Untersucht wurde der Zusammenhang zwischen der Proteinkinase A-vermittelten Ekto-Phosphorylierung des Membranproteins FAT/CD36 [Hatmi et al. 1996] und der initialen zellulären Aufnahme langkettiger Fettsäuren. Wir zeigten einen inhibitorischen Effekt auf die initiale Palmitinsäure-Aufnahme humaner Thrombozyten unter den Bedingungen der Ekto-Phosphorylierung von FAT/CD36. Damit kann erstmalig ein Mechanismus für die kurzfristige Regulation der proteinvermittelten Aufnahme langkettiger Fettsäuren vorgeschlagen werden. Für die Bearbeitung der Fragestellung wurden die Isolation "ruhender", morphologisch und funktionell intakter humaner Thrombozyten und eine Methode zur Messung der initialen Palmitinsäure-Aufnahme etabliert. Die Kinetik der Palmitinsäure-Aufnahme humaner Thrombozyten wurde charakterisiert und bestätigt, dass ein wesentlicher Anteil der initialen Aufnahme proteinvermittelt erfolgt. Die von Hatmi und Co-Autoren beschriebene Ekto-Proteinkinase A-vermittelte, cAMP-abhängige Phosphorylierung von FAT/CD36 [Hatmi et al. 1996] konnte unter unseren experimentellen Bedingungen nachvollzogen werden. Die Ekto-Phosphorylierung von FAT/CD36 ging mit einer signifikanten Abnahme der initialen Palmitinsäure-Aufnahme einher. Die maximale Abnahme auf 72 % des Kontrollwerts wurde bei einer extrazellulären ATP-Konzentration von 0,5 nM erreicht. Der inhibitorische Effekt liess sich durch Co-Inkubation mit dem spezifischen Proteinkinase A-Inhibitorpeptid PKI 5-24 oder mit beta-gamma-ATP aufheben. Der Effekt war durch Dephosphorylierung mit Alkalischer Phosphatase vollständig reversibel. Bei extrazellulären ATP-Konzentrationen zwischen 10 pM und 15 nM war der inhibitorische Effekt der Ekto-Phosphorylierung auf die Palmitinsäure-Aufnahme signifikant. ATP-Konzentrationen über 15 nM verminderten den Effekt, bei über 5 µM ATP war kein Effekt nachzuweisen. Wir konnten ausschliessen, dass die Aufhebung durch ATP-Abbauprodukte verursacht wurde. Unsere Beobachtungen deuten auf einen regulatorischen Einfluss höherer extrazellulärer ATP-Konzentrationen, der dem inhibitorischen Effekt der Ektophosphorylierung von FAT/CD36 auf die Fettsäure-Aufnahme entgegenwirkt. / We investigated the correlation between the ecto-protein kinase A-mediated phosphorylation of the membrane-associated protein FAT/CD36 [Hatmi et al. 1996] and the initial cellular long chain fatty acid uptake. Under the conditions of FAT/CD36-ecto-phosphorylation, an inhibitory effect on the initial palmitate uptake of human platelets could be shown. This is the first time that a mechanism for the short-term regulation of protein-mediated long chain fatty acid uptake can be proposed. The isolation of morphologically and functionally intact resting human platelets and a method for measuring the initial palmitate uptake were established. The kinetics of palmitate uptake by human platelets were characterised and it was shown that a substantial fraction of initial palmitate uptake is protein-mediated. The ecto-protein kinase A-mediated, cAMP-dependent phosphorylation of FAT/CD36 as described by Hatmi and co-authors could be demonstrated under our experimental conditions. The ecto-phosphorylation of FAT/CD36 was paralleled by a significant impairment of the initial palmitate uptake. Maximum inhibition was achieved at 0,5 nM extracellular ATP, when the palmitate uptake was decreased to 72 % compared to control. The inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI 5-24 or with beta-gamma-methylene-ATP, and was fully reversible upon addition of alkaline phosphatase. The inhibitory effect of the ecto-phosphorylation on the initial palmitate uptake was significant at extracellular ATP concentrations between 10 pM and 15 nM. ATP concentrations over 15 nM reduced the effect and concentrations over 5 µM completely abolished it. We could exclude that the abolishment was caused by ATP-derivates. Our data point to a regulatory influence of higher ATP concentrations, that antagonises the inhibitory effect of the ecto-phosphorylation of FAT/CD36 on the initial palmitate uptake.

The gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) require n-3 long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), for optimal growth and health. Due to the rapid growth of global aquaculture the quantity of marine oils used in aquafeeds has been limited, yet the overall quantity of oil in an aquafeed has increased by the addition of vegetable oil (VO) to supply dietary energy. For aquaculture to continue to grow more fish must be produced with less marine ingredients, yet EPA and DHA must be maintained at levels above fish requirements. This project set out to re-evaluate the requirement for EPA and DHA in gilthead seabream and European seabass. Two dose-response studies were designed and executed where juvenile seabream and seabass were fed one of six levels of EPA+DHA (0.2 – 3.2 % as fed). Biometric data were collected and analysed to determine new requirement estimates for EPA+DHA for fish of two weight ranges (24 – 80 g and 80 – 200 g). The effects of the dietary LC-PUFA gradient on lipid composition and metabolism were also considered.

X–linked adrenoleukodystrophy (X–ALD) is the most common peroxisomal enzyme deficiency disorder, characterized by inborn mutations in the ABCD1 gene, an ATP–binding cassette (ABC) half–transporter. The ABCD1 gene encodes the adrenoleukodystrophy protein (ALDP), the transporter for the very–long–chain fatty acids (VLCFA; C > 22:0) from the cytosol into the peroxisomes to enter the peroxisomal B–oxidation pathway. The diagnostic disease marker is the elevated levels of VLCFAs which accumulate in different tissues and body fluids, leading to inflammatory demyelination, neuro–deterioration and adrenocortical insufficiency. At present, there is no satisfactory therapy for X–ALD available. However, another peroxisomal ABC half–transporter, ALDRP can compensate for the functional loss of ALDP and is encoded by the ABCD2 gene. This prompted a new approach to treatment strategies. Phenylbutyric acid (PBA) over–expresses the ABCD2 gene, leading to an increased expression of ALDRP and PBA decreases VLCFA levels by increasing peroxisomal B–oxidation. This study had a dual aim: to determine the antioxidant capacity of PBA and to verify known and identify new metabolites of PBA. In vitro, HeLa cells were cultivated and treated with 0.5 mM, 1 mM, 2 mM and 5 mM PBA for 48 hours. The ROS, lipid peroxidation, apoptosis and cell viability were determined using fluorescein–based flow cytometry. Images were taken to visualize the peroxisome proliferation. In vivo, a vervet monkey was given a single dose of 130 mg/kg PBA. Blood was collected before treatment and 15 minutes, 30 minutes, 1, 2 and 3 hours after treatment. ROS, apoptosis and lipid peroxidation were determined by fluorescein–based flow cytometry. Urine was collected before treatment and 15 minutes, 30 minutes, 1, 2, 3, 7 and 24 hours after PBA treatment. A standardised method, employing gas chromatography–mass spectrometry (GC/MS), was used to analyse the organic acids in the urine and fatty acids in the blood. In vitro results showed decreased levels of ROS and lipid peroxidation with increased concentrations of PBA. PBA showed a protective effect towards the HeLa cells with reduced apoptosis and a high number of viable cells. In vivo levels of ROS en lipid peroxidation decreased over time of treatment with PBA. The fluorescence microscope images confirmed an increased number of peroxisomes after PBA treatment. The short term effect of PBA showed an initial, but small decrease in the levels of the fatty acids, suggesting induction over a longer period rather than activation of peroxisomal B–oxidation. New metabolites of phenylbutyrate were identified in the urine of a vervet monkey. These new metabolites originated from monooxygenase, N–phenylacetyl–glutamine synthases and B–oxidation byproducts. Recently discovered metabolites in humans and rats were also verified and confirmed in the vervet monkey. We therefore propose that treatment with PBA, on account of its beneficial effects of restoring VLCFA levels and reducing oxidative stress, could be considered a novel approach for the treatment of X–ALD. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

X–linked adrenoleukodystrophy (X–ALD) is the most common peroxisomal enzyme deficiency disorder, characterized by inborn mutations in the ABCD1 gene, an ATP–binding cassette (ABC) half–transporter. The ABCD1 gene encodes the adrenoleukodystrophy protein (ALDP), the transporter for the very–long–chain fatty acids (VLCFA; C > 22:0) from the cytosol into the peroxisomes to enter the peroxisomal B–oxidation pathway. The diagnostic disease marker is the elevated levels of VLCFAs which accumulate in different tissues and body fluids, leading to inflammatory demyelination, neuro–deterioration and adrenocortical insufficiency. At present, there is no satisfactory therapy for X–ALD available. However, another peroxisomal ABC half–transporter, ALDRP can compensate for the functional loss of ALDP and is encoded by the ABCD2 gene. This prompted a new approach to treatment strategies. Phenylbutyric acid (PBA) over–expresses the ABCD2 gene, leading to an increased expression of ALDRP and PBA decreases VLCFA levels by increasing peroxisomal B–oxidation. This study had a dual aim: to determine the