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Eukaryotic gene expression is regulated both at the transcription and the mRNA degradation levels. The implementation of functional genomics methods that allow the simultaneous measurement of transcription (TR) and degradation (DR) rates for thousands of mRNAs is a huge improvement in this field. One of the best established methods for mRNA stability determination is genomic run-on (GRO). It allows the measurement of DR, TR and mRNA levels during cell dynamic responses. Here, we offer a software package that provides improved algorithms for determination of mRNA stability during dynamic GRO experiments.

The model organism Saccharomyces cerevisiae has allowed the development of new functional genomics techniques devoted to the study of transcription in all its stages. With these techniques, it has been possible to find interesting new mechanisms to control gene expression that act at different levels and for different gene sets apart from the known cis-trans regulation in the transcription initiation step. Here we discuss a method developed in our laboratory, Genomic Run-On, and other new methods that have recently appeared with distinct technical features. A comparison between the datasets generated by them provides interesting genomic insights into the different layers of gene regulation in eukaryotes.

Thiolutin is commonly used as a general inhibitor of transcription in yeast. It has been used to calculate mRNA decay rates by stopping the transcription and then determining the relative abundance of individual mRNAs at different times after inhibition. We report here that thiolutin is also an inhibitor of mRNA degradation, and thus its use can lead to miscalculations of mRNA half-lives. The inhibition of mRNA decay seems to affect the mRNA degradation pathway without impeding poly(A) shortening, given that the decay rate of total poly(A) amount is not reduced by thiolutin. Moreover, the thiolutin-dependent inhibition of mRNA degradation has variable effects on different functional groups of genes, suggesting that they use various degradation pathways for their mRNAs.

Oenological traits, such as temperature profile and production of certain metabolites, were tested for four interspecifc hybrids obtained by"spore to spore" crossing between Saccharomyces cerevisiae and Saccharomyces uvarum strains and uniformity of their inheritance was found. PCR/RFLP analysis of ITS regions was carried out to confirm the hybrid nature of the strains. They showed an additive profile with five bands of the respective 325, 230, 170 and 125 bp. Finally gene expression study was performed by comparative DNA macroarray analysis of the hybrids and the preliminary results showed that the global gene expression patterns of hybrids are remarkably similar to one another. In conclusion, the data obtained by two different approaches, such as metabolic and transcriptomic strategies, suggest a large degree of homogeneity among interspecific hybrids between S. cerevisiae and S. uvarum. Moreover, the uniformity of F1 hybrids advises that the oenological trait inheritance mechanism is highly constant and reproducible.

HAT-B is a yeast histone acetyltransferase composed of Hat1, Hat2 and Hif1 proteins. We demonstrate that a hat2 mutant or a hat1hat2 double mutant, but not a hat1 mutant, have an extended life-span. Transcriptome analysis shows that the single hat mutants are not very different from wild type. However, the comparison of the hat1 and hat2 transcriptomes shows that they are different. The hat1hat2 double mutant shows a transcriptional phenotype similar to that of the hat1 mutant but strongly enhanced. These results indicate that Hat2p could have additional functions in the cell to those of Hat1p.

The Saccharomyces cerevisiae GRX5 gene codes for a mitochondrial glutaredoxin involved in the synthesis ofiron/sulfur clusters. Its absence prevents respiratory growth and causes the accumulation of iron inside cellsand constitutive oxidation of proteins. Null Δgrx5 mutants were used as an example of continuously oxidizedcells, as opposed to situations in which oxidative stress is instantaneously caused by addition of external oxidants.Whole transcriptome analysis was carried out in the mutant cells. The set of genes whose expression wasaffected by the absence of Grx5 does not significantly overlap with the set of genes affected in respiratorypetite mutants. Many Aft1-dependent genes involved in iron utilization that are up-regulated in a frataxin mutantwere also up-regulated in the absence of Grx5. BIO5 is another Aft1-dependent gene induced both upon irondeprivation and in Δgrx5 cells; this links iron and biotin metabolism. Other genes are specifically affected underthe oxidative conditions generated by the grx5 mutation. One of these is MLP1, which codes for a homologueof the Slt2 kinase. Cells lacking MLP1 and GRX5 are hypersensitive to oxidative stress caused by externalagents and exhibit increased protein oxidation in relation to single mutants. This in turn points to a role forMlp1 in protection against oxidative stress. The genes of the Hap4 regulon, which are involved in respiratorymetabolism, are down-regulated in Δgrx5 cells. This effect is suppressed by HAP4 overexpression. Inhibition ofrespiratory metabolism during continuous moderately oxidative conditions could be a protective response bythe cell.

Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus followed by mRNA export to the cytoplasm for translation. Here we report the identification of a protein, Sus1, which functions in both transcription and mRNA export. Sus1 is a nuclear protein with a concentration at the nuclear pores. Biochemical analyses show that Sus1 interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, and with the Sac3-Thp1 complex, which functions in mRNA export with specific nuclear pore proteins at the nuclear basket. DNA macroarray analysis revealed that Sus1 is required for transcription regulation. Moreover, chromatin immunoprecipitation showed that Sus1 is associated with the promoter of a SAGA-dependent gene during transcription activation. Finally, mRNA export is impaired in sus1 mutants. These data provide an unexpected connection between the SAGA histone acetylase complex and the mRNA export machinery.

In order to clarify their physiological functions, we have undertaken a characterization of the three-membered gene families SNZ13 and SNO13. In media lacking vitamin B6, SNZ1 and SNO1 were both required for growth in certain conditions, but neither SNZ2, SNZ3, SNO2 nor SNO3 were required. Copies 2 and 3 of the gene products have, in spite of their extremely close sequence similarity, slightly different functions in the cell. We have also found that copies 2 and 3 are activated by the lack of thiamine and that the Snz proteins physically interact with the thiamine biosynthesis Thi5 protein family. Whereas copy 1 is required for conditions in which B6 is essential for growth, copies 2 and 3 seem more related with B1 biosynthesis during the exponential phase.

Saccharomyces cerevisiae strains frequently exhibit rather specific phenotypic features needed for adaptation to a special environment. Wine yeast strains are able to ferment musts, for example, while other industrial or laboratory strains fail to do so. The genetic differences that characterize wine yeast strains are poorly understood, however. As a first search of genetic differences between wine and laboratory strains, we performed DNA-array analyses on the typical wine yeast strain T73 and the standard laboratory background in S288c. Our analysis shows that even under normal conditions, logarithmic growth in YPD medium, the two strains have expression patterns that differ significantly in more than 40 genes. Subsequent studies indicated that these differences correlate with small changes in promoter regions or variations in gene copy number. Blotting copy numbers vs. transcript levels produced patterns, which were specific for the individual strains and could be used for a characterization of unknown samples. Copyright © 2001 John Wiley & Sons, Ltd.

