Las lesiones severas de nervio periférico se caracterizan por la desconexión del soma neuronal con el órgano inervado. Esta desconexión causa una pérdida de control motor, sensitivo y autonómico que conducen a una situación incapacitante para el paciente. Después de una lesión el sistema nervioso periférico tiene la capacidad de regenerar, pero para ello se requiere de un proceso de limpieza en el fragmento distal a la lesión y la promoción de la regeneración a través del extremo proximal. En esta tesis hemos querido centrarnos en el papel de la matriz extracelular en la regeneración de nervio periférico. Para ello, en el primer capítulo nos hemos centrado en el papel de dichas proteínas en la promoción de regeneración selectiva de axones motores y sensoriales y si una matriz basada en colágeno resulta un vehículo adecuado para funcionalizar guías neurales artificiales para la reparación de lesiones nerviosas. Utilizamos un cultivo organotípico de secciones transversales de médula espinal y explantes de ganglio de la raíz dorsal de animales postnatales y contrastamos nuestros resultados con un modelo adulto in vivo. Nuestros resultados muestran que tanto las matrices enriquecidas con fibronectina o laminina incrementan la elongación de neuritas sensoriales mientras que las neuritas motoras solo respondían a las matrices enriquecidas con fibronectina, al compararlas con matrices controles de colágeno sin enriquecer in vitro. Además, en etapas postnatales las matrices enriquecidas con fibronectina promovían la regeneración específica de neuritas tanto motoras como sensoriales proprioceptivas, mientras que laminina actuaba sobre neuritas sensoriales cutáneas. Esta respuesta selectiva de las neuronas hacia proteínas de la matriz extracelular resulta importante para facilitar la organización de los axones regenerados hacia ramas musculares o cutáneas después de la lesión. Sin embargo, esta actividad preferencial mostrada se perdía en edad adulta. En segunda instancia buscamos la mejor alternativa quirúrgica al autoinjerto en la reparación de lesiones severas de nervio periférico. Nos centramos en mejorar la técnica de reparación por tubulización en un modelo crítico de regeneración de 15 mm en el nervio de rata, donde en general, la recuperación funcional, reinervación de órganos diana y el número de fibras mielínicas se ve comprometido con respecto al autoinjerto (todavía considerada como la mejor técnica en la práctica clínica). Los resultados muestran que la dificultad de los axones para regenerar en el interior de las guías neurales artificiales se relaciona con la capacidad limitada de dichos axones de crear un ambiente proregenerativo que les permita cruzar por el interior del lumen tubular. En el primer caso, la reparación con tubos de silicona resultó en un fracaso absoluto. Sin embargo, los tubos basados en quitosano, un material biodegradable y absorbible, mejoró el número de animales regenerados hasta porcentajes cercanos al 50%. A pesar de estos resultados esperanzadores, todavía nos encontrábamos lejos del 100% de éxito obtenido con el autoinjerto. Por ello, en el tercer capítulo, mejoramos el lumen tubular mediante la adición de las matrices enriquecidas con laminina o fibronectina estudiadas en el primer capítulo, tanto como hidrogeles hidratados como estabilizados. En este caso, el porcentaje de animales regenerados aumentó hasta un máximo del 75% en las matrices enriquecidas con fibronectina estabilizada. Con el objetivo final de imitar el ambiente regenerativo que encontramos en un fragmento distal de un nervio lesionado, en el último capítulo mejoramos el lumen tubular mediante la incorporación de células de soporte. Cultivamos células de Schwann y células madre mesenquimales y las integramos en matrices alineadas enriquecidas con fibronectina o laminina. Mediante la implementación de estas últimas matrices junto a la administración del agente inmunosupresor FK506, conseguimos obtener unos resultados similares a la reparación con autoinjerto. / Severe peripheral nerve injuries are characterized by the disconnection of the neural soma and the innervated organ. This disconnection causes the loss of motor, sensory or autonomic control which lead to a disabling situation for the patient. Regeneration can occur in the peripheral nervous system, but the clearance of the degenerated distal nerve and the promotion of regeneration of the proximal stump are needed to facilitate adequate reinnervation and functional recovery. In this thesis we wanted to focus in the role of the extracellular matrix (ECM) for peripheral nerve regeneration. For that in our first chapter we focused on the role of the ECM to promote selective regeneration of motor and sensory axons and if collagen-based scaffolds are suitable vehicles to functionalize artificial nerve conduits in limited peripheral nerve gaps. We used organotypic spinal cord slices and DRG explants of early postnatal and young adults animals, and we contrasted our results in the adult in vivo. Our results show that fibronectin and laminin enriched scaffolds increased the elongation of sensory neurites whereas motor neurites were more elongated in fibronectin compared to collagen-based scaffolds in vitro. Furthermore, in young postnatal stages, we were able to preferentially promote elongation of motor neurites and presumptive proprioceptive