antioxidant capacity of PBA and to verify known and identify new metabolites of PBA. In vitro, HeLa cells were cultivated and treated with 0.5 mM, 1 mM, 2 mM and 5 mM PBA for 48 hours. The ROS, lipid peroxidation, apoptosis and cell viability were determined using fluorescein–based flow cytometry. Images were taken to visualize the peroxisome proliferation. In vivo, a vervet monkey was given a single dose of 130 mg/kg PBA. Blood was collected before treatment and 15 minutes, 30 minutes, 1, 2 and 3 hours after treatment. ROS, apoptosis and lipid peroxidation were determined by fluorescein–based flow cytometry. Urine was collected before treatment and 15 minutes, 30 minutes, 1, 2, 3, 7 and 24 hours after PBA treatment. A standardised method, employing gas chromatography–mass spectrometry (GC/MS), was used to analyse the organic acids in the urine and fatty acids in the blood. In vitro results showed decreased levels of ROS and lipid peroxidation with increased concentrations of PBA. PBA showed a protective effect towards the HeLa cells with reduced apoptosis and a high number of viable cells. In vivo levels of ROS en lipid peroxidation decreased over time of treatment with PBA. The fluorescence microscope images confirmed an increased number of peroxisomes after PBA treatment. The short term effect of PBA showed an initial, but small decrease in the levels of the fatty acids, suggesting induction over a longer period rather than activation of peroxisomal B–oxidation. New metabolites of phenylbutyrate were identified in the urine of a vervet monkey. These new metabolites originated from monooxygenase, N–phenylacetyl–glutamine synthases and B–oxidation byproducts. Recently discovered metabolites in humans and rats were also verified and confirmed in the vervet monkey. We therefore propose that treatment with PBA, on account of its beneficial effects of restoring VLCFA levels and reducing oxidative stress, could be considered a novel approach for the treatment of X–ALD. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

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

Les techniques d’électrophysiologie chez les animaux et d’imagerie fonctionnelle chez l’Homme ont permis d’étudier le contrôle hédonique de la prise alimentaire. Ce contrôle hédonique n’a cependant jamais été exploré chez l’Homme par l’étude des potentiels évoqués gustatifs (PEG), de meilleure résolution temporelle que l’imagerie fonctionnelle. Le premier objectif de la thèse a été de mettre au point une technique fiable et reproductible de recueil des PEG, en regard des aires cérébrales gustatives, en réponse à une stimulation sapide. Le deuxième objectif a été d’étudier les variations des PEG en fonction de la valeur hédonique de la prise alimentaire. Le travail de thèse a permis de mettre au point l’enregistrement des PEG, réalisés chez plus de 100 jeunes sujets sains. Les comparaisons effectuées entre les enregistrements cérébraux obtenus en réponse à l’eau seule ou à l’huile de paraffine, solutions non palatables, et après stimulation par une solution sapide ont permis d’apporter des arguments forts en faveur de l’origine gustative des potentiels évoqués enregistrés. L’analyse des PEG a permis de mettre en évidence des modifications de l’activation cérébrale en fonction du plaisir alimentaire, traduites par des changements de latence ou d’amplitude des PEG. Plusieurs situations connues pour faire varier le plaisir alimentaire ont été étudiées : avant/après repas ; stimulation par des solutions sucrées d’intensités différentes ou de valeurs énergétiques différentes ; stimulation par des acides gras. Des PEG en réponse aux acides gras à longue chaine (acides linoléiques) ont été enregistrés par ce biais, renforçant l’hypothèse du «gras» en tant que sixième saveur primaire. / Hedonic control of food intake has been studied using neurophysiological investigations in animals and functional imaging in humans. Gustatory evoked potentials (GEPs), a higher time resolution technique than functional imaging, have never been used for this purpose. The first aim of this thesis was to establish a reliable recording of GEPs in humans, in response to a sapid stimulus. The second aim was to determine the GEPs modifications according to the hedonic value of food intake. GEPs recording was performed in response to an intermittent stimulation of a sapid solution in more than 100 young healthy subjects. The comparisons between cerebral recordings in response to water or paraffin oil, non palatable solutions, and in response to sapid solutions (sucrose, sodium chlorure and fatty acids) allow us to advance strong arguments for the gustative nature of the recorded evoked potentials. GEPs analysis underlined changes in cerebral activation according to the hedonic value of the stimulus. These changes in cerebral activation were highlighted by modifications of GEPs latency or amplitude. Several physiological situations, marked by different pleasantness of food stimulation, were studied: before/after food intake, stimulation by sweet solutions with different concentrations or different caloric contents, stimulation by fatty acids. Moreover, GEPs in response to long chain fatty acids (linoleic acids) were recorded, reinforcing the hypothesis that fatty acids could be the sixth primary flavor.

