tumorales. El objetivo de este estudio fue identificar cambios en la expresión de receptores para endocanabinoides en una línea celular de glioblastoma humano suplementada con DHA. Para este fin, se emplearon cultivos de la línea U87MG, que fueron suplementados con DHA a concentraciones de 50, 100 y 150 μM durante 24, 48 o 72 horas. La expresión de receptores para endocanabinoides se determinó por medio de inmunofluorescencia y western-blot. Los resultados obtenidos muestran un incremento significativo en la expresión de los receptores CB1 en las células de glioblastoma después del tratamiento con DHA a una concentración de 50 μM después de 72 horas de tratamiento; sin embargo, a concentraciones más elevadas, el tratamiento provocó una disminución significativa en la expresión de CB1 y CB2, correspondiendo con un aumento en la muerte celular. Los resultados sugieren que la suplementación con DHA puede modular la expresión de receptores para endocanabinoides en las células de glioblastoma humano, pero sus efectos son variables en relación con la concentración y tiempo de exposición empleados.
Proyecto financiado por la UAEM: 5026/2020CIB

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.

Il mesotelioma pleurico maligno (MPM) è un tumore molto aggressivo e rapidamente progressivo che si sviluppa a livello del mesotelio che compone la pleura; questa neoplasia può assumere diversi sottotipi istologici (epitelioide, bifasico e sarcomatoide), i quali sono strettamente correlati alla prognosi. Le modificazioni epigenetiche che avvengono nelle fasi di iniziazione e progressione del MPM possono svolgere un ruolo fondamentale nel regolare negativamente il crosstalk tra tumore e sistema immunitario, contribuendo a mantenere un microambiente tumorale immunosoppressivo. Conoscere più dettagliatamente il panorama epigenetico del MPM può contribuire a definire il razionale per nuove terapie antitumorali e porre le basi per studi di combinazione che prevedano l’utilizzo di farmaci epigenetici con farmaci immunoterapeutici. Con il presente studio abbiamo voluto valutare, in un primo momento, le modificazioni nel profilo di espressione genica di 10 linee di MPM, di diverso istotipo, trattate con la guadecitabina, un agente demetilante il DNA di seconda generazione, tramite la piattaforma nCounter di Nanostring. I risultati ottenuti tramite Ingenuity Pathway Analysis (IPA) hanno mostrato che la guadecitabina era in grado di indurre l’attivazione dei geni coinvolti nel crosstalk tra cellule dendritiche e natural killer nel 50% delle linee cellulari di MPM indagate, accompagnata dall’attivazione di altre componenti coinvolte nella risposta immunitaria a infezioni e infiammazioni. I fattori trascrizionali “upstream” più frequentemente attivati appartenevano al pathway di segnalazione dell’interferon (IFN)-γ. Inoltre, è stata riscontrata l’up-regolazione (fold change medio (mFC) ≥ 1.5) di molecole immuno-relate, come NY-ESO-1 (mFC=13.16), MAGE-B2 (mFC=13.09), CD70 (mFC=5.27) e CTLA-4 (mFC=4.81). Abbiamo inoltre effettuato analisi istotipo-specifiche per esplorare le modificazioni molecolari indotte dalla guadecitabina nei 3 sottotipi di MPM. La guadecitabina ha indotto l’up-regolazione dell’espressione di marcatori del fenotipo epiteliale (es. CDH1, EPCAM e PECAM1), osservata ad alti livelli nelle linee cellulari sarcomatoidi; ciò è stato associato alla down-regolazione di molecole di origine mesenchimale (es. CDH2 e NCAM) e induttori della cascata metastatica (es. CDH11). Successivamente abbiamo comparato gli effetti immunomodulatori della guadecitabina con quelli di altri farmaci epigenetici (gli inibitori delle iston acetiltransferasi (HDAC) VPA e SAHA o l’inibitore di EZH2 EPZ-6438) da soli o in combinazione con la guadecitabina in 5 linee cellulari di MPM (2 sarcomatoidi, 1 bifasica e 2 epitelioidi). Analisi citofluorimetriche e molecolari hanno rivelato che la guadecitabina up-regolava l’espressione delle molecole immuno-relate, quali HLA di classe I (mFC=1.59), ICAM-1 (mFC=3.27), PD-L1 (mFC=2.13), e NKG2DL (MICA mFC=1.88, MICB mFC=2.42, ULBP2 mFC=3.16), inducendo/up-regolando l’espressione dei Cancer Testit Antigens (CTA) NY-ESO-1, MAGE-A1 e MAGE-A3; il VPA up-regolava l’espressione degli antigeni di HLA di classe I (mFC=1.50), PD-L1 (mFC=2.76), NKG2DL (MICA mFC=1.69, MICB mFC=2.67, ULBP2 mFC=3.26) e quella dei CTA MAGE-A1 e MAGE-A3, rispettivamente in 2/5 e 3/5 linee cellulari di MPM; il SAHA up-regolava l’espressione di MICA (mFC=1.57), MICB (mFC=4.05) e MAGE-A1 e MAGE-A3, rispettivamente in 2/5 e 4/5 linee cellulari; per contro, l’EPZ-6438 ha mostrato minime capacità immunomodulanti, inducendo solamente NY-ESO-1 e up-regolando l’espressione di PD-L1, MICB e ULBP2 in 1 linea cellulare ciascuno. Contrariamente ai risultati eterogenei ottenuti dai singoli farmaci, l’associazione di VPA, SAHA o EPZ-6438 alla guadecitabine ha rafforzato le capacità immunomodulanti di quest’ultima, influenzando l’espressione di tutte le molecole indagate. Specificatamente, le combinazioni di guadecitabine con VPA, SAHA o EPZ-6438 up-regolavano l’espressione degli antigeni HLA di classe I (mFC=2.21, 2.03, o 2.29 rispettivamente), di ICAM-1 (mFC=4.09, 4.63, o 5.33), di PD-L1 (mFC=6.95, 2.42, o 2.50), di MIC-A (mFC=3.48, 2.00, o 2.23), di MIC-B (mFC=6.80, 2.48, o 2.81) e di ULBP2 (mFC=13.45, 3.40, o 4.11). Infine, livelli di up- regolazione/induzione maggiori sono stati osservati per i CTA a seguito di tutti e 3 i trattamenti combinati rispetto alla guadecitabina in singolo. La modulazione delle caderine è stata influenzata dal sottotipo istologico di MPM: l’espressione di CDH1 è stata indotta dalla guadecitabina in singolo e dalla sua combinazione con VPA, SAHA e EPZ-6438 nelle 2 linee cellulari sarcomatoidi, costitutivamente negative per l’espressione del gene; l’espressione di CDH2 è stata up-regolata dal VPA e dal SAHA singoli in 1/5 linee cellulari e dalle combinazioni di guadecitabina con VPA o SAHA, rispettivamente in 3/5 o 1/5 linee cellulari di MPM; ciononostante, non è stata osservata alcuna up-regolazione del gene nelle 2 linee cellulari epiteliodi, costitutivamente negative per l’espressione di CDH2. In conclusione, dalle analisi approfondite del pannello di espressione