neurites on fibronectin-enriched matrices, whereas laminin increased the elongation of presumptive cutaneous sensory neurites. This is important, since neuron type preference towards specific extracellular matrix components may facilitate the organization of regenerating axons in muscular or cutaneous nerve branches. However, this preferential activity was gradually decreased and finally lost in the adult. Second, we searched for a feasible surgical alternative to autograft in the repair of severe peripheral nerve injuries. For that we focused in the improvement of the tubulization repair technique for the repair of severe peripheral nerve defects (15 mm in the rat), where the general outcome achieved by this repair technique (degree of recovery, functional reinnervation and number of regenerated fibers) is still inferior to the autograft, (considered the gold standard technique for the clinical practice) despite assuming the loss of a secondary healthy nerve. The results show that the greatest difficulty of axons to regenerate trough tubular devices is related to the limited capacity of injured axons to create an enabling environment that allows regeneration in the lumen of the tubular devices. In the first case, the repair with silicone conduits resulted in a complete failure during the regeneration process. However, the use of chitosan-based guides, a biodegradable and absorbable material, improved the number of regenerated animals to a percentage close to 50%. Although these promising results for a hollow conduit, the percentage of regenerated animals was still far from the 100% found in the autograft. For that reason, in our third chapter we led to the implementation of intratubular content by introducing fibronectin and laminin enriched collagen scaffolds added, both as fully hydrated hydrogels or stabilized and rolled. In this case, the percentage of animals that regenerated increased to a maximum of 75% in the fibronectin-enriched, stabilized and rolled hydrogels. With the ultimate goal of mimicking what is occurring in a healthy nerve or the distal fragment of a degenerated nerve, in the fourth chapter we decided to fully functionalize the lumen of the tube by incorporating support cells. We harvested and cultured Schwann cells and Mesenchymal Stem cells which were embedded in a tethered-aligned collagen-based construct enriched with either fibronectin or laminin and implanted to the animals. By implementing the latter matrices together with the coadjuvant administration of the immunosuppressant agent FK506, we achieved a degree of recovery comparable to the autograft model.

Reproductive physiology has many applications for a successful management of fish population in aquaculture. In particular the stock management of endangered species might take advantage of the knowledge concerning the reproductive cycle in order to improve the protocols and restore the population for restock activities. The Adriatic sturgeon (Acipenser naccarrii) and the European eel (Anguilla anguilla) are two endemic species of the North Adriatic Sea and are both listed in the the IUCN Red List of threatened species as “critically endangered”. Both the species nowadays rely on the experimental activities and new technologies that try to implement the farming conditions for an improvement of the population management for restocking purposes. In the present study the onset of puberty was investigated through plasma Testosterone analysis in A. naccarii. With reference to A. Anguilla the effect of photoperiod was assessed on the endocrine profiles during hormonal induction. Furthermore a new tank design has lead to spontaneous spawning that was then compared to the manual stripping protocol.

La continua immissione in ambiente dei farmaci ad uso umano, e la loro incompleta rimozione nei depuratori rende questi composti pseudo-persistenti e potenzialmente pericolosi per gli organismi acquatici. La ricerca ha inteso valutare gli effetti sub-letali di farmaci di rilevanza ambientale su due organismi d’interesse commerciale: il mitilo mediterraneo e l’anguilla europea, tramite lo sviluppo e l’applicazione di indicatori biologici, in laboratorio e in campo. Gli studi sull’anguilla hanno permesso di identificare nella glicogenolisi, valutata in termini di glucosio rilasciato dagli epatociti, il parametro ideale per valutare alterazioni sul metabolismo glucidico. Con l’utilizzo di un metodo in vitro dinamico e sensibile, la perifusione di epatociti isolati in colonna, sono stati valutati gli effetti dei β-bloccanti propranololo ed atenololo sul metabolismo glucidico. I risultati hanno mostrato la maggior potenza del propranololo nell’alterare la glicogenolisi e la maggior sensibilità dei pesci quando esposti al farmaco. Questa metodologia potrebbe permettere di rilevare la presenza di composti adrenergici, noti e non, nelle matrici ambientali. La consolidata batteria di biomarker applicata sui mitili in condizioni controllate, ha permesso di valutare gli effetti specifici della caffeina dopo esposizione di 7 giorni a concentrazioni ambientali. I risultati hanno evidenziato una moderata induzione della sindrome da stress, confermando la bassa pericolosità della caffeina per gli organismi acquatici, rispetto ad altri farmaci. L’applicazione degli stessi biomarker in un sistema naturale soggetto ad effetti antropici (la laguna Piallassa Piombone), ha permesso di valutare la qualità biologica della laguna, ma non ha potuto imputare gli effetti osservati ai residui farmaceutici, risultati minoritari rispetto ad altri inquinanti. In conseguenza di effetti osservati a basse dosi, concludiamo che è necessario aumentare le conoscenze sugli effetti che questi contaminanti hanno sulle specie non target, e di sviluppare nuove e sensibili metodologie applicabili nella valutazione di rischio ambientale derivante da farmaci. / The continuous release and incomplete removal of human pharmaceuticals in the environment poses a risk for aquatic wildlife. The research aimed at evaluating sub-lethal effects of pharmaceuticals of environmental concern on two commercial aquatic species: the Mediterranean mussel and the European eel. The effects were assessed through the development and application of biological indicators in experiments under controlled conditions and in the field. Studies on eel allowed to identify that glycogenolysis, assessed in term of glucose released from the hepatocytes, is the best parameter to evaluate the alterations on hepatic glucose metabolism. The development of a sensitive in vitro dynamic method (perifusion of isolated hepatocytes on columns), permitted to assess the effects of two β-blockers (propranolol and atenolol) on fish glucose metabolism. The results showed that propranolol is more potent than atenolol in modifying the glycogenolysis. Moreover, fish resulted more sensitive when exposed to therapeutic concentrations of the pharmaceutical. The use of this technique could allow the search for unknown adrenergic compounds in environmental matrices. The well-established set of biomarkers used in controlled conditions, allowed the evaluation of the specific effects of caffeine on mussels health status, after 7-days exposure to environmentally relevant concentrations. Results showed a moderate induction of the stress syndrome, confirming the low risk posed by caffeine for aquatic species, in comparison with other pharmaceuticals. The application of the same set of biomarkers in a natural impacted ecosystem (the Piallassa Piombone lagoon), allowed to assess the biological quality of the lagoon, but could not attribute the observed effects to pharmaceuticals amongst other pollutants. Considering the effects observed at low pharmaceutical doses, we highlight the need for a wider knowledge regarding the effects that these contaminants on non-target species, and for implementing new and sensitive methodologies to be used in the environmental risk assessment posed by pharmaceuticals.

Starch is a polymer of D-glucose that plants accumulate as semi-crystalline and osmotically inert granules. Besides being the major energy storage in plants, starch is of primary importance also in human and animal diet, industry and biofuels production. Two kinds of starch, structurally indistinguishable, but different for location and rates of synthesis and degradation are found in plants: secondary starch (characterized by long term-accumulation and found in storage organs) and transitory starch (mainly located in chloroplasts, produced during the day and degraded the subsequent night to meet the energy demand of the plant). Due to its structure, several enzymes are required in starch biosynthesis and degradation, controlling distinct features of starch granules and conferring different physical-chemical properties. Here, the TILLING approach was used on the barley TILLMore population to identify new alleles in five genes related to secondary starch metabolism and known to be expressed in barley seeds. Moreover, the role in Arabidopsis development of the (phospho)glucan, water dikinase proteins (GWD1, GWD2, PWD), known to be involved in nighttime transitory starch degradation, was investigated. Other enzymes, such as β-amylase 1 (BAM1) and α-amylase 3 (AMY3), were demonstrated to be involved in diurnal transitory starch degradation in specialized cells or under stress conditions. Here, we demonstrated that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline, a compatible solute, required to face osmotic stress. Moreover, the behaviour of Arabidopsis BAM1 and AMY3 enzymes under oxidative treatments and the possible role of glutathionylation, a redox post-translational modification occurring mainly under stress conditions and promoted by ROS, were investigated. AtBAM1 and AtAMY3 were found to be sensitive to oxidants and glutathionylated, with a modulatory and protective effect on protein activity.

En els últims anys ha crescut considerablement l’interès per descobrir aliments naturals funcionals amb propietats beneficioses per a l’hoste. En aquest sentit, el cacau ha passat a ser un dels principals subjectes d’estudi pel seu contingut en flavonoides. Són molts els estudis que associen la ingesta de cacau amb efectes beneficiosos sobre la salut. A més, se li han atribuït propietats immunomoduladores en rata. En base a aquests efectes descrits, l’objectiu d’aquesta Tesi Doctoral va ser establir l’efecte de dietes enriquides amb cacau, flavonoides del cacau i fibra de cacau sobre la microbiota intestinal així com sobre la funció immunitària intestinals. Per tal d’assolir aquests objectius, s’han portat a terme estudis preclínics en rata amb una dieta enriquida amb cacau convencional al 10%, dietes elaborades