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

[ES] Los ácidos grasos de cadena muy larga (VLC-FA; >C24), aunque presentes en pequeñas cantidades, juegan un importante papel para el correcto desarrollo y funcionalidad de los tejidos neurales en vertebrados, especialmente durante su desarrollo temprano. Sin embargo, a pesar de su aparente importancia, los estudios sobre estos compuestos en peces son escasos. La biosíntesis de los VLC-FA se lleva a cabo mediante las denominadas proteínas de elongación de los ácidos grasos de cadena muy larga 4 (Elovl4) y, en consecuencia, la dotación y la función de estas enzimas determinan la capacidad endógena que una determinada especie tiene para satisfacer las demandas fisiológicas de VLC-FA, especialmente durante su desarrollo temprano. Además, esta producción endógena de los ácidos grasos poliinsaturados de cadena muy larga (VLC-PUFA) es dependiente de los sustratos. Así, para su biosíntesis se requiere de ácidos grasos más cortos, es decir ácidos grasos poliinsaturados de cadena larga (LC-PUFA; C20-24) que actúen como precursores, los cuales son incorporados principalmente a través de la dieta. Teniendo esto en mente, el presente trabajo de investigación tuvo como objetivos caracterizar los genes elovl4 en dorada (Sparus aurata) y en lenguado senegalés (Solea senegalensis), determinar la función de sus correspondientes proteínas codificadas, así como analizar el patrón de expresión tisular de elovl4. Además, se ha investigado la regulación nutricional de los genes implicados en la biosíntesis de VLC-PUFA (elovl4a, elovl4b) y LC-PUFA (fads2, elovl5) durante las primeras etapas del ciclo de vida de ambas especies (larvas y poslarvas), mediante el uso de dietas adaptadas a cada etapa del desarrollo. Los resultados confirmaron que ambas especies de peces poseen dos genes elovl4 distintos, denominados elovl4a y elovl4b según su homología con sus ortólogos de pez cebra. Asimismo, los ensayos funcionales de sus correspondientes proteínas, llevados a cabo en levaduras, indicaron que tanto Elovl4a como Elovl4b tienen la capacidad de elongar los ácidos grasos precursores (C20-24) hasta VLC-FA en ambas especies. Sin embargo, Elovl4b mostró mayor actividad que Elovl4a para elongar todos los ácidos grasos poliinsaturados a productos de cadena más larga, especialmente de la serie n-3. Además, los resultados de expresión génica indicaron que, aunque fueron detectados transcritos de elovl4 en la mayoría de los tejidos analizados, ambos genes elovl4 se expresaron más intensamente en los tejidos neurales de ambas especies, como el cerebro y los ojos, que mostraron los niveles de expresión más altos de elovl4a y elovl4b, respectivamente. Además, los resultados procedentes de los ensayos de regulación nutricional indicaron que los genes fads2, elovl5, elovl4a y elovl4b pueden ser regulados a través del contenido en LC-PUFA presente en la dieta. Es importante destacar que elovl4a y elovl4b fueron regulados de manera distinta según las hipotéticas necesidades de VLC-PUFA asociadas, de manera específica, con cada etapa del desarrollo temprano y dependiendo de disponibilidad dietaria de LC-PUFA. Estos hallazgos pueden contribuir a alcanzar una mejor comprensión de la vía biosintética de los VLC-FA en los teleósteos marinos, resaltando así el papel crucial que los productos de Elovl4 desempeñan para el correcto desarrollo y mantenimiento de las funciones neurofisiológicas durante las primeras etapas del desarrollo de los peces. Asimismo, estos resultados pueden ayudar a esclarecer el mecanismo molecular que controla la biosíntesis de VLC-PUFA, así como a establecer sus requerimientos específicos a lo largo del desarrollo de los teleosteos marinos en función de la especie. De esta manera, se abre la posibilidad de incorporar con éxito fuentes lipídicas alternativas a través de una programación nutricional temprana que estimule la biosíntesis de los VLC-PUFA durante las primeras etapas de alimentación exógena. / [CA] Els àcids grassos de cadena molt llarga (VLC-FA; >C24), encara que presents en petites quantitats, juguen un important