genica abbiamo confermato che la guadecitabina è in grado di up-regolare/indurre l’espressione di molecole immunitarie e immuno- relate cruciali per il crosstalk tra il tumore e il sistema immunitario; inoltre, abbiamo dimostrato che essa induce l’attivazione di geni correlati all’IFN, soprattutto nel fenotipo sarcomatoide, supportando l’ipotesi che i demetilanti possano aumentare la risposta immunitaria contro il MPM, potenzialmente anche del tipo istologico più aggressivo; la modulazione delle molecole di adesione tendente verso il fenotipo epitelioide suggerisce la possibilità di revertire la transizione epitelio-mesenchima, cruciale nel processo di metastatizzazione. Infine, combinando la guadecitabina con farmaci inibitori delle HDAC/EZH2 ha rafforzato la sua attività immunomodulante, fornendo il razionale per studi di associazione di farmaci epigenetici e agenti immunoterapici in modo da aumentare l’efficacia di questi ultimi nel trattamento del mesotelioma.
Malignant pleural mesothelioma (MPM) is a highly aggressive and rapidly progressive tumor that affect the mesothelium componing the pleura; it can acquire different histological subtypes (mainly epithelioid, biphasic, and sarcomatoid MPM), which are of prognostic significance. Epigenetic modifications occurring during MPM initiation and progression may play a relevant role in negatively regulating the crosstalk between the tumor and the immune system, as well as contributing to the highly immunosuppressive microenvironment. A better understanding of MPM epigenetics will contribute to refine antitumor strategies, laying the ground for epigenetic-based immunotherapy. The present study evaluated, in the first instance, changes in the gene expression fingerprint of 10 MPM cell lines of different phenotype treated with the second-generation DNA hypomethylating agent (DHA) guadecitabine, through the Nanostring Oncology panel with nCounter readout. Ingenuity pathway analysis results revealed that guadecitabine induced the activation of natural killer and dendritic cells signaling pathways in 50% of MPM cell lines, followed by the activation of other components involved in the immune system response to infections and inflammation. Besides, the most frequently activated upstream regulators belonging to the interferon (IFN)-γ signaling pathway. Also, the up- regulation (mean fold change (mFC) ≥ 1.5) of key immune-related molecules, such as the NY-ESO-1 (mFC=13.16), MAGE-B2 (mFC=13.09), CD70 (mFC=5.27), and CTLA-4 (mFC=4.81) was reported. We also performed histological type-specific investigations to explore molecular changes induced by guadecitabine among the 3 histotypes. Guadecitabine induced the up-regulation of the expression of epithelial markers (e.g., CDH1, EPCAM, PECAM1), observed at higher levels in sarcomatoid cell lines; this was accompanied by the down-regulation of mesenchymal origin molecules (e.g., CDH2, NCAM), and inductor of metastatic signals (e.g., CDH11). Secondly, the immunomodulatory effects of guadecitabine were compared to those of different epigenetic drugs (the histone deacetylase (HDAC) inhibitors VPA and SAHA, or the EZH2 EPZ- 6438), alone or in combination with guadecitabine, in 5 MPM cell lines (two sarcomatoid, one biphasic, and two epithelioid). We performed cytofluorimetric and molecular qRT-PCR analyses and, in this regard, results showed that guadecitabine up-regulated the expression of immune-related molecules, such as HLA class I antigens (mFC=1.59), ICAM-1 (mFC=3.27), PD-L1 (mFC=2.13), and NKG2DLs (MIC-A mFC=1.88, MIC-B mFC=2.42, and ULBP2 mFC=3.16), and up-regulated/induced Cancer Testis Antigens (CTA: NY-ESO-1, MAGE-A1, and MAGE-A3) expression; VPA up-regulated the expression of HLA class I antigens (mFC=1.50), PD-L1 (mFC=2.76), NKG2DLs (MIC-A mFC=1.69, MIC-B mFC=2.67, and ULBP2 mFC=3.26), and the expression of CTA MAGE-A1 and MAGE-A3 in 2/5 and 3/5 MPM cell lines, respectively; SAHA up- regulated the expression of MICA (mFC=1.57), MICB (mFC=4.05), MAGE-A1 and MAGE-A3 in 2/5and 4/5 MPM cell lines, respectively; conversely, EPZ-6438 induced minimal immunomodulatory effects, inducing only NY-ESO-1 and up-regulating PD-L1, MIC-B, and ULBP2 expression in 1 MPM cell line each. Despite the heterogeneous activities of single epigenetic drugs, the addition of both VPA, SAHA, and EPZ-6438 to guadecitabine strengthened the immunomodulatory effects of the latter, by affecting the expression of all investigated molecules. Specifically, guadecitabine plus VPA, SAHA, or EPZ-6438 upregulated the expression of HLA class I antigens mFC=2.21, 2.03, or 2.29; ICAM-1 mFC=4.09, 4.63, or 5.33; PD-L1 mFC=6.95, 2.42, or 2.50; MIC-A mFC=3.48, 2.00, or 2.23; MIC-B mFC=6.80, 2.48, or 2.81; ULBP2 mFC=13.45, 3.40, or 4.11, respectively. Lastly, higher levels of upregulated/induced CTA expression were observed after all 3 combination treatments versus guadecitabine alone. Cadherins modulation was MPM histotype-related: CDH1 expression was induced in the 2 constitutive-negative sarcomatoid MPM cell lines by guadecitabine alone or combined with VPA, SAHA, or EPZ-6438; CDH2 expression was upregulated by VPA or SAHA in 1/5 cell lines, and by guadecitabine plus VPA or SAHA in 3/5 or in 1/5 MPM cell lines, respectively; however, no induction of CDH2 have been reported in the constitutive negative epithelioid cell lines. Overall, from comprehensive gene expression panel analyses, we confirmed that guadecitabine induced/up-regulated the expression of immune and immune-related molecules, pivotal in the tumor- immune system crosstalk; also, we highlighted that guadecitabine-induced activation of IFN-related genes, especially in the sarcomatoid phenotype, supporting the hypothesis that DHA could increase the immune response against MPM, potentially also with sarcomatoid features; moreover, the modulation of adhesion molecules towards the epithelial type suggests the possibility to revert the epithelial-to- mesenchymal transition (EMT) event, crucial in the invasion-metastasis cascade. Also, combining guadecitabine with HDACi/EZH2i strengthened its immunomodulatory capabilities, laying the rationale for epigenetic drugs-based immunotherapies, to enhance efficacy of these strategy in the MPM clinic.