a partir de dos extractes de cacau no fermentats i una dieta amb fibra de cacau. Pel que fa als resultats de microbiota, es va observar diferent patró de composició després de la intervenció nutricional amb les dietes enriquides amb flavonoides del cacau però únicament la dieta de fibra de cacau va mostrar un efecte prebiòtic al promoure el creixement dels gèneres Bifidobacterium i Lactobacillus. A més, la dieta de fibra de cacau va comportar els canvis més pronunciats en la producció d’AGCC en femtes i contingut cecal. Particularment, va augmentar la concentració a nivell fecal i cecal dels àcids acètic, propiònic i butíric. A més, les dietes de cacau i fibra de cacau van modular de forma diferencial l’expressió gènica de TLR en còlon. Quant a les immunoglobulines en el compartiment mucosal, totes les dietes enriquides amb polifenols del cacau van modular la secreció d’IgA intestinal, tot i que de forma no proporcional al seu contingut en flavonoides. La fibra de cacau, depenent del compartiment intestinal estudiat, va exercir un efecte o un altre. Pel que fa al compartiment extraintestinal, tot i que la dieta de fibra de cacau va mostrar el mateix efecte atenuador de la síntesi d’IgA i d’IgM que la dieta de cacau, el mecanisme d’acció va ser diferent. A més, totes les dietes enriquides amb flavonoides del cacau, van disminuir la seva proporció de bacteris fecals units a IgA independentment del seu contingut en flavonoides, mentre que aquest percentatge va incrementar amb la dieta de fibra de cacau. Únicament la dieta al 10% de cacau provoca un alentiment en la corba ponderal. Aquest efecte es correlaciona amb els canvis produïts en la microbiota i es pot associar amb la modificació de l’expressió en còlon dels gens implicats en el metabolisme lipídic. Pel que fa al perfil metabòlic en orina, les dietes de cacau i fibra de cacau van provocar patrons diferencials, els quals poden ser usats com a marcadors d’ingesta. El perfil metabòlic es correlacionen amb els efectes del cacau sobre el pes corporal, amb les hormones metabòliques, amb la immunitat intestinal i amb la composició de la microbiota. A més, aquestes variables també mostren correlació entre si. Els efectes del cacau són el resultat de la suma del efectes del components bioactius present en el cacau: polifenols i fibra de cacau, els quals exerceixen efectes sinèrgics o bé contraris depenent de la variable estudiada. Altres components del cacau també estan involucrats en aquests efectes. / In the last few years, cocoa has become one of the main subjects of study due to its high content in flavonoids. Several studies have associated the cocoa intake with health benefits. Moreover, immunomodulatory properties in rats have been also attributed to cocoa. On this basis, the aim of the present thesis was to establish the impact of diets enriched with cocoa, cocoa flavonoids or cocoa fiber on the fecal microbiota composition and its activity as well as on the immune function in the gut. To achieve this objective, preclinical studies were carried out in rats fed a 10% conventional cocoa-enriched diet, diets elaborated with different amounts of non- fermented cocoa extracts and cocoa fiber diet. Regarding microbiota results, differential composition pattern was observed after all the experimental diets intake but only the cocoa fiber diet increased the Bifidobacterium spp. and Lactobacillus spp. proportion. In addition, the cocoa fiber diet was the one which caused the most pronounced changes in the short chain fatty acids (SCFA) production. Particularly, it increased the cecal and fecal concentration of acetic, propionic and butyric acids. Moreover, both the 10%-cocoa diet and cocoa fiber diets differentially modulated the TLR gene expression in the colon. Concerning the mucosal immunoglobulin production, all cocoa polyphenol-enriched diets modulated the intestinal IgA secretion although this effect was not proportional to their flavonoid content. The cocoa fiber diet also exerted an effect on intestinal IgA secretion but in a different way depending on the compartment. Focusing on the extraintestinal compartment, although the cocoa fiber diet showed the same down- modulatory effect on IgA and IgM secretion as the cocoa diet, its mechanisms were different. In addition, all cocoa flavonoid-enriched diets decreased IgA-coated bacteria proportion in a non-dose dependent manner whereas this percentage increased by the cocoa fiber intake The 10% cocoa diet was the only one that caused a slower body weight gain. This effect is correlated with the microbiota modulation. The change induced by cocoa diet on the expression of genes involved in the lipid metabolism in the colon could be also involved. Regarding the urinary metabolites, the cocoa and the coca fiber diets caused differential metabolic profile that can be used as consumption marker. The metabolic fingerprint correlated well with the body weight, the metabolic hormones, the intestinal immunity and the microbiota composition. Moreover, all these variables showed also an association between them. Therefore, the effects produced by cocoa intake are due to the differential effects caused by each one of its main bioactive compounds - polyphenols and fiber - which act in a synergistic or opposite manner depending on the variable. Other cocoa compounds are also involved in such effects.