paper per al correcte desenvolupament i funcionalitat dels teixits neurals en vertebrats, especialment durant el seu desenvolupament inicial. No obstant, malgrat la seua aparent importància, els estudis sobre aquests compostos en peixos son escassos. La biosíntesi dels VLC-FA es porta a terme mitjançant les denominades proteïnes d'elongació dels àcids grassos de cadena molt llarga 4 (Elovl4) i, en conseqüència, la dotació i la funció d'aquests enzims determinen la capacitat endògena que una determinada espècie té per a satisfer les demandes fisiològiques de VLC-FA, especialment durant el seu desenvolupament inicial. A més, aquesta producció endògena dels àcids grassos poliinsaturats de cadena molt llarga (VLC-PUFA) és depenent dels substrats. Així, per a la seua biosíntesi es requereix d'àcids grassos més curts, és a dir àcids grassos poliinsaturats de cadena llarga (LC-PUFA; C20-24) que actuen com a precursors, els quals són incorporats principalment a través de la dieta. Tenint això en compte, el present treball de recerca va tindre com a objectius caracteritzar els gens elovl4 en orada (Sparus aurata) i llenguado (Solea senegalensis), determinar la funció de les seues corresponents proteïnes codificades, així com analitzar el patró d'expressió tissular de elovl4. A més, es va investigar la regulació nutricional dels gens implicats en la biosíntesi de VLC-PUFA (elovl4a, elovl4b) i LC-PUFA (fads2, elovl5), durant les primeres etapes del cicle de vida d'ambdues espècies (larves i poslarves), mitjançant l'ús de dietes adaptades a cada etapa del desenvolupament. Els resultats van confirmar que totes dues espècies de peixos posseeixen dos gens elovl4 diferents, denominats elovl4a i elovl4b segons la seua homologia amb els seus ortòlegs en peix zebra. Així mateix, els assajos funcionals de les corresponents proteïnes, duts a terme en llevats, van indicar que tant Elovl4a com Elovl4b tenen la capacitat d'elongar els àcids grassos precursors (C20-24) fins a VLC-FA en totes dues espècies. No obstant això, Elovl4b va mostrar major activitat que Elovl4a per a elongar tots els àcids grassos poliinsaturats (substrats) fins a productes de cadena més llarga, especialment de la sèrie n-3. A més, els resultats d'expressió gènica van indicar que, encara que van ser detectats trànscrits de elovl4 en la majoria dels teixits analitzats, tots dos gens elovl4 es van expressar més en teixits neurals de totes dues espècies, com el cervell i els ulls, que van mostrar els nivells d'expressió més alts d'elovl4a i elovl4b, respectivament. A més, els resultats procedents dels assajos de regulació nutricional van indicar que els gens fads2, elovl5, elovl4a i elovl4b poden ser regulats a través del contingut en LC-PUFA present en la dieta. És important destacar que elovl4a i elovl4b van ser regulats de manera diferent segons les hipotètiques necessitats de VLC-PUFA associades, de manera específica, amb cada etapa del desenvolupament inicial i depenent de la disponibilitat dietaria de LC-PUFA. Aquestes troballes poden contribuir a tenir una millor comprensió de la via biosintètica dels VLC-FA en els teleostis marins, ressaltant així el paper crucial que els productes d'Elovl4 exerceixen en el correcte desenvolupament i manteniment de les funcions neurofisiològiques durant les primeres etapes del desenvolupament dels peixos. De la mateixa forma, aquests resultats poden ajudar a esclarir el mecanisme molecular que controla la síntesi endògena de VLC-PUFA, així com a establir els requisits específics de cada espècie al llarg del desenvolupament dels teleostis marins. D'aquesta manera, s'obri la possibilitat d'incorporar amb èxit fonts lipídiques alternatives a través d'una programació nutricional inicial que estimuli la biosíntesi de VLC-PUFA durant les primeres etapes d'alimentació exògena. / [EN] Very long-chain fatty acids (VLC-FA; >C24), although present in small amounts, play important roles for the correct development and functionality of neural tissues, especially during early development of vertebrates. However, despite their putative importance, their study in fish is