Cette thèse visait à évaluer l’effet d’une alimentation pourvue en graines de lin riches en acide a-linolénique (ALA) et en microalgues riches en acide docosahexaenoïque (DHA) sur le métabolisme des lipides et la qualité de la viande de porc. Les régimes n’ont pas eu d’effet sur les performances de croissance et les critères de qualité de la carcasse mesurés en abattoir. La digestibilité des acides gras polyinsaturés (AGPI) n-3, leurs quantités dans les régimes et les traitements technologiques subis par la graine de lin ont impacté le dépôt d’ALA et de DHA dans les lipides totaux et les fractions neutres et polaires des tissus. La quantité de malonaldéhyde, représentatif de la peroxydation des acides gras, du muscle longissimus dorsi et du tissu adipeux sous cutané dorsal (TAD) a été augmentée avec un apport de microalgues.Une odeur plus prononcée des rôtis issus du régime microalgues a été identifiée par un jury entrainé. Enfin, les activités et l’expression des gènes de la lipogenèse et de la synthèse des AGPI n-3 ont été diminuées au niveau hépatique avec l’apport de microalgues. Un second objectif visait à recenser les facteurs de variation du dépôt des AGPI n-3 dans les tissus porcins pour les intégrer dans un modèle prédictif de ce dépôt. En raison d’une grande variabilité entre individus, seuls des modèles linéaires ont pu être établis en considérant les quantités digestibles d’ALA et de DHA ingérées, le sexe des animaux et l’effet du lot. Enfin, la thèse a permis de proposer des équations de calibration de spectrométrie en proche infra-rouge (SPIR) afin de déterm
Pigs used in this thesis were fed linseed rich in a-linolenic acid (ALA) and microalgae rich in docosahexaenoic acid (DHA) to evaluate the effect of diet on lipid metabolism and pork quality. Fatty acid (FA) composition of the diets did not have any effect on pig performances and carcass parameters measured at slaughter. The digestive utilization, the quantity of n-3 polyunsaturated FA (PUFA) in the diet and the technological treatment applied to linseed were identified as modifiers of the deposition of ALA and DHA in total, neutral and polar lipids. They also had an impact on the activity of lipogenesis enzymes and on the gene expression involved in n-3 PUFA synthesis in the liver. The malonaldehyde content, representative of the FA lipoperoxydation, measured in longissimus dorsi muscle and subcutaneous adipose tissue of the back (SCB),significantly increased with the supply of microalgae and with linseed to a lesser extent. Finally, the odor of the meat from pigs fed microalgae was more pronounced than meat from pigs fed linseed or a mix 75%/25% of linseed and microalgae. From results obtained in animal experiments, linear models were built to predict n-3 PUFA deposition in pig tissues from quantities of digestible ALA and DHA ingested by animals. Finally, a last part of the thesis allowed characterizing the FA composition of the SCB by near infrared spectroscopy (NIRS) in order to quickly identify the meat enriched with n-3 PUFA

Over the past two decades, Americans' omega-3 FA intake has been decreasing while the U.S. rate of depression diagnoses and antidepressant prescriptions have been increasing. The purpose of this thesis was to examine the relationship between dietary omega-3 FA intake and depression scores using a sample data set of U.S. adult survey participants in the 2009-2010 National Health and Nutrition Examination Survey (NHANES). Specifically, this study examined the relationship between depression scores and dietary EPA, dietary DHA and total 30-day supplementation of polyunsaturated fatty acids. Results showed that there was a statistically significant relationship between each independent variable and total depression scores; furthermore, indicating that as dietary EPA, DHA and 30-day PUFA intakes increase, depression scores decrease. Although results were statistically significant, the R ² values suggest low predictive power; thus, results are not generalizable to the entire population.

Ce travail est une contribution à l'étude de la cristallisation de la dihydroxyacétone, agent auto-bronzant utilisé dans le domaine cosmétique depuis de nombreuses années pour ses propriétés de pigmentation de la peau. La dihydroxyacétone étant un sucre, l'étude de sa cristallisation doit tenir compte du rôle important de la viscosité liée à la nature même de ce composé. En effet, la haute valeur de la viscosité du milieu est à l'origine des difficultés rencontrées dans l'industrie sucrière car, cette propriété influe en particulier sur le phénomène de diffusion (transfert de matière sur la surface cristalline). Les conséquences sont un ralentissement de la vitesse de croissance et une accélération de la vitesse de nucléation. Aussi, afin de mieux comprendre ces phénomènes, dans la première partie de notre étude, nous présentons les propriétés de la dihydroxyacétone ainsi que les notions théoriques concernant la cristallisation. La seconde partie est consacrée à l'étude de la solubilité et de la cristallisation de la dihydroxyacétone. L'optimisation de sa cristallisation a ensuite conduit à la mise en oeuvre d'un nouveau procédé. Ce procédé est novateur dans son principe de contrôle de la viscosité pour faciliter la diffusion des particules, améliorant ainsi les résultats et les conditions de cristallisation. Ce procédé peut, en outre, être extrapolé sur d'autres milieux visqueux.

