Search

Tesina-Universidad de Murcia., Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TL 119.

La especie porcina es considerada el modelo animal idóneo para el estudio y desarrollo de terapias contra enfermedades humanas debido a sus similitudes genéticas, anatómicas y fisiológicas con el hombre. Sin embargo, la eficiencia de la producción in vitro (PIV) de embriones porcinos para utilizarlos con este fin sigue siendo muy baja, pues aun persisten los problemas de la fecundación polispérmica y las condiciones de cultivo subóptimas. Por estos motivos, se pretende profundizar en el conocimiento de los diferentes factores relacionados con el oviducto porcino que pudieran ser utilizados para modular el proceso de la fecundación in vitro (FIV) y contribuir a la mejora de los sistemas de PIV de embriones. La presente tesis aborda tres posibles vías de mejora diferentes en cuanto a complejidad e implicaciones fisiológicas consistentes en la adición al medio de cultivo de tres tipos de compuestos: fluido oviductal (FO), α-L-fucosidasa y óxido nítrico (NO). El objetivo del primer estudio fue determinar las condiciones óptimas de inclusión del FO en los protocolos de FIV. Para ello, se preincubaron ovocitos madurados in vitro con FO pre o postovulatorio y se realizó FIV. Se midieron las concentraciones de E2 y P4 en el FO. Finalmente se comparó la eficiencia de la FIV al utilizar semen fresco o congelado con ovocitos preincubados en FO preovulatorio. La eficiencia de la FIV porcina mejoró con la preincubación en FO preovulatorio. Cuando se utilizó semen congelado su capacidad de penetración aumentó comparando con semen fresco. La concentración de E2 en el FO preovulatorio fue mayor que la determinada en sangre para la misma fase del ciclo estral y la adición de E2 al medio de FIV a la concentración detectada en el oviducto produjo un efecto similar al de la adición de FO. Concluimos que la preincubación de los ovocitos en FO preovulatorio o la adición de E2 pueden considerarse protocolos adecuados para aumentar la eficiencia de la FIV porcina con semen fresco. En el segundo estudio se investigó el papel de la α-L-fucosidasa durante la FIV porcina. Para ello se añadió la enzima al medio de FIV y se observó un incremento en la penetración y en la unión de espermatozoides a la zona pelúcida (ZP). Se realizó inmunofluorescencia indirecta para detectar la α-L-fucosidasa espermática y finalmente se realizaron estudios de capacitación espermática, demostrándose que la α-L-fucosidasa aumenta los niveles de [Ca2+] intracelular y la fosforilación de tirosina. Por lo que concluímos que la α-L-fucosidasa estimula eventos relacionados con la capacitación en espermatozoides porcinos, dando lugar a una mayor capacidad de unión a la ZP y de penetración al ovocito. En el tercer estudio se investigó el papel del NO durante la maduración in vitro (MIV) porcina. Para ello se suplementó el medio de MIV con tres inhibidores distintos de la óxido nítrico sintasa (NOS) y con un donante de NO. Se estudió la expansión de las células del cúmulus y la reanudación de la meiosis, la resistencia de la ZP a la digestión enzimática y parámetros relacionados con la FIV. Los resultados mostraron que los tres inhibidores ejercían un efecto sobre la expansión de las células del cúmulus y que la reanudación de la meiosis solamente se vio detenida al añadir el inhibidor aminoguanidina. Posteriormente, se planteó la posibilidad de que la inhibición de las NOS afectara a la S-nitrosilación de proteínas del ovocito. Para ello se realizó nitrosilación in situ cuantificándose la intensidad de fluorescencia. De este modo, se demostró que la S-nitrosilación de proteínas es una de las vías por la que el NO ejerce su efecto en el ovocito, revelando la importancia del NO en la MIV., The porcine species, owing to their genetic, anatomic, and physiologic similarities with humans, is considered as the ideal animal model for studying and developing novel therapies against human diseases. However, the efficiency of porcine embryo in vitro production (IVP) is nowadays still unacceptably low, because the problem of polyspermic fertilization has still not been adequately worked out and the embryo in vitro culture conditions are still considered to be suboptimal. For these reasons, the present doctoral thesis aims to deepen the understanding of the different factors related to the physiology of the pig oviduct that could be used to modulate the in vitro fertilization (IVF) process and contribute to improve pig embryo IVP systems. Therefore, we investigate three different ways of improvement, in terms of complexity and physiological implications, such as: 1) the oviductal fluid (OF), 2) α-l-fucosidase, 3) nitric oxide (NO). The aim of the first study was to determine the optimal conditions for the OF inclusion in the IVF protocols. For this purpose, in vitro matured oocytes were pre-incubated in OF collected before or after ovulation and subsequently used for IVF. Steroids concentrations in OF were measured and used in IVF experiments. Finally the IVF efficiency was compared between fresh or frozen semen with oocytes preincubated in preovulatory OF. Results showed that pig IVF efficiency improved with preincubation in preovulatory OF. When frozen-thawed semen was used with preovulatory OF incubated oocytes, penetration ability and polyspermy increased. The estradiol (E2) concentration in preovulatory OF was greater than that determined in blood serum at the same phase of the estrous cycle. We conclude that preincubation of oocytes in preovulatory OF or the addition of E2 can be considered suitable tools to increase the efficiency of IVF, only with fresh semen, in the pig. In the second study the role of α-L-fucosidase in pig IVF was investigated. Therefore, the enzyme was added to the IVF medium and a significant increase in sperm binding to the zona pellucida (ZP) and penetration was observed. Thereafter, the sperm-associated α-L-fucosidase was detected by indirect immunofluorescence. In this case, neither the intensity of fluorescence nor the patterns of sperm-associated α-L-fucosidase distribution were affected. Finally, experiments focused on the study of the capacitation events were conducted. The results showed that the addition of exogenous α-L-fucosidase increased the sperm intracellular [Ca 2+] and P-tyrosine phosphorylation, suggesting a role in promoting capacitation and at the same time protecting the sperm cells from premature acrosome reaction. This sequence of events would, in turn, explain the increased ability of this population of treated spermatozoa to bind ZP and penetrate the oocyte. The third study arose to determine the effect of NO during pig oocyte maturation. In vitro maturation (IVM) medium was supplemented with three NOS inhibitors, and a NO donor. The effects on cumulus cells expansion, meiotic resumption, ZP resistance to enzymatic digestion and various parameters related to IVF were studied. The results showed that after IVM, the three NOS inhibitors exerted an inhibitory effect on cumulus cells expansion, while meiotic resumption was suppressed only when the inhibitor aminoguanidine was added. Subsequently, to determine whether the NOS inhibition affect the S-nitrosylation of oocyte proteins, we performed in situ S-nitrosylation and the fluorescence intensity was quantified. Thus, it was shown for the first time that the S-nitrosylation of proteins is one of the ways by which NO exerts its effect on porcine maturation, revealing the importance of NO in both IVM and subsequent fertilization.

El Octodon degus es un roedor endémico de Chile que ha sido caracterizado como diurno tanto en su medio natural como en condiciones de laboratorio. Sin embargo, se ha descrito que algunos individuos invierten su actividad a nocturna cuando se les permite hacer ejercicio en una rueda giratoria instalada en su jaula. Esta tesis doctoral tiene como objetivos: entender este proceso de nocturnalismo, caracterizar los mecanismos circadianos implicados y definir el papel que juega el marcapasos central circadiano. Los resultados apuntan a que el nocturnalismo en los degus se basa en una respuesta termorreguladora de evitación temporal frente a altas temperaturas diurnas. Esta actividad nocturna la consiguen mediante encarrilamiento estable del reloj circadiano pero también mediante la inhibición de la actividad locomotora ante la presencia de luz (masking). Además, la secreción endógena de melatonina, o su administración exógena, como el propio marcapasos circadiano no parecen estar implicados en esta respuesta., Octodon degus is an endemic Chilean rodent which has been characterized as diurnal in both natural habitat and laboratory conditions. However, a switch from diurnal to nocturnal activity has been described in some individuals when they are allowed to exercise in a wheel placed in their cage. The aims of this doctoral thesis are: understand this process of nocturnalism, characterize the circadian mechanisms involved in it and define the role of the circadian pacemaker. The results indicate that degus' nocturnalism is based on a thermorregulatory response of temporal avoidance against high diurnal temperature. This nocturnal activity is achieved by steady-entrainment of the central pacemaker but also by locomotor activity suppression induced by the presence of the light (masking). Moreover, both the endogenous secretion of melatonin, or its exogenous administration, and the circadian pacemaker seem not to be implicated in this response.

Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TL 201.

La luz y los patrones de alimentación influyeron sobre el comportamiento, fisiología y reloj molecular de dos peces teleósteos. Se clonaron dos criptocromos de lubina (cry1 y cry2), expresándose en todos los tejidos estudiados y mostrando ritmos diarios de expresión en cerebro, hígado y corazón (excepto cry2). La fase (día/noche) de alimentación a demanda en lubina afecta a los patrones diarios de actividad amilasa intestinal y expresión de period1 en tejidos periféricos (i.e. hígado), pero no en cerebro. Durante las inversiones estacionales de alimentación de la lubina, mostraron mayores valores de glucosa sanguínea durante la fase de alimentación, revelando también los mayores valores medios en lubinas nocturnas invierno. El pez cebra utilizó un nuevo comedero a demanda, mostrando independencia de fase entre ritmos circadianos de alimentación (nocturno) y locomoción (diurno), con diferentes periodicidades. Además la actividad y posición del pez cebra resultó espectro-dependiente, provocando mayores efectos las longitudes de onda cortas., Thesis summary in English Light and feeding patterns influence on the behaviour, physiology and molecular clock of two teleost fish. Two cryptochromes (cry1 and cry2) were cloned, being expressed in all studied tissues and showing daily rhythm of expression in brain, liver and heart (except cry2). The self-seabass feeding phase (day/night) affected on daily patterns of intestinal amylase activity and on the period1 expression in peripheral tissues (i.e. liver), but not in brain. During the seasonal feeding inversions in seabass, the highest blood glucose levels were shown during their feeding phase, revealing also the highest values in nocturnal seabass in winter. Zebrafish used a new self-feeding system, displaying phase-independence between feeding (nocturnal) and locomotor (diurnal) circadian rhythms, with different periodicities. Moreover, the zebrafish activity and vertical position were spectrum-dependent, being the main effects provoked by short-wavelengths.

Tesis-Universidad de Murcia., Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M. 2624.

Tesis-Universidad de Murcia., Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. D 602., Consulte la tesis en: BCA. GENERAL. DEPOSITO. T.M-1966.

Con el presente trabajo se ha pretendido investigar la influencia de diversos factores relacionados con el cocultivo de los gametos porcinos, sobre los resultados de la fecundación" in vitro" (AV), fundamentalmente con la intención de mejorar la eficacia actual del sistema de AV en cuanto a la consecución de embriones viables (fecundaciones monospérmicas). Para ello, se han utilizado 129 hembras porcinas prepúberes a las que se indujo la ovulación mediante un tratamiento con 1250 U.I. de PMSG y 750 U.I. de HCG. El tratameinto empleado resultó eficaz para los fines perseguidos en un 80'62% de las hembras y el número medio de ovocitos recogidos fue de 19'07 + 1'52 por animal, utilizándose un total de 1984 ovocitos.En relación a las condiciones del sistema de fecundación, el primer factor investigado ha sido el tiempo de cocultivo, entendido como tiempo de contacto entre los gametos. En las dos experiencias realizadas, utilizando tiempos de 4, 6 u 8 horas (experiencia la), o de 1, 2, 3 ó 4 horas (experiencia lb), los mejores resultados se obtuvieron tras 4 horas de cocultivo, ya que los porcentajes de penetración de mantuvieron altos con respecto al máximo alcanzado a las 8 horas (82,68 vs. 93,96%), mientras que los de monospermia no disminuyeron excesivamente con respecto a los obtenidos con tiempos de cocultivo menores, teniendo en cuenta que la concentración de espermatozoides empleada fue intencionadamente elevada (12 x 105 esp/ml). El segundo factor analizado fue la concentración espermática. Se utilizaron concentraciones de 3, 6 y 12 x 105 esp vivos/ml, deduciéndose de los resultados que la mayor efectividad en nuestro sistema correspondía a la concentración de 6 x lO 5 esp/ml, ya que los porcentajes de penetración fueron significativamente diferentes a los obtenidos con la concentración espermática más alta (71'62% vs. 76'83%), y los porcentajes de monospermia tampoco se diferenciaron de los obtenidos con la concentración espermática más baja (62'26 vs. 68'08%). El tercer factor estudiado ha sido la influencia de la presencia o ausencia en el medio de cocultivo del "cumulus" expandido que acompaña al ovocito en la ovulación. Por los resultados obtenidos, se puede pensar que la presencia estas células junto con la correspondiente matriz intrecelular de ácido hialúrico es altamente beneficiosa para la mejora del rendimiento de la FIV debido a que los porcentajes de penetración en los ovocitos denudados (53'69 fueron menores (pPor último, se ha investigado el efecto de la reducción del volumen medio de cocultivo más comúnmente utilizado (2 mI) a otro menor (0'4 n obteniéndose resultados equivalentes en ambos casos para los porcentajes penetración, pero mayores porcentajes de monospermia (pDel conjunto de los resultados se deduce que los porcentajes de penetración y polispermia en la AV porcina son consecuencia de la influencia de diferentes factores, entre los que se encuentran el tiempo de cocultivo, concentración espermática, la presencia del "cumulus oophorus" y el volumen de medio de cocultivo utilizados., In the present work, we have investigated the influence of differen factors, related to porcine gametes coculture, on the results of "in vitro" fertilization (IVF). We have try to improve the efficiency of the current system to get viable embryos (monospermic fertilizations). 129 prepuberal gilts have been used after the induction of ovulation by administration of 1250 I.U. of PMSG followed, 55 hours later, by 750 I.U. of HCG. The results showed that the best moment for the recovery of oocytes was 44 h after HCC administration. In the same way, the treatment followed was effective for the required objectives in 80.62% of the studied females and the medium number of recovered oocytes was 19.07 + 1.52 per animal, giving a total number 01 1984 oocytes used. In relation with the conditions of the fertilization system, the first investigated factor was the coculture time, understanding it as contact time between gametes. To study the effect of this factor, two experiences were realized; fot the first one, 4, 6 or 8 hours of coculture time were used (experience lb) The best results were obtained at 4 hours of coculture, because the percentage of penetration was maintained high (82.68%) and, at the same time, the percentage of monospermy increased (pThe second investigated factor was the sperm concentration. The results showed that, among the used concentrations (3, 6 and 12 x lO 5 alive spz/ml), the maximum effectiveness in our system was obtained for the concntration of 6 x lOS spz/ml, since the percentage of penetration was not signficatively different of that obtained with the highest sperm concentration The third studied factor was the influence of the presence or absence of the expanded ."cumulus", which is shed with the oocytes at the ovulation, in the coculture medium. The presence of these cells joined with the intercellular matrix of hialuronic acid was highly beneficious for the improvement of the IVF, because of the percentage of penetration with the denuded oocytes (53.69%) was lower (p Finally, the percentage of penetration and monspermy was investigated using two different coculture medium volume, one the commonly used by other authors (2 ml) and another minor volume (0.4 mI). The results showed that the percentage of monospermy was higher (p We may deduce from the total results that the percentages of and monospermy in porcine IVF are due to the influence of different factors, some of them being the coculture time, the sperm concentrarían, the presence of "cumulus". and the coculture medium volume.