scarce. Biosynthesis of VLC-FA is carried out by the so-called elongation of very long-chain fatty acid 4 (Elovl4) proteins and, consequently, the complement and function of these enzymes determine the endogenous capacity that a given species has for satisfying the physiological demands for VLC-FA, especially during its early development. Moreover, this endogenous production of very long-chain polyunsaturated fatty acid (VLC-PUFA) is substrate-dependent. Therefore, shorter fatty acid precursors, i.e. long-chain polyunsaturated fatty acids (LC-PUFA; C20-24), are required. These nutrients are mostly incorporated by the diet and their bioavailability can influence the capacity of Elovl4 for satisfying the physiological VLC-PUFA demands in marine fish. Thus, nutritional regulation of elovl4, as well as other elongase and desaturase genes involved in LC-PUFA biosynthesis (elovl5, fads2) has been proposed as a strategy to enhance endogenous production of LC-PUFA and VLC-PUFA in fish farming. The present thesis aimed to characterize elovl4 genes from the marine teleosts Sparus aurata and Solea senegalensis, to determine the function of the corresponding encoded proteins, and to analyze the tissue expression pattern of these genes. Moreover, we investigated the nutritional regulation of genes involved in the biosynthesis of the VLC-PUFA (elovl4a, elovl4b) and the LC-PUFA (fads2, elovl5) in early life-cycle stages (larvae and post-larvae) of both fish species fed diets adapted to each development stage. Live preys were supplied to larvae (early and late larvae) and microdiets for post-larvae, with a variable content in VLC-PUFA precursors, i.e. LC-PUFA. The results confirmed that both fish species possess two distinct elovl4 genes termed as elovl4a and elovl4b based on their homology to the zebrafish orthologs. Functional assays of the corresponding proteins in yeast denoted that Elovl4a and Elovl4b from both species have the capability to elongate C20-24 fatty acid precursors to VLC-FA products. However, Elovl4b appeared to have a higher activity than Elovl4a elongating all the polyunsaturated fatty acid substrates assayed to longer chain polyunsaturated products, especially those of the n-3 series. Moreover, gene expression results indicated that, although elovl4 transcripts were detected in most of the tissues analyzed, elovl4 genes were more strongly expressed in the neural tissues of both species, such as brain and eyes, which showed the highest expression levels of elovl4a and elovl4b, respectively. Furthermore, the results from nutritional regulation assays denoted that fads2, elovl5, elovl4a and elovl4b genes could be regulated by the dietary LC-PUFA content. It is important to highlight that elovl4a and elovl4b genes were differently regulated according to the species-specific VLC-PUFA putative needs, associated with each early life-stage and the LC-PUFA dietary availability. Importantly, these findings can contribute to a better understanding of the VLC-FA biosynthetic pathway in marine teleosts, highlighting the crucial role that the Elovl4 products carry out for the correct development and maintenance of neurophysiological functions during early stages of the fish development. Therefore, these results can help to elucidate the molecular mechanism controlling the VLC-PUFA biosynthesis and their species-specific requirements along the marine fish development, opening the possibility to incorporate successfully alternative lipid sources, through an early nutritional programming that stimulates the VLC-PUFA biosynthesis during the first exogenous feeding stages. / Para desarrollarel trabajo de investigación descrito en esta memoria, Miguel Torres Rodríguez recibió una beca predoctoral de la Excma. Diputación de Castellón. / Torres Rodríguez, M. (2021). Estudio de los patrones de expresión de genes implicados en la síntesis de ácidos grasos de cadena muy larga durante el desarrollo de la dorada y el lenguado, y su regulación nutricional [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166619 / TESIS

No description available.

No description available.