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Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de La Rioja -- 2019-02-26, One of the major problems of the wine industry in warm climate countries is the increasing alcohol content in wines, experienced during the last decades; which is the result of increasing sugar content in grapes at harvest time. This problem is mainly related with global climate change, but it is also connected to the changing preferences of consumers for full-bodied wines and strong aroma. However, due to health and road safety considerations, as well as to tax policies in some importing countries, the market is also demanding for wines with lower ethanol content. There are many points in the vine growing and winemaking workflow that can be targeted to reduce the alcohol content of the final wine. In this thesis I focused on the fermentation step, in which sugars are converted into ethanol by the activity of yeasts, mainly Saccharomyces cerevisiae. Previous work in this research group centered on respiration as the most promising yeast metabolic pathway that would have to be increased in order to divert carbon flow from ethanol production. Considering the Crabtree features of this yeast species, the use of non-Saccharomyces species was required. One major problem found to implement this approach at the industrial level was acetic acid production by S. cerevisiae, which is greatly enhanced in the presence of oxygen. This does not only affect wine fermentations inoculated with S. cerevisiae, since some S. cerevisiae cells will be naturally present, and tend to dominate the process. This would result in increased volatile acidity for aerated fermentations, whether they have been inoculated with S. cerevisiae or with non-Saccharomyces starters.

Trabajo presentado en Congreso Nacional de Biotecnología (BIOTEC 2019), celebrado en Vigo (España), del 10 al 13 de junio de 2019, Excessive acetic acid production is a key problem in winemaking processes aiming to reduce ethanol content of wine (1). It is related to a boost in acetate yields observed for Saccharomyces cerevisiae and some other yeasts under aerobic fermentation conditions. However, the biochemical mechanisms underlying this problem are unclear. We therefore used a genomic-scale modelling approach, combined with experimentally determined uptake/secretion rates, to gain insights into the metabolic rewiring of S. cerevisiae in response to oxygen under conditions relevant for winemaking. The model was also used to identify gene deletion targets affecting the production of acetic acid under aerobic conditions. Several gene knock-out with expected reduction in acetic acid production were thus identified as targets using MoMa and MiMBl algorithms. The predictions were tested using strains from the prototrophic yeast knockout collection for the fermentation of natural grape must under aerobic conditions. Although many of the prediction did not show any reduction in acetic acid yield, and increased yield was observed in some cases, significant acetate reduction was obtained for three gene deletion strains, ald6¿, mdh3¿ and rip1¿. The involvement of ALD6 on aerobic acetic acid production could be expected from results by other authors. However, the results for MDH3 and RIP1 could not be easily anticipated and thus provide new insights into yeast metabolism concerning acetic acid production, a major concern for the wine industry., We acknowledge funding by MINECO (AGL2015-63629-R and PhD training contract for AJR.