Objetivos: - Analizar las variaciones en la composición lipídica y el contenido de carbohidratos del músculo y de la glándula digestiva de O. vulgaris durante un periodo corto de ayuno, como aproximación a sus requerimientos nutricionales. - Conocer la aceptabilidad y crecimiento de O. vulgaris alimentado con dietas formuladas con diferentes ingredientes de origen vegetal y animal. - Elaborar piensos formulados en base a los resultados previos de aceptabilidad y comprobar su rendimiento en O. vulgaris mediante ensayos de engorde y digestibilidad. - Estimar los requerimientos lipídicos de O. vulgaris después de suministrar dietas con distinto contenido en aceite de pescado, comprobando los cambios que se producen en el crecimiento, aprovechamiento nutritivo y digestibilidad de la dieta. - Determinar la composición nutricional de O. vulgaris después de suministrar dietas formuladas con distinto contenido en aceite de pescado. - Estudiar los cambios que se producen en el crecimiento, composición nutricional, aprovechamiento nutritivo y digestibilidad de la dieta de O. vulgaris después de suministrar dietas formuladas que incluyen carbohidratos simples o complejos. Metodología: -Ensayos de ayuno: Se configuraron 5 grupos experimentales (n = 4). Cuatro grupos se sacrificaron a los días 1, 2, 4 y 8 después de suministrar la última comida y un grupo control fue alimentado durante los 8 días de duración del experimento. El día del sacrificio los animales se diseccionaron separando el tejido muscular y la glándula digestiva. Se determino el contenido de carbohidratos, los lípidos totales, y las clases lipídicas mediante HPTLC. -Aceptabilidad de ingredientes: Se prepararon diferentes dietas formuladas con un ingrediente a testar (30%), gelatina como aglomerante (20%), y agua (50%). Los ingredientes de la dieta se mezclaron al Baño María a 45ºC y se dejaron enfriar a 4ºC. Se utilizaron grupos experimentales de entre 6 y 8 individuos (887,0 ± 274,2 g) y se le suministró la dieta entre 3 y 15 días. Se calcularon las tasas de alimentación y crecimiento y el índice de eficacia alimentaria de cada dieta. -Ensayos de engorde: Se probaron 8 piensos diferentes elaborados con ingredientes secos o liofilizados. La duración de los ensayos fue de 42 a 56 días con ejemplares subadultos (700-800 g) mantenidos a 18ºC en recirculación con control de la temperatura. Se realizaron análisis de composición corporal (humedad, proteína, grasa, cenizas y MELN) de los animales, las heces y en las dietas suministradas. Con los datos obtenidos se calcularon las tasas de alimentación, crecimiento y digestibilidad. Resultados y Conclusiones En los animales sometidos a un periodo de ayuno se detectó un aumento en los monoglicéridos y el colesterol en el músculo y se movilizó un promedio de 0,23 g de lípidos por día de ayuno, principalmente ácidos grasos libres, ésteres de esterol y triglicéridos procedentes de la glándula digestiva. Los lípidos contribuyeron con un 26% y los carbohidratos con un 9,9% al gasto energético diario durante el ayuno en el pulpo de roca. Los piensos elaborados con liofilizados de pescado y la yema de huevo en polvo ofrecieron los mejores resultados, con tasas de alimentación y crecimiento similares a las obtenidas para dietas naturales a base de pescado. Las dietas artificiales semihúmedas basadas en una mezcla de ingredientes secos o liofilizados, que emplearon pota (T. sagittatus) como ingrediente principal, mostraron buenas tasas de alimentación, crecimiento, aprovechamiento nutritivo y digestibilidad, igualando o superando las dietas naturales a base de pescado. Se sugieren tasas óptimas de ingesta de lípidos para el pulpo en torno a 1 g al día, con porcentajes apropiados de grasa para dietas formuladas entre el 13-14% en sustancia seca, aunque estos porcentajes pueden variar en futuros estudios según las proporciones de lípidos polares y neutros de la dieta. La dieta mejor aceptada incluía almidón al 5%, aunque los mejores índices de aprovechamiento nutritivo se observaron en la dieta con glucosa al 5%, destacando un PPV del 72% e índices de conversión inferiores a 1, Aims: - To analyse the variations in lipid composition and carbohydrates content of muscle and digestive gland of O. vulgaris during short-term starvation as approximation to its nutritional requirements. - To determine the acceptability and growth of O. vulgaris fed formulated diets with different ingredients of vegetal and animal origin. - To prepare formulated diets based on the previous results of acceptability and check its efficiencies on O. vulgaris by means of ongrowing and digestibility experiments. - To estimate the lipid requirements of O. vulgaris after supplying diets with different content in fish oil, comparing the changes that take place in the growth, feed efficiency and digestibility of diet. - To determine the proximate and lipid classes composition of O. vulgaris after supplying formulated diets with different content in fish oil. - To study the changes that produce in the growth, nutritional composition, feed efficiency and digestibility of diet in O. vulgaris after supplying formulated diets including simple or complexes carbohydrates. Methodology: -Experiment of starvation: We configured 5 experimental groups (N = 4). Four groups were sacrificed 1, 2, 4 and 8 days after the last meal and a group control was fed throughout the 8 day experimental period. The day of the sacrifice the animals were dissected separating the muscular and the digestive gland. We determined the content of carbohydrates, total lipids, and lipid classes by means of HPTLC. -Acceptability of ingredients: We prepared different formulated diets with an ingredient to test (30%), gelatine as binder (20%) and water (50%). The ingredients of the diet were mixed at 45ºC and cool to 4 ºC. Each diet was tested in experimental groups of between 6 and 8 individuals (887.0 ± 274. g) during a period between 3 and 15 days. The feeding and growth rates and feed efficiency indices of each diet were calculated. -Ongrowing experiments: Eight different formulated diets including dry or freeze-dried ingredients were tested. The length of the experiments was of 42 to 56 days with subadults (700-800 g) kept to 18ºC in a recirculation seawater system with control of the temperature. The proximate composition (moisture, protein, lipid, ash and MELN) of the animals, the faeces and the diets supplied were analysed. The feeding and growth rates and digestibility were calculated with the data obtained. Results and Conclusions In the animals subjected to a starvation period detected an increase in the monoacylglycerols and the cholesterol in the muscle and 0.23 g of lipids by day of starvation, mainly free fatty acids, steryl esters and triacylglycerols were mobilized from the digestive gland. The lipids contributed with 26% and the carbohydrates with a 9.9 % of the energy costs of the animals during starvation in octopus. The elaborated diet based on freeze-dried fish and egg yolk powder offered the best results, with feeding and growth rates similar to the obtained for natural diets based on fish. The semi-moist formulated diets based on a mixture of dried or freeze-dried ingredients, composed of squid (T. sagittatus) as the main ingredient, showed good feeding and growth rates, feed efficiency and digestibility, similar or surpassing the natural diets based on fish. We propose optimal lipid feeding rates of around 1 g day-1, with appropriate percentages of lipid for formulated diets between the 13-14% dry matter, although these percentages can vary in future studies according to the proportions of polar and neutral lipids of the diet. The best acceptability was detected in the diet formulated with 5% of starch, although the best feed efficiency was observed in the diet with 5% of glucose, highlighting a productive protein value of 72% and a food conversion ratio lower than

La fecundación en mamíferos consiste básicamente en la fusión de los gametos masculino y femenino para formar un zigoto capaz de dar lugar a un nuevo individuo. Hasta ahora, la mayoría de las investigaciones encaminadas al esclarecimiento de este proceso se han centrado en el estudio de la biología de ambos gametos como células aisladas, o bien en el estudio de los diversos mecanismos que tienen lugar durante la interacción de ambos. Sin embargo, se ha dejado un poco de lado a otro elemento fundamental y sin el cual el resto de estudios pueden resultar incompletos: el microambiente en el que tiene lugar el encuentro entre los gametos, es decir, el fluido y las células oviductales que secretan ciertos componentes de dicho fluido. La fase folicular tardía o etapa inmediatamente preovulatoria, es el momento en el que el oviducto se encuentra preparado para el encuentro de los gametos y para que se produzca la fecundación. Debido a la importancia de los factor es implicados en la unión de gametos que han de estar presentes en este momento preciso del ciclo estral y a la escasez de información acerca de los mismos, centramos nuestro estudio en esta fase. Para ello, se abordó el análisis de los componentes oviductales y sus características fundamentalmente desde cuatro perspectivas: (1) efecto del fluido oviductal (FO) sobre la resistencia a la digestión enzimática de la zona pelúcida en 9 especies, (2) fraccionamiento del FO bovino en base a su capacidad de unión a heparina, (3) análisis proteómico del FO y (4) análisis de la expresión génica del oviducto porcino., Fertilization in mammals is basically the merging of male and female gametes to form a zygote that can give rise to a new individual. So far, most research aimed at clarifying this process have focused either on the study of the biology of the spermatozoon and the oocyte as isolated cells or in the study of the various mechanisms that occur during the interaction of both. However, it has been partially left to one side to another key element without which the other studies may be incomplete: the microenvironment in which the meeting takes place between the gametes, ie the fluid and the oviductal cells that secrete some components of such a fluid. The late follicular phase or immediately pre-ovulatory phase is the time when the oviduct is prepared for the meeting of gametes and fertilization occurs. Due to the importance of the factors involved in the gametes interaction which must be present at this precise moment of the estrous cycle and the scarcity of information about them, we focused our study on this phase. To do so, it was addressed the analysis of oviductal components and features mainly from four perspectives: (1) effect of oviductal fluid (OF) on the resistance to enzymatic digestion of the zona pellucida in 9 species, (2) fractionation of the bovine oviductal fluid based on their ability to bind to heparin, (3) proteomic analysis of OF and (4) gene expression analysis of pig oviduct.

Tesina-Universidad de Murcia., Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TM 3262., Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TL 184.

El objetivo de este trabajo consistió en describir el papel del sistema plasminógeno/plasmina (PLG/PLA) en la fecundación bovina y porcina. Mediante fecundación in vitro, demostramos que la presencia de PLG ó PLA en el medio de coincubación de los gametos disminuía la penetración de los espermatozoides en los ovocitos y su unión a la zona pelúcida (ZP). Esta disminución no se debía a alteraciones de la funcionalidad espermática ni a cambios en la resistencia de la ZP a la proteolisis, sino a que la PLA provocaba la liberación de los espermatozoides adheridos a la ZP. Mediante inmunofluorescencia indirecta detectamos la presencia de PLG y sus activadores en la ZP y en el oolema de los ovocitos antes de la fecundación. Tras la fecundación, dicha presencia disminuyó o desapareció por completo, por lo que proponemos que el sistema PLG/PLA se activa durante la interacción espermatozoide-ovocito y contribuye a regular la polispermia., The aim of this study was to describe the role of the plasminogen/plasmin system (PLG/PLA) in bovine and porcine fertilization. Through in vitro fertilization, we demonstrated that the presence of PLG or PLA in the incubation medium of gametes decreased penetration of oocytes and sperm binding to the zona pellucida (ZP). This decrease was not due to alterations in sperm function or changes in the ZP resistance to proteolysis, but the PLA caused the release of sperm previously bound to the ZP. By indirect immunofluorescence we detected the presence of PLG and its activators in the ZP and oolema of the oocytes before fertilization. After fertilization, this presence diminished or disappeared completely, so we propose that the PLG/PLA system is activated during sperm-oocyte interaction and contributes to the regulation of polyspermy.