El factor de inicio de traducción de eucariotas 5A (eIF5A) es una proteína esencial y conservada con funciones en las tres fases de la traducción. eIF5A está codificado por dos genes parálogos, TIF51A/TIF51B y EIF5A1/EIF5A2 en levadura y humanos respectivamente. eIF5A es la única proteína que contiene hipusina, una modificación esencial para su actividad, y se une a los ribosomas para facilitar la traducción de motivos con prolinas consecutivas o combinaciones de prolina con glicina y aminoácidos cargados. eIF5A participa en otras funciones y procesos como exporte de mRNAs nucleares, proliferación y apoptosis. Su asociación con enfermades como el cáncer es de interés. En esta tesis se han utilizado los organismos modelo Saccharomyces cerevisiae y Mus Musculus para adquirir conocimiento fundamental de las funciones de eIF5A. Se han identificado dos nuevas dianas de eIF5A para la traducción, una función básica nueva del factor y las bases moleculares de la regulación transcripcional de los dos genes de eIF5A. Nuestros resultados con levadura y células de mamífero demostraron que eIF5A hipusinado es necesario para la síntesis de colágeno I ya que la traducción se para en motivos colagénicos, enriquecidos en prolina y glicina, en deficiencia de eIF5A. Su inactivación redujo el contenido en colágeno I y produjo la retención de colágeno parcialmente sintetizado en el retículo endoplásmico y estrés. La sobreproducción de colágeno I células humanas hepáticas estrelladas tratadas con la citoquina profibrótica TGF-β1 dependía de eIF5A también. En cuanto a la regulación transcripcional de eIF5A, se determinó que la expresión génica de las dos isoformas respondía a las demandas energéticas y dependía de TORC1 y Hap1. En condiciones de respiración, Hap1 activaba TIF51A por unión directa y reprimía TIF51B indirectamente. Por el contrario, cuando la respiración era comprometida, Hap1 se convertía en un represor para inhibir la expresión de TIF51A e inducir TIF51B. Nuestros resultados demostraron el papel esencial de la isoforma Tif51A en la respiración mitocondrial. Se descubrió un nuevo mecanismo que conecta la actividad de eIF5A con las funciones mitocondriales. Se observó que eIF5A es necesario para la traducción de la región de prolinas de Tim50, una proteína esencial que reconoce proteínas mitocondriales y media su importe a la mitocondria. La inactivación de eIF5A inhibía el importe de proteínas y generaba la agregación de precursores y estrés. Finalmente, se reveló una nueva función de eIF5A en la regulación transcripcional de genes cuyos mRNAs lo requieren para la traducción. Nuestros resultados demostraron su unión a la cromatina y su inactivación aumentaba la transcripción de genes que codifican proteínas con motivos dependientes de eIF5A, sugiriendo un efecto transcripcional represor sobre sus dianas. Así, eIF5A no solo funciona en traducción sino también en el control de la síntesis de mRNA para mantener la homeostasis de proteína de sus dianas. The eukaryotic translation initiation factor 5A (eIF5A) is an essential, evolutionarily conserved protein with functions in the three stages of translation. eIF5A is codified by two paralog genes, TIF51A/TIF51B and EIF5A1/EIF5A2 in yeast and human respectively. eIF5A is the only known protein containing hypusine, an essential modification for its activity. eIF5A binds ribosomes to facilitate translation of motifs with consecutive prolines or combinations of proline with glycine and charged amino acids. eIF5A has been linked to other molecular functions and cellular processes such as nuclear mRNA export, proliferation and apoptosis. Its association with the pathogenesis of several diseases including cancer is of interest. In this thesis we have used Saccharomyces cerevisiae and Mus Musculus to gain fundamental knowledge on the functions of eIF5A. Here, we identified and characterized two novel targets requiring eIF5A for translation, a new basic function for the factor and the molecular bases of the transcriptional regulation of the two eIF5A genes. Our results in yeast and mammalian cells proved that hypusinated eIF5A is needed for synthesis of mammalian collagen I as translation stalls at collagenic motifs, enriched in proline and glycine, under eIF5A deficiency. eIF5A inactivation reduced collagen I and led to the retention of partially synthesized collagen I in the endoplasmic reticulum and to stress. The collagen I overproduction in human hepatic stellate cells treated with the profibrotic cytokine TGF-β1 was also dependent on eIF5A. The gene expression regulation of the two isoforms responded to the cellular energy demands and was found to be dependent on TORC1 and Hap1. Under respiratory conditions, Hap1 induced TIF51A through direct binding while indirectly repressed TIF51B. Conversely, when the respiration was compromised, Hap1 became a repressor to down-regulate TIF51A expression and up-regulate TIF51B. Our results demonstrated the essential role of the Tif51A isoform in the mitochondrial respiration. A novel mechanism connecting eIF5A activity with mitochondria functions was revealed. We showed that eIF5A is required for the translation of the proline-rich region of Tim50, an essential protein that specifically recognizes mitochondrial proteins and mediates their import into mitochondria. eIF5A inactivation inhibited mitochondrial protein import and caused precursors to aggregate and stress. Finally, a novel function of eIF5A was uncovered. eIF5A was found to participate in the transcriptional regulation of genes whose mRNAs require eIF5A for translation. Our findings proved the eIF5A binding to chromatin and its absence increases the transcription of specific genes encoding proteins with eIF5A-dependent motifs, suggesting a transcriptional repressor effect on its targets. Thus, our results indicated that eIF5A functions not only in translation but also in the control of mRNA synthesis to maintain protein homeostasis of its targets.