One of the major problems of the wine industry in warm climate countries is the increasing alcohol content in wines, experienced during the last decades; which is the result of increasing sugar content in grapes at harvest time. This problem is mainly related with global climate change, but it is also connected to the changing preferences of consumers for full-bodied wines and strong aroma. However, due to health and road safety considerations, as well as to tax policies in some importing countries, the market is also demanding for wines with lower ethanol content. There are many points in the vine growing and winemaking workflow that can be targeted to reduce the alcohol content of the final wine. In this thesis I focused on the fermentation step, in which sugars are converted into ethanol by the activity of yeasts, mainly Saccharomyces cerevisiae. Previous work in this research group centered on respiration as the most promising yeast metabolic pathway that would have to be increased in order to divert carbon flow from ethanol production. Considering the Crabtree features of this yeast species, the use of non-Saccharomyces species was required. One major problem found to implement this approach at the industrial level was acetic acid production by S. cerevisiae, which is greatly enhanced in the presence of oxygen. Since this species is usually present in grape must, even in the absence of inoculation, and tend to dominate after some hours of fermentation, providing oxygen to natural grape juice brings about the risk of excess volatile acidity of the final wines, which would preclude commercialization despite any eventual success concerning alcohol reduction. In order to advance in the development of efficient yeast strains and fermentation procedures aiming to alcohol reduction in wine, while avoiding the drawbacks related with acetic acid production, my PhD work targeted both S. cerevisiae and non-Saccharomyces species. The focus of my work on non-Saccharomyces yeast strains was on understanding the physiology of aerobic growth on grape must for these species, including factors that affect alcohol and acetate yields, and the impact of these growth conditions at the transcriptome level. In the case of S. cerevisiae, I tried to understand by a combination of computational biology and genetic engineering approaches the genetic determinants of excess acetate production when cultures are aerated. The results obtained indicate that environmental factors that can be easily manipulated during wine fermentation have a huge impact on the yields of acetic acid and alcohol for all yeast species tested. In addition, I was able to identify several genes whose deletion results in reducing the problem of acetic acid production by aerated cultures of S. cerevisiae. These results will serve to guide the development of fermentation procedures using some non-Saccharomyces species, aiming to alcohol level reduction by respiration. On the other side, the information is serving to develop non-GMO S. cerevisiae derivatives that are improved for acetic acid production (reduced yield) and can be combined with non-Saccharomyces yeasts during the aerated step or used as pure cultures for alcohol level reduction. Uno de los principales problemas de la industria enológica en países de clima cálido es el incremento que se ha producido en las últimas décadas en el contenido alcohólico de los vinos, que deriva a su vez del mayor contenido en azúcares de las uvas en el momento de la vendimia. Este problema está principalmente relacionado con el cambio climático global, pero también con el cambio en las preferencias de los consumidores hacia vinos con mayor cuerpo y potencial aromático. Sin embargo, teniendo en cuenta consideraciones de salud y seguridad vial, así como las políticas de impuestos sobre bebidas alcohólicas en algunos países importadores, el mercado está demandando a su vez vinos con menor contenido alcohólico. Hay varios puntos de la cadena de valor viña-vino que pueden ser objeto de mejora ara reducir el contenido alcohólico de los vinos. En esta tesis me he concentrado sobre la etapa de fermentación, en la cual los azúcares son transformados en etanol debido a la actividad de las levaduras, especialmente Saccharomyces cerevisiae. El trabajo previo en este grupo de investigación identificó la respiración como la vía metabólica más prometedora que se podría fomentar para desviar el flujo de carbono de la producción de etanol. Teniendo en cuenta las características Crabtree de esta especie de levadura, este objetivo requiere el uso de levaduras no-Saccharomyces. Uno de los principales problemas encontrados para implementar esta estrategia a escala industrial fue la producción de ácido acético por parte de S. cerevisiae, que se incrementa en gran manera en presencia de oxígeno. Dado que esta especie está presente en el mosto en prácticamente todas las ocasiones, incluso aunque no se inocule, y tiende a dominar el proceso al cabo de algunas horas de fermentación, proporcionar oxígeno al mosto natural, para favorecer la respiración de las levaduras non-Saccharomyces, casi siempre acarrea un riesgo de dar lugar a un exceso de acidez volátil que impediría la comercialización del vino, a pesar de la reducción de alcohol. Para avanzar en el desarrollo de levaduras y procesos de fermentación que permitan reducir el grado alcohólico, pero evitando el inconveniente del ácido acético, mi tesis trata tanto sobre S. cerevisiae como sobre otras especies de levaduras. En el caso de levaduras no-Saccharomyces he tratado de comprender su fisiología en mosto, en condiciones aeróbicas, incluyendo los factores que afectan al rendimiento en alcohol y acetato, y el impacto de estas condiciones sobre el transcriptoma. Para S. cerevisiae he utilizado una combinación de biología computacional e ingeniería genética para identificar los factores genéticos que influyen en la producción de acetato en cultivos aireados. Los resultados de la tesis indican que hay factores ambientales, fácilmente controlables en enología, que pueden tener un gran impacto sobre los rendimientos de alcohol y ácido acético para todas las especies de levaduras analizadas. Además, he podido identificar varios genes cuya deleción permite reducir el problema del ácido acético en cultivos aireados de S. cerevisiae. Mis resultados pueden servir de guía para el desarrollo de procesos fermentativos con levaduras no-Saccharomyces que permitan reducir el contenido alcohólico del vino. Por otro lado, esta información está sirviendo para el desarrollo de cepas no recombinantes de S. cerevisiae mejoradas respecto al problema del exceso de acético en aerobiosis que se podrían utilizar solas o en combinación con otras especies de levaduras para reducir el grado alcohólico.