Tesis-Universidad de Murcia., Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. D 252., Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M.-399.

La intervención de los seres humanos en los procesos de cría y recría en animales de producción pueden tener diferente impacto en virtud al tipo, momento y duración de la misma. El objetivo del trabajo fue evaluar si existen cambios en parámetros fisiológicos, sanguíneos y de comportamiento en cabritos Saanen en función al tipo de crianza, 10 criados con sus madres (CT) vs. 10 criados artificialmente (CA), en las etapas de lactante y recría. Semanalmente se midió: frecuencia cardiaca (FC), temperatura rectal (TR), frecuencia respiratoria (FR), predisposición a ser atrapado y actitud de patear. Previo se tomaron 10 ml de sangre para realización del leucograma. El diseño experimental fue el de mediciones repetidas en el tiempo. Los datos fueron analizados a través del programa InfoStat. Ni entre periodos ni entre tratamientos se detectaron diferencias significativas para FC como TR. En ambos periodos a los animales del CT resultó más difícil atraparlos (lactante: p

This study aimed to characterize the molecules involved in osteogenesis in seabream and establish using in vitro/in vivo approaches the responsiveness of selected key genes to temperature. The impact of a temperature drop from 23 to 13 degrees C was evaluated in juvenile fish thermally imprinted during embryogenesis. Both, in vitro/in vivo, Fib1a, appeared important in the first stages of bone formation, and Col1A1, ON and OP, in regulating matrix production and mineralization. OCN mRNA levels were up-regulated in the final larval stages when mineralization was more intense. Moreover, temperature-dependent differential gene expression was observed, with lower transcript levels in the larvae at 18 degrees C relative to those at 22 degrees C, suggesting bone formation was enhanced in the latter group. Results revealed that thermal imprinting affected the long-term regulation of osteogenesis. Specifically, juveniles under the low and low-to-high-temperature regimes had reduced levels of OCN when challenged, indicative of impaired bone development. In contrast, gene expression in fish from the high and high-to-low-temperature treatments was unchanged, suggesting imprinting may have a protective effect. Overall, the present study revealed that thermal imprinting modulates bone development in seabream larvae, and demonstrated the utility of the in vitro MSC culture as a reliable tool to investigate fish osteogenesis. "Ministerio de Economia y Competitividad" (MINECO) [BES-2015-074654]; Portuguese Science Foundation (FCT) [SFRH/BPD/111512/2015, SFRH/BD/81625/2011]; MINECO, Spain [AGL2010-17324, AGL2014-57974-R]; "Generalitat de Catalunya" (XRAq); Generalitat de Catalunya [2014SGR-01371]; FCT, Portugal [CCMAR/Multi/04326/2013]; European Union [LIFECYCLE EU-FP7 222719] info:eu-repo/semantics/publishedVersion

La reproducción ex situ es una de las medidas reconocidas por la comunidad científica y conservacionista para la conservación de especies amenazadas y su reintroducción en el medio natural. Sin embargo, la reproducción en cautividad de especies de aves amenazadas fracasa con frecuencia debido a la ausencia de estímulos ambientales asociada a la vida en cautividad. Esta circunstancia impide el desarrollo de los ciclos reproductivos fisiológicos en condiciones naturales y, por tanto, la obtención de progenie a partir de individuos valiosos desde el punto de vista de la diversidad genética y ecológica. De modo general y, en resumen, los estímulos ambientales, a través de un conjunto de eventos fisiológicos en el sistema nervioso central, desencadenan la liberación hipotalámica de la hormona liberadora de gonadotropinas (GnRH), que inducirá la síntesis y liberación de gonadotropinas (FSH y LH) en las células gonadotropas de la adenohipófisis. Tanto la FSH como la LH inducirán a nivel gonadal los cambios fisiológicos necesarios para la maduración de los gametos. La posibilidad de inducir farmacológicamente los ciclos reproductivos en aves podría resolver en gran medida las dificultades de la reproducción en cautividad en especies o individuos de alto valor ecológico. Por otra parte, la aportación de conocimiento a la fisiología reproductiva de las aves, y, concretamente, a los mecanismos hormonales que controlan estos procesos, sería relevante para la mejor comprensión tanto de los ciclos reproductivos en la naturaleza como para la aplicación de dicho conocimiento a la producción de aves y huevos para consumo humano. Los trabajos de esta tesis incluyeron la obtención de una FSH recombinante de codorniz en un sistema de expresión heterólogo de células de mamífero (CHO) y la purificación de LH a partir de adenohipófisis de pavo. Por tanto, el desarrollo de anticuerpos específicos para FSH recombinante de codorniz y FSH y LH hipofisarias de pavo, fue considerado una herramienta imprescindible para el seguimiento de estas hormonas en los procesos de laboratorio: expresión en el medio de cultivo o lisados celulares, purificación, concentración y diálisis. La producción de anticuerpos específicos contra las subunidades beta de FSH y LH permitió la identificación de la FSH recombinante de codorniz (ccrFSH) expresada en células de ovario de hámster chino (CHO) y la identificación y purificación de LH de pavo. En los ensayos biológicos in vitro realizados, ccrFSH indujo significativamente la secreción de progesterona en células de la granulosa de folículos F5. Sin embargo, no indujo la secreción de progesterona en células de la granulosa de folículos F1. Estos resultados demostraron la especificidad de acción de esta hormona. En los experimentos in vivo, la administración exclusiva de ccrFSH durante quince días en codornices prepúberes indujo de manera consistente el desarrollo de folículos de diámetro ≥ 1 mm. Sin embargo, este tratamiento no indujo el desarrollo folicular completo. La administración combinada de ccrFSH y LH o eCG sí indujo desarrollo folicular completo, la ovulación y la formación de huevos en codorniz. En estos experimentos se demostró la actividad biológica in vivo de ccrFSH y la necesidad de la actividad conjunta de FSH y LH en la inducción del ciclo en esta especie. La FSH recombinante de codorniz también fue eficaz, administrada junto a eCG, en la inducción del desarrollo y maduración folicular ovárica. La administración de extracto glicoproteico de adenohipófisis de pavo en los individuos con folículos ováricos maduros indujo la ovulación y la puesta de huevos. La inseminación artificial en estos individuos y la incubación de los huevos puestos resultaron en desarrollo embrionario y eclosión.El control del ciclo inducido se realizó mediante exploración ecográfica de la cavidad celómica y permitió el seguimiento del desarrollo folicular hasta la ovulación. La ecografía se mostró un método eficaz para el seguimiento del ciclo ovárico inducido. Estos resultados demuestran la eficacia de ccFSH en la inducción del ciclo ovárico en aves y la posibilidad de utilizar esta tecnología en los procesos de reproducción asistida en especies amenazadas para la obtención de progenie y su reintroducción en el medio natural. Ex situ reproduction is one of the measures recognized by the scientific and conservationist community for the conservation of threatened species and their reintroduction into the natural environment. However, captive breeding of endangered bird species often fails due to the absence of environmental stimuli associated with life in captivity. This circumstance prevents the development of physiological reproductive cycles in natural conditions and, therefore, the obtention of progeny from valuable individuals from the point of view of the genetic and ecological diversity. In general, and in summary, environmental stimuli, through a set of physiological events in the central nervous system, trigger the hypothalamic release of gonadotropin-releasing hormone (GnRH), which will induce the synthesis and release of gonadotropins (FSH) and LH) in gonadotropic cells of the adenohypophysis. Both FSH and LH will induce at the gonadal level the physiological changes for the maturation of gametes. The possibility of pharmacologically inducing reproductive cycles in birds could resolve to a large extent the difficulties of captive breeding in species or individuals of high ecological value. On the other hand, the contribution of knowledge on the reproductive physiology of birds, and, specifically, to the hormonal mechanisms that control these processes, would be relevant for the better understanding both of the reproductive cycles in nature and the application of said knowledge to the production of birds and eggs for human consumption. The work of this thesis included the expression of a recombinant quail FSH in a heterologous mammalian cell expression system (CHO) and the purification of LH from turkey adenohypophysis. Therefore, the development of specific antibodies for recombinant quail FSH and pituitary FSH and LH, was considered an essential tool for monitoring these hormones in laboratory processes: expression in the culture medium or cell lysates, purification, concentration and dialysis. The production of specific antibodies against the beta subunits of FSH and LH allowed the identification of recombinant quail FSH (ccrFSH) expressed in Chinese hamster ovary cells (CHO) and the identification and purification of turkey LH. At the in vitro biological tests performed, ccrFSH significantly induced progesterone secretion in granulosa cells of F5 follicles. However, it did not induced progesterone secretion in granulosa cells of F1 follicles. These results demonstrated the specificity of action of this hormone. At the in vivo experiments, the exclusive administration of ccrFSH for fifteen days in prepubertal quail consistently induced the development of follicles with a diameter ≥ 1 mm. However, this treatment did not induce complete follicular development. The combined administration of ccrFSH and LH or eCG did induce complete follicular development, ovulation and egg formation in quail. These experiments demonstrated the in vivo biological activity of ccrFSH and the need for the joint activity of FSH and LH in the induction of the cycle in this species. Recombinant quail FSH was also effective, administered together with eCG, in the induction of ovarian follicular development and maturation. The administration of turkey adenohypophysis glycoprotein extract in individuals with mature ovarian follicles induced ovulation and egg laying. The artificial insemination in these individuals and the incubation of the laid eggs resulted in embryonic development and hatching. The control of the induced cycle was performed by ultrasound exploration of the coelomic cavity and allowed the follow-up of the follicular development until ovulation. Ultrasound showed an effective method for monitoring the induced ovarian cycle. These results demonstrate the efficacy of ccFSH in the induction of the ovarian cycle in birds and the possibility of using this technology in assisted reproduction processes in endangered species to obtain progeny and their reintroduction into the wild.

RESUMO O objetivo deste trabalho foi avaliar as respostas hematológicas do acari-bola Peckoltia oligospila submetido ao estresse de transporte. Variações nos parâmetros de sangue foram analisadas às zero, seis, 24, 48, 72 e 96 horas após o transporte. Respostas ao estresse foram observadas entre zero e seis horas do transporte, mas a maioria dos parâmetros retornou aos valores basais em 24 horas. O tempo de zero hora (momento imediato após transporte) foi o mais crítico, com valores elevados de glicemia, eritrócitos e eritroblastos. Respostas secundárias tardias foram observadas para a proteína plasmática total, o volume corpuscular médio (VCM) e a hemoglobina corpuscular média (HCM) em seis horas após o transporte dos peixes, retornando aos valores basais após esse período. O número de leucócitos não sofreu alterações após o transporte. O estresse de transporte não comprometeu a fisiologia de P. oligospila, o que indica que esse peixe é resistente ao estresse se comparado com outras espécies. Porém, recomenda-se que não se realize qualquer outro procedimento estressante durante pelo menos 24 horas da recuperação dos peixes após transporte, para garantir a saúde e a sobrevivência dos animais transportados. ABSTRACT The objective of this work was to evaluate the hematological responses of bola-pleco (Peckoltia oligospila) undergoing the stress of transportation. Variations on blood parameters were analyzed at 0, 6, 24, 48, 72 and 96h after transportation. Responses to stress were detected from 0 to 6h after the transportation of fish, however, most parameters returned to baselines values within 24h of transportation. The moment of 0h was the most critical, presenting higher values of glycemia, erythroblasts and erythrocytes. Late secondary responses were observed to total plasmatic protein, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) at 6h, returning to baselines values after this time. Leukocyte number was not affected by stress of transportation. The stress by transportation was not severe to influence the health of P. oligospila, indicating that fish is resistant to stress if compared to other species. However, we recommended no stressful procedures for at least 24 hours for recovery, in order to ensure health and survival of fish.

146 p. ÁMBITO DE APLICACIÓN: los flavonoides y compuestos relacionados parecen tener efectos favorables en la progresión de la enfermedad del hígado graso no alcohólico (NAFLD), aunque los mecanismos exactos implicados son poco conocidos. En este estudio, el objetivo fue investigar el efecto de la quercetina de flanovol en la desregulación de la expresión génica involucrada en el desarrollo de NAFLD, así como la posible implicación de la fosfatidilinositol 3-quinasa (PI3K) / AKT modulación de la vía. MÉTODOS Y RESULTADOS: utilizamos un modelo in vivo basado en ratones alimentados con dieta con deficiencia de metionina y colina (MCD) y un modelo in vitro que consiste en células Huh7 incubadas con medio MCD. Los ratones alimentados con MCD mostraron características patofisiológicas clásicas de la esteatohepatitis no alcohólica, asociadas con la regulación transcripcional alterada de la expresión de genes relacionados con la captación y el tráfico de ácidos grasos, con un aumento de la lipoperoxidación. La vía de PI3K / AKT se activó por MCD y desencadenó la desregulación de los genes, lo que provocó la activación o inhibición de todos los genes estudiados, como se demostró a través de la incubación de células con el inhibidor de PI3K LY294002. El tratamiento con quercetina redujo la fosforilación de AKT y los genes relacionados con el estrés oxidativo / nitrosativo, la inflamación y el metabolismo de los lípidos mostraron una tendencia a normalizarse tanto en modelos in vivo como in vitro. CONCLUSIÓN: Estos resultados ubican a la quercetina como una posible estrategia terapéutica para prevenir la progresión de la NAFLD al atenuar la desregulación de la expresión génica, al menos en parte a través de la inactivación de la vía PI3K / AKT.