També adscrita al Dept. Farmàcia i Tecnologia Farmacèutica / En los últimos años, ha incrementado la importancia del estudio de los efectos cronobiológicos con respecto a la farmacología y el diagnóstico, no obstante, la aplicación de la cronobiología en la dermatología no ha sido prácticamente estudiada. Los ritmos circadianos pueden influir en la permeación y la absorción a través de la piel de fármacos administrados por vía dérmica y transdérmica o en la manifestación de enfermedades de la piel. Por otro lado, la melatonina es una sustancia que tiene propiedades demostradas muy diversas. Es la hormona principal en la regulación del ritmo de sueño-vigilia, pero también posee una potente actividad antioxidante y por tanto puede utilizarse para evitar la oxidación en los tratamientos con radioterapia y quimioterapia o por exposición a la radiación ultravioleta del Sol. En estos casos, pese a que la vía oral podría ser de elección, la piel sufre directamente la radiación UV y la radiación usada en la radioterapia. Conociendo la existencia de la cronofarmacocinética y de la cronofarmacología, ¿Sería lo mismo administrar una formulación con melatonina transdérmicamente a una hora del día que a otra? ¿Influyen los ritmo de la piel en cómo se absorbe la melatonina? Con el fin de responder a las preguntas planteadas, los objetivos de esta tesis doctoral han sido (1) la elaboración y caracterización de formulaciones con melatonina y otros compuestos para administración por vía dérmica/transdérmica capaces de ejercer una acción local como antioxidante y que pudiera absorberse para alcanzar la circulación sistémica y ejercer una acción reguladora de ritmos biológicos o antioxidante. (2) el estudio de variables funcionales de la piel en un modelo animal seleccionado para conocer si presentan un ritmo y si éstos siguen la ritmicidad del reloj biológico interno o bien tienen un funcionamiento libre, en distintas condiciones lumínicas y ambientales. (3) En base a los resultados del objetivo anterior, realizar un estudio cronofarmacocinético para evaluar si la presencia de los ritmos funcionales de la piel y del organismo tienen influencia en la farmacocinética de la melatonina. Los resultados obtenidos demuestran la presencia de ritmos en las variables funcionales de la piel analizadas y que éstos influyen en como la melatonina se absorbe a través de la piel, juntamente con el resto de variables del organismo que modulan los procesos de ADME, haciendo que durante la fase de reposo, la biodisponibilidad de la melatonina sea mayor. Por lo tanto, en el modelo animal utilizado, la fase de reposo es la recomendada para aplicar la melatonina por vía transdérmica tanto si se busca un efecto local como sistémico. / Circadian rhythms can influence the permeation and absorption of drugs through skin that are dermally or transdermally administered. Furthermore, melatonin is a substance that has very diverse properties demonstrated. It is the main hormone in regulating the rhythm of sleep-wake, but also has a potent antioxidant activity, therefore can be used to prevent the oxidation caused by treatment with radiation and chemotherapy or by exposure to ultraviolet radiation from the sun. In these cases, although the oral route may be preferred, the skin directly suffers the UV radiation and the radiation used in radiotherapy. Knowing the existence of chronopharmacokinetics and chronopharmacology, would it be the same to administer transdermally a formulation with melatonin at a time of the day or at another? Do the rhythms of the skin influence in the rate of melatonin that it is absorbed? In order to answer these questions, the objectives of this thesis were (1) the preparation and characterization of formulations with melatonin and other compounds for dermal/transdermal administration that would be able to exert a local action as an antioxidant, and that could be absorbed to reach the systemic circulation and exert a systemic antioxidant action or regulate biological rhythms (2) the study of skin function variables in an animal model, in different light and environmental conditions, to see if they are rhythmic and if they follow the rhythmicity of the internal biological clock, or on the contrary have a free run (3) based on the results of the previous objective, to perform a chronopharmacokinetics study to evaluate whether the presence of rhythms in the skin functions and in the body influence the pharmacokinetics of melatonin. The results have demonstrated the presence of circadian rhythms in skin functions for the parameters analyzed, and thus influence in melatonin absorption through the skin, along with other variables of the organism that modulate ADME processes. In the case of the rats, during the rest phase, the bioavailability of melatonin is greater than during activity phase. Therefore, in this animal model, the resting phase is the recommended for applying melatonin transdermally for obtaining a local or systemic effect.

The present work takes into account three posterior parietal areas, V6, V6A, and PEc, all operating on different subsets of signals (visual, somatic, motor). The work focuses on the study of their functional properties, to better understand their respective contribution in the neuronal circuits that make possible the interactions between subject and external environment. In the caudalmost pole of parietal lobe there is area V6. Functional data suggest that this area is related to the encoding of both objects motion and ego-motion. However, the sensitivity of V6 neurons to optic flow stimulations has been tested only in human fMRI experiments. Here we addressed this issue by applying on monkey the same experimental protocol used in human studies. The visual stimulation obtained with the Flow Fields stimulus was the most effective and powerful to activate area V6 in monkey, further strengthening this homology between the two primates. The neighboring areas, V6A and PEc, show different cytoarchitecture and connectivity profiles, but are both involved in the control of reaches. We studied the sensory responses present in these areas, and directly compared these.. We also studied the motor related discharges of PEc neurons during reaching movements in 3D space comparing also the direction and depth tuning of PEc cells with those of V6A. The results show that area PEc and V6A share several functional properties. Area PEc, unlike V6A, contains a richer and more complex somatosensory input, and a poorer, although complex visual one. Differences emerged also comparing the motor-related properties for reaches in depth: the incidence of depth modulations in PEc and the temporal pattern of modulation for depth and direction allow to delineate a trend among the two parietal visuomotor areas.