Candida albicans es el patógeno humano más prevalente. A pesar de ser un organismo comensal, también es un hongo oportunista que puede causar infecciones de seria gravedad en personas inmunodeprimidas. Aunque los principales factores de virulencia han sido caracterizados, el proceso exacto de patogénesis en Candida todavía se desconoce. Los mecanismos de adaptación a los distintos ambientes a los que se enfrenta durante la invasión del cuerpo humano todavía no están suficientemente caracterizados. En este trabajo se usaron análisis genómicos funcionales para el estudio de la respuesta global de C. albicans a dos estreses que encuentra durante la invasión del cuerpo humano: estrés oxidativo e hipóxico. En el análisis se encontraron varios genes implicados en la adaptación a estos estreses, lo cual se vio reflejado en la regulación de sus niveles de RNA mensajero y proteína. Un hallazgo interesante de este estudio fue que la regulación de los genes implicados en la respuesta a estrés oxidativo ocurre principalmente a nivel post-transcripcional. Para estudiar la posible implicación de RNAs no codificantes en la respuesta a estos estreses, se creó una aplicación web que permitió la detección de aquellos ncRNAs cuya expresión se vio alterada durante el estrés. Dos grupos de 154 y 159 nRNAs, incluyendo intergénicos y antisentido a ORFs, se identificaron como implicados en la respuesta a estrés hipóxico y oxidativo, respectivamente. Para el estudio de cambios en transcripción naciente durante estos estreses, la técnica de Genomic run-on (GRO), ya establecida para el estudio del nascentoma de Saccharomyces cerevisiae, se implementó para su uso en C. albicans en su versión más actualizada, el BioGROseq. La técnica de GRO fue después usada para el estudio de cambios en parámetros de estudio de la expresión génica a distintas temperaturas de crecimiento, tales como la densidad de RNApol II o la tasa de traducción, usando el organismo modelo S. cerevisiae como herramienta. Los resultados mostraron que la densidad de RNApol II disminuye con el incremento en la temperatura de crecimiento, y que esta disminución en densidad se compensa con un incremento en la velocidad de elongación de la RNApol II para mantener la homeostasis de los niveles de mRNA. Esta compensación está regulada a nivel de iniciación de la RNApol. Dentro del rango óptimo de temperaturas de crecimiento en S. cerevisiae (26-34 oC), tanto los niveles de RNA total como los de mRNA se mantienen en homeostasis, disminuyendo marcadamente a temperaturas inferiores o superiores. Finalmente, con el aumento en la temperatura de crecimiento se observó un aumento en la tasa de traducción. En general, los resultados presentados en este trabajo proporcionan nuevos hallazgos sobre los mecanismos de regulación que las levaduras utilizan para su crecimiento a distintas temperaturas, y ayudan a entender cómo C. albicans sobrevive durante su invasión en el cuerpo humano. Candida albicans is the most prevalent human fungal pathogen. Despite being a harmless commensal organism, it is also an opportunistic fungus which can cause life-threatening infections in immunocompromised people. Although major virulence factors have been characterized, the exact mechanism of Candida pathogenesis still remains unknown. Mechanisms of adaptation under the different environments it faces during body invasion are yet poorly characterized. In this thesis, functional genomic analyses were utilized to study the global response of C. albicans to two environmental insults it faces during invasion of the human body: oxidative and hypoxic stress. Several genes were found to be involved in the adaptation to these stresses, as reflected by the up or down regulation of their mRNA and protein products. Interestingly, the expression of the genes involved in the response to oxidative stress was found to be regulated mainly at the post-transcriptional level. To explore the possible implication of non-coding RNAs in the response to these stresses, an online web application was created to enable the detection of ncRNAs which levels change under stress conditions. Two groups of 154 and 159 ncRNAs, including intergenic and antisense to ORFs, were found to be involved in the responses to hypoxia and oxidative stress respectively. To study changes in nascent transcription under these stresses, Genomic run-on (GRO), a well-established technique used for the study of the nascentome in Saccharomyces cerevisiae, was implemented for its use in C. albicans in its most up-to-date version, BioGROseq. The GRO technique was later used for the study of changes in gene expression parameters at different growth temperatures, such as RNApol II density or translation rate, using the model yeast S. cerevisiae as a tool. The results showed that RNA pol II density decreases as growth temperature increases, and that this decrease in density is compensated with an increase in RNA pol II speed to maintain mRNA homeostatic levels, compensation that is regulated at the RNA pol II initiation level. Within the optimal range of growth temperatures in S. cerevisiae (26-34 oC) both total RNA and mRNA amounts were found to be kept at homeostatic levels, decreasing markedly at temperatures above and below. Finally, an increase in translation rate was observed with the increase in growth temperatures. Overall, the results presented in this thesis provide clues into the regulatory mechanisms that yeast utilize to grow at different temperatures and contribute to the understanding of how C. albicans thrives during infection of the human body.

En eucariontes, el mRNA es transcrito en el núcleo, exportado hacia el citoplasma, traducido en proteínas y finalmente degradado. De esta manera, la expresión génica se describe como un sistema lineal. Un aspecto importante de la expresión génica consiste en mantener los niveles del mRNA, los cuales son el resultado de la interacción dinámica entre la tasa de transcripción y de degradación. Para mantener el equilibrio entre ambas tareas, es necesaria la existencia de mecanismos que comuniquen ambos procesos. Utilizando la levadura S. cerevisiae y mediante la técnica llamada Genomic Run On, estudié el recambio del mRNA.ElGenomic Run On permite medir a escala genómica la tasa de transcripción y la cantidad de mRNA maduro de todos los genes de S. cerevisiae simultáneamente. Eliminando factores que participan de la transcripción, analicé cómo se afecta la síntesis, la degradación y los niveles de mRNA maduro. Los resultados mostraron que cuando se afecta la transcripción, los niveles de mRNA se compensan mediante cambios en la estabilidad de los mensajeros. Similares resultados se obtuvieron eliminando factores envueltos en la degradación. Al afectarse la degradación se espera que ocurra una acumulación del mRNA no degradado, sin embargo, nuestros resultados mostraron que el nivel de mRNA maduro se mantiene dentro de ciertos niveles. Como consecuencia, la tasa de transcripción disminuyó, compensando esto con un aumento en la estabilidad del mRNA, pero no en su cantidad. Estos resultados indican que la regulación génica es un proceso circularmente regulado, en donde hay mecanismos aún no descritos que comunican los procesos de degradación y transcripción de manera cruzada. Finalmente, estudiamos el transcriptoma naciente a escala genómica de un conjunto de cepas de S. cerevisiae con cambios importantes en el volumen celular. Nuestros resultados indican que la transcripción del rRNA y tRNAs escalan con el incremento del volumen celular para mantener sus niveles constantes, mientras que la transcripción del mRNA no se ajusta a los cambios del volumen celular. Esto indica que la célula compensa la concentración de sus proteínas aumentando la transcripción de los componentes de la maquinaria de traducción.

Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica Hoje em dia, a procura de leveduras com elevada capacidade fermentativae que tenham impacto favorável sobre a qualidade do vinho, é um dos principais objetivos na indústria enológica.Nesta linha, o estudo do impacto do azoto na qualidade do vinho é de grande relevância, uma vez que é importante não só para assegurar o crescimento de leveduras e cinética fermentação adequados, mas também por poder afetar a produção dos principais metabolitos resultantes da fermentação dos açúcares.Neste trabalho, explorámos primeiro a diversidade fenotípica de 20 estirpes de Saccharomyces cerevisiae de origem geográfica diferente em termos de resistência a várias condições de “stress” normalmente encontradas durante a fermentação alcoólica, bem como do seu potencial para a produção de compostos aromáticos. Com o objetivo de avaliar como o azoto afeta, não apenas o crescimento e atividade fermentativa das leveduras, mas também a qualidade do produto final, oito estirpes selecionadas foram usados numa abordagem experimental fatorial (concentração de azoto x fonte de azoto x estirpe de levedura). Os resultados sugerem que o uso de diferentes fontes e concentrações de azoto resulta na produção de vinhos com perfis aromáticos divergentes, e altamente dependentes da estirpe utilizada. A determinação de metionina e a sua administração antes da fermentação são cruciais para suprimir a formação de H2S e permitir a obtenção de vinhos com diferentes perfis sensoriais. A diversidade exo‐metabólica encontrada entre as estirpes de levedura mostrou que o “pré‐screening” utilizado pode ser empregue na selecção da estirpe de levedura mais adequada a ser utilizada, dependendo tanto da composição em azoto do kosto, bem como do estilo de vinho pretendido. Com base nos dados obtidos relativamente aos requisitos em azoto, cinética de crescimento, desempenho fermentativo e perfis metabólicos, três estirpes distintas de S. cerevisiae foram selecionadas para análise funcional comparativa em duas condições de fermentação divergentes no que respeita à disponibilidade em azoto. A correlação entre expressão genéticae parâmetros fisiológicos encontrada neste estudo revelou que a maior expressão de genes de resposta ao “stress” está associada a uma alta capacidade fermentativa. Além disso, a descoberta de genes comuns correlacionados, tanto com a atividade de fermentação bem como com o consumo de azoto, sublinham o papel deste nutriente na capacidade fermentativa das leveduras. As enormes diferenças encontradas na expressão genéticadas três estirpes de leveduras ressaltam a importância de se considerarem leveduras com diferente “background” genético na procura de biomarcadores de deficiência de azoto. No entanto, parte dos resultados apresentados são uma previsão da classificação de resposta comum à limitação de azoto, o que facilitará o diagnóstico de deficiência deste nutriente nos mostos e no desenvolvimento de estratégias para otimizar o desempenho fermentativo de leveduras em fermentações industriais. A introdução de culturas “starter” de leveduras, consistindo numa mistura de estirpes de leveduras de S. cerevisiae e não‐Saccharomyces, está a emergir como uma opção interessante na indústria enológica mundial, com vista a satisfazer as exigências dos consumidores de vinhos com alta complexidade de sabor e distinção estilística. No entanto, não há dados sobre o impacto desta estratégia no metabolismo do azoto da principal levedura de fermentação, S. cerevisiae. Neste trabalho, pretendeu avaliar‐se o efeito do nível de azotodos mostos no comportamento fermentativo e metabólico de S. cerevisiae, tanto em cultura pura como em cultura mista com uma estirpe de Hanseniaspora guiliermondii. Os resultados mostraram claramente que a sua presença compromete o crescimento e desempenho fermentativo da estirpe de S. cerevisiae. Considerou‐seque a potencial interação levedura‐levedura poderia também ser revelada pela expressão diferencial de genes em ambos os tipos de fermentação. Várias alterações detectadas na expressão genéticade S. cerevisiae parecem responder a mudanças na disponibilidade de nutrientes no mosto, principalmente azoto e vitaminas, e que estão potencialmente associados à atividade metabólica de H. guilliermondii. Observou‐se que as diferenças na expressão de genes de S. cerevisiae envolvidos na formação de compostos aromáticos poderiam explicar os diferentes perfis de aroma obtidos em culturapura e mista. Estes resultados levantam a questão de perceber se o impacto de estirpes de não‐Saccharomycesno perfil aromático são resultado de uma contribuição direta com produção dos compostos e/ou de influência no comportamento metabólico da levedura S. cerevisiae. Em suma, e abrindo perspectivas interessantes para futuras pesquisas na biologia de levedura, os dados apresentados nesta tese fornecem informações importantes para uma compreensão mais profunda do papel do azoto na fermentação alcoólica, que podem ajudar os enólogos na obtenção de vinhos de alta qualidade. Nowadays, the pursuit for yeast strains displaying both high potential fitness to conduct alcoholic fermentations and favourable influence on the wine quality is one of the major goals in wine making industry.In this line, the study of the impact of nitrogen on wine quality is of major relevance since it is not only important for ensuring adequate yeast growth and fermentation kinetics, but also can affect the production of the major metabolites arising from sugar fermentation. In this work, we first explored the phenotypic diversity among 20 Saccharomyces cerevisiae strains from different geographic origin in terms of their resistance to several stress conditions usually found during alcoholic fermentation, as well as of their potential to produce aroma compounds. Aiming to assess how nitrogen affects not only yeast cells growth and fermentative activity, but also the quality of the final product, eight selected strains were used in a factorial design experimental approach (N concentration x N source x Yeast strain). Our results suggest that the use of different nitrogen sources and different nitrogen concentrations result in the production of wines with divergent aroma profiles. We found that methionine determination and its management prior to fermentation are crucial for suppressing H2S and for endowing beverages with diverse sensory traits. The exo‐metabolic diversity found among the yeast strains showed that the prescreening used can be employed inthe selection of the most suitable yeaststrainto be used depending on both grape‐juice nitrogen composition and wine style envisioned. Based on the data obtained regarding nitrogen requirements, growth kinetics, fermentative fitness, and metabolic profiles, three contrastingS. cerevisiae strains were selected for comparative functional analysis throughout two divergent fermentation conditions of nitrogen availability. The correlations of gene expression with the physiological parameters found in our study revealed that higher expression of stress responsive genes is associated with a high fermentative capacity. Also the finding of common genes correlated with both fermentation activity and nitrogen up‐takeunderlie the role of nitrogen on yeast fermentative fitness. The huge dissimilarities found in gene expression among the three yeast strains underscore the importance of considering different yeast strain backgrounds in the search for biomarkers of nitrogen deficiency. Nevertheless, the results presented are a preview on classification of common nitrogen limitation response, which will facilitate diagnosis of deficiency of this nutrient in the musts and the development of strategies to optimize yeast performance in industrial fermentations. The introduction of yeast starter cultures consisting in a blend of S. cerevisiae and non‐Saccharomyces yeast strains is emerging as an interesting option for production of wines complexity of flavour and stylistic distinction. However, there is no data regarding the impact of this strategy on nitrogen metabolism of the main fermenting yeast, S. cerevisiae. In this work, we aimed at evaluate the effect of nitrogen level of grape‐musts on fermentative and metabolic behaviour of S. cerevisiae in mixed culture with a non‐ Sacharomyces strain of Hanseniaspora guiliermondii. The results have clearly shown that its presence compromise the growth and fermentation fitness of S. cerevisiae. We reasoned that potential yeast‐yeast interaction would also be revealed by the differential expression of genes in both fermentations. Several changes in S. cerevisiae gene expression detected appear to respond to changes in nutrient availability, mainly nitrogen and vitamins, in the fermenting must that are potentially linked to Hanseniaspora guilliermondii metabolic activity. We found that differences in the expression of S. cerevisiae genes involved in aroma compounds formation could explain the differences in the aroma profiles obtained in single and mixed‐culture. These results raises the question whether the impact of non‐Saccharomyces in the sensorial profile in co‐culture fermentations results from a direct contribution with the production of aromatic compounds and/or from influencing the metabolic behaviour of the fermentative yeast S. cerevisiae. In sum, while opening interesting perspectives for future research in yeast biology, the data presented in this thesis clearly provides important information towards the deeper understanding of the role of nitrogen in alcoholic fermentation that can assist winemakers in the pursuit of high quality wines. Foi financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE (FCOMP‐01‐0124‐FEDER‐041576), por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PTDC/AGR‐ALI/111224/2009, pelo projeto ENOEXEL ‐ FROM VINEYARD TO WINE: TARGETING GRAPE AND WINE EXCELLENCY ‐ NORTE‐07‐0124‐ FEDER‐000032 financiado pelo Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER), e ainda por fundos nacionais (PIDDAC)através da Fundação para a Ciência e a Tecnologia (FCT/MEC) e pela bolsa de doutoramento SFRH/BD/61881/2009, atribuída pela FCT e financiada pelo Programa Operacional Potencial Humano do QREN‐Portugal 2007‐2013 e por verbas do Orçamento de Estado do MCTES.

