Abiotic stress responses in mould and non-conventional yeasts

Debaryomyces hansenii es una levadura ascomicética, de un gran potencial biotecnológico, que se encuentra habitualmente en ambientes salinos como pueden ser el agua de mar o algunos embutidos. Sin embargo, su caracterización molecular se ha visto ralentizada por algunas peculiaridades como el hecho de que la mayoría de las cepas son aneuploides, de ser una levadura difícil de transformar, ó mostrar un bajo porcentaje de recombinación homóloga. En este trabajo de tesis doctoral se han abordado diversos aspectos de la fisiología de D. hansenii tanto a nivel molecular cómo a nivel más aplicado. Primero estudiamos las posibles relaciones entre las respuestas a estrés salino y oxidativo tanto a nivel molecular como enzimático o metabólico. Nuestros resultados muestran como la expresión de genes relacionados con estrés oxidativo se indujo por exposición a estrés salino (NaCl y KCl), y viceversa, que la transcripción de algunos genes relacionados con el estrés osmótico/salino fue inducida por exposición a H2O2. Estos efectos también se han demostrado a nivel enzimático donde el estrés salino indujo la actividad de las enzimas catalasa y glutatión reductasa, típicamente consideradas defensas antioxidantes. Además, a nivel metabólico, ambos estreses indujeron la producción de ROS, y la exposición a peróxido de hidrogeno influyó en la acumulación intracelular de sodio. Por otra parte, hemos abordado el tema del posible carácter halotolerante o halófilo de D. hansenii desde una aproximación novedosa, puesto que por primera vez se han utilizado biorreactores para este estudio que nos han permitido crecer las células bajo condiciones controladas. Nuestros resultados han demostrado que la presencia de altas concentraciones de NaCl o KCl fue beneficiosa para la velocidad de crecimiento de la levadura en comparación con los experimentos control (sin sales), tanto utilizando para su estimación la cosecha de biomasa como la producción de CO2. De hecho, en condiciones de limitación de nutrientes como la glucosa, la presencia de NaCl produjo una mayor cantidad de biomasa que el KCl. En su conjunto estos resultados apoyan el carácter halófilo y no sólo halotolerante de esta levadura. En todos estos experimentos no se produjo etanol en ninguna de las condiciones de trabajo, lo que indica un metabolismo completamente respiratorio. Finalmente, en la última parte de este trabajo se ha utilizado una cepa de D. hansenii aislada de embutidos de la zona del Valle de los Pedroches y se ha inoculado sobre la superficie de lomo ibérico con el fin de estudiar su posible efecto sobre las características fisicoquímicas del embutido. En todas las condiciones estudiadas la levadura fue capaz de implantarse en la superficie de las muestras de lomo y afectar a sus características de manera que estas muestras mantenían una mayor actividad de agua, un pH más alto, y un menor contenido en sodio al final de los tres meses de maduración del producto. Aunque la levadura no cambio el perfil general de ácidos grasos sí que afectó a los niveles de compuestos volátiles y aromáticos. Hay que señalar que las catas realizadas indican una tendencia a una mayor aceptación de los lomos inoculados específicamente con levadura por parte de los consumidores.Debaryomyces hansenii is an ascomycetic yeast with a high biotechnological potential that can be usually found in salty environments such as seawater or several sausages. However, its molecular characterization has been slowed down due to some peculiarities: most strains are aneuploid, horizontal gene transfer is difficult, and homologous recombination is low. In this thesis, we have studied several molecular and physiological aspects of D. hansenii using both basic and applied approaches. We have studied possible interconnections between the responses to salt and oxidative stress at molecular, enzymatic and metabolic levels. Our results show that expression of genes related to oxidative stress was induced by NaCl and KCl. On the other hand transcription of genes related to osmotic/salt stress was induced by H2O2. These effects have been also demonstrated at the enzymatic level since salt stress induced the activity of antioxidants defenses such as catalase and glutathione reductase. Moreover, at metabolic level both stresses induced an increase in intracellular ROS, and H2O2 exposure influenced intracellular sodium accumulation. Also, we have addressed the controversy over whether D. hansenii is halotolerant or halophilic from a new approach since, as far as we know, controlled growth in bioreactors has been used to study the physiology of this yeast. Our results have shown that the presence of NaCl or KCl improved the growth rate of the yeast since it increased total biomass or CO2 production. In fact, under limiting glucose content, the presence of NaCl produced a higher amount of biomass than the presence of KCl. All together, these results support the halophilic behavior of this yeast. We did not detect significant amounts of ethanol under any of the different conditions used in this work, suggesting a fully respiratory metabolism in D. hansenii. Finally we have used a D. hansenii wild terroir (Valle de los Pedroches) strain to inoculate the surface of Iberian pork loins in order to study its effect on the physicochemical characteristics in the product. Under all the conditions tested, this yeast was able to grow on the surface of the pork loin modifying its characteristics. All inoculated samples showed higher water activity and pH, and less sodium content after the three months ripening period. The yeast did not change the fatty acid profile but it affected the level of volatile and aromatics compounds. Finally, the acceptability tests indicated a tendency to a better consumer acceptance of the pork loin specifically inoculated with the yeast