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de La Rioja -- 2019-02-26 One of the major problems of the wine industry in warm climate countries is the increasing alcohol content in wines, experienced during the last decades; which is the result of increasing sugar content in grapes at harvest time. This problem is mainly related with global climate change, but it is also connected to the changing preferences of consumers for full-bodied wines and strong aroma. However, due to health and road safety considerations, as well as to tax policies in some importing countries, the market is also demanding for wines with lower ethanol content. There are many points in the vine growing and winemaking workflow that can be targeted to reduce the alcohol content of the final wine. In this thesis I focused on the fermentation step, in which sugars are converted into ethanol by the activity of yeasts, mainly Saccharomyces cerevisiae. Previous work in this research group centered on respiration as the most promising yeast metabolic pathway that would have to be increased in order to divert carbon flow from ethanol production. Considering the Crabtree features of this yeast species, the use of non-Saccharomyces species was required. One major problem found to implement this approach at the industrial level was acetic acid production by S. cerevisiae, which is greatly enhanced in the presence of oxygen. This does not only affect wine fermentations inoculated with S. cerevisiae, since some S. cerevisiae cells will be naturally present, and tend to dominate the process. This would result in increased volatile acidity for aerated fermentations, whether they have been inoculated with S. cerevisiae or with non-Saccharomyces starters.

Trabajo presentado en la 7th Conference on Physiology of Yeast and Filamentous, celebrado en Milán (Italia), del 24 al 27 de junio de 2019, Excess acetate yield under aerobiosis is a major drawback for S. cerevisiae and some other yeast species to be used in winemaking processes aiming to reduce ethanol content of wine (1). Yet, the biochemical mechanisms underlying the observed excess acetate production under aerobic conditions are unclear. We therefore used a genomic-scale modelling approach, combined with experimentally determined uptake/secretion rates, to gain insights into the metabolic reorganization in S. cerevisiae in response to oxygen under conditions relevant for winemaking. The model was also used to identify gene deletion targets towards altering the production of acetic acid in the presence of oxygen. Several gene knock-out targets with expected reduction in acetic acid production were thus obtained using for MoMa and MiMBl algorithms. The model predictions were tested in the fermentation of natural grape must under aerobic conditions with strains from the prototrophic yeast knockout collection. Although many of the prediction did not show any reduction in acetic acid yield, and increased yield was observed in some cases, acetate reduction was obtained for three gene deletion strains, ald6¿, mdh3¿ and rip1¿. The involvement of ALD6 on aerobic acetic acid production could be expected from published results. The results for MDH3 and RIP1 could not be easily anticipated and thus provide new insights into yeast metabolism concerning acetate production., We acknowledge funding by MINECO (AGL2015-63629-R and PhD training contract for AJR.

Durante los últimos años se menciona a menudo el incremento del grado alcohólico de los vinos, sobre todo en las regiones vitivinícolas de clima cálido, como un problema para la comercialización y la exportación. Este aumento se debe, obviamente, al creciente contenido en azúcar de la uva en el momento de la vendimia. Y a su vez, éste último tiene dos causas principales, el cambio climático, y la demanda de los consumidores por vinos bien estructurados y de alta expresión. El grupo Microwine, del Instituto de Ciencias de la Vid y del Vino (ICVV), trata de mejorar el conocimiento sobre la fisiología de las levaduras enológicas, y utilizarlo para conseguir reducir el grado alcohólico. El concepto clave es “reducir el rendimiento alcohólico”. Es decir, conseguir que las levaduras produzcan menos etanol por cada gramo de azúcar consumido. De modo que, en el caso de la mayor parte de los estilos de vinificación, en los que el azúcar del mosto debe ser consumido en su práctica totalidad, de conseguiría un vino igualmente “seco”, pero con un contenido menor de alcohol., El trabajo en el grupo MICROWINE en esta línea ha sido financiado por proyectos y contratos de formación del Ministerio de Ciencia Innovación y Universidades que se detallan a continuación: AGL2012-32064, RTC2014-2186-2, AGL2015-63629-R, y PCI2018-092949 (CoolWine: ERACoBiotech), así como los contratos predoctoral de Alda J. Rodrigues, y postdoctoral de José A. Curiel. J. Tronchoni está actualmente financiado por el programa COMFUTURO de la FGCSIC.

