La fraîcheur est un paramètre clé de la qualité du poisson. Les méthodes actuelles appliquées en routine pour déterminer la fraîcheur du poisson ne sont pas applicables à toutes les espèces et reflètent davantage un début d'altération du produit. Ainsi, la recherche d'indicateurs précoces de fraîcheur du poisson représente encore un défi majeur et d'actualité dans l'industrie de la pêche. Le but de ces travaux de thèse était de démontrer que les fonctions et l'intégrité mitochondriales étaient susceptibles de constituer des indicateurs précoces de la fraîcheur de filets de poisson. En effet, la mitochondrie est la "centrale" énergétique de la cellule eucaryote et joue un rôle clef dans les mécanismes de mort cellulaire tels que l'apoptose et la nécrose. Les fonctions et l'intégrité mitochondriales de cellules musculaires de filets de poisson ont été étudiées à différents temps de conservation post mortem à 4°C. Le modèle d'étude était la daurade royale (Sparus aurata) (lignée cellulaire de fibroblastes (SAF-1) et muscles de poisson). Dans un premier temps, la structure des mitochondries de poisson a été étudiée par microscopie électronique à transmission. De nombreuses dégradations de la structure des mitochondries ont été observées dans les filets à partir de 72 heures (J3) de conservation à 4°C. Ces altérations se sont accentuées à J4 et J6. La fonctionnalité des mitochondries a ensuite été évaluée selon deux approches : la respiration mitochondriale (oxygraphie) et le potentiel membranaire mitochondrial (ΔΨₘ) estimé avec la sonde fluorescente Rhodamine 123. A partir de 96 heures de conservation à 4°C (J4), ces deux paramètres ont été significativement impactés témoignant d'une altération des fonctions et de l'intégrité mitochondriales.Ces résultats sont ainsi en corrélation avec l'altération structurale observée par microscopie. En parallèle, une méthode d'évaluation du potentiel membranaire a été développée avec un fluorimètre à microvolume à partir d'un modèle bactérien puis de mitochondries isolées. Ces travaux de thèse ont démontré que l'étude des fonctionnalités mitochondriales constitue un marqueur fiable et précoce de la fraîcheur des filets de poisson. Des connaissances supplémentaires sur les mécanismes cellulaires post mortem ont également été apportées. Ces résultats constituent ainsi le point de départ pour le développement d'un kit d'évaluation de la fraîcheur et ouvrent la voie pour la recherche de marqueurs de fraîcheur et de congélation/décongélation basés sur les fonctionnalités et intégrité mitochondriales. / Freshness is a key parameter of fish quality. Current routine techniques to determine fish freshness are not applicable to all species and reflect a late stage of alteration. Thus, research on early indicators of fish freshness still represents a major and topical challenge in fishing industry. This PhD research project aimed to demonstrate that mitochondrial functions and integrity constitute early indicators of fish fillet freshness. Mitochondria are the powerhouse of the cell and play a central role in cell death mecanisms such as apoptosis and necrosis. Mitochondrial function and integrity in fish filet muscle cells were studied at different times of storage post mortem at 4°C. The species studied as a model was the gilthead seabream (Sparus aurata) (gilthead seabream fibroblast cell line (SAF-1) and fish fillets). Firstly, the structure of fish mitochondria was studied by transmission electron microscopy. Numerous mitochondrial structural alterations have been observed in fish fillet from 72 hours (D3) of storage at 4°C. These alterations were more pronounced at D4 and D6. Then, mitochondrial functionality was assessed with two approaches: mitochondrial respiration (oxygraphy) and mitochondrial membrane potential (ΔΨₘ) estimated with the fluorescent probe Rh123. From 96 hours of storage at 4°C (D4), these two parameters were significantly disrupted demonstrating the alteration of mitochondrial function and integrity. The results are in correlation with the mitochondrial structural alterations described by microscopy. In parallel, a method of mitochondrial membrane potential evaluation has been developed with a micro-volume fluorimeter, first using bacteria and then isolated mitochondria. This work demonstrated that the mitochondrial functionality study constitutes a reliable and early fish filet freshness indicator. Additional knowledge on cell mechanisms in post mortem condition has been brought. These results constitute the starting point for the development of a fish freshness assay kit and pave the way to research on others freshness and freeze-thawing indicators based on mitochondrial integrity and functionality.