We aimed to determine the effects of different intensities of acute exercise on Hcy plasma levels, and the exercise-induced changes in Hcy liver metabolism.First, thirty-two Wistar rats were randomly submitted to an acute bout of swimming exercise carrying a load of 2% (n=8), 4% (n=8) and 6% (n=8) of their total body weight attached in their tail. Control rats remained rested (n=8). Blood samples were taken from tail vein for plasma S-containing amino acids determination before (Rest) and post, 1, 2, 3, 4, 6, and 10h after acute swimming exercise. Second, 56 exercised rats (4% loads) were euthanized before (Rest) and1, 2, 3, 4, 6, and 10h after acute swimming exercise. Blood and liver samples were collected for amino acids and keys genes involved in the Hcy metabolism assay.Acute exercise increases (P0.05) plasma Hcy concentration in an intensity-dependent manner (rest 7.7±0.8; 6% load 13.8±3.6; 4% load 12.2±2.9±and 2% load 10.1±2.6, μmol/L); this increase is transient and does not promote hyperhomocysteinemia (15μmol/L).Exercise-induced increased plasma Hcywas accompanied by the decreased liver S-adenosylmethionine/S-adenosylhomocysteine ratio and elevated MAT1a mRNA content. Acute exercise also caused elevated mRNA of key enzymes of transsulfuration (CBS) and remethylation (BHMT and the MTRR).Our data provided evidence that acute exercise increases plasma Hcy concentration due to the augmented requirement for methylated compounds that increases liver SAM consumption. Also, Hcy remethylation and transsulfuration are coordinately regulated to maintain methyl balance.

Durante a história de vida dos organismos, diferentes vias metabólicas devem ser otimizadas para garantir a ocorrência exitosa de processos fisiológicos distintos, como a reprodução e o investimento somático (manutenção). Neste viés, as reservas energéticas do animal nem sempre são suficientes para sustentar estes dois processos em paralelo, fazendo com que ocorra o trade-off entre a reprodução e a sobrevivência/longevidade, ou seja, animais que investem em reprodução, como típico de fêmeas, dispõe de menos energia para investir em reparos, acelerando o envelhecimento e reduzindo o tempo de vida, como mostrado por alguns estudos. Este fenômeno também é conhecido como "custo da reprodução". Porém, distintos resultados vêm sendo encontrados ao relacionar estes aspectos, pois além da reprodução propriamente dita, o momento em que ela ocorre na história de vida também tem relação com a longevidade. Com o avanço da idade, a resposta das fêmeas de grilo ao chamado para a cópula emitido pelos machos é menor, podendo ser o resultado do envelhecimento de estruturas cruciais para a captação do som nestes insetos, o tímpano. Estudos acerca do envelhecimento morfológico deste órgão são inexistentes. Quando iniciada, a reprodução altera a taxa metabólica dos organismos pelo aumento da demanda energética, refletindo seus custos sobre o acúmulo de um pigmento derivado principalmente da oxidação de macromoléculas, denominado lipofuscina (LF). Neste estudo, grupos experimentais com fêmeas de Gryllus assimilis foram formados para determinar os efeitos da reprodução sobre a sobrevivência, ajustando-se o modelo de Gompertz e sobre o envelhecimento fisiológico, por meio da quantificação de neurolipofuscina (nLF), além do registro de danos morfológicos no tímpano causados ao longo do envelhecimento por meio de análise ultraestrutural em microscopia eletrônica de varredura (MEV). Para isto, um grupo foi privado de cópula, um grupo teve reprodução normal (como início aos 14 dias de vida) e outro, reprodução tardia (com início aos 30 dias de vida). Diferenças na longevidade máxima entre os grupos não foram observadas, porém em fêmeas virgens houve um atraso no início da queda exponencial da sobrevivência, enquanto em fêmeas de reprodução normal esta queda ocorreu mais cedo. Fêmeas de reprodução tardia ficaram em situação intermediária. Não foram encontradas diferenças significativas na taxa de acúmulo de nLF entre os grupos ao longo do tempo. Estes resultados podem ser explicados pela produção de ovos ocorrer em todas as condições reprodutivas, tendo custos semelhantes, refletidos no acúmulo de nLF, além de terem sido alimentadas ad libitum, mascarando maiores efeitos do trade-off entre reprodução/sobrevivência. No entanto, efeitos deletérios da cópula sobre a longevidade de fêmeas de G. assimilis não foram encontrados. Relativo ao envelhecimento morfológico do tímpano, cinco lesões foram descritas pela primeira vez: excrescência, perfuração, fungos, formação de placas e rachaduras, sendo as três primeiras significativas em relação ao tempo, possivelmente prejudicando o funcionamento do aparelho auditivo. Não houve diferenças nos danos encontrados entre os grupos experimentais, embora o aumento destes acentuou-se após o período reprodutivo (senescência). Portanto, a reprodução parece modular a história de vida das fêmeas de grilo e interferir na manutenção de estruturas importantes para este processo, como o tímpano. During the life history of organisms, different metabolic pathways must be optimised to ensure a successful occurrence of distinct physiological traits, such as reproduction and somatic maintenance. In this way, the energy reserves of an animal are not enough to sustain these two processes simultaneously, leading to a trade-off between reproduction and survival, in other words, animals that invest in reproduction, as typical of females, have less energy to invest in self-repairs, accelerating ageing and reducing lifespan, as shown by some studies. This phenomenon is also known as "cost of reproduction". However, several results have been found showing that the moment when the start of reproduction in the life-history (i.e, either early or late reproduction) is also related to longevity. Because of the ageing process, the response of cricket females to the calling song emitted by males is slow, it could be the result of damages on fundamental structures of sound reception, i.e., the tympanal membrane. Studies on the morphological ageing of this organ are non-existent. When reproduction starts, it changes the metabolic rate of the organisms by increasing energy demand, reflecting its costs on the accumulation of a pigment derived mainly from oxidation of macromolecules, called lipofuscin (LF). This is an autofluorescent pigment already validated as a marker of physiological ageing in several animal species. In the present investigation, experimental groups with female crickets (Gryllus assimilis) were established to determine the effects of reproduction on survival, applying Gompertz model, and to quantify physiological ageing through neurolipofuscin (nLF) accumulation in the brain, and by recording morphological damages (tympanum) during ageing process by ultrastructural analysis using scanning electron microscopy (SEM). For this, a group was deprived from copula, one had normal reproduction (young adults) and another, late reproduction (old adults). No differences were found in maximum longevity among groups, but in virgin females it was observed a delay to initiate exponential decline in survival when compared to reproductive groups (normal and late). Late-reproducing females had an intermediate onset of mortality. For the first time the nLF accumulation was quantified in crickets, although significant differences in the rate of accumulation of this between groups over time were not found. These results are likely due to the similar costs exhibited by all reproductive conditions, reflecting in the accumulation of nLF, besides being fed ad libitum, what probably masked a traditional trade-off observation between reproduction and survival. Deleterious effects of copula on the longevity of G. assimilis females were not seen. Regarding to the morphological ageing of the tympanal membrane, five lesions were described for the first time: excrescence, perforation, fungi, plaque formation and cracking; the first three were time-dependent and impaired the functioning of the auditory organ in these insects. However, the increase of these damages accentuated after the reproductive period (senescence). Therefore, reproduction seems to modulate the life history of cricket females and interfere with the maintenance of important structures for this process, such as tympanum.

El objetivo principal fue mejorar el conocimiento actual de la criopreservación espermática en porcino. Se propusieron tres objetivos concretos: (1) Determinar el papel de los antioxidantes del plasma seminal (PS) en la congelabilidad espermática (exp. 1); (2) evaluar si el PS de la fracción post-espermática es el único responsable de que los espermatozoides del eyaculado completo congelen peor que los de la fracción rica en espermatozoides (FR) (exp. 2); y (3) analizar si el tiempo de conservación en nitrógeno líquido (NL) de las dosis de semen congeladas influye en la calidad espermática a la descongelación (exp. 3). En el exp. 1, se utilizaron diez eyaculados recogidos manualmente en fracciones: los 10 primeros ml de la FR (P1), el resto de la FR (P2) y la post-FR. Los espermatozoides de una parte de cada fracción fueron criopreservados utilizando un protocolo estándar para pajuelas de 0,5 ml. El resto fue utilizado para recoger el PS. Los espermatozoides de P1 y P2 mostraron mejor motilidad y viabilidad que los de la post-FR (P

En resumen, esta tesis aporta nuevas evidencias de los efectos sub-letales de la depredación y parasitismo de cría sobre las decisiones previas a la reproducción de las aves en ambientes naturales. Estos efectos se han mostrado a través de cambios en la elección de los territorios de reproducción y tienen efectos indirectos sobre la estructura, composición y nivel de interacción entre gremios dentro de la comunidad. Encontramos también efectos del riesgo de depredación percibido durante la reproducción mediante cambios en el cuidado parental. Sin embargo, no hemos encontrado evidencias claras de los efectos del riesgo de parasitismo sobre rasgos reproductivos y fisiológicos en un hospedador de un parásito obligado de cría una vez comenzada la reproducción. Finalmente, los resultados sugieren que la actividad humana también puede ser percibida como un factor de riesgo modulando el estado fisiológico de especies en declive poblacional como consecuencia de la alteración de sus hábitats. Por tanto, los resultados conjuntos de esta tesis ofrecen nuevos avances en el estudio del riesgo de depredación y parasitismo de cría en tiempo ecológico de gran interés tanto en el campo de la Ecología Evolutiva y del Comportamiento como por su aplicación en la Conservación y Gestión Animal. Además, los resultados de la tesis en su conjunto sugieren que el estudio de múltiples pistas es fundamental para llegar a una compresión profunda de los efectos del riesgo puesto que las aves tienen la capacidad de evaluarlo en base a pistas que perciben por distintos canales de comunicación., In summary, this thesis provides new evidence of the sub-lethal effects of predation and inter-specific brood parasitism on decisions prior to the reproduction of birds in natural environments. These effects have been shown through changes in nest-site choice and have indirect effects on the structure, composition and level of interaction between guilds within the community. We also found effects of the risk of nest predation perceived during reproduction through changes in parental care. However, we have not found clear evidence of the effects of the risk of parasitism on reproductive and physiological traits in a host of an obligate brood parasite, once reproduction has begun. Finally, the results suggest that human activity can also be perceived as a risk factor modulating the physiological state of species under population decline, as a consequence of the alteration and disturbances in their habitats. Therefore, the results of this thesis offer new advances in the study of the risk of predation and the risk of cuckoo parasitism in ecological time of great interest both in the field of Evolutionary Ecology and Behavior and its application in Animal Conservation and Management. In addition, the results of the thesis suggest that the study of multiple cues is necessary to deeply understand the effects of risk, given that birds may evaluate threats perceived by cues through different communication channels., Tesis Univ. Granada., Beca predoctoral de formación de personal investigador (FPI, BES-2012- 051898) con fondos FEDER (CGL2011-27561/BOS and CGL2014- 56769-P) del Ministerio de Economía y Competitividad.

This article aims to review the work of the German anatomist Joseph Disse (1852-1912), specifically with regard to the contents of his ,, Ueber die Lymphbahnen der Laugethierleber". In this he described a thin space, until that moment not referred, located between the hepatocyte and the sinusoidal membrane (sinusoids).

Alcohol is a serious public health concern that has a differential impact on individuals depending upon age and sex. Patterns of alcohol consumption have recently changed: heavy episodic drinking—known as binge-drinking—has become most popular among the youth. Herein, we aimed to investigate the consequences of intermittent adolescent alcohol consumption in male and female animals. Thus, Wistar rats were given free access to ethanol (20% in drinking water) or tap water for 2-h sessions during 3 days, and for an additional 4-h session on the 4th day; every week during adolescence, from postnatal day (pnd) 28–52. During this period, animals consumed a moderate amount of alcohol despite blood ethanol concentration (BEC) did not achieve binge-drinking levels. No withdrawal signs were observed: no changes were observed regarding anxiety-like responses in the elevated plus-maze or plasma corticosterone levels (pnd 53–54). In the novel object recognition (NOR) test (pnd 63), a significant deficit in recognition memory was observed in both male and female rats. Western Blot analyses resulted in an increase in the expression of synaptophysin in the frontal cortex (FC) of male and female animals, together with a decrease in the expression of the CB2R in the same brain region. In addition, adolescent alcohol induced, exclusively among females, a decrease in several markers of dopaminergic and serotonergic neurotransmission, in which epigenetic mechanisms, i.e., histone acetylation, might be involved. Taken together, further research is still needed to specifically correlate sex-specific brain and behavioral consequences of adolescent alcohol exposure.

Inbred mouse strains, living in simple laboratory environments far removed from nature, have been shown to vary consistently in their immune response. However, wildlife populations are typically outbreeding and face a multiplicity of challenges, parasitological and otherwise. In this study we seek evidence of consistent difference in immunological profile amongst individuals in the wild. We apply a novel method in this context, using longitudinal (repeated capture) data from natural populations of field voles, Microtus agrestis, on a range of life history and infection metrics, and on gene expression levels. We focus on three immune genes, IFN-γ, Gata3, and IL-10, representing respectively the Th1, Th2 and regulatory elements of the immune response. Our results show that there was clear evidence of consistent differences between individuals in their typical level of expression of at least one immune gene, and at most all three immune genes, after other measured sources of variation had been taken into account. Furthermore, individuals that responded to changing circumstances by increasing expression levels of Gata3 had a correlated increase in expression levels of IFN-γ. Our work stresses the importance of acknowledging immunological variation amongst individuals in studies of parasitological and infectious disease risk in wildlife populations.