Trabajo presentado en el XIV Congreso Nacional de Investigación Enológica (GIENOL 2018), celebrado en Ciudad Real (España), del 26 al 29 de junio de 2018

Trabajo presentado en el 34th International Specialized Symposium on Yeasts (ISSY34), celebrado en Bariloche (Argentina), del 1 al 4 de octubre de 2018

Trabajo presentado en el International Congress of Grapevine and Wine Sciences, celebrado en Logroño (España), del 7 al 9 de noviembre de 2018, Recent reports indicate [GAR+] prion-like elements could influence ethanol yield during wine fermentation. These prion-like elements partially relieve carbon catabolite repression, and increased respiration was one the proposed mechanisms. However, supporting data are not truly consistent (1,2). We have obtained and characterized [GAR+] derivatives from wine yeasts with different genetic backgrounds. The characterization included carbon catabolite repression, different factors affecting prion state appearance and stability, and the physiological impact in terms of growth and metabolic yields under aerobic and anaerobic fermentation conditions with natural grape must. Our thorough characterization of different yeasts strains shows clearly different frequencies of prion apparition, and prion stability in desiccation experiments; highlighting the importance of the genetic background. However, concerning natural grape must fermentation, no advantage for [GAR+] strains was detected. For instance, in some prion strains, the [GAR+] phenotype is undistinguishable from that of the [gar-] version. However, for other strains, fermentations of natural grape must inoculated with [GAR+] yeast had a reduced fermentative performance in terms of residual sugar or CO2 release; while the impact on ethanol content was null or almost negligible. Our results do not support a practical use of [GAR+] in winemaking; and rule out prions as an explanation for sluggish or stuck fermentation., The financial support of MINECO trough grant AGL2015-63629-R, and PhD training contracts for AJR and AM, is acknowledged.

Trabajo presentado en el XXI Congreso Nacional de Microbiología de los Alimentos, celebrado en Tarragona (España), del 17 al 20 de septiembre de 2018, [GAR+] es un elemento epigenético de tipo prion que alivia la represión por catabolito en Saccharomyces cerevisiae. La represión por catabolito de carbono es un fenómeno común en muchos microorganismos por el cual la transcripción de los genes implicados en la utilización de fuentes de carbono alternativas está reprimida en presencia de algunos azúcares, fundamentalmente la glucosa y la fructosa ya que éstos son metabolizados con mejor eficiencia. Tras su descubrimiento, se propuso que este elemento podía tener relevancia tanto sobre la supervivencia de las levaduras enológicas y su eficiencia en la utilización de sustratos, como sobre el rendimiento alcohólico durante la fermentación. Sin embargo, otros resultados de los mismos grupos de investigación no parecen apoyar estas expectativas [1,2]. Asimismo, también se ha propuesto su implicación en paradas de fermentación., Este trabajo ha sido financiado por el Ministerio de Economía, Industria y Competitividad (AGL2015-63629-R; PhD AM, AJR; PostDoc JAC).

Trabajo presentado en el 10th In Vino Analytica Scientia Symposium, celebrado en Salamanca (España), del 17 al 20 de julio de 2017

Trabajo presentado en el 33rd International Specialised Symposium on Yeast (ISSY33), celebrado en Cork (Irlanda), del 26 al 29 de junio de 2017, Our group is interested in reducing ethanol yield during wine fermentation, to counter the growing sugar content in grapes due to climate change, and avoid the negative impact of increasing ethanol content in the wine market. While respiratory metabolism appears as an attractive alternative to pure fermentation, to redirect carbon flux towards sensory neutral products (CO2), both the Crabtree effect of Saccharomyces cerevisiae and the tendency to increased acetic acid production during aeration (as we have previously shown), constitute a hurdle to industrial application of respiration compatible fermentation conditions. However, mechanisms involved in acetic acid production by S. cerevisiae in the presence of oxygen are yet poorly understood. We addressed this topic by deletion of several genes, HXK2, REG1, PDE2, PDC1 (homozygous) and PYK1 (heterozygous) in the wine yeast strain FX0 (Laffort) background. Some of them had been previously related with the Crabtree effect. Our results show that oxygenation helps reducing ethanol content of wines. However, there is little impact of the deletions on this reduction. Interestingly, some of these deletions do results in a reduction in acetate yields during oxygenated fermentation of natural grape must., MINECO(AGL2015-63629-R, RTC-2014-2186-2) EU(7FP-IRSES-GA612441); Junta de Andalucía(P10-AGR6544)

