Caloric restriction (CR) extends lifespan from yeast to mammals. In budding yeast, inhibition of the conserved TOR and/or PKA pathways has been shown to mediate lifespan extension by CR partly through the activation of stress response. However, how the stress response is regulated at the systems level is poorly understood. In this study, by using fluorescent reporters whose expression is dependent on the transcription factors Msn2/4 and Gis1, two separate screenings were conducted to reveal novel regulators of the stress response induced by starvation. A 'focused' screening on the 272 'signalling' mutants revealed that, apart from the previously identified Rim15, Yak1 and Mck1 kinases, the SNF1/AMPK complex, the cell wall integrity (CWI) pathway and a number of cell cycle regulators are necessary to elicit appropriate stress response. The chronological lifespan (CLS) of these signalling mutants correlates well with the amount of accumulated storage carbohydrates but poorly with transition-phase cell cycle status. Subsequent analyses reveal that the levels of intracellular reactive oxygen species are controlled by Rim15, Yak1 and Mck1. Furthermore, CLS extension enabled by tor1 deletion is dependent on the above three kinases. These data suggest that the signalling pathways (SNF1 and CWI) and the kinases downstream of TOR/PKA (Rim15, Yak1 and Mck1) coordinate the metabolic reprogramming (to accumulate storage carbohydrates) and the activation of anti-oxidant defence systems (to control ROS levels) to extend chronological lifespan. A 'genome-wide' screening of a haploid deletion library indicates that less than 10% of the non-essential genes are implicated in the regulation of starvation-induced stress response. Gene ontology analysis suggests that they can be grouped into major clusters including mitochondrial function, r-RNA processing, DNA damage and repair, transcription from RNA polymerase and cell cycle regulation. Further phenotypic assays confirm the previous observation that CLS extension is mostly correlated with the accumulation of storage carbohydrates. Compromised expression of stress response reporters is confirmed by FACS in a variety of mitochondrial mutants, suggesting that mitochondrial respiration also plays a key role in the activation of stress response. Put together, the above findings indicate that stress response and metabolic reprogramming induced by glucose starvation are coordinated by multiple signalling pathways and the activation of mitochondrial respiration is essential to both cellular processes and to CLS extension.

No description available.

Uma dieta rica em carboidratos aparece como um dos poucos fatores ambientais capazes de interferir tanto na longevidade quanto no envelhecimento de um organismo. Neste sentido, a geração aumentada e o acúmulo dos AGEs (do inglês Advanced glycation end-products), formados por reações não enzimáticas entre os monossacarídeos glicose e frutose e/ou seus intermediários metabólicos com os ácidos nucleicos e grupos amina de proteínas, determinam a importância que estes produtos representam para a duração do ciclo de vida dos organismos. Os AGEs aparecem associados a uma série de patologias relacionadas com a longevidade e com a ocorrência do envelhecimento precoce, aparecendo em número aumentado nos casos de diabetes melito e nas doenças neurodegenerativas como o Alzheimer e o Parkinson. Nesta dissertação de mestrado buscou-se mostrar uma associação entre as proteínas do metabolismo de carboidratos e as vias de reparação do DNA. Os dados obtidos evidenciaram uma importante interação entre as principais enzimas do metabolismo de carboidratos com as proteínas de reparação de DNA e mostraram que ambos parecem ser essenciais para a manutenção da integridade genômica em leveduras. Nesta dissertação também foi verificado o tempo de vida cronológico de diferentes linhagens da levedura Saccharomyces cerevisiae frente a diferentes fontes de carbono e os resultados obtidos foram relacionados com a atuação do metabolismo de carboidratos e com as vias de reparação do DNA. Soma-se ao trabalho os dados de idade cronológica associados com a ausência do complexo Tor1 (Target of Rapamycin), uma via relacionada com a indução de autofagia e que está associada com os mecanismos promotores da longevidade e do envelhecimento. Os dados gerados por esta dissertação também permitiram relacionar informações importantes sobre o metabolismo de carboidratos e sua interferência nos mecanismos genéticos e bioquímicos associados com a longevidade e com o envelhecimento de leveduras, abrindo caminhos para a realização de novos estudos que visem buscar maiores conhecimentos sobre a caracterização dos mecanismos genéticos associados com a longevidade dos organismos. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-05-22T17:00:00Z No. of bitstreams: 1 Dissertacao Fernanda Barea.pdf: 625512 bytes, checksum: 6e37386b24cc1eb8e2338a52150f535d (MD5) / Made available in DSpace on 2014-05-22T17:00:00Z (GMT). No. of bitstreams: 1 Dissertacao Fernanda Barea.pdf: 625512 bytes, checksum: 6e37386b24cc1eb8e2338a52150f535d (MD5) / A diet rich in carbohydrates is one of the few environmental factors capable of interfering in longevity and in an aging of organisms. In this sense, the increased generation and/or accumulation of AGES (Advanced glycation end-products) formed by reactions between monosaccharides glucose and fructose and/or their metabolic intermediates with nucleic acid and amine group of proteins determine the importance of these products for the lifespan. The AGES appear to be associated with a series of diseases related with longevity and aging. In addition, it has been reported an increased in AGEs in diabetes mellitus and neurodegenerative diseases, e.g. Alzheimer´s and Parkinson´s diseases. In this work we search for an association between carbohydrate metabolism and DNA repair mechanisms. The data showed a significant interaction between key enzymes of carbohydrates metabolism with DNA repair mechanisms and indicated that both processes seems to be essential for the maintenance of genomic integrity in yeast. In this work it was also verificated the chronological lifespan (CLS) in several strains of yeast Saccharomyces cerevisiae grown on different carbon sources. The results generated were related with carbohydrate metabolism and DNA repair mechanisms. In addition, CLS was associated with the absence of Tor1 (Target Of Rapamycin), a genetic mechanism that has been associated with aging and longevity. Furthermore, the data generated by the current study allowed to obtain important data about carbohydrate metabolism and its association with longevity and the aging of yeast.

Uma dieta rica em carboidratos aparece como um dos poucos fatores ambientais capazes de interferir tanto na longevidade quanto no envelhecimento de um organismo. Neste sentido, a geração aumentada e o acúmulo dos AGEs (do inglês Advanced glycation end-products), formados por reações não enzimáticas entre os monossacarídeos glicose e frutose e/ou seus intermediários metabólicos com os ácidos nucleicos e grupos amina de proteínas, determinam a importância que estes produtos representam para a duração do ciclo de vida dos organismos. Os AGEs aparecem associados a uma série de patologias relacionadas com a longevidade e com a ocorrência do envelhecimento precoce, aparecendo em número aumentado nos casos de diabetes melito e nas doenças neurodegenerativas como o Alzheimer e o Parkinson. Nesta dissertação de mestrado buscou-se mostrar uma associação entre as proteínas do metabolismo de carboidratos e as vias de reparação do DNA. Os dados obtidos evidenciaram uma importante interação entre as principais enzimas do metabolismo de carboidratos com as proteínas de reparação de DNA e mostraram que ambos parecem ser essenciais para a manutenção da integridade genômica em leveduras. Nesta dissertação também foi verificado o tempo de vida cronológico de diferentes linhagens da levedura Saccharomyces cerevisiae frente a diferentes fontes de carbono e os resultados obtidos foram relacionados com a atuação do metabolismo de carboidratos e com as vias de reparação do DNA. Soma-se ao trabalho os dados de idade cronológica associados com a ausência do complexo Tor1 (Target of Rapamycin), uma via relacionada com a indução de autofagia e que está associada com os mecanismos promotores da longevidade e do envelhecimento. Os dados gerados por esta dissertação também permitiram relacionar informações importantes sobre o metabolismo de carboidratos e sua interferência nos mecanismos genéticos e bioquímicos associados com a longevidade e com o envelhecimento de leveduras, abrindo caminhos para a realização de novos estudos que visem buscar maiores conhecimentos sobre a caracterização dos mecanismos genéticos associados com a longevidade dos organismos. / A diet rich in