The redox properties of Copper (Cu) make it both an ideal cofactor for many enzymes, and, in its free form, a highly toxic molecule capable of stimulating production of reactive oxygen species or binding to protein thiol groups. Therefore, living organisms have evolved homeostatic systems to “handle” Cu avoiding dangerous and wasteful aspecific interactions. These systems comprise uptake, carrier, storage and excretion proteins. The importance of Cu-homeostatic systems was initially discovered in humans where alterations of Cu-excretory proteins were shown to be responsible for two lethal genetic disorders; the Wilson and Menkes diseases. The levels of bioavailable Cu in the aquatic environment is important because concentrations in oceanic waters tend to be minute, whilst in some fresh and coastal waters, particularly around areas of mineral extraction, viniculture and farming operations, concentrations can be excessive. In contrast to terrestrial vertebrates, fish are not only exposed to dietary sources of copper but are also exposed to dissolved ionic copper that may enter via the skin and gills. Indeed, the latter route is important in fish and it has been demonstrated in physiological studies that under conditions of dietary deficiency, fish can satisfy their own body requirements by uptake from water. Therefore, fish must have systems relating to both gill and gut to enable maintenance of body homeostasis of this essential, yet toxic, metal. In an attempt to understand the mechanisms of Cu homeostasis in fish, whether under conditions of deficiency, adequacy or excess, it is essential to consider the expression of known Cu-homeostasis proteins. Thus, cDNAs for sea bream (Sparus aurata) homologues of copper transporter 1 (Ctr1), antioxidant protein 1 (Atox1), Menkes protein (ATP7A), Wilson protein (ATP7B), and metallothionein (MT), which are responsible for the uptake, delivery to the secretory pathway and scavenging of intracellular Cu, were cloned and their mRNA tissue expression levels measured. To investigate the molecular basis of the different homeostatic and toxic responses to waterborne or dietary Cu, sea bream were exposed to sub-toxic levels of Cu in the diet (130 mg/Kg of dry diet) or water (0.3 mg/L) and tissue mRNA and Cu levels were measured. Moreover, to discriminate between the effect of different metals on the transcriptional regulation of Cu homeostasis genes in fish, Sparus aurata fibroblast (SAF1) cells were exposed to sub-toxic levels of Cu (25 μM), Zn (100 μM) and Cd (10 μM). In addition, a microarray was used to gain a broader overview of the transcriptional response of SAF1 cells to Cu (25 μM). Waterborne or dietary Cu resulted in distinct expression profiles of Cu-homeostasis genes and markers of oxidative stress. After dietary exposure, Cu increased in intestine and liver, whilst after waterborne exposure Cu increased in gill and liver. Exposure to dietary Cu resulted in decreases in Ctr1 and ATP7A mRNA in both liver and intestine. Renal Ctr1 levels remained unchanged, whilst ATP7A mRNA decreased. In contrast, waterborne Cu exposure increased intestinal Ctr1 and ATP7A mRNA, and increased renal Ctr1 and decreased renal ATP7A mRNA. Both dietary and waterborne Cu increased ATP7B mRNA in liver. Metallothionein (MT) mRNA increased in liver and gill after waterborne Cu. Glutathione reductase (GR), a marker of oxidative stress, increased expression in liver and gill after waterborne Cu exposure, but decreased in intestine. Thus, exposure to Cu via water or diet has different, often opposite effects on Cu-homeostasis genes. The decrease in expression of both Cu-transport genes in intestine after dietary exposure may indicate a defensive mechanism to limit uptake of Cu. The opposite effects in intestine after waterborne exposure are more difficult to explain, but again may reflect a defence mechanism against excess bloodborne Cu coming from the gill. Since both dietary and waterborne Cu increased Cu levels in liver and increased hepatic ATP7B it is likely that well-characterised mammalian route of Cu excretion to bile is active in sea bream. However, only hepatic Cu derived from gill increased the expression of the stress markers MT and GR. This suggests that Cu is delivered to liver in a different form from gill as that from intestine, the intestinally derived pool being less toxic. Thus the increase in copper transport gene expression in intestine after gill exposure might be a mechanism to enable incorporation of excess bloodborne Cu into the intestinal pathway of Cu delivery to liver, thus