Trabajo presentado en YeSVitE Final Conference (Yeasts for the Sustainability in Viticulture and Oenology), celebrado en Milán (Italia), el 4 de diciembre de 2017

Trabajo presentado en YeSVitE Final Conference (Yeasts for the Sustainability in Viticulture and Oenology), celebrada en Milán (Italia), el 4 de diciembre de 2017

Trabajo presentado en el Congreso Nacional de Biotecnología (BIOTEC), celebrado en Murcia (España), del 18 al 21 de junio de 2017., Saccharomyces cerevisiae is the main yeast species involved in winemaking. However, other yeast play a relevant role in this process, including, Hanseniaspora/Kloeckera, Pichia, Candida or Metschnikowia strains, the main species during the initial stages of spontaneous grape wine fermentation. Nowadays oenologists are trying to recover some of the fragrances of traditional wines by gathering together the advantages of microbiological control and those of the metabolic diversity of natural fermentation, by developing yeast starters from wine yeast species alternative to S. cerevisiae (collectively known as non-Saccharomyces in the field). These starters are used in combination with conventional S. cerevisiae starters to ensure complete sugar fermentation. Physiological and ecological interactions between the different yeast species and strains acquire a high relevance under this new scenario. In this context, our research group is exploring and exploiting the metabolic diversity of non-Saccharomyces wine yeasts to address one of the major current challenges of the winemaking industry, especially in warm climate wine producing regions, the steady increase in ethanol content of wines experienced over the last 30 years. This problem is related to global climate changes, as well as new wine stiles, both contributing to increasing sugar content of grapes at harvest. Our approach consists in using selected strains of, preferably, Crabtree negative yeast species, and controlled aeration during the first stages of fermentation, in order to favour sugar consumption by respiratory metabolism, rather than by fermentation. Final alcohol content of wine is reduced in the same percentage as that of sugar consumed by this pathway. Development of such alternative wine fermentation procedures requires a better understanding of factors affecting respiro-fermentative balance in different wine yeast species, the impact of environmental factors on acetic acid production (the major potential drawback of oxygenation), competition of different starters for scarce nutrients (nitrogen and vitamins), or chemical oxidation of quality related wine components. In addition, we are using transcriptomic approaches to elucidate some of the mechanisms underlying wine yeast inter-specific interactions. Our findings indicate that S. cerevisiae responds to the presence of yeasts belonging to other species by further activating its already quick sugar consumption pathways2 . In addition, nitrogen catabolite repression seems to be partially relieved (higher expression of genes under the control of Gln3p; see Figure 1), apparently helping balance sugar and nitrogen consumption for biomass production. The nitrogen utilization response of S. cerevisiae, is stronger for yeasts showing a closer phylogenetic relationship. At least in the case of Torulaspora delbrueckii, there is a reciprocal response in terms of the activation of glucose consuming pathways. However, this response is delayed, as compared to S. cerevisiae., This s work was partially supported by MINECO (AGL2015-63629-R, RTC-2014-2186- 2, JdC for JAC, FPI for AJR), EU (7FP-IRSES-GA612441).

The purpose of this paper is the dissemination of the Rock Art Virtual Corpus of the Northwest of Portugal (CVARN), a database dedicated to the theme of Post- Palaeolithic rock art in the Northwest of Portugal, which has been available online since the end of September 2014. This software tool is being built and developed under the ENARDAS project, a scientific project financed by the Thematic Factors of Competitiveness Operational Programme and by the European Regional Development Fund, and has been on-going since 2011, but is now open to the entire scientific community. Apart from being a first compilation of different “styles” of rock arts that occur between the western façade of Iberia, between the basins of the Minho and Vouga rivers, it will have both social and scientific functions. In social terms, the results obtained may contribute to regional development, especially regarding development from the perspective of tourism. In scientific terms, it will allow us to promote scientific research in this field of knowledge and to enable scientifically validated arguments for the integration of rock art sites into tourism evaluation projects., info:eu-repo/semantics/acceptedVersion