La présente étude évalue la capacité de la formulation d’oméga-3 EPA:DHA 6:1, une formulation capable d’induire la formation continue de monoxyde d’azote par la NO synthase endothéliale, à améliorer la dysfonction endothéliale liée à l’âge établie chez le rat. La dysfonction endothéliale liée à l’âge est caractérisée par une altération des composantes de la relaxation et une augmentation des réponses contractiles dépendantes de l’endothélium. L’âge augmente le stress oxydant vasculaire, l’expression de la NADPH oxydase, COX-2, eNOS, ACE, AT1R, et des marqueurs de senescence, alors que la COX-1 est sous-exprimé. La formulation EPA:DHA 6:1 améliore la composante NO, diminue l’EDCF et le stress oxydant vasculaire, et normalise l’expression des protéines cibles. En conclusion, la consommation chronique de EPA:DHA 6:1 améliore la dysfonction endothéliale liée à l’âge chez le rat, probablement en prévenant l’activation du système angiotensine locale et le stress oxydant en résultant
EPA:DHA 6:1 omega-3 formulation has been shown to induce a sustained endothelial NO synthase-derived formation of nitric oxide. This study examined if the intake of EPA:DHA 6:1 improves an established ageing-related endothelial dysfunction. Ageing-related endothelial dysfunction was characterized by a blunted NO-mediated component of relaxation, abolished EDH-mediated component and increased COX-derived endothelium-dependent contractile responses. Ageing increased vascular oxidative stress, expression of NADPH oxidase subunits, COX-2, eNOS, ACE, AT1R, and senescence markers, whereas COX-1 was down-regulated. Chronic intake of EPA:DHA 6:1 improved the NO-mediated relaxations, reduced EDCFs, vascular oxidative stress and normalized the expression of protein markers. In conclusion, chronic intake of EPA:DHA 6:1 prevented the ageing-related endothelial dysfunction in old rats, most likely by preventing activation of the local angiotensin system and the subsequent vascular oxidative stress

Introduction: La consommation de poissons gras semble diminuer le risque de déclin cognitif lors du vieillissement. Cet effet serait potentiellement attribuable à deux acides gras oméga-3 concentrés dans le poisson: l'acide docosahexaénoïque (DHA) et l'acide eicosapentaénoïque (EPA). Cependant, ce lien n'est pas valide chez les personnes porteuses du génotype de l'apolipoprotéine E epsilon 4 (ApoE4), le facteur de risque génétique le plus important pour la maladie d'Alzheimer. Ces données suggèrent un débalancement dans le métabolisme du DHA chez les porteurs de l'ApoE4. Objectif: Évaluer si le polymorphisme de l'ApoE4 débalance le métabolisme d'un traceur DHA marqué au carbone 13 ([indice supérieur 13] C-DHA). Méthodologie: Quarante participants de plus de 50 ans ont été recrutés. De ce nombre, 14 sont des hommes et 26 sont des femmes. Ils ont consommé une dose unique de 50 mg de [indice supérieur 13] C-DHA afin de suivre son métabolisme sur une période de 28 j. L'incorporation plasmatique ainsi que la p-oxydation du [indice supérieur 13] C-DHA ont été suivis à l'aide d'échantillons de plasma et d'haleine prélevés avant la prise du [indice supérieur 13] C-DHA et 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 7 j, 14 j, 21 j et 28 j post-consommation. Résultats: 6 participants étaient porteurs de l'ApoE4 et 34 étaient non-porteurs. L'incorporation plasmatique du [indice supérieur 13] C-DHA était 3,7 fois moins grande chez les porteurs de l'ApoE4 2 h post-consommation (p = 0,04) et 1,9 fois moins grande 4 h post-consommation (p = 0,04) comparativement aux non-porteurs de l'ApoE4. La beta-oxydation du [indice supérieur 13] C-DHA était 1,8 fois plus grande chez les porteurs de l'ApoE4 7 j post-consommation (p = 0,05), 2,4 fois plus grande 21 j post-consommation (p = 0,02) et 2,3 fois plus grande 28 j post-consommation comparativement aux non- porteurs. Discussion/conclusion: Ces résultats suggèrent que les porteurs de l'ApoE4 montrent un débalancement dans leur métabolisme du [indice supérieur 13] C-DHA. Ceci pourrait modifier l'apport recommandé en DHA chez les porteurs de l'ApoE4 afin que ceux-ci soient protégés contre le déclin cognitif par la consommation d'acides gras oméga-3.