International audience; The liver is the most important link between the circadian system and metabolism. As a food-entrainable oscillator, the hepatic clock needs to be entrained by food-related signals. The objective of the present study was to investigate the possible role of ghrelin (an orexigenic peptide mainly synthesized in the gastrointestinal tract) as an endogenous synchronizer of the liver oscillator in teleosts. To achieve this aim, we first examined the presence of ghrelin receptors in the liver of goldfish. Then, the ghrelin regulation of clock gene expression in the goldfish liver was studied. Finally, the possible involvement of the phospholipase C/ protein kinase C (PLC/ PKC) and adenylate cyclase/protein kinase A (AC/PKA) intracellular signalling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of goldfish hepatic cells. Ghrelin induced the mRNA expression of the positive (gbmal1a, gclock1a) and negative (gper genes) elements of the main loop of the molecular clock machinery, as well as grev-erba (auxiliary loop) in cultured liver. These effects were blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a PLC inhibitor (U73122), a PKC activator (phorbol 12-myristate 13-acetate) and a PKC inhibitor (chelerythrine chloride) demonstrated that the PLC/ PKC pathway mediates such ghrelin actions. Experiments with an AC activator (forskolin) and a PKA inhibitor (H89) showed that grev-erba regulation could be due to activation of PKA. Taken together, the present results show for the first time in vertebrates a direct action of ghrelin on hepatic clock genes and support a role for this hormone as a temporal messenger in the entrainment of liver circadian functions.

Made available in DSpace on 2018-12-11T17:04:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-04-01 Fundação de Amparo à Pesquisa do Estado de Goiás In rheophilic tropical fish, the use of carp pituitary extract (CPE) is still the main protocol since the use of gonadotropin-releasing hormone (GnRH) induces ovulation, but usually does not provide viable embryos. Thus, in order to generate knowledge to support the establishment of a successful protocol using GnRH, we compared the ovulatory process and reproductive performance of Leporinus macrocephalus using CPE (0.5 and 5.0 mg kg−1) and mGnRHa (7 µg kg−1) associated with metoclopramide. Females of both treatments ovulated, but the mGnRHa treatment did not provide viable embryos, which was associated with a more potent ovulation, lower latency period and less intense hydration of eggs, suggesting a possible treatment overstimulation. This is the first report associating a less intense egg hydration process to a failure in obtaining viable eggs in induced fish spawning. Considering that the mGnRHa doses applied here were quite low, it is possible that L. macrocephalus as well as other rheophilic tropical species present a higher sensibility to mGnRHa and/or metoclopramide (or derived substances) and future approaches must consider using even lower doses of these substances as well as testing different types of GnRH and dopamine inhibitors. Centro de Aquicultura Universidade Estadual Paulista (UNESP), Campus de Jaboticabal Instituto de Biociências Universidade de São Paulo (USP), Campus de São Paulo Centro de Aquicultura Universidade Estadual Paulista (UNESP), Campus de Jaboticabal Fundação de Amparo à Pesquisa do Estado de Goiás: 2010/08334-7

Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish. Fil: Blanco, Ayelén Melisa. Universidad Complutense de Madrid; España. University of Saskatchewan; Canadá Fil: Bertucci, Juan Ignacio. University of Saskatchewan; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Ramesh, Naresh. University of Saskatchewan; Canadá Fil: Delgado, Mariá Jesús. Universidad Complutense de Madrid; España Fil: Valenciano, Ana Isabel. Universidad Complutense de Madrid; España Fil: Unniappan, Suraj. University of Saskatchewan; Canadá

O incremento no uso de pesticidas vem ocasionando a degrada??o de diferentes ecossistemas, sobretudo aqu?ticos. Apesar de possu?rem aplica??es espec?ficas a determinados organismos, sabe-se que os agrot?xicos s?o capazes de ocasionar altera??es metab?licas e funcionais em diversos organismos n?o alvo. Os anf?bios constituem a Classe de vertebrados mais amea?ada globalmente, sendo que os decl?nios populacionais observados no mundo todo s?o decorrentes da a??o sin?rgica de diversos fatores, incluindo a fragmenta??o e degrada??o de habitat, principalmente para fins agr?colas, a exposi??o a contaminantes derivados destas atividades, dentre outros. Neste contexto, Melanophryniscus admirabilis ? uma esp?cie de sapo bufon?deo micro end?mica, descrita atualmente para apenas uma localidade no estado do Rio Grande do Sul (Brasil), e avaliada como Criticamente em Perigo de extin??o, em n?vel regional, nacional e global. O alto grau de amea?a desta esp?cie deve-se a um conjunto de fatores, como a fragmenta??o e perda de qualidade do habitat, principalmente devido ? convers?o de ?reas para agricultura e ? larga utiliza??o de agrot?xicos no entorno da ?rea de ocorr?ncia da esp?cie. Com isso, o objetivo do presente estudo foi analisar poss?veis altera??es em par?metros metab?licos e do balan?o oxidativo em homogeneizado total de girinos de M. admirabilis expostos a duas concentra??es de formula??o comercial contendo Sulfentrazone (Boral? 500 SC) e outras duas contendo Glifosato (Roundup? Original). Foram analisados os n?veis totais de glicog?nio, prote?nas e ?cido ?rico; a poss?vel ocorr?ncia de lipoperoxida??o (LPO) atrav?s dos n?veis de TBARS; e o n?vel de atividade das enzimas Super?xido Dismutase (SOD), Catalase (CAT) e Glutationa S-Transferase (GST). Os resultados obtidos evidenciaram altera??es significativas nos par?metros mensurados, nos grupos expostos ?s diferentes concentra??es dos herbicidas Sulfentrazone e Glifosato. A partir disto, sugere-se que a mobiliza??o conjunta das enzimas e metab?litos analisados possivelmente foi capaz de conter o dano oxidativo nestes animais. Entretanto, cabe ressaltar que as altera??es observadas podem vir a afetar estes animais ao longo do ciclo de vida, especialmente durante e ap?s o per?odo metam?rfico. A aparente inibi??o enzim?tica observada nos grupos expostos aos herbicidas, al?m de indicar grande suscetibilidade dos girinos frente ? exposi??o a estes agentes, pode refletir um preju?zo da capacidade destes animais em lidar com situa??es ambientais adversas e sin?rgicas, como a fragmenta??o de habitat, radia??o UV e exposi??o a outros poluentes. A aus?ncia de mortalidade nos girinos de M. admirabilis sugere resist?ncia ?s concentra??es dos herbicidas testados; contudo, caberia a compara??o da capacidade antioxidante desta esp?cie com outras esp?cies taxonomicamente pr?ximas. Assim, este estudo demonstrou que a exposi??o de girinos de M. admirabilis a concentra??es subletais dos herbicidas Sulfentrazone e Glifosato conduziu a uma ruptura da homeostase que pode afetar a sobreviv?ncia da esp?cie em etapas futuras de seu ciclo de vida. Seriam relevantes estudos futuros visando ? compreens?o dos efeitos de outras concentra??es e herbicidas, bem como a an?lise de outros metab?litos e componentes do sistema antioxidante desta esp?cie. The increased use of pesticides has caused the degradation of a variety of ecosystems, especially aquatic environments. Despite specific targets, agrochemicals are able to cause metabolic and functional alterations including in non-target organisms. Amphibians constitute the most globally threatened Class of vertebrates, and the population declines observed around the world are due to a synergy of different factors, including habitat degradation and fragmentation, mostly to agricultural purposes, and the exposure to contaminants derived of these activities, among others. Within this context, Melanophryniscus admirabilis is a micro endemic bufonid toad species, with occurrence registered to only one area in Rio Grande do Sul state (Brazil). This species has a critically endangered (CR) status, at regional, national and global levels. The great danger of extinction is due to several factors, including habitat fragmentation (especially for agricultural purposes) and the decreased habitat quality, mainly because of extensive use of agrochemicals in areas near of the natural habitat of this species. In view of the foregoing, the objective of the present study was to analyze possible alterations in metabolic and oxidative parameters in total homogenate of M. admirabilis tadpoles exposed to two different concentrations of commercial formulation containing Sulfentrazone (Boral? 500 SC) and two concentrations containing Glyphosate (Roundup? Original). The total levels of glycogen, proteins and uric acid were analyzed; as well as the possible occurrence of lipid peroxidation (LPO), expressed by TBARS levels; and the enzymatic activity of Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione S-Transferase (GST). The results showed significant alterations in metabolic and oxidative parameters, in groups exposed to Sulfentrazone and Glyphosate herbicides. Analyzing these results, we hypothesize that the associated mobilization of enzymes and metabolic parameters presumably was capable of counter the oxidative lipid damage in these animals. Despite of that, the observed alterations may affect these animals during its life cycle, especially during and after metamorphic period. The apparent enzymatic inhibition observed in groups exposed to the herbicides, in addition to indicate the great susceptibility of tadpoles to these agents, reflects an impairment of the ability of these animals to cope with adverse and synergistic environmental situations, such as habitat fragmentation, UV radiation and exposure to other pollutants. The absence of mortality suggests resistance of M. admirabilis tadpoles to the tested concentrations of herbicides; nevertheless, the comparison of the antioxidant capacity of this species against taxonomically related species would be relevant. Therefore, the present study has demonstrated that the exposure of M. admirabilis tadpoles to sub lethal concentrations of Sulfentrazone and Glyphosate herbicides has affected the homeostasis, thus affecting the survival, of these animals in later life cycle stages. However, further studies would be necessary, including other concentrations and herbicides, as well as the analysis of further metabolites and components of the antioxidant system of this species. Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq

Intensive culture of Senegalese sole, Solea senegalensis, represents an option to diversify Mediterranean aquaculture. As a flatfish species, the fish body presents marked asymmetry between the dorsal or ocular side and the ventral or blind side. Taking into account that different surroundings come into contact with each side, in the present study we aimed to compare the properties of skin mucus from both sides, which is the most external barrier and protection for the fish. Skin mucus has recently been proposed as a novel minimally invasive target for the study of physiological responses. Here, a group of ten Senegalese sole were used to measure basal mucus parameters; then four additional groups of ten specimens each were used to study a hypoxia stress challenge (post-stress groups: 15 min-PS, 1 h-PS, 6 h-PS and 24 h-PS), consisting of a 3-minute air exposure. Physicochemical parameters (viscosity, osmolality, pH and main osmotic ions) were analysed in dorsal and ventral mucus. Thereby, mucus components related to stress responses such as cortisol, glucose, lactate, soluble protein and antioxidant power, were measured and compared between the mucus types and in response to hypoxia. Rheograms revealed similar non-Newtonian behaviour of dorsal and ventral mucus viscosities, which suddenly increased in the 15 min-PS group, thus providing fish with immediate insulating and mechanical protection. However, ventral side viscosity of the 24 h-PS group was lower than basal values and its functionality was compromised. Whereas chemical parameters did not differ between sides or in response to hypoxia, components that are soluble in mucus showed asymmetry with soluble protein and lactate being twofold higher in ventral mucus. The classical stress response of increased plasma cortisol accompanying glucose and lactate release was observed. In parallel, mucus glucose and lactate showed rapid but transitory exudation in the 15 min-PS group, while cortisol was detected at minimal levels in skin mucus, and therefore is not recommended as a mucus indicator for this species. Mucus-soluble protein also rose in the 15 min-PS group, which indicates greater exudation of defensive-related proteins. Finally, the antioxidant power of dorsal mucus increased and accumulated, without returning to basal levels. All these findings indicate that flatfish skin mucus properties depend on animal side, and further studies will be necessary to elucidate whether other mucus defensive parameters show this asymmetry and their relevance in these benthonic species.

Objetivou-se, com este trabalho, analisar as respostas fisiológicas de duas raças de caprinos nativos do semiárido brasileiro submetidos a ambiente com temperatura e umidade relativa controladas; utilizaram-se 12 animais machos, 6 Canindés e 6 Moxotós, com pesos médios iniciais de 19,6 ± 1,9 kg e 20,4 ± 1,6 kg, distribuídos em delineamento inteiramente casualizado em esquema fatorial (2 x 4), duas raças e quatro níveis de temperatura (20,6, 24,8, 27,8 e 31,6°C), com 6 repetições, e umidade relativa média do ar de 60 ± 5% e velocidade do vento de 0,5m s-1. Na temperatura entre 20,6 e 27,8°C a média da frequência respiratória (25,7 mov min-1) e frequência cardíaca (76,7 mov min-1) dos animais ficaram dentro da normalidade, sendo que a 31,6°C ocorreu elevação da frequência respiratória (134,5 mov min-1) e da frequência cardíaca (104,3 mov min-1). A temperatura retal foi semelhante em todas as temperaturas com média de 39,5°C, dentro da faixa de normalidade para a espécie. A temperatura superficial elevou-se com o aumento da temperatura ambiente. A faixa de temperatura de 20,6 a 27,8 °C pode ser considerada a zona de conforto térmico para caprinos nativos Moxotó e Canindé.The objective of this study was to determine the physiological response of two caprine races from the Brazilian semiarid, submitted to a controlled thermal conditions in a climatic chamber. Twelve male animals were used, 6 Canindés e 6 Moxotós, with mean weight of 19,6 ± 1,9 kg and 20,4 ± 1,6 kg distributed in a completely randomized design, in factorial scheme (2 x 4), two races and four temperature levels (20.6, 24.8, 27.8 and 31,6°C), with six repetitions, with mean relative air humidity of 60 ± 5% and wind speed of 0.5 m s-1. In the temperature between 20.6 and 27.8°C the mean respiratory frequency (25.7 mov min-1) and cardiac frequency (76.7 mov min-1) of the animals were within normality, however, at 31.6°C, occurred elevation to 134.5 and 104.3 mov min-1 for the respiratory and cardiac frequency, respectively. Rectal temperature was similar in all temperatures with average of 39.5°C, within the range of normality for the species. The superficial temperature raised with the increase of the environment temperature. The range of temperature between 20.6 and 27.8 °C can be considered within the thermal comfort zone for native Moxotó and Canindé caprines.