Esta tesis parte de la existencia de una técnica genómica para el estudio de la transcripción naciente en levadura ampliamente utilizada y contrastada: el Genomic run-on, basada en la utilización de macrochips de las ORFs completas del genoma de S. cerevisiae. Debido a la aparición progresiva de nuevas plataformas que permiten interrogar la totalidad de las regiones del genoma, y a una resolución mayor, como los microchips de embaldosado o tiling arrays, el objetivo principal de esta tesis es la puesta a punto de una técnica adaptada a ellas que permita un análisis detallado de la transcripción naciente. Los objetivos concretos que se marcaron fueron: -Desarrollar un nuevo procedimiento de run-on a escala genómica que sustituya el uso de las plataformas basadas en radiactividad y aproveche las plataformas de más resolución, así como de las herramientas bioinformáticas necesarias para el análisis de los datos generados. -Estudiar los perfiles globales de transcripción naciente aprovechando el carácter específico de hebra de los datos para estudiar las dinámicas del transcriptoma global de levadura. Comparar y evaluar la complementariedad de los datos con otras medidas alternativas de tasas de transcripción existentes en la actualidad. -Aplicar la técnica al estudio del efecto de mutantes relacionados con el ciclo de síntesis y degradación del RNA para poder extraer información sobre el funcionamiento de la maquinaria transcripcional y su regulación. -Detectar posibles patrones de actividad de las RNA Polimerasas a lo largo de los transcritos y de las zonas flanqueantes que pudieran obedecer a condicionantes impuestas, tanto por su contexto cromatínico, como por otros factores. -Caracterizar la transcripción naciente producida por las otras RNAP nucleares de levadura. -Desarrollar un protocolo que permita analizar los RNAs nacientes a la máxima resolución mediante secuenciación masiva.