Trabajo presentado en el Simposio Internacional 2Las levaduras como bancos de prueba en las Ciencias de la Vida", celebrado en Madrid el 17 y 18 de octubre de 2016., Saccharomyces cerevisiae is the main yeast species involved in traditional or industrial fermentation of sugar rich substrates, including the production of fermented foods and bioethanol. However, other yeast, as well as bacteria, play a relevant role in these fermentation processes, especially fermented foods prepared with non-sterilized raw materials. For example, Hanseniaspora/Kloeckera, Pichia, Candida or Metschnikowia strains predominate during the initial stages of spontaneous grape wine fermentation. Some decades ago oenologists learned to master wine fermentation processes by using S. cerevisiae starter yeasts. Despite the gain in reliability of the fermentation process and overall quality of wines, the rather quick replacement of natural microbiota during the first stages of grape juice fermentation have leaded to a relative uniformity of the sensorial profile of commercial wines. Evidence has also been accumulated during the last twenty years on the positive contribution to wine quality of some of these indigenous yeast species during spontaneous fermentation. In this context, one of the late trends in wine microbiology is gathering together the advantages of microbiological control and those of the metabolic diversity of natural fermentation, by developing yeast starters from wine yeast species alternative to S. cerevisiae. In addition to their contribution to the aromatic profile, the metabolic features of some of these yeasts can eventually be managed to reduce alcohol content of wines through aerobic respiration. Industrial use of non-conventional yeasts and/or non-conventional fermentation conditions, including aerated fermentation (to allow for partial respiration of sugars) and multispecies starters (either in sequential or simultaneous inoculation), opens new questions whose answers will be interesting both from an academic or an applied perspective. This talk will deal with some of these topics, including the impact of oxygenation on the metabolism of some yeast species, beyond the direct impact on respiration; the relationship between fermentation under micro-oxygenated conditions and acetic acid production; or the early responses of yeast strains to the presence of potential competitors in the same culture medium.

Trabajo presentado en el Simposio Internacional 2Las levaduras como bancos de prueba en las Ciencias de la Vida", celebrado en Madrid el 17 y 18 de octubre de 2016. Saccharomyces cerevisiae is the main yeast species involved in traditional or industrial fermentation of sugar rich substrates, including the production of fermented foods and bioethanol. However, other yeast, as well as bacteria, play a relevant role in these fermentation processes, especially fermented foods prepared with non-sterilized raw materials. For example, Hanseniaspora/Kloeckera, Pichia, Candida or Metschnikowia strains predominate during the initial stages of spontaneous grape wine fermentation. Some decades ago oenologists learned to master wine fermentation processes by using S. cerevisiae starter yeasts. Despite the gain in reliability of the fermentation process and overall quality of wines, the rather quick replacement of natural microbiota during the first stages of grape juice fermentation have leaded to a relative uniformity of the sensorial profile of commercial wines. Evidence has also been accumulated during the last twenty years on the positive contribution to wine quality of some of these indigenous yeast species during spontaneous fermentation. In this context, one of the late trends in wine microbiology is gathering together the advantages of microbiological control and those of the metabolic diversity of natural fermentation, by developing yeast starters from wine yeast species alternative to S. cerevisiae. In addition to their contribution to the aromatic profile, the metabolic features of some of these yeasts can eventually be managed to reduce alcohol content of wines through aerobic respiration. Industrial use of non-conventional yeasts and/or non-conventional fermentation conditions, including aerated fermentation (to allow for partial respiration of sugars) and multispecies starters (either in sequential or simultaneous inoculation), opens new questions whose answers will be interesting both from an academic or an applied perspective. This talk will deal with some of these topics, including the impact of oxygenation on the metabolism of some yeast species, beyond the direct impact on respiration; the relationship between fermentation under micro-oxygenated conditions and acetic acid production; or the early responses of yeast strains to the presence of potential competitors in the same culture medium.

Additional file 3: Table S1. Primers used in this study.

Póster presentado en el XXV Congreso de la Sociedad Española de Microbiología (SEM), celebrado en Logroño del 7 al 10 de julio de 2015.

Trabajo presentado en la 3rd International Conference on Microbial Diversity (Microbial Diversity 2015. The challenge of Complexity), celebrada en Perugia del 27 al 29 de octubre de 2015., The steady increase in alcohol levels in wine is one of the main challenges faced by the oenological industry. High alcoholic strength might impair wine sensory quality, or discourage consumers, concerned about health or road safety. The problem is complex and is being addressed by researchers involved in all the stages of wine production (Teissedre, 2013). In this context, development of low alcohol yield Saccharomyces cerevisiae yeast strains has been a hot topic for several years (Cadiere et al. 2011; Varela et al. 2012; Tilloy et al. 2014), but modifying ethanol yields in this species has proven to be tough. The current interest of the wine industry on alternative yeasts, and the metabolic diversity they offer, open interesting alte rnatives to genetic modification. Our research group proposed using the respiratory metabolism of these alternative yeast species for reducing the alcohol content of wine (Gonzalez, et al. 2013). We have now addressed the impact of three easily manageable environmental factors (temperature, nitrogen source, and oxygen supply level) on the aerobic metabolism in synthetic must of some of these yeasts.