El objetivo de este estudio fue evaluar la presentación clínica, perfil bioquímico, y la respuesta al tratamiento en los caballos con hipocalcemia inducida experimentalmente. Doce yeguas se distribuyeron en un grupo control (n = 5) y un grupo tratado (n = 7). Ácido etilendiaminotetraacético disódico (Na2EDTA 5%) se infundió a los animales hasta que presentaron signos clínicos de hipocalcemia, momento en el que fueron tratados con una solución comercial con calcio, fósforo, magnesio, y la glucosa en una dosis de 1 mL/ kg / PV de 30 minutos. El grupo control recibió 0,9% solución salina en el mismo volumen. Exámenes físicos y colección de muestras sanguíneas se realizaron en cuatro oportunidades: T0 (basal), T1 (al final de la infusión de Na2EDTA), T2 (al final del tratamiento), y T3 (24 horas después del fin del experimento). Todas las yeguas presentaron una disminución temporal en las concentraciones de calcio total y de magnesio y se observaron signos clínicos de hipocalcemia. En el curso del tratamiento, la taquicardia, taquipnea, hipofonesis, y la atonía del ciego desaparecieron, así como aumentaron las concentraciones de calcio total magnesio. El tratamiento efectuado posibilitó la recuperación de la hipocalcemia clínica dentro de 30 minutos y retorno de las concentraciones de los principales parámetros bioquímicos a los valores basales., The aim of this study was to examine the clinical presentation, biochemical profile and response to treatment among horses with experimentally-induced hypocalcemia. Twelve adult, mixed breed mares were used. A 5% ethylenediaminetetraacetic acid disodium (Na2EDTA) solution was infused into all the mares until the animals presented clinical signs of hypocalcemia, at which point they were divided into a control group (n = 5) and a treatment group (n = 7). The treated group received an infusion of calcium, phosphorus, magnesium and glucose at a dosage of 1 mL/kg/BW during 30 minutes. The control group received 0.9% saline solution at the same dosage. Clinical examination and blood sample collection were performed at the times T0 (baseline- thirty minutes before beginning the Na2EDTA), T1 (at the end of the Na2EDTA infusion), T2 (at the end of the treatment) and T3 (24 hours after the end of the experiment). Serum calcium and magnesium concentrations decreased in response to Na2EDTA administration. The clinical signs of hypocalcemia in the mares included tachycardia, tachypnea, hypophonesis and cecal atony. These signs disappeared over the course of treatment, while total calcium and magnesium increased. Treatment produced recovery from clinical hypocalcemia within 30 minutes and promoted return of the main biochemical parameters to baseline values.

The House Sparrow is a globally distributed species and is closely associated with anthropised environments. They are well-adapted to urban life; therefore the decline of their populations in Europe represents an unexpected event that demands an investigation into its causes. Causes that have promoted this decline are not well-known, but one of the highlighted hypotheses is an increase of oxidative stress linked to the toxicity of pollution in urban areas. From an ecophysiological perspective, oxidative damage, antioxidant defense, and oxidative balance are considered reliable indicators of environmental stressors such as pollutants. To carry out this study, blood samples were collected from House Sparrows in three different habitats that varied in terms of urbanization degree: urban, suburban, and rural; during the winter and breeding season. According to our results, urban sparrows showed higher levels of oxidative damage and higher activity of antioxidant enzymes, but lower antioxidant capacity in comparison with the rural birds; and these differences especially increase during the breeding season. The maintenance of oxidative balance increases in an urban environment in comparison to a rural one; we suggest that the high level of pollution and the poor quality diet linked to urban environments. The breeding season is expected to be particularly challenging for the oxidative balance of urban birds, when the reallocation of resources between self-maintenance and reproduction may be critical due to the scarcity of antioxidants found in urban areas. This study may contribute to determining the causes of the population decrease of House Sparrows in cities.

Os compostos organoestânicos (OTs) apresentam uma variedade de químicos amplamente empregados na agricultura e na indústria em geral, sendo amplamente usados. Estudos vêm demonstrando que os OTs causam sério impacto ambiental, como o caso do hidróxido de trifenilestanho (TPT), de modo que se torna importante a realização de estudos que avaliem as alterações fisiológicas causadas por estes compostos, principalmente no que se refere as espécies nativas de regiões portuárias e de atividade agrícola. Do ponto de vista toxicológico, um aspecto importante diz respeito aos possíveis efeitos do TPT sobre o sistema reprodutivo. O presente estudo objetiva avaliar a influência da exposição ao TPT sobre a fisiologia reprodutiva de machos da espécie Calomys laucha, verificando os efeitos sobre a atividade espermática nas fases pré-púbere e adulta. Para tanto, este estudo foi dividido em dois experimentos visando verificar: Exp.1: Efeitos do TPT sobre a atividade espermática em indivíduos expostos via amamentação, onde as fêmeas lactantes foram organizadas em 5 grupos (n =5), sendo um controle água e um controle do veículo (carboximetilcelulose 1%) e três grupos com diferentes concentrações de exposição ao contaminante 0,5; 2,5 e 5,0 mg TPT/kg. As fêmeas parturientes foram expostas por gavagem ao TPT durante 20 dias, do 1º dia pós-parto até o 21º dia pós-natal (PND). Após este período os filhotes foram alojados separadamente e aos 100 (±5) PND foram eutanasiados e realizada a avaliação da atividade espermática. Na avaliação da cinética espermática foi constatada uma redução na motilidade total nos grupos de 0,5 e 5,0 mg TPT/kg; na motilidade progressiva houve uma diminuição em todos os grupos tradados; constatou-se ainda a diminuição nas distâncias e velocidades média percorrida, retilínea e curvilínea, principalmente nas doses de 2,5 e 5,0 mg/kg, bem como na retilinearidade, linearidade e oscilação. Nos resultados obtidos na citometria de fluxo, foi evidenciada uma redução na integridade de membrana, conforme o aumento das doses; a funcionalidade de mitocôndria apresentou redução nos grupos de 0,5 e 5,0 mg TPT/kg; foi constatado um aumento na ruptura de membrana nos grupos de maior dose 2,5 e 5,0 mg TPT/kg; a reação de acrossoma apresentou níveis mais elevados de acrossomas não íntegros nas doses de 0,5 e 5,0 mg TPT/kg. Com base nos resultados obtidos, podemos concluir que o TPT causa comprometimento da atividade espermática em indivíduos expostos na fase de amamentação, principalmente no que se refere as motilidades total e progressiva, integridade de membrana e a funcionalidade de mitocôndria os quais apresentam danos persistentes observados quando avaliados na fase adulta, aos 100 (±5) dias de vida. Exp.2: Efeitos do TPT sobre a atividade espermática em indivíduos expostos na fase adulta. Os animais foram divididos em 6 grupos (n =7), sendo um controle água e um controle do veículo (carboximetilcelulose 1%) e quatro grupos com diferentes concentrações de exposição ao contaminante 0,5; 2,5; 5,0 e 10,0 mg TPT/kg. Os animais foram expostos por gavagem ao TPT durante 21 dias, do 50º (±3) ao 71º (±3) dia de vida. Os animais expostos a maior dose (10 mg/kg) apresentaram sinais de intoxicação grave em apenas 7 (sete) dias de exposição. Os dados referentes a esta dose foram analisados separadamente por se tratar de uma exposição a curto prazo e os resultados obtidos sugerem que a exposição nesta dose foi severamente prejudicial à atividade espermática, principalmente no que se refere a motilidade espermática, funcionalidade mitocôndria, integridade e fluidez de membrana. Nas outras dosagens, a principal alteração constatada na avaliação da cinética espermática, foi a redução nas motilidades total e progressiva a partir do grupo 0,5 mg TPT/kg. Nos dados obtidos na avaliação por citometria de fluxo, foi observada uma diminuição na funcionalidade de mitocôndria, conforme o aumento das doses, bem como um aumento na fluidez de membrana a partir da dose de 5,0 mg TPT/kg, a reação de acrossoma apresentou maiores valores de acrossomas não íntegros nas doses de 0,5 e 5,0 mg TPT/kg. Com base nos resultados obtidos, podemos concluir que o TPT causa comprometimento da atividade espermática em indivíduos expostos na fase adulta, principalmente no que se refere as motilidades total e progressiva e a funcionalidade de mitocôndria. Deste modo, concluímos que o TPT pode ser excretado através do leite materno em fêmeas expostas por via oral, e que em machos expostos nas fases pré-pubere e adulta, por via oral, promove prejuízos na atividade espermática. Triphenyltin (TPT) is an organotin compound (OT), used primarily in agriculture as a fungicide, herbicide and pesticide, and has been used in the composition of antifouling paints for ships worldwide. Over the years, studies have demonstrated its effect as an endocrine disrupter in several organisms, by preventing enzymatic expression and causing reproductive toxicity. Native species of port regions and agricultural activity are susceptible to exposure to TPT, so they deserve special attention on the impacts of this exposure on their physiology, especially on the possible effects on the reproductive system. This study aimed to evaluate the effects of exposure to TPT, via breastfeeding, on reproductive physiology in the Calomys laucha species, verifyingits influence on sperm activity in adulthood, since this species inhabits regions prone to exposure to this contaminant. The experimental design was of 5 groups (n = 5), one water control and one vehicle control (carboxymethylcellulose 1%) and three groups with different doses of exposure to the contaminant 0.5; 2.5 and 5.0 mg TPT/kg, which females were exposed by gavage to the TPT for 20 days, from the 1st day postpartum to the 21st postnatal day (PND). After this period, the pups were housed separately and at 100 (± 5) PND were euthanized and the kinetics, organelles and biochemistry of the sperm were evaluated. Among the main results obtained in the evaluation of sperm kinetics, a reduction in total motility was observed in the groups of 0.5 and 5.0 mg TPT/ kg, being more pronounced in the group 0.5 mg TPT / kg; in progressive motility there was a decrease in all the traded groups, but as in total motility, the group with the most exacerbated reduction was 0.5 mg TPT/kg; it was also observed the decrease in the distances and velocities traveled, rectilinearity and curvilinearity, mainly in the doses of 2.5 and 5.0 mg/kg, as well as in the oscillation. In the results obtained in the evaluation of cellular organelles, a reduction in membrane integrity was evidenced, as the doses increased; the functionality of mitochondria showed reduction in the groups of 0.5 and 5.0 mg TPT/kg; increased membrane rupture in the higher dose 2.5 and 5.0 mg TPT / kg groups; increased the acrosome reaction at doses of 0.5 and 5.0 mg TPT/kg. Based on the results obtained, we can conclude that the TPT causes alteration of the reproductive parameters, decreasing the activity and sperm quality, in individuals exposed in the breastfeeding phase, mainly regarding total and progressive motilities, membrane integrity and the functionality of mitochondria.

El envejecimiento es un proceso universal, intrínseco, progresivo y deletéreo que podría definirse como el conjunto de cambios que afectan a todas nuestras células y tejidos al avanzar la edad, llevando a una pérdida progresiva de nuestras capacidades fisiológicas así como a una incapacidad para mantener la homeocinesis. Así mismo, el envejecimiento también se caracteriza por su heterogeneidad, de manera que no todos los cambios que suceden en los sistemas de un organismo se producen a la misma velocidad, así como tampoco lo hacen los individuos con una misma edad cronológica, lo que hace surgir el concepto de “edad biológica”, considerado un mejor indicador de la velocidad a la que envejece un individuo que la edad cronológica y siendo, por tanto, determinante de su esperanza de vida. Según la teoría de la oxidación-inflamación, este deterioro asociado con la edad sería consecuencia de un estrés oxidativo crónico asociado a un estrés inflamatorio, los cuales afectarían a todas las células, pero de manera más relevante a las de los sistemas reguladores, el nervioso, el endocrino y el inmunitario. La afectación de estos sistemas y, consecuentemente, de la comunicación entre ellos (comunicación psiconeuroinmunoendocrina) deteriora la homeocinesis y aumenta la morbilidad y mortalidad. Además, en esta teoría se propone que dadas las características del sistema inmunitario, el cual necesita producir oxidantes y compuestos inflamatorios en su función defensiva frente a patógenos y cánceres, si dicha producción no está bien regulada, puede entrar en un círculo vicioso que aumente el estrés oxidativo e inflamatorio del organismo. De esta forma, se sugiere que el sistema inmunitario no sólo puede modular la velocidad de envejecimiento de cada individuo sino que su capacidad funcional supone un excelente marcador del estado de salud. Los cambios que afectan al sistema inmunitario al envejecer se recogen bajo el término de inmunosenescencia y abarcan todos los niveles de organización de dicho sistema, desde genético hasta funcional...