En este trabajo se ha realizado un estudio exhaustivo sobre el recambio de los mRNAs a escala genómica en la levadura Saccharomyces cerevisiae.Se ha confirmado que la asunción de estado estacionario para la expresión génica en condiciones de crecimiento exponencial, y por lo tanto la validez del cálculo indirecto de valores de estabilidad de mRNAs a partir de datos de cantidad y tasa de transcripción. También se han caracterizado ligeras desviaciones del estado estacionario específicas de grupos funcionales y se ha calculado la contribución a estas variaciones de mRNA dependientes de cambios en la transcripción o en la estabilidad de mRNAs.Una vez comprobada la validez del estado estacionario para la expresión génica en las condiciones estudiadas, se ha realizado un cálculo alternativo de estabilidades de mRNAs usando un método directo. Concretamente se ha realizado una parada general de la transcripción usando tiolutina y se ha cuantificado la desaparición de los mRNAs. Durante este experimento se detectó un efecto inhibitorio de la tiolutina sobre la degradación de los mRNAs. Los datos obtenidos por cada procedimiento, directo e indirecto, se han comparado.Por otra parte, se ha desarrollado un método para medir la tasa de transcripción a escala genómica basado en la inmunoprecipitación de cromatina (RPCC). Este método ha permitido descubrir y corregir alguno de los sesgos técnicos presentes en el GRO contribuyendo a generar unos datos genómicos de transcripción en levadura más robustos y fiables. Además, la comparación de estas dos técnicas también ha permitido descubrir diferencias biológicas en la forma que tiene la célula de transcribir los genes pertenecientes a diferentes grupos funcionales. Se ha profundizado en el estudio de la regulación de estas diferencias y se ha descrito un posible mecanismo de acción para el caso de los genes de las proteínas ribosómicas.Finalmente se ha puesto a punto una variante del GRO basada en la purificación selectiva de mRNAs nacientes. Esta alternativa permitirá en un futuro el estudio de la transcripción a una mayor resolución usando chips de DNA de embaldosado u otras técnicas genómicas de nueva generación., We have confirmed the assumption of a steady state for gene expression during exponential growth in the yeast Saccharomyces cerevisiae. Therefore, we also have confirmed that it is possible to compute the mRNA stability for all yeast genes in an indirect way using mRNA amounts and transcription rates data. We also have studied minor gene group-specific deviations from the steady state, and have determined witch part of the mRNA amount variation is due to changes in the transcription rate or in the mRNA stability.The mRNA stabilities have also been studied genome wide by using a direct method: inhibition the mRNA transcription with thiolutin and measurement of the mRNA decay. During this study we have been able to characterize a secondary effect of the thiolutin inhibiting the mRNA degradation. We also have done a comparison between the different genomic methods used to study the mRNA decay.On the other hand, we have implemented a new method for measuring the transcription at genomic level using chromatin immunoprecipitation (RNA Pol ChIP-on-chip, RPCC). This method has allowed us to improve the quality of the transcription rate data obtained by GRO. Moreover, using the comparison between the GRO and RPCC methods we have been able to discover transcription elongation differences specific for gene groups. The case of the ribosomal proteins genes has been studied with more detail and we have postulated a model for its transcription based in the repressive effect of Rap1 protein.Finally, we have developed a new version of the GRO technique using a selective purification of the nascent mRNAs that allows fluorescent labeling. This variation will allow high resolution studies of the transcription using tiling arrays or massive parallel DNA sequencing.