Debaryomyces hansenii Is a Real Tool to Improve a Diversity of Characteristics in Sausages and Dry-Meat Products

Debaryomyces hansenii yeast represents a promising target for basic and applied biotechnological research It is known that D. hansenii is abundant in sausages and dry-meat products, but information regarding its contribution to their characteristics is blurry and contradictory. The main goal in this review was to define the biological contribution of D. hansenii to the final features of these products. Depending on multiple factors, D. hansenii may affect diverse physicochemical characteristics of meat products. However, there is general agreement about the significant generation of volatile and aromatic compounds caused by the metabolic activities of this yeast, which consequently provide a tendency for improved consumer acceptance. We also summarize current evidence highlighting that it is not possible to predict what the results would be after the inoculation of a meat product with a selected D. hansenii strain without a pivotal previous study. The use of D. hansenii as a biocontrol agent and to manufacture new meat products by decreasing preservatives are examples of exploring research lines that will complement current knowledge and contribute to prepare new and more ecological products

Candida albicans Potassium Transporters

Potassium is basic for life. All living organisms require high amounts of intracellular potassium, which fulfils multiple functions. To reach efficient potassium homeostasis, eukaryotic cells have developed a complex and tightly regulated system of transporters present both in the plasma membrane and in the membranes of internal organelles that allow correct intracellular potassium content and distribution. We review the information available on the pathogenic yeast Candida albicans. While some of the plasma membrane potassium transporters are relatively well known and experimental data about their nature, function or regulation have been published, in the case of most of the transporters present in intracellular membranes, their existence and even function have just been deduced because of their homology with those present in other yeasts, such as Saccharomyces cerevisiae. Finally, we analyse the possible links between pathogenicity and potassium homeostasis. We comment on the possibility of using some of these transporters as tentative targets in the search for new antifungal drugs

Use of room temperature ionic liquids for the selective fractionation of bioactive ketoses from aldoses

This work deals with the effective fractionation of bioactive ketoses, i.e. lactulose and tagatose, from their corresponding aldoses, lactose and galactose, in equimolar binary mixtures driven by room temperature ionic liquids, i.e. 1-ethyl-3-methylimidazolium dicyanamide ([EMIM][DCA]) and 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO4]), respectively. Under assayed conditions, tagatose was found to be 6-fold more soluble on [BMIM][MeSO4] than galactose; meanwhile lactulose was 3 times more soluble than lactose on [EMIM][DCA]. As an application example in a more complex sample, a lactose isomerization mixture containing in addition lactulose and monosaccharides was enriched in this ketose by using [EMIM][DCA]. Carbohydrates were then successfully recovered from the ionic liquid following an activated charcoal-based treatment. Overall, lactulose content was enriched from a 24% in the initial isomerization reaction mixture to a 62% in the purified sample. These experimental results demonstrated the potential of ionic liquids as green alternative solvents for the selective fractionation of bioactive ketoses from their corresponding aldoses in food and beverage production.Authors thank Ministerio de Ciencia e Innovación (projects AGL2009-11909 and AGL2011-27884), Ministerio de Economía y Competitividad (project CTQ2012-32957) and the European founding from FEDER program for financial support. L. Ruiz-Aceituno is supported by CSIC by a JAE-Pre grant, co-financed by the European Social Fund (ESF). C. Carrero-Carralero thanks Ministerio de Ciencia e Innovación (Spain) for a predoctoral contract.Peer Reviewe

Engineered axon tracts within tubular biohybrid scaffolds

Injuries to the nervous system that involve the disruption of axonal pathways are devastating to the individual and require specific tissue engineering strategies. Here we analyse a cells-biomaterials strategy to overcome the obstacles limiting axon regenerationin vivo, based on the combination of a hyaluronic acid (HA) single-channel tubular conduit filled with poly-L-lactide acid (PLA) fibres in its lumen, with pre-cultured Schwann cells (SCs) as cells supportive of axon extension. The HA conduit and PLA fibres sustain the proliferation of SC, which enhance axon growth acting as a feeder layer and growth factor pumps. The parallel unidirectional ensemble formed by PLA fibres and SC tries to recapitulate the directional features of axonal pathways in the nervous system. A dorsal root ganglion (DRG) explant is planted on one of the conduit's ends to follow axon outgrowth from the DRG. After a 21 d co-culture of the DRG + SC-seeded conduit ensemble, we analyse the axonal extension throughout the conduit by scanning, transmission electronic and confocal microscopy, in order to study the features of SC and the grown axons and their association. The separate effects of SC and PLA fibres on the axon growth are also experimentally addressed. The biohybrid thus produced may be considered a synthetic axonal pathway, and the results could be of use in strategies for the regeneration of axonal tracts