minimizing toxicity. The in vitro exposure of SAF1 cells to Cu showed a similar response to liver of fish exposed to waterborne Cu indicating similar Cu availability and complexation. ATP7A mRNA levels were induced by Cu but not by Zn or Cd suggesting Cu-specific regulation. Conversely, MT and GR were induced by all metals tested. The transcriptomic analysis highlighted that the biological processes most significantly affected by Cu were secretion, protein trafficking and stress. Overall, these results show that in fish copper has distinct effects on tissue Cu transporter genes and oxidative stress depending on whether it is taken up via the gill or gut and that intestinal absorption may be required for normal uptake and metabolism of Cu, regardless of the route of uptake. Moreover, changes in mRNA levels indicate that Cu homeostasis genes, at least in fish, may be regulated at the transcriptional level. Although more work needs to be done to identify genes that are robust predictors of Cu toxicity, the microarray results presented here show a clear transcriptional fingerprint which may characterize Cu toxicity in fish.

Gilthead seabream (Sparus aurata) is one of the most important species of intensively reared fish in the Mediterranean region. Its short history of domestication, along with the need to develop markets, new products and efficiency in the production process, has resulted in an increased interest in the potential genetic improvement of this species. Little work has so far been directed at establishing the procedures for selective breeding in gilthead seabream at a commercial level, although genetic parameters calculated in other studies have indicated that there is a large potential for improvement in certain traits. Selective breeding of commercial gilthead seabream populations is constrained by aspects of the biology that complicate the production of genetic groups and the maintenance of same-age offspring populations. The aim of this thesis was to develop a protocol for the selective breeding of gilthead seabream, specifically to serve a commercial hatchery and on-growing unit in Cyprus, where the fieldwork was carried out. The hatchery broodstock was monitored over a three-year period to identify the rate of sex reversal in introduced fish and to quantify the sex ratio of the stock over time. The analysis of the egg production records was used to evaluate the success of photoperiod manipulation in each group. Size variation in the larval and juvenile stages is a common problem in the rearing of gilthead seabream, leading to cannibalism and labour-intensive sorting operations. Studies on larval populations, from first feeding through to metamorphosis, indicated the origin of size variation was the differences in early feeding ability. The size advantages could be maintained throughout the larval period. During the juvenile stage of the farm production system, a method to standardise the size sorting of populations by grading was developed in order to counter environmental effects of separating groups of fish. Using this method, grading would be suitable to form the first stage of a selection programme for growth rate. The potential gain of selection for growth rate during the on-growing stage was very high, using a simulated criterion and previous estimates of heritability. Other possible quality traits for selection were also examined and quantified in the hatchery populations. Existing and specifically developed microsatellite markers were used for the assignment of offspring to parents from mass spawning of the hatchery broodstock. The effective population size of single spawning events were found to be low and determined by a high variation in contribution to mass spawning. Contribution was found to be significantly linked to body size, which led to the formation of a replacement policy for the broodstock to maximise spawning performance. Survival of individual families through the larval period was also examined. Based on the results of the experimental work, a two-stage selection programme was designed, along with the presentation of specific procedures for each stage of the production system. This project makes recommendations on various strategies that can increase the effective population size within a selection programme and these are discussed as part of the genetic management of hatchery populations. Significant progress has also been made in the use of genetic markers in monitoring the rate of inbreeding and contribution of individual broodfish, which are considered essential in this species.