Memoria para optar al Título de Bioquímico
El cáncer es una patología que ha concentrado por muchos años, una gran atención y constante investigación en búsqueda de nuevos fármacos para su tratamiento. Con el objeto de desarrollar nuevas terapias contra esta enfermedad, analizamos el potencial antineoplásico de dos nuevos compuestos orgánicos: 4,4- Dimetil-5-8-dihidroxinaftaleno-1-ona (DHN) y 9,10-dihidroxi-4,4-dimetil-5,8-dihidro- 1(4H)-antraceno (DHA). En primera instancia estudiamos el efecto de estos compuestos sobre la proliferación de células tumorales humanas U937 y K562, mediante el método de exclusión del colorante azul de tripan. Los resultados mostraron en ambas líneas celulares que DHN y DHA provocaron una disminución de la viabilidad en forma dosis-dependiente, con selectividad sobre células tumorales y no sobre células normales (PBMC) con un IC50 a 72 horas de 7,96 μM para DHA y 40,39 μM para DHN en U937 y de 0,68 μM para DHA y 32,10 uM para DHN en K562. Sabiendo que señales proinflamatorias favorecen el proceso carcinogénico, se estudió el efecto que tendrían ambos compuestos sobre la expresión de Cox-2 en células U937, previamente diferenciadas a macrófago con TPA y estimuladas con LPS. Los inmunowesterblot mostraron una disminución de la inducción y expresión de dicha enzima por acción de DHA a una concentración de 10 uM. Mediante ensayos de viabilidad se observó que ambos compuestos en combinación con extractos naturales no exhiben mayor efecto inhibitorio sobre la viabilidad; sin embargo, con indometacina un antiinflamatorio conocido, se potencia el efecto inhibitorio que tiene DHA. Por otra parte, mediante citometria de flujo se midió el efecto de DHN y DHA sobre el potencial de membrana mitocondrial con rodaminal23 y la generación de ROS mediante CM-H2DCFDA. Los resultados muestran una despolarización de la membrana por acción de ambos compuestos en especial por DHA y una baja generación de ROS por parte de ambos. Dado que las células NK constituyen un importante mecanismo de defensa antitumoral, se evaluó el efecto de DHN y DHA sobre la actividad citotóxica mediada por células NK midiendo la liberación de 51Cr desde células blanco K562. Los resultados indicaron que ambos compuestos no alteran la respuesta normal de las células NK. De acuerdo a estos resultados podemos inferir que DHA y DHN ejercen una importante acción antitumoral inhibiendo la proliferación de células tumorales. Sin embargo el efecto de DHA es mayor al de DHN, esto podría explicarse probablemente debido a un efecto citotóxico mediado por la despolarización de la membrana mitocondrial, y/o inhibiendo la respuesta proinflamatoria mediada por Cox-2, sin generación de especies reactivas por parte de ningún compuesto y sin afectar la respuesta inmune innata
Cancer is a pathology that has concentrated, a great deal of attention in biological research aimed principally to find new drugs for its treatment. For this reason we decided to study the antitumoral effects of two new organic compounds: 4,4-Dimetil-5-8-dihidroxinaftaleno-1-ona (DHN) and 9,10-dihidroxi-4,4-dimetil-5,8- dihidro-1(4H)-anthraceno (DHA). In the first place we studied the effect of these compounds on the proliferation of two human tumor cells lines, U937 and K562, using the trypan blue dye exclusion method. Our data showed a decrease on the cell viability in a dose-dependent manner. Tumor cell lines resulted more sensitive to these compounds. Thus the IC50 values were 7.96 μM for DHA and 40.39 μM for DHN in U937; 0.68 μM for DHA and 32.10 uM for DHN in K562. Interestingly PBMC viability was not affected by these compounds. It has been shown that proinflammatory signals favor the carcinogenic process. Thus we evaluated the effect of DHN and DHA on the in vitro LPS-induced COX-2 expression. Our results showed that both compounds partially inhibited LPSinduced expression of COX-2. A 60 % and 80% suppressed expression of COX-2 were observed by DHN and DHA respectively. On the other hand, these compounds in combination with anti-inflammatory natural plants extracts did not shown any additive effect upon tumour cell viability. Nevertheless, indometacina a well known antiinflammatory drug, in combination with DHA increased the tumour cell citotoxicity effect. In order to understand the mechanism of action of these compounds we evaluated whether the mitochondrial membrane potential of K562 cells could change due to DHA and DHN. Our results showed that treatment with both compounds induced a depolarization of the mitochondrial membrane as assessed with rhodamine123. Moreover, the depolarization induced by DHA was higher than DHN. On the other hand, when we study the effects of DHA and DHN on the intracellular formation of reactive oxygen species no ROS generation was detectable by any of these compounds. Morover our findings show that both compounds could protect from ROS at 25 μM. NK cells constitute an important mechanism of antitumor defense. For this reason it is desirable that DHN and DHA did not affect at all the NK cell function. Thus, NK citotoxic activity was measured using the 51Cr release assay from the target cell K562. Our results indicated that neither DHA nor DHN affected NK citotoxic activity. According to these results we can conclude that DHA exerts an important antitumor upon U937 and K562 tumor cells, probably due to the depolarization of the mitochondrial membrane, and/or inhibiting the expression of Cox-2, without generation of reactive species and without affecting the innate immune response

In the present work we focus into the potential relevance of PUFAs in some models and human samples from patients suffering amyotrophic lateral sclerosis (ALS). Due to its pathological implication, oxidative stress was our first goal. We started from simple oxidative methodology screening to search for an antioxidant substance (among 21 different candidates) available in a Mediterranean diet. The results demonstrated high heterogeneity in carbonyl (measured by DNP) accumulation, regarding the oxidative source, substrate suffering it and the antioxidant structure. Further, thanks to GC/MS and LCQTOF, we detailed the protection over specific accrual of protein and lipid peroxidation markers as well as lipid profile modifications (as % of total fatty acids -FA) in oxLDL thanks to those dietary compounds. Moreover, we demonstrated its in vitro relevance, in terms of survival, when two cell lines (HMEC-1, HepG2) were treated with this oxidized (and protected) compounds, and finally address in vivo importance of those findings, demonstrating decreased carbonyl and oxidative accumulation in hamsters under an atherogenic diet supplemented with antioxidants. Once described the protective effect of antioxidants and specific signatures found regarding lipid oxidation markers, we extend the study focusing in different ALS samples. From previous work, we demonstrated an altered docosohexaenoic acid (DHA) composition in different location for patients suffering sporadic ALS. Hence, we though necessary to define whether the enzymatic machinery aimed to synthesize DHA from its precursors, are affected in sALS. Interestingly, we found a tissue specific variation (spinal cord vs cortex), compatible with our previous FA results. Further, thanks to inmunohistochemistry, differential involvement was unveiled for motor neurons (MN) and surrounding glia. Therefore, trying to depict cellular contribution, we switch to a neuronal model (N2A under oxidative stressors and/or aggregation-prone-TDP-43) and a tissular one (OT). There, we showed decreased desaturase (Δ6) and drebrin expression as well as increased DHA synthesis and an unreported inverse correlation of drebrin loss and aberrant p-TDP-43 expression under oxidative conditions in the cell culture. In the OT model, lipidomic analysis showed specific accretion of 8-iso-PGF2α and NPD1 as well as increased DHA (and dramatically decreased precursors) and reduced AA concentrations (GC measured). Analysis of slice O2 consumption showed decreased O2 levels under excitotoxic treatment and alleviation by antioxidant (tocopherol) addition. Treatment of OT slices with Ω-3 precursors (better than final products) and DHA plus tocopherol ameliorated MNs number. Finally, we wanted to disclose PUFA’s implication and phenothype of an animal model (SODG93A) under a dietary intervention with opposed FA unsaturation levels. Not surprisingly, FA profile was difficult to be altered in nervous system, although subtle specific variations were found. More importantly, differences in survival and clinical manifestations, UPR (Ubiquitin inclusions), mt-DNA (8- oxo-dG) and protein oxidative modifications revealed sex as a relevant factor in lipid handling for this model. Hence, whereas male under a low PUFA diet showed increased survival, females lack this beneficial outcome. Last but not least, we wanted to dig deeper regarding this sexual dimorphism. For this purpose, we focused in mitochondria and analyzed spinal cord oxygen consumption, oxidative damage to proteins and lipid profile along disease progression and also in a neuronal model (N2A overexpressing SODG93A, treated with 17β-estradiol).We could demonstrate a clear sexual implication, with females having late onset clinical symptoms concomitant to an upgraded mitochondrial function and lower protein and mitochondrial damaged proteins compared with males. Finally, to further confirmed steroid potential as a protective element in disease progression, in vitro estradiol pretreatmet of N2A showed increased oxygen consumption, with no relation with the mitochondrial complex expression.
