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The potential anti-inflammatory effect of plant sterols (PS) enriched milk-based fruit beverages (PS, 1 g/100 mL) (MfB) with/without galactooligosaccharides (GOS, 2 g/100 mL) (MfB-G) in an experimental mice model of chronic ulcerative colitis was evaluated. Beverages were orally administered to mice every day by gavage to achieve PS and GOS doses of 35 and 90 mg/kg, respectively, and experimental colitis was induced by giving mice drinking water ad libitum containing 2% (w/v) dextran sulphate sodium (DSS) for 7 days, alternating with periods without DSS up to the end of the study (56 days). MfB beverage showed significant reduction of symptoms associated to ulcerative colitis and improved the colon shortening and mucosal colonic damage, but it was not able to reduce the increase of myeloperoxidase levels produced by DSS. MfB-G showed higher incidence of bloody feces and loss of stool consistency than MfB, as well as high levels of immune cells infiltration in colon tissue and myeloperoxidase. Therefore, PS-enriched milk-based fruit beverage could be an interesting healthy food to extend the remission periods of the diseases and the need to evaluate, in a pre-clinical model, the anti-inflammatory effect of the combination of bioactive compounds in the context of a whole food matrix.

Las bebidas a base de zumo de frutas y leche son matrices adecuadas para el enriquecimiento con esteroles vegetales (EV) debido a su perfil nutricional saludable y aporte de compuestos antioxidantes (β-criptoxantina (β-Cx), polifenoles y vitamina C). Aunque el efecto más estudiado de los EV es el hipocolesterolemiante, recientes estudios apuntan a que podrían tener beneficios adicionales sobre la defensa frente a estrés oxidativo, procesos inflamatorios a nivel intestinal y sistémico y actividad antiproliferativa en células de cáncer de colon. El enriquecimiento con galactooligosacáridos (GOS) podría ser una estrategia para mejorar la funcionalidad de las bebidas enriquecidas en EV, dado su conocido efecto beneficioso sobre la microbiota intestinal y procesos inflamatorios colónicos, si bien se ha indicado que la adición de fibra soluble puede reducir la bioaccesibilidad de los esteroles en modelos de digestión gastrointestinal simulada. El objetivo general de la Tesis Doctoral es evaluar la influencia que ejercen los GOS sobre la bioaccesibilidad de los esteroles y los efectos beneficiosos asociados al consumo regular de bebidas a base de zumo de frutas y leche conteniendo EV, β-Cx y GOS con un abordaje triple: in vitro (Caco- 2), ex vivo (eritrocitos humanos) e in vivo (Caenorhabditis elegans y ratones). La adición de GOS a bebidas enriquecidas con EV (2,5 g/250 ml) (MfB) a dosis de 2,3 y 4,5 g/250 ml (MfB-G2 y MfB-G5, respectivamente) no afecta a la bioaccesibilidad del colesterol (78-88%) y EV totales (~37%) evaluada tras la aplicación de una digestión gastrointestinal micelar, por lo que se selecciona la bebida con mayor contenido de GOS para posteriores estudios de bioactividad (MfBG5). La aplicación del método estandarizado de digestión Infogest a la bebida sin GOS no permite la cuantificación del colesterol en la fracción bioaccesible y requiere un cambio en el origen de las sales biliares utilizadas (bovinas en lugar de porcinas) o reducir la concentración de sales biliares de origen porcino (10 mM a 1,4 mM). Los resultados obtenidos en los ensayos de citoprotección en células Caco-2 diferenciadas (modelo de epitelio intestinal) indican que la pre y coincubación con las fracciones bioaccesibles de las bebidas objeto de estudio protegen frente al estrés oxidativo y la apoptosis inducida por óxidos del colesterol (COPs). El efecto citoprotector observado se confirma en el modelo de C. elegans en el que las bebidas, además de reducir el estrés oxidativo producido por los COPs y el H2O2, incrementan la longevidad del nematodo. En general, la bebida con GOS no potencia el efecto citoprotector. En células Caco-2 diferenciadas y en modelo murino con colitis crónica, se constata que la presencia de GOS reduce el efecto beneficioso de las bebidas sobre mediadores proinflamatorios (IL-6, IL-8 y factor nuclear-κB (NF-κB)), los síntomas clínicos asociados a la colitis y el daño del tejido colónico. 