<p> </p><p> </p><p> </p><p><p>The main</p><p>objective of this thesis is to study the polarization effects of the beetle <em>Cetonia aurata </em>using Mueller-matrix ellipsometry. The outer shell of the beetle consists of complex microstructures which control the polarization of the reflected light. It has metallic appearance which originates from helicoidal structures. When these microstructures are exposed to polarized or unpolarized light, only left-handed circularly polarized light is reflected. Moreover, the exo-skeleton of the beetle absorbs right-handed polarized light. Multichannel Mueller-matrix ellipsometer or dual rotating compensator ellipsometer, called RC2, from J.A.Woollam is used to measure the polarization caused by different parts of beetle’s body. The 16 Mueller matrix elements are measured in the spectral range 400-800 nm at multiple angles of incidencein the range 40⁰-70⁰. An Optical model is developed to help us understand the nature and type of microstructure which only reflects the green colour circularly polarized light. With the help of multiparametric modeling, we were able to find optical properties and structural parameters. The parameters are: the number of layers, the numbers of sub-layers, their thicknesses, and the orientation with respect to optical axes. This optical model describes the nanostructures which provide the reflection properties similar to the nanostructure found in the beetle <em>Cetonia aurata. </em>The model is also useful for analysis of the optical response data of different materials with multilayer structures.</p></p><p> </p>

The main objective of this thesis is to study the polarization effects of the beetle Cetonia aurata using Mueller-matrix ellipsometry. The outer shell of the beetle consists of complex microstructures which control the polarization of the reflected light. It has metallic appearance which originates from helicoidal structures. When these microstructures are exposed to polarized or unpolarized light, only left-handed circularly polarized light is reflected. Moreover, the exo-skeleton of the beetle absorbs right-handed polarized light. Multichannel Mueller-matrix ellipsometer or dual rotating compensator ellipsometer, called RC2, from J.A.Woollam is used to measure the polarization caused by different parts of beetle’s body. The 16 Mueller matrix elements are measured in the spectral range 400-800 nm at multiple angles of incidencein the range 400-700. An Optical model is developed to help us understand the nature and type of microstructure which only reflects the green colour circularly polarized light. With the help of multiparametric modeling, we were able to find optical properties and structural parameters. The parameters are: the number of layers, the numbers of sub-layers, their thicknesses, and the orientation with respect to optical axes. This optical model describes the nanostructures which provide the reflection properties similar to the nanostructure found in the beetle Cetonia aurata. The model is also useful for analysis of the optical response data of different materials with multilayer structures.