Cocoa is a well recognized source of polyphenols, but studies on their consumption refer that it contributes with a low proportion to the daily polyphenol consumption. Cocoa polyphenol content is plenty recognized and the benefits from its consumption in human health are established. However, the contribution of cocoa to daily polyphenol intake is not totally clear due to the lack of studies estimating cocoa consumption. Taking these facts into account, the hypothesis of this thesis was that cocoa consumption is underestimated in overall food intake studies and perhaps it can be associated with healthy status. Therefore, the present thesis was carried out on the following aims: 1) to develop and validate a food frequency questionnaire (FFQ) including the main cocoa food products common in Catalan and Portuguese dietary habits in a young population; 2) to establish the consumption of cocoa in university students of Catalonia and Portugal; and 3) to establish the relationship between cocoa intake and healthy status, including the presence of allergies, and healthy lifestyle practices. The FFQ developed as well as the other questionnaires to validate them have been applied to a sample of 50 students from the University of Barcelona and to a sample of 70 students from Egas Moniz Health Sciences Institute. Afterwards, validated FFQ together questionnaires inquiring about physical activity and also health status were applied to a sample of 270 students from both institutions. The main results of this thesis consist in the development and validation of a FFQ devoted to know the real cocoa intake in University students in Spanish and Portuguese languages. This FFQ allows the assessment of more cocoa/chocolate product intake than others FFQ because it considered a wide range of products which were not included in others FFQ (dairy, pastry, desserts, cereals and spreads). According the developed FFQ, students from the University of Barcelona use to eat 2.5 portions of products containing cocoa or chocolate per day that represents 12 g of cocoa per day, whereas the students of the Egas Moniz Health Science Institute consume more than 3 products per day which represent 14 g of cocoa per day. Moreover, differential cocoa consumption patterns between both cohorts have been found. In a cohort of 270 students from both Universities it has been established correlations between cocoa consumption and physical activity, body mass index, blood pressure, recent illness and also allergic processes. In addition, the values of these variables were assessed in students grouped according their cocoa consumption (low, moderate and high consumers). There was not a significant correlation of cocoa intake with physical activity and blood pressure, but there was a significantly lower proportion of overweight individuals in the high cocoa consumers group. The proportion of allergic people in the moderate and high cocoa consumers resulted statistically lower than that in the students in the low cocoa consumer group. Moreover, the cocoa intake, especially moderate consumption, was also associated with lower presence of allergic derived symptoms. In summary, the present study evidences the underestimation of cocoa consumption due to approach limitations and has generated a valid tool for estimating real cocoa consumption. Cocoa intake in University students has been assessed, and different patterns of consumption have been found between Portuguese and Spanish university students regarding the type of cocoa source more frequently consumed. Finally, some clear associations between cocoa consumption and health status have been found. Therefore, this study can be the first part of a new study using the same methodology in a higher number of participants or different type of population. Alternatively, these health improvements by cocoa consumption showed here can be confirmed by an interventional clinical study. / El cacao posee efectos beneficiosos para el organismo, si bien estudios sobre ingesta de alimentos revelan su bajo consumo. Los objetivos de esta tesis han sido: 1) desarrollar y validar una encuesta de frecuencia de consumo alimentario que incluya los alimentos con cacao frecuentes en la dieta de Cataluña y Portugal; 2) estimar la ingesta de cacao en estudiantes universitarios de los dos países; y 3) evaluar la asociación entre ingesta de cacao e indicadores de actividad física y de salud, incluyendo las alergias. El FFQ desarrollado se ha validado con 50 estudiantes de la Universidad de Barcelona y 70 estudiantes de Egas Moniz Health Sciences Institute. Posteriormente, se ha aplicado a 270 estudiantes de ambas instituciones junto con cuestionarios acerca de su actividad física y su estado de salud. Los resultados muestran el desarrollo y validación de un FFQ dedicado a conocer la ingesta real de cacao en los estudiantes universitarios. Su aplicación ha permitido evaluar la ingesta de más productos de cacao/chocolate que otros FFQ, puesto que considera una amplia gama de productos. Según el FFQ desarrollado, los estudiantes de la Universidad de Barcelona ingieren 2,5 porciones de productos con cacao/chocolate por día, lo que representa unos 12 g/día, mientras que los estudiantes del Egas Moniz Health Sciences Institute consumen más de 3 productos por día, que representan unos 14 g/día. Se han estudiado la relación entre el consumo de cacao y la actividad física, el índice de masa corporal, la presión arterial, una enfermedad reciente y también procesos alérgicos. No se ha hallado una correlación significativa entre la ingesta de cacao y la actividad física y la presión arterial, pero existe una proporción menor de personas con sobrepeso en el grupo de estudiantes con elevado consumo de cacao. El porcentaje de personas alérgicas en el grupo con moderado y elevado consumo de cacao ha resultado ser inferior a la de los estudiantes en el grupo de baja ingesta de cacao. Por otra parte, el consumo de cacao, especialmente el consumo moderado, también se ha podido asociar a una menor presencia de manifestaciones alérgicas.

