Thèses sur le sujet « Saccharomyces cerevisiae, healthy aging »

Tutti gli organismi viventi col passare del tempo invecchiano, ossia vanno incontro ad un progressivo ed irreversibile declino funzionale/fisiologico, accompagnato da un aumentato rischio di contrarre malattie. Tra i diversi fattori coinvolti nell’invecchiamento, i nutrient-sensing pathway di TORC1/Sch9 e Ras/PKA e le Sirtuine, una famiglia di deacetilasi NAD+-dipendenti, svolgono un ruolo prioritario. Essi sono evolutivamente conservati dal lievito all’uomo, e risultano, inoltre, mediare alcuni degli effetti della Calorie Restriction (CR), un intervento che consiste nel limitare l’apporto di nutrienti senza incorrere in malnutrizione ed è noto estendere la longevità di molti organismi. Nell’ambito della ricerca sull’invecchiamento, il lievito Saccharomyces cerevisiae è un utile sistema sperimentale. In particolare, la Chronological LifeSpan (CLS), definita come il tempo che una popolazione di cellule quiescenti può sopravvivere durante la fase stazionaria, rappresenta un modello per lo studio dell’invecchiamento di cellule post-mitotiche di mammifero, quali i neuroni e i miociti. Infatti, le cellule in fase stazionaria, pur non proliferando, rimangono metabolicamente attive e responsive agli stimoli. Nel lavoro di questa tesi è emerso che nell’invecchiamento cronologico il metabolismo ossidativo attraverso la respirazione può esercitare effetti negativi sulla funzionalità mitocondriale, producendo ROS, e ridurre la CLS. Al contrario, un metabolismo gluconeogenetico favorisce l’instaurarsi di una condizione vantaggiosa per la sopravvivenza anche durante la CR. Abbiamo visto che Sir2, il capostipite delle Sirtuine, rappresenta un elemento chiave in questo contesto in quanto, regolando lo stato di acetilazione di Pck1, l’enzima “rate-limiting step” della gluconeogenesi, ne controlla l’attività. La mancanza di Sir2 determina infatti un aumento di Pck1 acetilata che correla con un incremento della sua attività e quindi con un incremento della gluconeogenesi. Sulla base di questo, abbiamo valutato gli effetti sulla CLS e sul metabolismo della Nicotinammide (inibitore fisiologico di Sir2) e di due sostanze che sembrerebbero mimare la CR, Resveratrolo e Quercitina. L’utilizzo di queste tre sostanze, presenti in alcuni alimenti e bevande, potrebbe esercitare un’azione preventiva, favorendo quello che viene definito “healthy aging”.
All living organisms undergo a functional/physiological decline with age, which is progressive and irreversible, and it is associated to an increased risk of the development of many diseases. Among the factors involved in aging, TORC1/Sch9 and Ras/PKA nutrient-sensing pathways and Sirtuins, a family of NAD+-dependent deacetylases, play a prominent role. They are evolutionarily conserved from yeast to humans, and they also mediate some of the effects of Calorie Restriction (CR), an intervention consisting in a reduction in calorie intake without malnutrition, known to extend longevity in many organisms. In the field of aging research, the yeast Saccharomyces cerevisiae is a useful experimental system. In particular, the Chronological LifeSpan (CLS), defined as the time that a population of quiescent cells can survive in stationary phase, represents a model for studying aging of post-mitotic mammalian cells, such as neurons and myocytes. Although cells do not proliferate during the stationary phase, they remain metabolically active and responsive to stimuli. In this thesis work, it emerged that, in the chronological aging, oxidative metabolism through respiration can have negative effects on the mitochondrial functionality, due to ROS production, and can reduce CLS. On the contrary, a metabolism based on gluconeogenesis allows for the establishment of a condition favorable to survival even in the CR regimen. Sir2, the founding member of the Sirtuin family, is a key element in this context as it controls the activity of Pck1, the "rate-limiting step" enzyme of gluconeogenesis, by regulating its acetylation state. Lack of Sir2, in fact, determines an increase of the acetylated form of Pck1 that correlates with an increase of its activity and, thus, with an enhanced gluconeogenesis. On this basis, we evaluated the effects of Nicotinamide (a physiological inhibitor of Sir2) and two substances that seem to mimic CR, Resveratrol and Quercetin, on CLS and metabolism. Using these three substances, present in some foods and drinks, may exert a preventive effect, favoring the so- called "healthy aging".