En el trabajo que aquí se presenta se ha profundizado en la relevancia que los ácidos grasos poliinsaturados (PUFA) puedan tener en el tratamiento de la esclerosis lateral amiotrófica (ALS). Dada su implicación en el desarrollo de la patología, el estudio del estrés oxidativo asociado fue uno de nuestros primeros objetivos. Para ello hemos empezado por un cribado metodológico simplista, tratando de encontrar una sustancia antioxidante (entre 21), biodisponible en una dieta Mediterránea equilibrada y que fuese capaz de reducir un daño oxidativo generado desde diversos frentes (medido como acumulación de carbonilos) y sobre diferentes substratos. Los resultados demostraron una alta heterogeneidad, dificultando así la elección de un único antioxidante. Aún así, gracias a la GCMS y la LCQTOF pudimos detallar la acumulación específica diferenciada de marcadores de daño oxidativo proteico y daño lipoxidativo producido cuando partículas de lipoproteínas de baja densidad (LDL) son oxidadas bajo la acción de diversos compuestos. Además se pudo objetivar el cambio en la composición lipídica de estas LDL (medida como % del total presente) y su relevancia biológica in vitro, medida en términos de supervivencia, cuando se exponen a dos líneas celulares (HMEC-1, HepG2). Por último se demostró la importancia in vivo, puesto que se pudo observar una menor acumulación de productos carbonílicos en hámsters alimentados con una dieta aterogénica, pero suplementada con antioxidantes. Una vez se demostró el papel jugado por estos antioxidantes en la acumulación diferenciada de productos de oxidación, extendimos el estudio a muestras y modelos de ALS. En trabajos previos habíamos evidenciado una composición tisular diferenciada en diversas localizaciones del sistema nervioso en pacientes diagnosticados de ALS. Por ello consideramos interesante el estudio de la expresión de la maquinaria enzimática necesaria para la síntesis lipídica. De un modo destacado, pudimos ver de nuevo una variación tisular, compatible con niveles reducidos de docosohexaenoico (DHA), y gracias a la inmunohistoquímica también se observaron diferencias entre las motoneuronas (MNs) y la glia circundante. Así pues, para poder revelar las aportaciones diferenciales de los distintos tipos celulares, utilizamos una línea celular (N2A) a la que sometíamos a diferentes estresores (daño oxidativo y sobreexpresión de una versión de TPD-43 que causa agregados) y a un cultivo tisular de médula espinal (OT), dónde se produce una muerta progresiva y selectiva de las MNs. Tras estos experimentos, observamos un descenso tanto en la expresión de la Δ6 desaturasa como de drebrin (marcador presináptico) tras la sobreexpresión de TDP-43, así como una mayor síntesis de DHA y una correlación inversa entre la pérdida de drebrina y la expresión de pTDP- 43 bajo condiciones de estrés oxidativo. Por otro lado, en el modelo OT, el análisis lipidómico reveló la acumulación especifica de 8-iso-PGF2α y NDPD1 (posiblemente en respuesta a un incremento del daño oxidativo) así como el aumento en la concentración de DHA (con un descenso muy marcado de sus precursores) y el descenso de araquidónico. Quisimos analizar también el consumo de oxígeno, tanto en tejido intacto como permeabilizado, pudiendo observar como la excitoxicidad reducía considerablemente su capacidad y como ésta era, en parte, rescatada con el uso de tocoferol. Además, el tratamiento del OT con precursores Ω-3 mejoró también el número de MNs. Por último, quisimos caracterizar la implicación que los PUFA dietarios podrían tener en un modelo animal bien conocido (SODG93A). No fue sorprendente encontrar que el perfil lipídico en el sistema nervioso fue muy difícil de alterar. Aún así, se observaron diferencias en supervivencia, en el devenir clínico, la respuesta UPR (con acumulaciones de Ub), el daño al DNA mitocondrial (8-oxo-dG) y modificaciones oxidativas en las proteínas y cómo éstas tenían un grado de afectación diferencial cuando considerábamos el sexo de los animales. Esto es, mientras que los machos sometidos a una dieta baja en PUFAs de cadena larga demostraron una mayor supervivencia, en las hembras no se apreció mejoría. Por último, pero no menos importante, quisimos profundizar más respecto a este dimorfismo. Centrándonos en la mitocondria, pudimos hacer un seguimiento del consumo de oxígeno a lo largo de la enfermedad, el daño oxidativo a proteínas y el perfil lipídico. Por lo tanto pudimos demostrar una clara implicación sexual, siendo las hembras las que más tarde comienzan su manifestación clínica, con mejores funciones mitocondriales asociadas a un menor daño oxidativo. Finalmente, la relevancia del papel protector de los estrógenos se pudo comprobar in vitro, mediante el pretratamiento con 17β-estradiol en la línea N2A que sobreexpresa SOD1G93A, relacionado con la ALS familiar, proponiéndose el estradiol como un nuevo elemento que juega un papel relevante en el desarrollo de la enfermedad.