Póster presentado en el EMBO/EMBL Symposium (Enabling Technologies for Eukaryotic Synthetic Biology), celebrado en Heidelberg (Alemania) del 21 al 23 de junio de 2015.

Tese de doutoramento, Estudos de Literatura e de Cultura (Teoria da Literatura), Universidade de Lisboa, Faculdade de Letras, 2015 Submitted by Amelia Janeiro (ajaneiro@reitoria.ul.pt) on 2015-08-14T16:39:12Z No. of bitstreams: 1 ulsd071202_td_Alda_Rodrigues.pdf: 879526 bytes, checksum: c60c1ebf7b7593f1b0d429a4883b2545 (MD5) Made available in DSpace on 2015-08-18T16:52:08Z (GMT). No. of bitstreams: 1 ulsd071202_td_Alda_Rodrigues.pdf: 879526 bytes, checksum: c60c1ebf7b7593f1b0d429a4883b2545 (MD5) Previous issue date: 2015

Póster presentado en el XXV Congreso de la Sociedad Española de Microbiología (SEM), celebrado en Logroño del 7 al 10 de julio de 2015.

Rotavirus diarrhea is still an important cause of mortality in children worldwide, responsible each year for about 500,000 deaths. The introduction of the vaccine against the virus could lead to a reduction in morbidity and mortality associated with rotavirus. In Brazil, the Rotarix ® rotavirus vaccine, which contains the genotype G1P [8], was included in the national programme of immunization since March 2006. This project aims to evaluate clinical and epidemiological data related to infection by RVA, rotavirus genotypes prevalent. The research project is a cross-sectional study in which children with acute diarrhoea were recorded prospectively from 2010 to 2012. The location of collection of fecal samples was the Hospital of urgency of Sergipe in Pediatric Emergency sector, in Aracaju/SE. The clinical and epidemiological information was obtained through a questionnaire. The clinical severity was determined by a scoring system 20, calculated from the survey point. It was verified the episodes of acute diarrhea and stool samples collected for research and genotyping of rotavirus by ELISA and RT-PCR method.Descriptive statistical calculations were carried out to define the epidemiology of rotavirus. EIA positive results were found in 78 of the 790 samples. The most frequent genotype was G2 P [4], followed by the G8 genotype P [4], P [8] G1, G3 P [8], G1 P [6]. Mixed infections were detected G2 P [4] P [8], P [4] and G1G2 G2G8 P [4] and in general there was a cocirculação of different genotypes in the years studied, moreover, shows an alternation between the genotypes every year. The results obtained in this study observed a variability of positive cases distributed, confirming that the seasonality in the region is not remarkable. Therefore, new strains of rotavirus are emerging and associated with severe diarrhea. For this reason, monitoring is required to follow changes in the epidemiology of rotavirus. A diarreia por rotavírus ainda é uma importante causa de mortalidade infantil em todo o mundo, sendo responsável a cada ano por cerca de 500.000 mortes. A introdução da vacina contra o vírus pode levar a uma redução da morbidade e mortalidade associadas ao rotavírus. No Brasil, a Rotarix®, a vacina contra o rotavírus, que contém o genótipo G1P[8], foi incluído no programa nacional de imunizações em março de 2006. Este projeto teve como objetivo avaliar dados clínicos, epidemiológicos e genótipos predominantes relacionados à infecção por RV-A. O projeto de pesquisa é um estudo transversal em que as crianças com diarreia aguda foram registrados prospectivamente a partir de 2010 a 2012. O local de coleta das amostras fecais foi o Hospital de Urgência de Sergipe no setor de Urgência Pediátrica, em Aracaju/SE. A gravidade clínica foi determinada por um sistema de pontuação 20, calculado a partir do ponto de questionário. Foram verificados os episódios de diarreia aguda e coletadas amostras de fezes para pesquisa e genotipagem de rotavírus pelo método ELISA e RT-PCR. Cálculos estatísticos descritivos foram realizados para definir a epidemiologia do rotavírus. Resultados positivos foram encontrados em 78 das 790 amostras. O genótipo mais frequente foi G2P[4], seguido do genótipo G8P[4], G1P[8], G3P[8], G1P[6]. Foram detectadas infecções mistas G2 P[4] P[8], G1G2 P[4] e G2G8 P[4] e de um modo geral observou-se uma cocirculação de distintos genótipos nos anos estudados, além disso, nota-se uma alternância entre os genótipos a cada ano. O resultado obtido neste estudo observou uma variabilidade dos casos positivos distribuídos, confirmando que a sazonalidade na região não é marcante. Portanto, novas cepas de rotavírus estão surgindo e associados à diarreia grave. Por esta razão, a vigilância contínua é necessária para acompanhar as mudanças na epidemiologia do rotavírus.