Els peixos carnívors presenten baixa capacitat per metabolitzar carbohidrats i per mantenir la glucèmia. En aquests organismes, la baixa capacitat per metabolitzar carbohidrats condueix a estats d’hiperglicèmia més marcats i sostinguts que els descrits per a mamífers, després de l’administració de glucosa o la ingesta de dietes amb elevat contingut de carbohidrats. L’alanina aminotransferasa (ALT) catalitza la transaminació reversible entre L-alanina i α-cetoglutarat per formar L-glutamat i piruvat. Mitjançant la interconversió d’aquests quatre metabòlits, l’ALT esdevé un nexe d’unió entre el metabolisme d’aminoàcids i el de carbohidrats. En estudis previs del nostre grup es va descriure la presència de tres isoformes ALT en S. aurata, els isoenzims citosòlics, cALT1 i cALT2, i la isoforma mitocondrial mALT. L’expressió hepàtica de cALT2 incrementa en situacions gluconeogèniques, mentre que la de cALT1 és predominant durant el període postprandial per a l’utilització dels nutrients de la dieta. L’objectiu d’aquest treball és incrementar el coneixement del metabolisme intermediari en peixos i així permetre realitzar futures intervencions biotecnològiques amb la intenció de millorar la utilització metabòlica dels nutrients de la dieta. Per comprendre millor la funció d’mALT vam estudiar la distribució tissular, la caracterització cinètica i la regulació nutricional i hormonal de l’expressió d’mALT en S. aurata. Addicionalment, vam analitzar l’expressió tissular i la regulació hormonal dels enzims aspartat aminotransferasa mitocondrial (AST2), glutamat deshidrogenasa (GDH) i glutamina sintetasa (GlnS), relacionats tots ells amb el metabolisme d’aminoàcids. En orades alimentades, mALT, GDH i GlnS s’expressen majoritàriment a fetge, intestí i ronyó. Els nostres estudis indiquen que l’expressió de mALT, GDH, AST2 i GlnS és elevada en animals alimentats, mentre que disminueix en condicions associades amb la gluconegènesi, com ara el dejú i el tractament amb estreptozotocina (STZ). Estudis cinètics de l’activitat enzimàtica mALT indiquen que l’enzim catalitza de manera més eficient la conversió d’L-alanina a piruvat, que no pas la reacció inversa. Per conèixer el mecanisme molecular implicat en la regulació transcripcional de l’expressió d’mALT, vam aïllar el promotor mALT d’orada, i vam mostrar que HNF4α incrementa la transcipció d’mALT per unió a una caixa HRE. El dejú i l’administració d’STZ disminuïren els nivells d’HNF4α en ronyó d’S. aurata, portant a un descens en la transcripció d’mALT. Els nostres resultats suggereixen que HNF4α té un paper important en la regulació transcripcional del gen mALT en ronyó d’S. aurata. En conclusió, els nostres resultats suggereixen que mALT i cALT1, que presenten una distribució tissular i un patró d’expressió en dejú i tractament amb STZ similars, podrien cooperar per direccionar els aminoàcids de la dieta al mitocondri per destinar-los a l’obtenció d’energia. Per tant, ALT es podria utilitzar com a diana per realitzar una intervenció biotecnològica a fi de reduir la utilització de proteïnes amb finalitats energètiques i optimitzar així l’ús dels nutrients de la dieta en el cultiu de peixos. / Carnivorous fish have poor ability to use dietary carbohydrates and to control the blood glucose levels. Compared with mammals, these animals show prolonged hyperglycemia after a glucose load or when feeding on high carbohydrate diets. Alanine aminotransferase (ALT) links carbohydrate and amino acid metabolism through catalyzing the reversible transamination between L-alanine and 2-oxoglutarate to form pyruvate and L-glutamate. Our group, in previous studies showed the presence of three ALT isoforms in Sparus aurata: the cytosolic isoenzymes cALT1 and cALT2 and a mitochondrial isoform, mALT. In fish liver, increased expression of cALT2 is associated to enhanced gluconeogenesis while cALT1 is predominant during the postprandial utilization of dietary nutrients. The aim of the present study was to increase the current knowledge of fish intermediary metabolism to allow future biotechnological actions in order to improve metabolic utilization of dietary nutrients. To better understand the functional role of mALT we analysed the tissue distribution, kinetic characterization and nutritional and hormonal regulation of mALT expression in S. aurata. Furthermore, cloning and characterization of the mALT promoter was also addressed. Additionally, tissue expression and nutritional regulation of glutamate dehydrogenase (GDH), mitochondrial aspartate aminotransferase (AST2) and glutamine synthetase (GlnS), also involved in amino acid metabolism, was followed. In S. aurata under feeding conditions, mALT, GDH and GlnS are mainly expressed in liver, intestine and kidney. Our studies indicate that the expression of mALT, GDH, AST2 and GlnS is increased in fed animals, while decreased in conditions associated with gluconeogenesis, such as fasting or treatment with streptozotocin (STZ). Kinetic analysis of mALT enzyme activity indicated that this enzyme catalyses more efficiently the conversion of L-alanine to pyruvate than the reverse reaction. To understand the molecular mechanism underlying the transcriptional regulation of mALT expression, we isolated the S. aurata mALT promoter, and showed that HNF4α enhances mALT transcription through binding to an HRE box. Starvation and administration of STZ decreased HNF4α levels in the kidney of S. aurata, leading to downregulation of mALT transcription. Our results suggest that HNF4α may play an important role in the transcriptional regulation of mALT gene in kidney of S. aurata. In conclusion, our findings suggest that mALT and cALT1, which present a similar tissue distribution and pattern expression under starvation and STZ-treatment, can cooperate to redirect dietary amino acids to the mitochondria for energetic pourposes. This points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.