4 Los EV de forma individual o como mezcla, a concentraciones colónicas, muestran un efecto antiproliferativo en células Caco-2 no diferenciadas, siendo más efectivos cuando se incuba como mezcla de EV. Así mismo, se observa que los EV a concentraciones séricas, estimadas a partir de la ingesta de una bebida enriquecida en EV y β-Cx, atenúan la activación de procesos eriptóticos y previenen completamente la hemólisis en eritrocitos humanos inducida por la β-Cx con y sin estrés oxidativo. Por lo tanto, los resultados obtenidos a partir de los diferentes modelos utilizados indican que la adición de GOS a las bebidas a base de zumo de fruta y leche enriquecidas en EV no modifican la bioaccesibilidad de los EV. Además, no mejoran los efectos biológicos relativos a citoprotección e inflamación in vitro e in vivo. Milk-based fruit beverages are suitable matrices for enrichment with plant sterols (PS) due to their healthy nutritional profile and presence of antioxidant compounds (β-cryptoxanthin (β-Cx), polyphenols and vitamin C). Although the cholesterol-lowering effect of PS is the most studied, recent studies suggest that PS could have additional benefits on defence against oxidative stress, inflammatory processes at intestinal and systemic levels and anti-proliferative activity in colon cancer cells. The enrichment with galactooligosaccharides (GOS) could be a strategy to improve the functionality of PS-enriched beverages, given its known beneficial effect on the intestinal microbiota and colonic inflammatory processes. However, it has been indicated that the addition of soluble fibre can reduce bioaccessibility of sterols in simulated gastrointestinal digestion models. The aim of the Doctoral Thesis is to evaluate the influence of GOS on the bioaccessibility of sterols and the beneficial effects associated with regular consumption of milk-based fruit beverages containing PS, β-Cx and GOS with triple approach; in vitro (Caco-2), ex vivo (human erythrocytes) and in vivo (Caenorhabditis elegans and mice). The addition of GOS to PS-enriched beverages (2.5 g / 250 ml) (MfB) at doses of 2.3 and 4.5 g/250 ml (MfB-G2 and MfB-G5, respectively) does not affect the evaluated bioaccessibility of cholesterol (78-88%) and total PS (~ 37%) after micellar gastrointestinal digestion. Therefore, the beverage with the highest GOS content is selected for subsequent bioactivity studies (MfB-G5). The application of the standardised digestion method of Infogest to the beverage without GOS does not allow the quantification of cholesterol in the bioaccessible fraction and requires a change in the origin of the bile salts used (bovine instead of porcine) or a reduction in the concentration of porcine bile salts (10 mM to 1.4 mM). The results obtained from the cytoprotective assays in differentiated Caco-2 cells (intestinal epithelial model) indicate that pre- and co-incubation with the bioaccessible fractions of the beverages tested protects against oxidative stress and apoptosis induced by cholesterol oxidation products (COPs). The cytoprotective effect observed is confirmed in the C. elegans model in which beverages, in addition to reducing the oxidative stress produced by COPs and H2O2, increase the longevity of the nematode. In general, the beverage with GOS does not enhance the cytoprotective effect. In differentiated Caco-2 cells and in the murine model with chronic colitis, the presence of GOS reduces the beneficial effect of beverages on pro-inflammatory mediators (IL-6, IL-8 and nuclear factor-κB (NF-κB)), as well as the clinical symptoms associated with colitis and colonic tissue damage. Individual or mixed PS, at colonic concentrations, show an anti-proliferative effect on undifferentiated Caco-2 cells, being more effective when incubated as a PS mixture. Likewise, PS at 6 serum concentrations, estimated from the intake of a beverage enriched in PS and β-Cx, attenuate the activation of eryptotic processes and completely prevent haemolysis in human erythrocytes induced by β-Cx with and without oxidative stress. Therefore, the results obtained from the different models indicate that the addition of GOS to PSenriched milk-based fruit beverages does not modify the bioaccessibility of PS. In addition, they do not improve the biological effects related to cytoprotection and inflammation in vitro and in vivo.