RESUMEN En este trabajo se ha realizado un estudio exhaustivo sobre el recambio de los mRNAs a escala genómica en la levadura Saccharomyces cerevisiae. Se ha confirmado que la asunción de estado estacionario para la expresión génica en condiciones de crecimiento exponencial, y por lo tanto la validez del cálculo indirecto de valores de estabilidad de mRNAs a partir de datos de cantidad y tasa de transcripción. También se han caracterizado ligeras desviaciones del estado estacionario específicas de grupos funcionales y se ha calculado la contribución a estas variaciones de mRNA dependientes de cambios en la transcripción o en la estabilidad de mRNAs. Una vez comprobada la validez del estado estacionario para la expresión génica en las condiciones estudiadas, se ha realizado un cálculo alternativo de estabilidades de mRNAs usando un método directo. Concretamente se ha realizado una parada general de la transcripción usando tiolutina y se ha cuantificado la desaparición de los mRNAs. Durante este experimento se detectó un efecto inhibitorio de la tiolutina sobre la degradación de los mRNAs. Los datos obtenidos por cada procedimiento, directo e indirecto, se han comparado. Por otra parte, se ha desarrollado un método para medir la tasa de transcripción a escala genómica basado en la inmunoprecipitación de cromatina (RPCC). Este método ha permitido descubrir y corregir alguno de los sesgos técnicos presentes en el GRO contribuyendo a generar unos datos genómicos de transcripción en levadura más robustos y fiables. Además, la comparación de estas dos técnicas también ha permitido descubrir diferencias biológicas en la forma que tiene la célula de transcribir los genes pertenecientes a diferentes grupos funcionales. Se ha profundizado en el estudio de la regulación de estas diferencias y se ha descrito un posible mecanismo de acción para el caso de los genes de las proteínas ribosómicas. Finalmente se ha puesto a punto una variante del GRO basada en la purificación selectiva de mRNAs nacientes. Esta alternativa permitirá en un futuro el estudio de la transcripción a una mayor resolución usando chips de DNA de embaldosado u otras técnicas genómicas de nueva generación. __________________________________________________________________________________________________ We have confirmed the assumption of a steady state for gene expression during exponential growth in the yeast Saccharomyces cerevisiae. Therefore, we also have confirmed that it is possible to compute the mRNA stability for all yeast genes in an indirect way using mRNA amounts and transcription rates data. We also have studied minor gene group-specific deviations from the steady state, and have determined witch part of the mRNA amount variation is due to changes in the transcription rate or in the mRNA stability. The mRNA stabilities have also been studied genome wide by using a direct method: inhibition the mRNA transcription with thiolutin and measurement of the mRNA decay. During this study we have been able to characterize a secondary effect of the thiolutin inhibiting the mRNA degradation. We also have done a comparison between the different genomic methods used to study the mRNA decay. On the other hand, we have implemented a new method for measuring the transcription at genomic level using chromatin immunoprecipitation (RNA Pol ChIP-on-chip, RPCC). This method has allowed us to improve the quality of the transcription rate data obtained by GRO. Moreover, using the comparison between the GRO and RPCC methods we have been able to discover transcription elongation differences specific for gene groups. The case of the ribosomal proteins genes has been studied with more detail and we have postulated a model for its transcription based in the repressive effect of Rap1 protein. Finally, we have developed a new version of the GRO technique using a selective purification of the nascent mRNAs that allows fluorescent labeling. This variation will allow high resolution studies of the transcription using tiling arrays or massive parallel DNA sequencing.