En aquest treball s’ha intentat profunditzar en la possible rellevància dels àcids grassos poliinsaturats (PUFA) en el tractament de l’esclerosi lateral amiotròfica (ELA). Atesa la seva implicació en el desenvolupament de la patologia, l’estudi de l’estrès oxidatiu associat fou un dels primers objectius. Per això, es va començar amb un cribratge metodològic simplista, intentant trobar una substància antioxidant biodisponible en una dieta mediterrània equilibrada i que fos capaç de reduir el dany oxidatiu generat des de diferents fronts i sobre diferents substrats. Els resultats van demostrar una alta heterogeneïtat, dificultant així l’elecció d’un únic antioxidant. Malgrat això, mercès a tècniques de GC-MS i LC-QTOF, es va poder detallar l’acumulació específica diferenciada de marcadors de dany oxidatiu proteic i dany lipoxidatiu produït quan partícules de lipoproteïna de baixa densitat (LDL) s’oxiden per l’acció de diversos compostos. A més, es va poder objectivar el canvi en la composició lipídica d’aquestes LDL i la seva rellevància in vitro, mesurada en termes de supervivència, quan es cocultiven amb les línies cel·lulars. Per últim, es va demostrar la importància in vivo, atès que es va observar una menor acumulació de productes carbonílics en hàmsters alimentats amb una dieta aterogènica suplementada amb antioxidants. Un cop es va demostrar el paper d’aquests antioxidants en l’acumulació diferenciada de productes d’oxidació, es va estendre l’estudi a mostres i models d’ELA. En treballs previs s’havia evidenciat una composició tissular diferenciada en diverses localitzacions del sistema nerviós central en pacients d’ELA (respecte l’àcid docosahexaenoic (DHA), depleció en medul·la espinal i acumulació en còrtex). Per aquesta raó es va considerar interessant l’estudi de l’expressió de la maquinària enzimàtica necessària per la síntesi lipídica. D’una manera destacada, es va poder veure de nou una variació tissular, compatible amb nivells reduïts de DHA i, per tècniques d’immunohistoquímica, es van observar diferències entre les motoneurones i la glia circumdant. Per tant, per poder revelar les aportacions diferencials dels diferents tipus cel·lulars, es va utilitzar la línia cel·lular N2A, sotmesa a diferents estressos (dany oxidatiu i sobreexpressió d’una forma de TDP-43 que causa agregats) i un cultiu tissular de medul·la espinal, en el que es produeix una mort progressiva i selectiva de les motoneurones. Es va observar un descens en l’expressió de FADS2 i de drebrina, un marcador sinàptic, així com una major síntesi de DHA i una correlació inversa entre la pèrdua de drebrina i l’expressió de TDP-43 sota condicions d’estrès oxidatiu. Per altra banda, en el model organotípic, l’anàlisi lipidòmica va revelar l’acumulació específica de 8-isoPGF2α i NDPD1, així com l’augment de la concentració de DHA i el descens motl marcat del seus precursors a mes del àcid araquidònic. Es va mesurar també el metabolisme oxidatiu, observant-se que l’excitotoxicitat reduïa considerablement la seva capacitat i, en part, es rescatava amb l’ús de tocoferol. A més, el tractament dels cultius organotípics amb precursors d’àcids grassos n-3 va millorar el nombre de motoneurones. Per últim, es va caracteritzar la implicació dels PUFA dietaris en un model animal d’ELA. Malgrat que el perfil lipídic del sistema nerviós central era difícil d’alterar, es van observar diferències en supervivència, fenotip clínic, resposta al malplegament de proteïnes (UPR) amb acumulació d’ubiquitina, dany en el DNA mitocondrial i modificacions oxidatives en les proteïnes, amb un grau d’afectació diferencial quan es considerava el sexe dels animals. En aquest sentit, mentre que els mascles sotmesos a una dieta baixa en PUFA de cadena llarga van mostra una major supervivència, en les femelles nomes va apreciar cap efecte millora. Per profunditzar en el dimorfisme sexual en ELA, i especialment la disfunció mitocondrial, es va analitzar mitjançant respirometria d’alta resolució la medul·la espinal durant tot el desenvolupament de la malaltia. Es va revelar una clara diferència de gènere, amb una manifestació clínica més tardana en femelles que correlaciona amb una millor conservació de la funció mitocondrial i un menor dany oxidatiu. El possible paper protector dels estrògens es va demostrar in vitro mitjançant el pretractament amb estradiol de cèl·lules N2A que sobreexpressen una forma de SOD1 humana mutada associada a l’ELA familiar (G93A-SOD1).