Uno de los problemas más importantes en los sistemas de realidad virtual es la diversidad de los dispositivos visuales y de interacción que existen en la actualidad. Junto a esto, la heterogeneidad de los motores gráficos, los motores físicos y los motores de inteligencia artificial, propicia que no exista un modelo que aúne todos estos aspectos de una forma integral y coherente. Con el objetivo de unificar toda esta diversidad, presentamos un modelo formal que afronta de forma integral el problema de la diversidad en los sistemas de realidad virtual, así como la definición de los módulos principales que los constituyen. El modelo propuesto se basa en la definición de varias gramáticas que integra su actividad, su visualización y su interacción con el usuario. La descripción de un mundo se presenta como una cadena de un lenguaje L(M) que representa una secuencia ordenada de agentes que aceptan eventos y que definen la actividad del sistema. Por otro lado, la visualización del estado actual del sistema se traduce en otra cadena del lenguaje de visualización L(V) y que está compuesta de primitivas y transformaciones. Los conceptos de primitiva y transformación son mucho más amplios de lo que es habitual en estos sistemas. Las primitivas no son simples primitivas de dibujo sino acciones que se deben ejecutar en un determinado sistema de representación que es definida mediante otro lenguaje L(G) y que representa las acciones ordenadas en un determinado dispositivo visual o no. Las transformaciones, por otro lado, modifican las primitivas, dependiendo de su naturaleza. Los agentes desarrollan una o varias actividades en el mundo virtual, se visualizan mediante primitivas y transformaciones, y usan eventos que también se definen en sentido amplio. El modelo presentado tiene como virtud la separación de la actividad del sistema de los dispositivos visuales y de interacción concretos que lo componen. Esto supone varias ventajas: los dispositivos pueden ser sustituidos por otros dispositivos o por simulaciones de estos, los elementos del sistema pueden ser reutilizados, y la representación gráfica puede ser diferente dependiendo del dispositivo visual. En definitiva, se ha pretendido diseñar un sistema integral, adaptativo, reutilizable y genérico. Por último, se presentan varios casos prácticos que permiten concretar cómo se utiliza el modelo propuesto.

The potential of integrating multiagent systems and virtual environments has not been exploited to its whole extent. This paper proposes a model based on grammars, called Minerva, to construct complex virtual environments that integrate the features of agents. A virtual world is described as a set of dynamic and static elements. The static part is represented by a sequence of primitives and transformations and the dynamic elements by a series of agents. Agent activation and communication is achieved using events, created by the so-called event generators. The grammar defines a descriptive language with a simple syntax and a semantics, defined by functions. The semantics functions allow the scene to be displayed in a graphics device, and the description of the activities of the agents, including artificial intelligence algorithms and reactions to physical phenomena. To illustrate the use of Minerva, a practical example is presented: a simple robot simulator that considers the basic features of a typical robot. The result is a functional simple simulator. Minerva is a reusable, integral, and generic system, which can be easily scaled, adapted, and improved. The description of the virtual scene is independent from its representation and the elements that it interacts with.

Microgreens constitute an emerging class of fresh, healthy foods due to their nutritional composition. In this study the content of minerals and antioxidant bioactive compounds, and for the first time bioaccessibility, were evaluated in broccoli (Brassica oleracea L. var. italica Plenck), green curly kale (Brassica oleracea var. sabellica L.), red mustard (Brassica juncea (L.) Czern.) and radish (Raphanus sativus L.) hydroponic microgreens. Macro- (K, Ca, Mg) and oligo-elements (Fe, Zn), ascorbic acid, total soluble polyphenols, total carotenoids, total anthocyanins, total isothiocyanates and total antioxidant capacity (Trolox Equivalent Antioxidant Capacity and Oxygen Radical Absorbance Capacity) were determined before and after the standardized simulated gastrointestinal digestion process. All microgreens provided relevant amounts of vitamin C (31&ndash, 56 mg/100 g fresh weight) and total carotenoids (162&ndash, 224 mg &beta, carotene/100 g dry weight). Mineral content was comparable to that normally found in hydroponic microgreens and the low potassium levels observed would allow their dietetic recommendation for patients with impaired kidney function. Both total soluble polyphenols and total isothiocyanates were the greatest contributors to the total antioxidant capacity after digestion (43&ndash, 70% and 31&ndash, 63% bioaccessibility, respectively) while macroelements showed an important bioaccessibility (34&ndash, 90%). In general, radish and mustard presented the highest bioaccessibility of bioactive compounds and minerals. Overall, the four hydroponic Brassicaceae microgreens present a wide array of antioxidant bioactive compounds.

The eryptotic and hemolytic effects of a phytosterol (PS) mixture (β-sitosterol, campesterol, stigmasterol) or β-cryptoxanthin (β-Cx) at physiological serum concentration and their effect against oxidative stress induced by tert-butylhydroperoxide (tBOOH) (75 and 300 μM) were evaluated. β-Cryptoxanthin produced an increase in eryptotic cells, cell volume, hemolysis, and glutathione depletion (GSH) without ROS overproduction and intracellular Ca2+influx. Co-incubation of both bioactive compounds protected against β-Cx-induced eryptosis. Under tBOOH stress, PS prevented eryptosis, reducing Ca2+influx, ROS overproduction and GSH depletion at 75 μM, and hemolysis at both tBOOH concentrations. β-Cryptoxanthin showed no cytoprotective effect. Co-incubation with both bioactive compounds completely prevented hemolysis and partially prevented eryptosis as well as GSH depletion induced by β-Cx plus tBOOH. Phytosterols at physiological serum concentrations help to prevent pro-eryptotic and hemolytic effects and are promising candidate compounds for ameliorating eryptosis-associated diseases.