carbohydrates is one of the few environmental factors capable of interfering in longevity and in an aging of organisms. In this sense, the increased generation and/or accumulation of AGES (Advanced glycation end-products) formed by reactions between monosaccharides glucose and fructose and/or their metabolic intermediates with nucleic acid and amine group of proteins determine the importance of these products for the lifespan. The AGES appear to be associated with a series of diseases related with longevity and aging. In addition, it has been reported an increased in AGEs in diabetes mellitus and neurodegenerative diseases, e.g. Alzheimer´s and Parkinson´s diseases. In this work we search for an association between carbohydrate metabolism and DNA repair mechanisms. The data showed a significant interaction between key enzymes of carbohydrates metabolism with DNA repair mechanisms and indicated that both processes seems to be essential for the maintenance of genomic integrity in yeast. In this work it was also verificated the chronological lifespan (CLS) in several strains of yeast Saccharomyces cerevisiae grown on different carbon sources. The results generated were related with carbohydrate metabolism and DNA repair mechanisms. In addition, CLS was associated with the absence of Tor1 (Target Of Rapamycin), a genetic mechanism that has been associated with aging and longevity. Furthermore, the data generated by the current study allowed to obtain important data about carbohydrate metabolism and its association with longevity and the aging of yeast.

Le vieillissement est un processus complexe déterminé par des facteurs génétiques et environnementaux qui varie d’un individu à l’autre. Bien que le vieillissement soit la cause principale de nombreuses maladies, nos connaissances sur le sujet sont relativement limitées. Tout au long de ce travail, j’ai utilisé la levure bourgeonnante Saccharomyces cerevisiae pour identifier les facteurs génétiques et environnementaux influant sur le vieillissement et pour comprendre les interactions qu’ils entretiennent entre eux. Jusqu’à présent, les approches classiques de génétique ont permis de découvrir un certain nombre de gènes impliqués dans la régulation du vieillissement chronologique de la levure (CLS), basé sur la longévité de celle-ci en conditions non-prolifératives. Or, ces approches se sont essentiellement centrées sur des souches de laboratoire et n’ont que très peu exploité les richesses de la biodiversité. Dans une première partie, j’ai utilisé une large cohorte de levures composée de plus de 1000 souches naturelles de S. cerevisiae afin d’estimer la variabilité de longévité existant au sein de l’espèce. Leur longévité a été étudiée dans différentes conditions connues pour freiner le vieillissement : sous restriction calorique ou en présence d’un agoniste de la restriction calorique, la molécule rapamycine, qui inhibe directement la voie de signalisation TOR. Les microorganismes passent la majeure partie de leur vie dans des environnements défavorables, pauvres en ressources nutritives. Leur capacité à survivre à ces périodes de restriction (CLS) est donc primordiale. J’ai observé que les souches sauvages ont tendance à spontanément initier le programme de méiose aboutissant à la formation de spores lorsque les conditions environnementales deviennent restreintes. En revanche, les souches domestiques préfèrent entrer en quiescence, ce qui leur confère une viabilité et une résistance accrues. De plus, en ayant recours à une approche basée sur des gènes présélectionnés et à une étude d’association pangénomique, j’ai observé que la variabilité de longévité entre les différentes souches est déterminée par un large spectre de polymorphismes génétiques, tels que des mutations non-synonymes ou non-sens, et par l’absence ou la présence de certains gènes. Toutes ces composantes génétiques interagissent pleinement avec l’environnement. Dans une deuxième partie, j’ai réalisé une analyse de liaison génétique grâce à 1056 souches descendantes de deux souches parentales. La longévité (CLS) de ces 1056 souches a été mesurée dans le but d’identifier des locus de caractères quantitatifs (QTLs). Le vieillissement chronologique a été déterminé à la fois à partir d’un milieu riche, d’un milieu restreint en calories, ou en présence de rapamycine. J’ai identifié 30 QTLs distincts, certains d’entre eux sont communs et récurrents dans plusieurs environnements, tandis