Programa de Doctorado en Estudios Medioambientales, Nature is complex and organisms commonly need to rapidly be able to detect and respond to environmental inputs in order to increase their survival odds. The ability of a given genotype to alter its morphology, behavior or development against changing environments is known as phenotypic plasticity, which is adaptive when the induced phenotypes confer increased fitness in the altered environment. Adaptive plasticity favors phenotypic diversity and increases population viability, as well as facilitates the maintenance of genetic variation reducing the severity of bottleneck events during rapid environmental change, and also by shielding genetic variants from selection. In particular, phenotypic plasticity is essential for species with low vagility and high philopatry, as is the case of most amphibian species. The life-cycle of amphibians is often a complex one that includes an aquatic larval stage that gives rise to a terrestrial stage through metamorphosis. Metamorphosis is thus a key ontogenetic switch point that entails vast anatomical, physiological and ecological changes in the organism. The timing and body condition at which metamorphosis occurs largely determine the likelihood of survival in larval and juvenile amphibians. Growth and development are remarkably decoupled over long periods of the amphibian larval ontogeny. This allows amphibian larvae to grow without advancing in development under benign conditions of high food availability, reduced competition, and abundant water, or else accelerate development at the expense of truncating growth when conditions worsen, as when at risk of pond drying. Such fine-tuning of growth and development relies on the ability of amphibian larvae to sense their environment and is regulated by neuroendocrine pathways, which can activate/repress multiple metabolic cascades, which in turn can involve short and long-term consequences for body condition and even life span. The main objective of this thesis is to understand the physiological mechanisms enabling developmental and growth plasticity in amphibians, and their consequences. Firstly, several simultaneous experiments were conducted to study the physiological alterations inflicted by common potential external stressors on amphibian larvae. In particular, we studied the effect of varying levels of salinity, herbicide, water pH, types of predators, and temperature in spadefoot toad larvae (Pelobates cultripes) evaluating their changes in corticosterone level, metabolic rate, activity of various antioxidant enzymes, lipid peroxidation, and immune state. Most of the studied levels factors caused some physiological imbalances in tadpoles, although high levels of salinity and herbicide caused the most dramatic physiological alterations. Tadpoles showed decreased levels of corticosterone when exposed to native predators, congruent with the common reduction in foraging and metabolic activity in the presence of predators. Interestingly, however, tadpoles did not reduce corticosterone in the presence of invasive predators, indicating a lack of innate recognition. Furthermore, corticosterone and the antioxidant enzyme glutathione reductase were the most sensitive parameters against the studied factors, and hence good candidates for further use in physiological monitoring of natural populations. Pond drying and predators are two of the main risks for amphibian larvae in temporary ponds. Physiological consequences of plastic responses in tadpoles after being exposed to both factors may allow us to understand short-term consequences of the developmental alterations induced, as well as to predict their long-term effects. We crossed the presence/absence of predators with permanent and drop-down water levels in a 2x2 experimental design simulating natural conditions in large outdoor mesocosms to test for induced changes in life-history traits, and also assessed their effects on fat reserves, oxidative stress, and telomere length of the surviving Pelobates cultripes juveniles that survived to both risks. Tadpoles accelerated their development in response to decreased water level, but at the expense of metamorphosing smaller and greatly depleting their fat reserves. Cellular oxidative stress due to developmental acceleration was successfully buffered by increased antioxidant enzyme activity, and telomere length remained unchanged. On the other hand, predators greatly reduced larval density, which relaxed competition and allowed survivors to develop fast, grow bigger and accumulate fat. However, tadpoles showed signs of oxidative stress and experienced telomere shortening. Telomere length is reduced during cell replication, and telomere shortening is known to be associated with senescence and reduced life span. Therefore, regarding body size and fat reserves at metamorphosis, developmental acceleration in response to pond drying compromises short-term survival, although its consequences are reversible in the long run. In turn, oxidative stress and telomere shortening due to rapid growth when tadpoles survived predators are likely reduce their long-term survival. Even within species, organisms do not respond phenotypically to the same extent against environmental inputs. In fact, natural populations often harbor substantial variation in their degree of phenotypic plasticity. Theoretical studies suggest that the evolution of plasticity is limited by costs of maintaining the machinery needed to detect and respond to environmental cues. However, only a few studies have empirically detected maintenance costs of plasticity. In the third chapter of this thesis, we tested for physiological costs of maintaining developmental, growth, and morphological plasticity in Pelobates cultripes larvae in response to both pond drying and predators. For this purpose, we first determined the degree of developmental plasticity of a total of twenty families (sibships) from various populations in response to these two environmental factors. Simultaneously, we assessed among families variation in body mass, fat reserves, metabolic rate, antioxidant enzyme activities, lipid peroxidation, reduced/oxidized glutathione, and immune state under benign control conditions. We tested the existence of costs of plasticity by testing for an association between the degree of plasticity of each family and the molecular markers of physiological stress. We found maintenance costs of plasticity associated to the degree of developmental and growth plasticity induced by predators, in terms of increased glutathione reductase activity and granulocyte to lymphocyte ratio, respectively. Morphological plasticity in response to both pond drying and predators were also linked to levels of antioxidant enzymes, lipid peroxidation, immune state or growth. Also, we detected trade-offs between the developmental responses of larvae to each factor so that genotypes (families) that readily accelerated development in response to pond drying were not effective in delaying metamorphosis in the presence of predators. Such a trade-off suggests possible constrains on the evolution of adaptive plasticity to conflicting environmental stimuli. Environmental heterogeneity can affect the degree of adaptive plasticity, which may also imply short and long-term consequences. In amphibians, the ability to accelerate development commonly results adaptive, since larvae usually inhabit pools in which water availability is seasonal and heterogeneous. Adaptive developmental plasticity is expected to evolve despite its possible costs when environmental heterogeneity precludes a single phenotype to maximize fitness across all conditions. Swedish Rana temporaria island populations show marked differences among populations in developmental rate and their responsiveness to pond drying so that populations with more variable durations in pond hydroperiod tend to show higher levels of developmental plasticity. In the fourth chapter, we study the physiological mechanisms and consequences of divergent developmental plasticity among some of these R. temporaria populations. We exposed larvae from six populations from three different island habitat types differing in their pool drying regime to simulated desiccation to determine their developmental plasticity. Populations from islands with only ephemeral pools showed higher developmental plasticity than populations from islands with permanent or long-lasting ponds. Individuals from islands with ephemeral ponds experience physiological alterations indicative of physiological costs of increased plasticity, such as altered catalase and glutathione reductase activities, and reduced telomere length. Elevated antioxidant activities indicate metabolic costs associated to increased developmental plasticity which may also compromise the health and lifespan of individuals and the viability of those populations, as shortened telomeres suggested. During the course of this thesis, we have also evaluated the suitability of some methodological aspects. (I) We evaluated the performance of three commonly used procedures for corticosterone determination using Xenopus laevis tadpoles: radioimmunoassay (RIA) in whole-body homogenates, enzyme immunoassay (EIA) on whole-body, and EIA on plasma. Each procedure presented advantages and disadvantages regarding sensitivity, the use of radioactivity, sampling size, or handling time. RIA is preferred in small-bodied animals from which blood cannot be obtained. EIA in plasma resulted a good non-radioactive alternative when blood sampling is possible. EIA on whole-body homogenates was the less sensitive procedure, although it may be a non-radioactive useful alternative to assess qualitative changes in corticosterone in small individuals when considerable differences are expected. (II) Immune response in amphibians has been commonly evaluated through indirect methods like phytohemagglutinin (PHA) injections or by direct like cell counts from blood smears. Here, we validated immunological evaluations in amphibians by means of flow cytometry. The immunological state of Pelobates cultripes tadpoles were experimentally altered by exposing them to exogenous corticosterone. Then, leukocyte proportions were quantified through both blood smears and flow cytometry. Both techniques showed similar patterns of leukocyte proportions. Once validated, flow cytometry also allowed quantification of changes in absolute number of leukocytes. The suitability of both techniques attending to accuracy, body size requirements, or the useful in field studies was also discussed. The results obtained in this thesis highlight the key role of physiological mechanisms in amphibian larvae plasticity and in its evolution. Therefore, for a holistic knowledge of ecological and evolutionary process results essential to understand the physiology underlying them., Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería Bioquímica, Postprint

274 p. Animal farming systems, including fish farming, are regarded to be a major cause of problems such as resource (water and land) depletion and climate change. Their required intensification represents a relevant cause of environmental concern at global level. In aquaculture, the option for reducing the environmental footprint of aquatic animal production and mitigating many of the impacts associated with traditional commercial fish culture technologies (i.e. net pens, ponds, flow-through systems) is Recirculating Aquaculture System (RAS) technology. The present work studies the feasibility of using RAS to rear cold water species such as Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) in the Basque coastal area. The study is made from four different perspectives: technical, economic, environmental and societal. The viability of the technology is tested coupled with the surrounding environment; the cost/benefits of producing fish in the local market are analyzed; the environmental performance and the created impacts are studied; and the final product¿s acceptability is studied among local consumers and seafood experts. Moreover, a new environmental assessment is presented and detailed knowledge of the main issues and future challenges are obtained. AZTI TEKNALIA

The obesity epidemic is a priority concern, as its relationship with cardiovascular diseases and type 2 diabetes, is associated with mortality and with elevated costs for public health systems across the globe. Epidemiological data higlights the fact that the obesity epidemic is likely to increase in the near future, as younger cohorts present with, even increasing, high rates of early obesity onset and prevalence. Although there is a broadly understood adult etiology for obesity, substantial data from epidemiological and experimental studies indicate the role of the perinatal environment in promoting obesity and related metabolic syndrome. Postnatal early overfeeding, induced by maternal high fat consumption or by early exposure to overfeeding during the suckling period, lead mammals to demonstrate a precocious onset of obesity. Infants and childrens who are obese are highly susceptible to remain obese into adult life. In a previous study, our group demonstrated that postnatal early overfeeding resulted in rats becoming obese and presenting an impaired interscapular brown adipose tissue (iBAT) thermogenic function, producing less heat than the control animals both, during day and night-time periods. Brown adipose tissue (BAT) is present in adult humans and BAT thermogenesis increase energy expenditure through uncoupling-protein-1 (UCP1) activity, which disrupts the normal mitochondrial energy system, resulting in a lower production of ATP and diversion of energy as heat. Given the potential of BAT to impact upon energy balance, revert metabolic program and augment energy expenditure by BAT to reduce fat storage and counter obesity, is a desirable target. The regular practice of physical exercise is one physiological approach that results in increased BAT activity. Few studies have considered the important role of BAT on the energetic metabolism, as a mechanism to reverse the long-term programmed effects of the early overfeeding on animals. The possible effects of physical exercise upon metabolism and BAT in programmed animals have also received scant attention. In this thesis we investigated the effects of early overfeeding by maternal high fat consumption on the adipose tissue at early stages of life and tissue development, moreover, we tested the effectiveness of a moderate and lowfrequency exercise protocol on the thermogenesis function of iBAT of adult rats, which were metabolic programmed by early overnutrition. Manuscript 1: To investigate, at postnatal day 10, the morphology and gene expression in subcutaneous WAT and interscapular BAT from pups born to High Fat Fed (HFD) dams. Manuscript 2: To investigate the potential of a moderate and low-frequency exercise protocol to deprogram the function of iBAT in adult male rats made obese by exposre to overfeeding during lactation. Manuscript 1: Nine-week-old female Sprague Dawley rats were divided into 2 groups: control normal fat diet (NFD N=6), or high-fat diet (HFD N=10). Rats were kept on their respective diet for 4 weeks prior to conception, through gestation and after birth, until postnatal day 10 (PN10), when pups were weighed and euthanized. Adipose tissue was collected from subcutaneous and interscapular depots for histology and gene expression studies. Manuscript 2: Wistar rats litters were delivered normally but at PN2, litters were adjusted to 9 pups per dam (normal litters - NL) for control, or 3 pups (small litter SL) per dam to induce overfeeding during early life. At PN21, all offspring were weaned and randomly divided into exercise or sedentary groups (NL SED, NL EXE, SL SED, and SL EXE). An exercise protocol commenced at PN30. Animals were initially subjected to an effort test on a treadmill. Training sessions (45minutes) were then performed 3 times per week, at 60% of the final workload achieved in the effort test. The effort test was repeated each 15 days to adjust the training workload. At PN80, animals ceased training and at PN81, underwent surgery to implant a temperature transponder underneath interscapular BAT (iBAT). The iBAT temperature was measured during light and dark periods from PN87 to PN90. At PN91 animals were euthanized, blood samples and fat pads collected for further analysis. IBAT nerve activity was recorded from a separated set of (non operated) animals at PN90. Manuscript 1: Subcutaneous white adipose tissue (sWAT) adipocytes from HFD offspring were larger compared to controls (p

© The Author(s) 2017, Cardiolipin (CL) is a mitochondrial-specific phospholipid. CL content and acyl chain composition are crucial for energy production. Given that estradiol induces CL synthesis in neurons, we aimed to assess CL metabolism in the cerebral cortex (CC) of male and female mice during early postnatal life, when sex steroids induce sex-dimorphic maturation of the brain. Despite the fact that total amount of CL was similar, its fatty acid composition differed between males and females at birth. In males, CL was more mature (lower saturation ratio) and the expression of the enzymes involved in synthetic and remodeling pathways was higher, compared to females. Importantly, the sex differences found in CL metabolism were due to the testosterone peak that male mice experience perinatally. These changes were associated with a higher expression of UCP-2 and its activators in the CC of males. Overall, our results suggest that the perinatal testosterone surge in male mice regulates CL biosynthesis and remodeling in the CC, inducing a sex-dimorphic fatty acid composition. In male’s CC, CL is more susceptible to peroxidation, likely explaining the testosterone-dependent induction of neuroprotective molecules such as UCP-2. These differences may account for the sex-dependent mitochondrial susceptibility after perinatal hypoxia/ischemia., This work was supported by a grant from the Ministerio de Economía y Competividad, Spain (BFU2014-51836-C2-1-R) to LMGS and a young researchers grant from the National Agency for Scientific and Technological Promotion (PICT 2014-1549), Argentina to MA.