Consequences of the Lack of IL-10 in Different Endotoxin Effects and its Relationship With Glucocorticoids

Sepsis constitutes one of the major causes of death in ICUs. In sepsis induced by gram-negative, although lipopolysaccharide (LPS) initially induces an exacerbated secretion of proinflammatory cytokines leading to endotoxic shock and death resembling a septic shock, it is also capable of inducing refractoriness to subsequent challenge with LPS, a state known as endotoxin tolerance, which is considered the initial step of the immunosuppression found in septic patients. As we previously demonstrated the importance of glucocorticoids in endotoxin tolerance, the aim of this study was to evaluate the contribution of Interleukin-10 (IL-10) both in the endotoxic shock and in the development of the tolerance and its relationship with glucocorticoids. Our results show that, upon LPS challenge, IL-10 knockout mice (KO) mice had an enhanced LPS sensitivity, along with elevated levels of proinflammatory cytokines as tumor necrosis factor-α, IL-12 and interferon-γ, and enhanced tissue damage, despite the high levels of glucocorticoids. This effect may be because, in part, of the higher expression of tumor necrosis factor receptors in IL-10 KO mice. Further, the injection of dexamethasone did not protect IL-10 KO mice from a LPS lethal challenge. Although tolerance was achieved in the absence of IL-10, it was weaker and the elevated levels of glucocorticoids were not able to reverse the high sensitivity of IL-10 KO mice to LPS. Nevertheless, glucocorticoids would play a pivotal role in the establishment and maintenance of this partial tolerance in IL-10 KO mice. Finally, our results show that IL-10 and glucocorticoids could act in a bidirectional way influencing the inflammatory and anti-inflammatory periods.Fil: Córdoba Moreno, Marlina Olyissa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Todero, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fontanals, Adriana Mirta. Fundación Instituto Leloir; ArgentinaFil: Pineda, Gonzalo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Montagna, Daniela Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Yokobori, Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ramos, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Barrientos, Gabriela Laura. Hospital Alemán. Laboratorio de Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Toblli, Jorge Eduardo. Hospital Alemán. Laboratorio de Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Isturiz, Martín Amadeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rearte, María Bárbara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Spatiotemporal genetic structure in the Daphnia pulex complex from Sierra Nevada lakes (Spain): reproductive mode and first record of North American D. cf. pulex in European alpine lakes

Daphnia is a good model organism for studying factors affecting dispersal and patterns of genetic diversity. Within this genus, the Daphnia pulex species complex includes lineages from North America and Europe, with some considered invaders in various continents, although their colonization history is poorly known. We used mitochondrial DNA and microsatellite markers to identify the D. pulex complex lineages in Sierra Nevada, determine their reproductive mode and reconstruct their genetic history (over the past ∼25 to 65 years). We present the first recording of North American (NA) D. cf. pulex in a European high-mountain lake, showing its arrival ∼65 years ago in lake Borreguil without temporal changes in its genetic structure. European (Eu) D. cf. pulicaria is the only lineage present in other Sierra Nevada lakes and also showed no genetic change over time. The results for both species are congruent with obligate parthenogenetic reproduction mode. Moreover, water mineralization may influence the clonal distribution of the D. pulex complex in Sierra Nevada, without ruling out dispersal limitation and/or founder effects. Although NA D. cf. pulex had not spread to other Sierra Nevada lakes, it could threaten Eu D. cf. pulicaria in Sierra Nevada and other European alpine lakes

Design of the Refurbishment of Historic Buildings with a Cost-Optimal Methodology: A Case Study

The transformation of existing buildings into Near Zero Energy Buildings or even positive energy buildings remains a major challenge. In particular, historic buildings are an important cultural heritage that, in most cases, may be rehabilitated and reused for new purposes. However, achieving higher e ciencies in those buildings presents many di culties, since there is a need to preserve aesthetic values and minimize impact on the buildings’ initial construction. In this work, a roadmap that allows rehabilitating a building from the eighteenth century is developed, turning it into a landmark building, to be used as a museum in the near future. The procedure is based on 3D models using REVIT software and detailed energy simulations supported by a cost-optimal methodology. The results reveal how conventional methodologies shown in the literature may improve the energy performance of the buildings during the heating regime, but performance may deteriorate during the cooling season. For that reason, the present study includes the design of a night ventilation system which allows not only solving this problem but also to reducing the cooling demands by more than 43% with little additional costs. In conclusion, historic buildings (which traditionally have a high thermal mass) have increased thermal storage potential by using the structures of the buildings themselves as well as passive cooling techniques