que d’autres sont plus spécifiques et occasionnels. Les deux QTLs principaux, associés aux gènes HPF1 et FLO11, codent tous deux des protéines du mur cellulaire, et sont jusqu’à présent non reconnus comme régulateurs du vieillissement. Etonnement, ces deux gènes contiennent des répétitions d’ADN en tandem qui s’avèrent être massivement amplifiées dans une des deux souches parentales d’origine. Alors que les allèles courts de HPF1 et FLO11 n’ont pas d’effet sur le vieillissement, les allèles longs sont relativement délétères, hormis en présence de rapamycine. Après investigation, il semble que la forme allongée de HPF1 provoque la flottaison des cellules de levure au cours de la phase de croissance, les exposants à des taux plus élevés d’oxygène. / Aging is a classical complex trait varying quantitatively among individuals and affected by both the genetic background and the environment. While aging is the highest risk factor for a large number of diseases, little is known about the underlying molecular mechanisms. Identifying the causal genetic variants underlying natural variation in longevity and understanding their interaction with the genetic background and the environment remains a major challenge. In this work, I used the budding yeast, Saccharomyces cerevisiae, to identify environmental and genetic factors contributing to aging. While extensive classical genetic studies discovered several genes involved in the regulation of chronological lifespan (CLS), which measures cell viability dynamic in non-dividing condition, using laboratory strains in standard conditions, there are only few studies exploiting variations in natural populations. In the first part, I used a large cohort of more than 1000 sequenced natural S. cerevisiae strains to provide a species-wide overview of CLS variability. Longevity was measured in different environments, including calorie restriction (CR), a natural intervention known to increase lifespan, and in the presence of rapamycin (RM), a drug that mimics CR by downregulating the TOR pathway. Unicellular microorganisms spend most of their lifetime in harsh restricted environments interrupted by short windows of growth, making CLS an important and likely adaptive trait. I found that wild strains subjected to CLS tend to trigger the meiotic developmental process leading to the formation of gametes wrapped into a very resistant cell wall. In contrast, domesticated strains tend to enter quiescence state when starved and display a tremendous variability in their survival capacity. Moreover, using both candidate gene approach and genome-wide association studies (GWAS), I demonstrated that variability in CLS is determined by a full spectrum of genetic variant that include gene presence/absence, copy number variation, non-synonymous SNPs and loss of function. All these genetic features were strongly regulated by the environment. In the second part, I performed linkage analysis using 1056 diploid segregants derived from a two parent advanced intercross. These 1056 diploid segregants were phenotyped for CLS to map quantitative trait loci (QTLs). The CLS was measured in complete media, CR and RM environments across multiple time points. I mapped 30 distinct QTLs, with some shared across different environments and time points, while others were unique to a specific condition. The two major effect size QTLs were linked with natural variation in the cell wall glycoproteins FLO11 and HPF1, previously unknown to regulate CLS. Interestingly, both genes presented massive intragenic tandem repeat expansions in one of the founder strain used in the crossing scheme. While the short versions of FLO11 and HPF1 alleles did not impact CLS, tandem repeat expansions within those genes were sufficient to confer a dominant detrimental effect that was partially buffered by rapamycin treatment. Further investigation revealed that the extended form of HPF1 makes cells floating during exponential phase, exposing them to higher oxygen rates, and leading to perturbation of redox homeostasis, activation of misfolded protein response, and alteration of multiple genes involved in methionine, ribosome and lipid biosynthesis, eventually contributing to CLS shortening. Taken together, my work provided an unprecedented overview of natural variation in CLS in a genetic model system and revealed multiple genetic and environmental factors that shape the species phenotypic variation.