El rotavirus (RV) és el principal causant de diarrea greu en nens menors de cinc anys i, encara que es necessiten més estudis estandarditzats, hi ha evidència de que els probiòtics poden ajudar a lluitar contra el RV i contra altres patologies infeccioses i intestinals. Sobre aquesta base, l'objectiu de la present tesi va ser el d’avaluar l'efecte protector de productes relacionats amb els probiòtics i els prebiòtics contra les infeccions per rotavirus, utilitzant models experimentals en rata lactant. Per aconseguir aquest objectiu, es va utilitzar un model d’infecció simple per RV ja existent, amb algunes noves variables. D'altra banda, es va desenvolupar un nou model de doble infecció per rotavirus en rates. Diverses variables immunològiques van demostrar que és adequat per a la realització d'estudis intervencionals, i sembla que les accions moduladores sobre la primera infecció tenen una influència sobre la resposta immunitària desenvolupada enfront a una segona infecció per RV. Això s'ha demostrat amb l'administració d’un calostre boví hiperimmune contra el RV (HBC) durant la primera infecció. Pel que fa a la suplementació amb la soca probiòtica Bifidobacterium breve M-16V durant la lactància, s’ha observat que millora el desenvolupament de la immunitat de la mucosa en rates per mitjà de la modulació de l'expressió de TLR, la millora del procés de reclutament dels limfòcits T verges als GLM i la retenció dels limfòcits activats al compartiment intraepitelial, així com la potenciació de la síntesi d’IgA intestinal. D'altra banda, modula la infecció i la reinfecció per RV en el model d’infecció simple i doble, respectivament, alleugerint les manifestacions clíniques durant la primera infecció, però permetent a l'hoste elaborar la pròpia resposta immunitària, que l'ajudarà a controlar una segona infecció. En quant a la suplementació prebiòtica durant la lactància en rates, la barreja prebiòtica scGOS/lcFOS 9:1 (Immunofortis), tot i presentar un efecte directe sobre la consistència de les femtes, ha mostrat efectes beneficiosos contra la infecció per RV i ha modulat la reinfecció en el model de doble infecció, mostrant una important acció immunomoduladora. Aquests efectes han estat comparables als del prebiòtic Bimuno® GOS. Com a simbiòtic, quan s’ha avaluat en combinació amb el probiòtic B. breve M-16V, s’ha mostrat molt eficaç en la modulació de la diarrea induïda per RV, així com en la modulació de la resposta immunitària i de la reinfecció per RV en el model preclínic de doble infecció. Per contra, la combinació d’Immunofortis® amb oligosacàrids acídics derivats de pectina (pAOS) no ha potenciat el seu efecte preventiu sobre el model de gastroenteritis per RV en rata. D'altra banda, la suplementació amb un postbiòtic (una llet fermentada per B. breve i Streptococcus thermophilus) durant la lactància en rates, ha estat capaç de prevenir gairebé totes les manifestacions derivades de la diarrea induïda per RV, mentre que també ha modulat la resposta immunitària anti-RV. L’addició d’Immunofortis® a aquest postbiòtic no ha mostrat efectes clars de sinèrgia. Com a conclusió general, tots els productes estudiats, essent el probiòtic i el postbiòtic els més eficaços, han mostrat efectes beneficiosos sobre la gastroenteritis induïda per RV en el model de rata lactant, modulant les variables clíniques i resposta immunitària en primeres etapes de vida. Així i tot, calen més estudis per comprendre millor el seu mecanisme d'acció i per a ser considerats per a la seva inclusió en preparats per a lactants o suplements, com estratègies per a la protecció contra la diarrea induïda per RV en nens., Rotavirus (RV) is the leading cause of severe diarrhoea among infants and young children and there is evidence that probiotics can help to fight against RV infection. The aim of the present thesis was to evaluate the protective effect of probiotics, prebiotics and other related products against RV infections using the suckling rat as experimental model. To achieve this objective, an updated model of simple RV infection and a new RV double-infection model in rat were used. Regarding the probiotic effect, the supplementation with Bifidobacterium breve M-16V improved the development of mucosal immunity in early-life rats. Moreover, it attenuated RV infection and reinfection by ameliorating diarrhoea during first infection but allowing the host to elaborate its own immune response, which seems to help control the second infection. Regarding the prebiotic effect, scGOS/lcFOS 9:1 (Immunofortis) ameliorated RV infection in the single-infection model and modulated reinfection in the double RV infection model, showing a high immunomodulatory action. Its effect was comparable to that of the prebiotic Bimuno GOS. A synbiotic combining the scGOS/lcFOS prebiotic with the probiotic Bifidobacterium breve M-16V, was highly effective in modulating RV-induced diarrhoea as well as modulating immune response and RV reinfection in the double-infection preclinical model. Conversely, the combination of Immunofortis with pectin-derived acidic oligosaccharides (pAOS) did not potentiate its preventive effect. The postbiotic supplementation with a Bifidobacterium breve and Streptococcus thermophilus-fermented formula, and its combination with scGOS/lcFOS were able to prevent almost all features derived from the RV-induced diarrhoea and they also modulated the anti-RV immune response. Overall, all tested products showed beneficial effects on the RV-induced gastroenteritis in the neonatal rat model, modulating clinical biomarkers and immune system responses early in life, with the probiotic and the postbiotic being the most effective. Further studies are needed in order to better understand their mechanism of action and for them to be considered for inclusion in infant formulas or supplements as strategies for protecting against human RV-induced diarrhoea in children.

The blood-brain barrier is a well-coordinated and highly selective barrier whose main function is to regulate brain homeostasis and the transport of endogenous and exogenous compounds between the blood and the brain. It permits the selective brain uptake of nutrients and impedes the entrance of potentially harmful substances and pathogenic organisms into the brain. Due to this restrictive nature of the blood-brain barrier, the transport of neurotherapeutics from the blood to the brain results extremely difficult, and has become a major pharmaceutical challenge in recent decades. Only lipophilic molecules with a molecular weight under 400-600 Da are able to cross the blood-brain barrier. For this reason, 98% of all small molecule drugs and almost 100% of large molecule drugs are unable to cross the blood-brain barrier, and hence, most neurological and neurodegenerative diseases currently have few or no treatment options. During the last decades, numerous strategies have been proposed to overcome the blood-brain barrier and efficiently deliver therapeutic agents to the brain. One of these strategies consists in linking the pharmacologically active substance to a molecular vector that acts as a molecular Trojan Horse and is capable of crossing the blood-brain barrier using a receptor-mediated transcellular transport system of the brain capillary endothelial cells. These molecular vectors can be natural ligands, peptides or monoclonal antibodies (mAbs) that bind to a particular receptor and trigger endocytosis or transcytosis processes. Several mAbs directed against the transferrin receptor (TfR), which is abundant in brain capillaries, have been extensively studied. However, there is still no consensus regarding their transcytotic capacity and their ability to transport substances across the blood-brain barrier. Moreover, the intracellular mechanisms that these antibodies or constructs undergo inside the endothelial cells remain unclear. To gain insight on this strategy, this thesis aimed to study the potential of the 8D3 monoclonal antibody, directed against the murine TfR, to transport substances across the blood-brain barrier in mice. On this basis, a series of experiments were performed where the 8D3 antibody was conjugated to different cargoes, the resulting constructs were administered in vivo to mice, and the distribution and intracellular mechanisms that these constructs undergo at the blood-brain barrier were studied. Overall results suggest, firstly, that the 8D3 antibody is able to bind to the TfR and trigger endocytosis of the 8D3-TfR complex, but does not complete transcytosis except in rare occasions. Secondly, the 8D3 antibody is capable of internalizing the conjugated cargo inside the brain capillary endothelial cells through a clathrin-dependent endocytosis process, and hence, of overcoming the first obstacle in the transport across these cells. However, once inside the endothelial cells, the constructs tend to progressively accumulate in mature endosomal structures of large size and great complexity. These results can be mainly explained by the high affinity of the peptidomimetic mAbs for their receptors and their consequent difficulty to dissociate from them. Only a small percentage of the endocytic vesicles fuse with the abluminal membrane and open up to the basal lamina. Thus, in these cases, the constructs complete transcytosis. Nevertheless, they remain attached to the abluminal membrane and never reach the brain parenchyma, probably, in this case as well, because of the high affinity of the 8D3 for the TfR. Based on this premise, different aspects can be approached regarding the design of the constructs. Nanocarrier functionalization, development of acid-cleavable linkages between mAb and cargo, and reducing mAb’s affinity for the target receptor are some of the ideas that are currently under development for the optimization of this receptor-mediated transport based strategy to overcome the blood-brain barrier. These aspects are today main focus of research and starting point towards new perspectives.

254 p.-117 fig.-11 tab.-anexos., [EN] In this PhD we wanted to understand the role of autophagy and mitophagy in the developing mouse retina. Our data show that autophagy is an active process during development of the mouse retina, especially in the early embryonic stages, being retinal ganglion cells which have a greater autophagy flux. This process has several functions during embryonic retina physiology. First, we have shown that there are differences in mitochondrial mass levels in the retinas of E13.5 embryos due to degradation of mitochondria by mitophagy only in the central region of the retina in that stage. Furthermore we have shown that autophagy is necessary to maintain ATP levels suitable for the exposure of phosphatidylserine on the surface of apoptotic cells, allowing their recognition and degradation. This function is not universal, since inhibition of autophagy doesn´t lead to an accumulation of apoptotic cells in the central region where the mitochondrial mass is lower. Furthermore, the mitophagy that occurs in the central area of the retina contributes to early neural cell death located in that region. Moreover, the mitochondrial degradation occurs widely in the entire retina in a more advanced stage of development, E15.5 due to activation of mitophagy process regulated by hypoxia and NIX. In this case, mitophagy not only contributes to the physiological cell death of the retina, if not it is also responsible for a metabolic shift towards glycolysis required for proper differentiation of retinal ganglion cells. Furthermore, there is a close relationship between the metabolism, mitochondrial mass and differentiation, since direct modulation of glycolysis lead to changes in mitochondrial levels and disturb the differentiation of retinal ganglion cells. That metabolic change mediated by mitophagy also may be involved in macrophage polarization to proinflammatory type M1 and to early differentiation of stem cells., [ES] En esta Tesis Doctoral hemos querido entender el papel de la autofagia y la mitofagia en el desarrollo de la retina de ratón. Nuestros datos muestran que la autofagia es un proceso activo durante el desarrollo de la retina de ratón, sobre todo en los estadios embrionarios iniciales, siendo las células ganglionares las que presentan un mayor flujo de autofagia. Este proceso presenta diversas funciones durante la fisiología de la retina embrionaria. En primer lugar, hemos demostrado que existen diferencias en los niveles de masa mitocondrial en las retinas de embriones en E13.5 debido a la degradación de mitocondrias por mitofagia únicamente en la región central de la retina en dicho estadio. Por otro lado hemos demostrado que la autofagia es necesaria para mantener unos niveles de ATP adecuados para la exposición de fosfatidil-serina en la superficie de las células apoptóticas, lo que permite su reconocimiento y degradación. Esta función no es universal, ya que la inhibición de autofagia no produce una acumulación de células apoptóticas en la región central, donde la masa mitocondrial es menor. Por otro lado, la mitofagia que se produce en la zona central de la retina contribuye a la muerte neural temprana localizada en dicha región. Además, esa degradación mitocondrial se produce de manera generalizada en toda la retina en un estadio del desarrollo mas avanzado, E15.5, debido a la activación del proceso de mitofagia regulado por hipoxia y NIX. En este caso, la mitofagia no solo contribuye a la muerte fisiológica de la retina, si no que también es responsable de un cambio metabólico hacia glicólisis necesario para la correcta diferenciación de las células ganglionares de la retina. Además, existe una estrecha relación entre el metabolismo, la masa mitocondrial y la diferenciación, ya que la modulación directa de la glicólisis produce cambios en los niveles de mitocondrias y altera la diferenciación de células ganglionares. Ese cambio metabólico mediado por mitofagia, también podría estar implicado en la polarización de macrófagos hacia tipo proinflamatorio M1 y en la diferenciación temprana de células stem.

Pontes et al. show that plasma membrane mechanics exerts an upstream control during cell motility. Variations in plasma membrane tension orchestrate the behavior of the cell leading edge, with increase–decrease cycles in tension promoting adhesion row positioning., Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II–independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.