Diferencias de género en la imagen corporal y su importancia en el control de peso

Adolescence is the most vulnerable stage for the emergence of body image issues. This study examined the differences between boys and girls (ranging from 13 to 18 years old) in Body Mass Index (BMI), perception of and satisfaction with their body image, and dieting to lose weight. The results show that girls, although being less overweight and obese than boys, were more dissatisfied with their body image, had a greater perception of obesity, and tended to diet to control their weight with greater frequency. In both groups, body image perception was more relevant than the adjusted bodyweight calculated by BMI in predicting behaviour regarding dieting to lose weight. Furthermore, body image satisfaction was also more relevant than BMI in predicting dieting in girls. The results highlight the importance of taking into account adolescents’ perceptions of their body image when designing, developing, and implementing intervention programs aimed at the prevention and treatment of eating disorders. Moreover, the gender differences found imply the need to take into account the differing perceptions of boys and girls to improve the likelihood of success of interventions.La adolescencia es la etapa más vulnerable para la aparición de problemas relacionados con la imagen corporal. Este estudio examinó las diferencias entre chicos y chicas adolescentes (13-18 años) en el Índice de Masa Corporal (IMC), la percepción y satisfacción con la imagen corporal, y hacer dieta para perder peso. Los resultados muestran que las chicas, a pesar de presentar menor sobrepeso y obesidad que los chicos varones, estaban más insatisfechas con su imagen corporal, se percibían más gordas y tendían a realizar dietas para controlar su peso con mayor frecuencia que los chicos. Por otro lado, tanto en chicos como en chicas, la percepción corporal tomó mayor importancia a la hora de predecir la conducta de hacer dieta que la adecuación del peso en base al IMC. Además, en las chicas, la satisfacción corporal se colocaba también por delante del IMC en dicha predicción. Los resultados de este estudio ponen de relieve la importancia de tener en cuenta las percepciones de los adolescentes sobre su imagen corporal a la hora de diseñar, desarrollar e implantar programas de intervención destinados a la prevención y tratamiento de trastornos alimenticios. Así mismo, las diferencias de género encontradas, apuntan a la necesidad de tener en cuenta las distintas percepciones de chicas y chicos para aumentar las probabilidades de éxito de las intervenciones

Gender differences in body image and its relevance in body weight control

La adolescencia es la etapa más vulnerable para la aparición de problemas relacionados con la imagen corporal. Este estudio examinó las diferencias entre chicos y chicas adolescentes (13-18 años) en el Índice de Masa Corporal (IMC), la percepción y satisfacción con la imagen corporal, y hacer dieta para perder peso. Los resultados muestran que las chicas, a pesar de presentar menor sobrepeso y obesidad que los chicos varones, estaban más insatisfechas con su imagen corporal, se percibían más gordas y tendían a realizar dietas para controlar su peso con mayor frecuencia que los chicos. Por otro lado, tanto en chicos como en chicas, la percepción corporal tomó mayor importancia a la hora de predecir la conducta de hacer dieta que la adecuación del peso en base al IMC. Además, en las chicas, la satisfacción corporal se colocaba también por delante del IMC en dicha predicción. Los resultados de este estudio ponen de relieve la importancia de tener en cuenta las percepciones de los adolescentes sobre su imagen corporal a la hora de diseñar, desarrollar e implantar programas de intervención destinados a la prevención y tratamiento de trastornos alimenticios. Así mismo, las diferencias de género encontradas, apuntan a la necesidad de tener en cuenta las distintas percepciones de chicas y chicos para aumentar las probabilidades de éxito de las intervenciones.Adolescence is the most vulnerable stage for the emergence of body image issues. This study examined the differences between boys and girls (ranging from 13 to 18 years old) in Body Mass Index (BMI), perception of and satisfaction with their body image, and dieting to lose weight. The results show that girls, although being less overweight and obese than boys, were more dissatisfied with their body image, had a greater perception of obesity, and tended to diet to control their weight with greater frequency. In both groups, body image perception was more relevant than the adjusted bodyweight calculated by BMI in predicting behaviour regarding dieting to lose weight. Furthermore, body image satisfaction was also more relevant than BMI in predicting dieting in girls. The results highlight the importance of taking into account adolescents' perceptions of their body image when designing, developing, and implementing intervention programs aimed at the prevention and treatment of eating disorders. Moreover, the gender differences found imply the need to take into account the differing perceptions of boys and girls to improve the likelihood of success of interventions