La acuicultura en su conjunto produce hoy en día más de la mitad del pescado consumido en el mundo. El objetivo de la presente tesis doctoral es estudiar la aplicación de diferentes tecnologías para incrementar y diversificar productos procedentes de acuicultura. Concretamente se han analizado las tecnologías de cocción y fritura a vacío, biopreservación y elaboración de texturizados a partir de dorada (Sparus aurata). Para ello en tres capítulos se aborda el estudio de estas tecnologías para el desarrollo de productos alternativos elaborados a base de dorada que permitan la diversificación del sector. El primer capítulo se centra en el estudio del proceso de fritura y cocción a vacío de filetes de dorada para evaluar aspectos relacionados con su calidad como ganancia de aceite, pérdida de agua, cambios de color, etc. con vistas a definir los tiempos óptimos de procesado del producto. En el segundo capítulo se estudia el desarrollo de texturizados, para ello se pretende analizar el efecto de diferentes formulaciones con o sin la adición de fibras sobre las características, fisicoquímicas, sensoriales y microbiológicas de los productos obtenidos con o sin cocción de los mismos. Por último, en el tercer capítulo se analiza la aplicación de técnicas de biopreservación con las que se pretende determinar las condiciones de preparación de los agentes biopreservantes, su forma de aplicación y el efecto sobre la vida útil de los filetes de dorada. Los resultados obtenidos muestran que el tratamiento de fritura a vacío a 110 ºC y 25 kPa con un tiempo comprendido entre tres y cinco minutos aporta un menor contenido graso con unas pérdidas de humedad, peso y encogimiento razonables, por lo que éstas podrían ser unas condiciones adecuadas para la aplicación de dicho tratamiento. Los tratamientos estudiados de cocción a vacío mostraron que no afectan al contenido total en proteínas y grasas del pescado, pero sí que pueden resultar interesantes para la conservación de los ácidos grasos monoinsaturados ya que además mantienen valores más elevados de humedad y presentan un menor encogimiento. / Andrés Bello, MD. (2012). Aplicación de nuevas tecnologías para el diseño y desarrollo de productos de dorada (Sparus aurata) procedente de acuicultura [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/15757 / Palancia

The aim of the work presented in this thesis is to contribute to a fundamental understanding of polarizing phenomena in some scarab beetles. The aim is also to study the beetle structures as inspiration in fabrication of artificially sculptured films. The three investigated species Cetonia aurata, Potosia cuprea and Liocola marmorata are of the family Scarabaediae and subfamily Cetoniianae (Guldbaggar). They were all collected at Swedish locations and are the only species of Cetoniinae scarabs in Sweden. This work reports on their optical properties represented by Mueller matrix elements, degree of polarization data and trace curves in the Cartesian complex plane representation of polarized light. From these results we verifyan earlier structural model for the Cetonia aurata and make way for similar models of the other two species. The ellipsometer used in this work is of dual rotating compensator type from which the complete Mueller-matrix for the medium examined can be obtained. The ellipsometric measurements were conducted on the scutellum for four different angles of incidence, 45°, 55°, 65° and 75° over a wave-length range of 245-1000 nm. Common for all examined species is that left polarization is observed in the wavelength range of 400 800 nm. For most of these species the polarization state is close to circular at some wavelengths especially at smaller angles of incidence. In general the degree of polarization is high (above 50%) when the polarization is near-circular. The degree of polarization also shows a clear dependence on the angle of incidence. The earlier model for Cetonia aurata shows a good agreement with the experimental data of this work. The model is also found as a good basis to work from to create models for the other two species.