La exposición a una sesión breve de choques eléctricos en un contexto determinado induce el desarrollo de miedo a dicho contexto, fenómeno conocido como condicionamiento de miedo al contexto. Además, produce a largo plazo una inhibición de la actividad en ambientes nuevos, efecto no necesariamente asociado a un aumento de la ansiedad, por lo que la interpretación de esta hipo-actividad se desconoce. Los resultados obtenidos indican que la aparición de la hipo-actividad inducida por la exposición previa a una sesión breve de choques eléctricos, sólo aparece si se ha podido desarrollar previamente un condicionamiento del miedo al contexto. Por otro lado, no observamos ninguna evidencia de un aumento de la conducta ansiosa. Este hallazgo sugiere que los animales que han podido desarrollar una generalización del miedo. Además, dicha hipo-actividad no iba asociada a un aumento de la respuesta del eje HPA y podía ser en gran medida revertida mediante la extinción del miedo condicionado. En el estudio de los efectos de la manipulación de la intensidad y el número de choques sobre los efectos condicionado, pudimos observar que desde un punto de vista conductual, la manipulación de los parámetros resultó poco efectiva, lo que contrasta con lo observado en la inducción de la hipoactividad, donde la variación de la intensidad si resultó relevante. Sin embargo, se observó una respuesta endocrina intensidad-dependiente delante del contexto condicionado. Por otro lado, la sensibilización hormonal observada tras la exposición a un ambiente nuevo está relacionada con la intensidad del choque recibido. Por último, el estudio de la IMO como estímulo estresante no mostró evidencias de condicionamiento ni a nivel conductual ni endocrino. Por lo tanto, concluimos que el desarrollo del condicionamiento del miedo, evaluado mediante medidas conductuales y endocrinas, no es una propiedad universal compartida por todos los estímulos estresantes, lo que ayudaría a explicar las divergencias en los efectos a largo plazo obtenidos con diferentes estímulos estresantes. / Exposure to a single session of footshock in absence of any specific cues results in the development of fear to the shock chamber, this is called contextual fear conditioning, that it is usually evaluated by time spend freezing. Furthermore, this shock exposure induces long-lasting inhibition of activity in unknown environments, these effects are not necessarily associated to an enhanced anxiety and the interpretation of the hypoactivity remains unclear. However, the development of contextual fear conditioning using other stressors remains poorly studied. Our results indicated that hypoactivity and a certain level of generalization of fear to the unknown environment only appeared when animals are able to developed fear conditioning, but no evidence for enhanced anxiety was found. These results suggest that if animals are able to associate an aversive experience with unknown environments, they display more caution behaviors in any unknown environments. In addition, this hypoactivity was not associated with greater HPA response in any different environment and it could be partially reversed by an extinction protocol. Moreover, the effect of shock intensity and number of shocks on both conditioned and unconditioned consequences was also studied. From the behavioural point of view the manipulation of intensity and number of shocks does not appear to be very relevant. But, an intensity-dependent endocrine response is observed when animals were exposed to the conditioned context. On the other hand, the endocrine sensitization after novel environments exposure is related to the high response showed after shock exposure. Regarding the use of other stressors to induce contextual fear conditioning, despite shock and IMO showed similar severity, no behavioural or endocrine evidence of conditioning was found if IMO was used as a stressor. Therefore, it can be concluded that development of fear conditioning, as evaluated by either behavioural or endocrine measures, appears to be dependent on the nature of the aversive stimuli. These phenomena could help to explain the divergences in long-lasting effects of each stressor.