Alterações epigenéticas, como metilação do DNA e modificações pós traducionais em histonas, tem importante papel na carcinogênese mamária. A modulação de eventos epigenéticos constitui relevante alvo terapêutico, devido ao seu caráter reversível. Experimentalmente, o ácido docosahexaenoico (DHA), um membro da família dos ácidos graxos ômega-3, é capaz de diminuir proliferação, induzir morte celular e diminuir o potencial invasivo de células tumorais de mama. No entanto, os mecanismos antitumorais do DHA e sua capacidade de modular eventos epigenéticos ainda não estão totalmente elucidados. Nosso objetivo foi verificar, in vitro, a ação do DHA em eventos epigenéticos em diferentes linhagens de carcinoma mamário humano. Três linhagens celulares de câncer de mama (MDA-MB-231, SKBR-3 e MCF-7) foram tratadas durante 72 horas com 100 ?M de DHA ou etanol (controle). As modificações pós traducionais em histonas, acetilação no resíduo de lisina 9 da histona 3 (H3K9ac) e no resíduo 16 da histona 4 (H4K16ac), bem como trimetilação no resíduo de lisina 9 da histona 3 (H3K9me3) e no resíduo de lisina 27 da histona 3 (H3K27me3) foram avaliadas pela técnica de western blot. A análise da expressão do genes RASSF1A, DNMT1, DNMT3A e DNMT3B foi feita pela técnica da reação em cadeia da polimerase quantitativa via transcriptase reversa (RT-qPCR). A avaliação do padrão de metilação de região promotora do gene RASSF1A foi realizada pela técnica de reação em cadeia da polimerase metilação específica (MS-PCR). Foram também analisadas as fases do ciclo celular por citometria de fluxo. Comparado ao controle, o DHA induziu a acetilação no resíduo 16 da histona 4 (H4K16ac) nas linhagens MCF7 (p = 0,04) e MDA-MB-231 (p = 0,03). Observamos que a H3K9me3 foi parcialmente inibida nas linhagens MDA-MB-231 e SKBR-3, após o tratamento com DHA, mas sem alcançar valor estatisticamente significante. Encontramos também diminuição dos níveis de H3K27me3 após o tratamento com DHA nas três linhagens estudadas, porém não foi estatisticamente significativo. O DHA aumentou a expressão do gene RASSF1A na linhagem MCF-7 (1,98 vezes, p = 0,03), mas não nas linhagens MDA-MB-231 e SKBR-3. Não houve mudanças estatisticamente significativas na expressão dos genes DNMT1, DNMT3A e DNMT3B. As análises qualitativas da metilação demonstraram que a região promotora analisada do gene RASSF1A apresentou-se hipermetilada nas três linhagens celulares. Após o tratamento com DHA, houve tendência de desmetilação na região promotora do RASSF1A na linhagem MCF-7 e SKBR3, mas não na linhagem MDA-MB-231. Não houve diferença significativa na porcentagem de morte e distribuição das células MDA-MB-231, SKBR-3 e MCF-7 nas diferentes fases do ciclo celular após tratamento com DHA. Em conclusão, o DHA pode atuar em mecanismos epigenéticos e, ainda, reativar o gene supressor de tumor, como RASSF1A, anteriormente silenciado por hipermetilação, em células MCF-7. Espera-se que esses resultados contribuam para melhor compreensão do potencial papel anticâncer do DHA no câncer de mama
Epigenetic changes, such as DNA methylation and post-translational histone modifications, play an important role in mammary tumorigenesis. Epigenetic events are important as therapeutic targets, because of its reversible nature. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids family, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanism of DHA and its ability to modulate epigenetic events are not completely understood. Our objective was to verify, in vitro, the action of DHA in epigenetic events related to transcriptional reactivation of tumor suppressor gene, such as RASSF1A, in different human breast cancer cell lines. Three breast cancer cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated with DHA (100 ?M) or vehicle (ethanol) for 72 hours. Western blot was used to analyze histone modification, as histone H3 lysine 9 (H3K9ac) and histone H4 lysine 16 (H4K16ac) acetylation, H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3). Real time quantitative PCR (RT-qPCR) was performed for gene expression quantification of RASSF1A, DNMT1, DNMT3A and DNMT3B. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR (MS-PCR). Moreover, we evaluated the phases of the cell cycle by flow cytometry. Compared to control cells, DHA induced H4K16ac in MCF-7 (p=0.04) and MDA-MB-231 (p=0.03). We observed that H3K9me3 was partially inhibited in MDA-MB-231 and SKBR-3 cells, after treatment with DHA, but did not reach a statistically significant value. We also found decreased levels of H3K27me3 after treatment with DHA in the three cell lines studied, but not statistically significant. DHA increased RASSF1A expression on MCF-7 (1.98 fold; p=0.03), but not in MDA-MB-231 and in SKBR-3 cells. There were no statistically significant changes in expression of genes DNMT1, DNMT3A and DNMT3B. These three breast cancer cells lines show methylation in specific region of RASSF1A promoter. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7 and SKBR-3. No significant difference was observed in the percentage of cell death nor cell distribution of MDA-MB-231, SKBR-3 and MCF-7 at different stages of the cell cycle after treatment with DHA. In conclusion, we suggest that DHA may act beneficially in epigenetic mechanisms and reactivation of tumor suppressor gene, RASSF1A as previously silenced by hypermethylation. It is hoped that these results can contribute to better understanding of the anticancer role of DHA in breast cancer

Os efeitos de EPA e DHA sobre a função de neutrófilos foram comparados. Para isso, foram realizados experimentos em neutrófilos isolados de ratos. Foram analisadas células com a membrana plasmática íntegra e fragmentação de DNA com o intuito de determinar as concentrações não tóxicas de EPA e DHA. Aumento da produção de peróxido de hidrogênio ocorreu a partir de concentrações menores de DHA (50 mM comparado com 100 mM de EPA). Já para a produção de ânion superóxido, EPA estimulou em doses menores (12.5 mM e o DHA em 100 mM). Ambos AGs aumentaram a síntese e liberação das citocinas CINC-2 e TNF-α e não modificaram a produção de IL1-b e óxido nítrico após incubação das células por 18 horas. Somente DHA elevou a capacidade fagocitária e a atividade fungicida dos neutrófilos. EPA e DHA apresentaram efeitos distintos na produção de citocinas, fagocitose e atividade fungicida dos neutrófilos. Já na produção das EROS, EPA e DHA apresentaram efeitos similares, embora em concentrações diferentes.
The effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) on neutrophil function were compared. For this purpose, experiments were performed in isolated rat neutrophils. Cells with intact plasma membrane and DNA fragmentation were analyzed in order to determine the non-toxic concentrations of EPA and DHA. Production of H2O2 was increased in lower concentrations of DHA (50 mM compared to 100 mM of EPA). For production of O2 -, EPA stimulated at lower doses (12.5 mM compared to 100 mM of DHA). Both FAs increased synthesis and release of cytokines, CINC-2 and TNF-α, and did not change the production of IL-1b and nitric oxide after incubation of the cells for 18 hours. Only DHA increased the phagocytic capacity and fungicidal activity of neutrophils. These FAs showed distinct effects on cytokine production, phagocytosis capacity and fungicidal activity by neutrophils. For production of ROS, both EPA and DHA had similar actions, although at different concentrations.