Los peces carnívoros presentan baja capacidad para utilizar carbohidratos provenientes de la dieta y controlar los niveles de glucosa en sangre. En comparación con los mamíferos, estos animales tras la ingesta de glucosa o de dietas con alto contenido en carbohidratos, muestran una hiperglucemia mas prolongada. La alanina aminotransferasa (ALT) constituye un nexo de interacción entre el metabolismo de aminoácidos y el de carbohidratos al catalizar la reacción de la transaminación reversible entre L-alanina y 2-oxoglutarato para formar piruvato y L-glutamato. Estudios previos de nuestro grupo indicaron la presencia de tres isoformas ALT en dorada (Sparus aurata): las isoenzimas citosólicas cALT1 y cALT2 y una isoforma mitocondrial, mALT. En hígado de dorada, la expresión de cALT2 incrementa en situación de gluconeogénesis mientras que cALT1 predomina durante el período postprandial para la utilización de los nutrientes de la dieta. El objetivo general del presente estudio es comprender a nivel molecular los efectos metabólicos derivados de la inhibición de ALT en peces para ayudar a establecer nuevas aplicaciones biotecnológicas orientadas a mejorar la utilización de los nutrientes de la dieta. Así, en acuicultura, identificar los efectos metabólicos asociados a la modulación de la actividad ALT constituye un punto de interés para conocer si es posible efectuar una sustitución parcial de las proteínas de la dieta por carbohidratos u otros nutrientes, a fin de reducir el coste de la producción en acuicultura y disminuir la eutrofización de las aguas del entorno. Nuestros estudios muestran que la inyección intraperitoneal de doradas con nanopartículas del complejo pCpG-siRNA-quitosán resultó adecuada para promover la expresión de un siRNA para bloquear la expresión de cALT1 en hígado de Sparus aurata. La inyección intraperitoneal de nanopartículas de pCpGsi1sh1-quitosán promovió la silenciación de cALT1 a nivel de mRNA y actividad enzimática en hígado de dorada. Por otra parte, hemos analizado la inhibición postranscripcional de la actividad ALT in vivo e in vitro con el compuesto aminooxiacetato (AOA) y analizado los cambios promovidos en metabolitos y enzimas clave en el metabolismo intermediario de carbohidratos y proteínas en hígado de Sparus aurata, tras la ingesta del AOA con dietas de diferente composición. In vitro, el AOA ejerce una inhibición dependiente de dosis sobre la actividad ALT hepática citosólica y mitocondrial. In vivo, el AOA se comportó como inhibidor de la actividad ALT citosólica hepática, pero no de la mitocondrial. Una exposición a largo plazo a AOA promovió un aumento de la actividad piruvato quinasa en el hígado de dorada, independientemente de la composición de la dieta suministrada a los peces. Los estudios de 1H-RMN mostraron que la inclusión de AOA en la dieta promueve una disminución en los niveles hepáticos de alanina, glutamato y glucógeno. Adicionalmente, los análisis de 2H-RMN indicaron una tasa de renovación más alta de alanina en el hígado de los peces alimentados con una dieta con un contenido alto en carbohidratos y bajo en proteínas y que el AOA disminuye el enriquecimiento de alanina en 2H independientemente de la composición de la dieta. Los estudios derivados de esta tesis indican que la inhibición dependiente de AOA de la actividad de la ALT citosólica podría contribuir a aumentar el uso de nutrientes por carbohidratos de la dieta de Sparus aurata. / Carnivorous fish have low ability to utilize dietary carbohydrates and controlling blood glucose levels. Compared with mammals, these animals after ingestion of glucose or diets high in carbohydrates show a more prolonged hyperglycemia. Alanine aminotransferase (ALT) links carbohydrate and amino acid metabolism through catalysing the reversible transamination between L-alanine and 2-oxoglutarate to form