Artigo bilingue, em português e inglês, Notice about the schemathic rock carving of Fieiral, in Northwestern Portugal., This work was develloped in the scope of the project Espaços naturais, arquiteturas, arte rupestre e deposições na pré-história recente da fachada ocidental do centro-norte português: das acções aos significados – ENARDAS / Natural spaces, architecture, rock art and depositions from the Late Prehistory of the Western front of Central and Northern Portugal: from actions to meanings (reference PTDC/HIS-ARQ/112983/2009) financed by the Operational Programme “Thematic Factors of Competitiveness” (COMPETE) and by the European Regional Development Fund (Fundo Europeu de Desenvolvimento Regional - FEDER)., info:eu-repo/semantics/publishedVersion

Tese de mestrado em Teoria da Literatura apresentada à Faculdade de Letras da Universidade de Lisboa, 2009 Nível de preservação: critical, Criado em: 2009-04-15 13:10:06, Criado por: super:ULB53, Alterado em: 2009-08-03 12:39:41, Modificado por: super:ULB59 Made available in DSpace on 2010-06-22T09:22:18Z (GMT). No. of bitstreams: 2 17304_MicrosoftWordTESEMESTRADO.pdf: 416038 bytes, checksum: 9b0e1a7f9af69c4c4e388ad6d58f9d65 (MD5) 17304.xml: 6703 bytes, checksum: a16b2915f55964c4f136599e99644850 (MD5) Previous issue date: 2008

Apesar das dificuldades interpretativas resultantes, em parte, da escassez de alguns dados ambientais, o cruzamento interdisciplinar entre o resultado dos ecofactos e vestígios arqueológicos permitiu-nos colocar a hipótese de que, a partir do Calcolítico, ocorrem comunidades com um sistema agro-silvo-pastoril estável, baseado no cultivo de cereais e de leguminosas, na exploração da floresta climácica e na criação de ovicaprinos. Esta situação parece manter-se durante as Idades do Bronze Inicial e Médio onde, a par da manutenção do cultivo do trigo de grão nu e da cevada, aparecem, pela primeira vez, o milho miúdo e as crucíferas, a indiciar uma maior especialização agrícola e uma intensificação desta práticas. Os resultados das colunas polínicas realizadas para o Noroeste Peninsular permitem as mesmas considerações. Apesar da amostra arqueozoológica ser reduzida, não deixa de ser curioso o facto de aparecerem, no Calcolítico e na Idade do Bronze, restos de cozinha maioritariamente compostos por ovicaprínos.Durante o Calcolítico denotam-se, também, evidências da caça ao cervo. Em termos do aproveitamento dos recursos do coberto vegetal, a recolecção de frutos é ainda uma actividade importante, assim como a exploração dos carvalhos e das fabáceas., Fundação para a Ciência e a Tecnologia (FCT)

Este trabalho dá a conhecer o lugar cerimonial de Vale Ferreiro, em Fafe, NW de Portugal, onde se depositaram corpos ao longo da Idade do Bronze em diferentes tipos de estruturas., O lugar onde se implantaram estas estruturas e deposições de âmbito funerário tem grande visibilidade sobre o meio, é atravessado por veios de quartzo e tem relação com águas termais que aí correm subterraneamente, características que não foram ignoradas pelos frequentadores deste local durante a Idade do Bronze, numa interrelação com o meio reveladora do grande simbolismo deste loci.