El estrés oxidativo (OS) es característico de muchas enfermedades y se produce cuando hay un desequilibrio entre oxidantes y antioxidantes, lo cual favorece un estado oxidativo que genera especies reactivas de oxígeno y de nitrógeno. Los lípidos de la membrana plasmática son dianas preferentes del OS y ello tiene como consecuencia la peroxidación lipídica. Este proceso modifica propiedades de la membrana tales como su fluidez, característica física muy importante conocida por modular la localización de las proteínas de membrana y las uniones receptor-ligando. Objetivos: 1) Evaluar el efecto del OS en la regionalización de la fluidez en la membrana plasmática de células vivas tales como macrófagos THP-1 y linfocitos MEC-1, de manera individualizada. 2) Analizar, en estas células, la relación entre la peroxidación lipídica y la fluidez de membrana. 3) Estudiar el efecto del OS sobre la unión receptor-ligando y sobre la fluidez de membrana: lipopolisacárido/receptores de tipo Toll (TLR2/4) en macrófagos y progesterone-induced blocking factor (PIBF)/PIBF-receptor en linfocitos. Material y Métodos: Se estandarizó la metodología two-photon microscopy por primera vez en la Universidad Autónoma de Barcelona, para analizar la fluidez de membrana en células vivas individuales. Conjuntamente se ha desarrollado un nuevo software capaz de medir el tamaño y el número de los dominios lipídicos de membrana. El OS se indujo mediante H2O2 y se empleó la sonda fluorescente Laurdan para detectar las diferencias de fluidez en la membrana plasmática. Se utilizaron LPS y PIBF soluble en macrófagos y linfocitos respectivamente, para analizar interacciones receptor-ligando en condiciones OS. Resultados: En los macrófagos se observó un aumento significativo, dependiente de la concentración de H2O2, en la frecuencia de regiones lipídicas rígidas principalmente compuestas por dominios lipid raft, a expensas de las regiones de fluidez intermedia. Asimismo, se detectó en condiciones de OS, un mayor número, aunque no un mayor tamaño, de dominios lipid raft. La activación de macrófagos con LPS incrementó la frecuencia de regiones fluidas en las membranas, efecto que fue inhibido en condiciones de OS. En cuanto a la función de los macrófagos, se detectó una disminución en la secreción de TNFα en condiciones oxidantes. En los linfocitos se observó un aumentó significativo en la frecuencia de regiones lipídicas rígidas, a expensas de las regiones fluidas, en condiciones de OS. Por otro lado, la unión del PIBF a su receptor, provocó un aumentó en la rigidez de la membrana plasmática debido al clustering de dominios lipid raft. Por el contrario, cuando se indujo OS en linfocitos en presencia de PIBF, se inhibió el clustering de dominios lipid raft y también disminuyó el reconocimiento del receptor de PIBF a su ligando. Conclusiones: 1) Se ha evaluado en células vivas, de forma individual, la dinámica lipídica de la membrana plasmática. 2) Una consecuencia general importante es que, durante el OS, tanto en macrófagos como en linfocitos la membrana plasmática se vuelve más rígida. 3) La fluidez de membrana cambia de forma distinta en los dos tipos celulares estudiados, como consecuencia de las interacciones receptor-ligando: durante la unión LPS-TLR2/4 se observó un aumento en la fluidez de la membrana plasmática de los macrófagos y, por el contrario, durante la unión PIBF/PIBF-R la membrana de los linfocitos se rigidificó, aumentando el clustering de los dominios lipid raft. 4) No obstante, en ambos casos el OS inhibió los cambios en la fluidez de membrana inducidos por la unión receptor-ligando. / Oxidative stress is present in many diseases and it is produced in cells when an imbalance between oxidants and antioxidants occurs, favoring an oxidant status which produce reactive oxygen and nitrogen species. Lipids in plasma membrane are one of the preferential targets giving rise to lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and receptor-ligand binding. Aims: 1) To evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages and MEC-1 lymphocytes. 2) To analyze, in these cells, the relationship between lipid peroxidation and membrane fluidity. 3) To study the effect of oxidative stress on receptor-ligand binding and membrane fluidity: lipopolysaccharide/toll-like receptors (TLR2/4) in macrophages and progesterone-induced blocking factor (PIBF)/PIBF-receptor in lymphocytes. Material and Methods: Two-photon microscopy was standardized for the first time in Universidad Autónoma de Barcelona by our laboratory, to analyze membrane fluidity in single living cells. It was also developed a new software application to analyze membrane lipid domain size and number. Cellular oxidative stress was induced by H2O2; the fluorescent probe Laurdan was applied to evaluate plasma membrane fluidity changes. LPS in macrophages or soluble PIBF in lymphocytes were used to analyze receptor-ligand interactions under oxidative stress. Results: Macrophages showed a significant H2O2 concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. Under oxidative stress conditions, an increase in number, but not in size, of lipid raft domains was detected. Macrophage activation by LPS increase the frequency of fluid regions, which was inhibited by oxidative stress. Concerning macrophage function, secretion of TNFα under oxidative conditions was decreased. Lymphocytes showed a significant increase in the frequency of rigid lipid regions, at the expense of fluid regions, under oxidative stress conditions. Upon PIBF binding to its receptor, lymphocyte plasma membrane became more rigid due to clustering of lipid rafts. However, when PIBF bound lymphocytes were placed in oxidizing conditions, lipid raft clustering was inhibited and PIBF binding to its receptor was also decreased. Conclusions: 1) In single living cells plasma membrane lipid dynamics was evaluated. 2) An important general consequence of oxidative stress is that both in macrophages and lymphocytes plasma membrane becomes more rigid. 3) Receptor-ligand interactions have an effect on membrane fluidity, which vary greatly between the two cell types studied: macrophages and lymphocytes. Upon receptor-ligand binding, macrophage plasma membrane became more fluid while lymphocytes plasma membrane became more rigid. Our results suggest that lipid raft clustering is linked to cell function: upon PIBF binding to its receptor lipid raft clustering occurs in lymphocytes; however, upon LPS/TLR2/4 lipid raft clustering does not occur in macrophages. 4) Nevertheless, the effect induced by receptor-ligand binding on membrane fluidity was inhibited during oxidative stress in both cases.