pyruvate and L-glutamate. Previous studies from our group indicated the presence of three isoforms ALT bream (Sparus aurata): cALT1 cytosolic isoenzymes and cALT2 and a mitochondrial isoform, mALT. In the liver os Sparus aurata cALT2 expression increases gluconeogenesis situation prevails while cALT1 during the postprandial period for the utilization of dietary nutrients. The overall objective of this study is to understand at a molecular level the resulting metabolic effects from the inhibition of ALT in fish to help establish new biotechnological applications aimed to improve the use of dietary nutrients. Thus in aquaculture, identifying metabolic effects associated with ALT activity modulation is a point of interest to know if it is possible to perform a partial replacement of dietary proteins by carbohydrate or other nutrients, to reduce the cost of Aquaculture production and reduce eutrophication of the environment. Our studies show that intraperitoneal injection of a chitosan-pCpG-siRNA nanoparticles complex to Gilthead seabream proved to be suitable for the expression of a siRNA to silence the expression of liver cALT1 in Sparus aurata. Intraperitoneal injection of pCpGsi1sh1-chitosan nanoparticles complex promoted cALT1 silencing at the mRNA level and a decrease of liver enzyme activity of Gilthead seabream. In the present study amino-oxyacetate (AOA) was used to evaluate its effect on liver ALT activity of the carnivorous fish Sparus aurata. Moreover, the derived metabolic effects on metabolites and other key enzymes of glycolysis, gluconeogenesis and the pentose phosphate pathway were also studied. A dose-effect-dependent inhibition of AOA on hepatic cytosolic and mitochondrial ALT activity was observed in vitro. In vivo, AOA behaved as an inhibitor of hepatic cytosolic ALT activity. A long-term exposure to AOA increased pyruvate kinase activity in the liver irrespective of the composition of the diet supplied to fish. 1H NMR studies showed that inclusion of AOA to the diet decreased the hepatic levels of alanine, glutamate and glycogen. Moreover, 2H NMR analysis indicated a higher renewal rate for alanine in the liver of fish fed with a high-carbohydrate/low-protein diet, while AOA decreased alanine 2H-enrichment irrespective of the diet. The present study indicates that AOA-dependent inhibition of the cytosolic ALT activity could help to increase the use of dietary carbohydrate nutrients by the Sparus aurata fish.

En la presente Tesis Doctoral se ha estudiado la presencia y bioacumulación de compuestos organoclorados en la piscicultura del Mediterráneo occidental, así como en el entorno marino circundante. La investigación realizada se estructura en tres partes diferenciadas. En la primera parte se ha utilizado la técnica GC-MS para el estudio de la presencia de los contaminantes organoclorados en productos de la acuicultura marina, investigándose el comportamiento de los mismos a lo largo del ciclo biológico de la dorada (Sparus aurata), y comparando los resultados obtenidos en ejemplares salvajes y cultivados. Además, se ha realizado un estudio específico sobre la transferencia maternal de organoclorados a los ovocitos de doradas salvajes y cultivadas. En la segunda parte se han determinado las relaciones entre isótopos estables de carbono y nitrógeno con el objetivo de estudiar las diferentes características alimentarias de las doradas salvajes y cultivadas, relacionándolas con el comportamiento de los compuestos organoclorados en las cadenas tróficas estudiadas. Por último, se ha desarrollado metodología analítica para la determinación de Dioxin-Like PCBs mediante GC-MS/MS (trampa iónica), en muestras procedentes de la acuicultura marina, estudiando en detalle las etapas de purificación de los extractos y el fraccionamiento de los analitos previamente a la determinación analítica.