L’aumento dell’aspettativa di vita non è associato con un altrettanto aumento delle condizioni di salute nella popolazione anziana. Oggigiorno, un’ampia parte di popolazione al di sopra dei 65 anni soffre di molteplici malattie, molte delle quali debilitanti, come le malattie cardiovascolari, i tumori o i disordini neurodegenerativi. Questo aspetto ha aumentato l’interesse per le tematiche legate all’invecchiamento, enfatizzando l’importanza di ridurre il gap tra longevità salute durante l’invecchiamento. A questo proposito, gli sforzi di molte linee di ricerca sono focalizzati nel tentativo di comprendere quali sono i principali fattori che influenzano l’invecchiamento, allo scopo di sviluppare approcci capaci di mitigare gli effetti dannosi dell’invecchiamento sulla salute. Molti pathway associati all’invecchiamento sono evolutivamente conservati dagli organismi unicellulari a quelli più complessi. Questo ci ha permesso di semplici organismi modello per studiare questo complesso fenomeno biologico. In questo lavoro abbiamo utilizzato l’eucariote unicellulare Saccharomyces cerevisiae, che va incontro sia all’invecchiamento replicativo che a quello cronologico, due modelli complementari di invecchiamento, che rispettivamente simulano il processo di invecchiamento delle cellule mitoticamente attive e quello delle cellule post-mitotiche. In questo contesto la replicative lifespan (RLS) è definita come il numero di cellule figlie generate da una cellula madre in presenza di nutrienti prima della morte. Al contrario, la chronological lifespan (CLS) è il periodo di sopravvivenza medio e massimo di una popolazione di cellule di lievito in fase stazionaria. Essa è determinata, partendo tre giorni dallo shift diauxico, dalla capacità di cellule quiescenti di riprendere la crescita una volta tornate su terreno fresco ricco. Considerata l’esistenza di una forte connessione tra invecchiamento cellulare, nutrienti e metabolismo, abbiamo studiato i possibili effetti di alcuni composti nutraceutici, allo scopo di identificare molecole per sviluppare interventi anti-aging, oltre che aggiungere informazioni utili per comprendere meglio il processo di invecchiamento. A questo scopo, durante il primo e il secondo anno del mio progetto di dottorato, ho studiato gli effetti del resveratrolo (RSV) sulla CLS. RSV è un composto polifenolico annoverato tra i composti attivatori delle Sirtuine (STAC) ed è riconosciuto per conferire benefici su molte patologie legate all’invecchiamento. Le Sirtuine sono una famiglia di deacetilasi NAD+-dipendenti, il cui capostipite è Sir2 di S. cerevisiae, la cui attività è coinvolta sia nell’RLS che nella CLS. Inaspettatamente, abbiamo osservato che il trattamento con RSV incrementava lo stress ossidativo, in concomitanza con una notevole riduzione del pathway anti-aging della gluconeogenesi. L’attività deacetilasica di Sir2 sul suo target gluconeogenico Pck1 era incrementata, determinandone la sua inattivazione e indicando che RSV effettivamente agisce come STAC. Come conseguenza, questo causava effetti negativi sul metabolismo, determinando un fenotipo short-lived. Successivamente, ci siamo focalizzati sulla quercitina (QUER), un composto nutraceutico con proprietà benefiche su diverse patologie, incluse le malattie cardiovascolari, il cancro e la dislipidemia. Ciononostante, i target cellulari della QUER devono essere ancora esplorati. Abbiamo visto che la QUER possiede proprietà anti-aging che favoriscono un’estensione della CLS. Tutti i dati indicano un’inibizione dell’attività deacetilasica di Sir2 a seguito del trattamento con la QUER, determinando un incremento dei livelli di acetilazione e di attività di Pck1. Questo determina un rimodellamento metabolico a favore del pathway della gluconeogenesi, incrementando le riserve di trealosio e garantendo un miglioramento del processo di invecchiamento.
Since the second half of past century in many developed Countries, life expectancy has gradually increased, reaching, and in some extreme cases exceeding, the threshold of 85 years. However, the increase of life expectancy is not associated with a corresponding increment of healthy conditions for the older population. Nowadays, a huge part of population over 65 years suffers a multitude of diseases, most of them highly disabling, like cardiovascular diseases, tumour or neurodegenerative disorders. This aspect has increased the interest on age-related issues, emphasizing the importance of reducing the gap between longevity and health during aging. For this purpose, efforts of many research lines have focused on studying which are the main factors that affect aging, in order to develop approaches that mitigate the detrimental effects of aging on health. Many aging-related pathways are evolutionarily conserved from some single-celled organisms to complex multicellular ones. Such knowledge has allowed us the use of simple model organisms to study this complex biological phenomenon. In this work we used the single-celled eukaryote Saccharomyces cerevisiae, which undergoes both replicative and chronological aging, two complementary models of aging, which respectively resemble the aging process of mitotically active and post-mitotic mammalian cells. In this context, replicative lifespan (RLS) is defined as the number of buds generated by a single mother cell in the presence of nutrients before death. On the contrary, chronological lifespan (CLS) is the mean and maximum period of time of surviving cells in stationary phase. It is determined, starting three days from the diauxic shift, by the capability of quiescent cells to resume growth once returning to rich fresh medium. Considering that there is a strong connection between cellular aging, nutrients and metabolism, we investigated the possible effects of some nutraceutical compounds, in order to identify molecules for anti-aging interventions, as well as add useful information to understand the aging process. To this end, during the first and second year of my PhD project, I studied the effects of resveratrol (RSV) on CLS. RSV is a polyphenolic compound counted among the Sirtuin Activator Compounds (STACs), which has been proposed to confer health benefits on different age-related diseases. Sirtuins are a family of NAD+-dependent deacetylases, the founding member of which is Sir2 of S. cerevisiae, whose activity is involved in both RLS and CLS. Unexpectedly, we found that RSV supplementation increased oxidative stress in concert with a strong reduction of the anti-aging gluconeogenesis pathway. The deacetylase activity of Sir2 on its gluconeogenic target Pck1 was enhanced, resulting in its inactivation and indicating that RSV really acts as a STAC. As a consequence, this brought about detrimental effects on the survival metabolism resulting in a short-lived phenotype. Next, we focused on the study of quercetin (QUER), a nutraceutical compound with health-promoting properties on different pathologies, including cardiovascular disorders, cancer and dyslipidaemia. Nevertheless, QUER cellular targets are still being explored. We found that QUER displays anti-aging properties favouring CLS extension. All data point to an inhibition of the deacetylase activity of Sir2 following QUER supplementation, resulting in increased levels of acetylation and activity of Pck1. This determines a metabolic remodelling in favour of the pro-longevity gluconeogenesis pathway, increasing trehalose storage and ensuring healthy aging improvement.

Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 117 pages. Includes Vita. Includes bibliographical references.

In yeast, aging appears to be marked by a progressive impairment in cellular mechanisms, resulting in irreversible changes in physiology and morphology. To date, very little has been reported about the biochemical changes that occur in yeast as a function of individual cell aging. To investigate this further, six generations of a brewing yeast strain of Saccharomyces cerevisiae (NCYC 1239) were separated according to cellular age using continuous phased culturing and biotin-streptavidin magnetic cell sorting.
To obtain cells with no bud scars (virgin cells), a concentrated yeast slurry was layered onto sucrose density gradients and centrifuged. The uppermost band from the gradients was collected and cells were biotinylated with biotinamidocaproate- N-hydroxysuccinimide ester, that covalently binds to lysine residues on the yeast cell wall. For continuous phased culturing, biotinylated cells were added to a carbon-limited nutrient medium and growth was synchronized using the doubling time of the cells. Harvested cells were incubated with streptavidin superparamagnetic beads and sorted with a strong permanent magnet. In total, approximately 75% of the biotinylated cells were recovered. Viability testing was conducted using vital staining and plate counts, with >98% viability reported with the vital stain and 37% viability with the agar plates.
In conclusion, continuous phased culture, together with magnetic cell sorting has the potential to become a powerful tool for the study of age-related biochemical changes in yeast. Further studies will focus on ensuring the reproducibility of the method and using the recovered cells to study biochemical changes occurring during yeasts' replicative lifespan.

This thesis concerns the asymmetric mechanism by which the "molecular aging clock" is reset in the budding yeast Saccharomyces cerevisiae, which is of great interest considering that many organisms' cells--including human stem cells--undergo this process. When yeast divides, it ages a generation, while daughter cells begin life at generation zero. One theory surrounding this process in yeast is the extrachromosomal rDNA circle (ERC) aging theory. ERCs are generated spontaneously in mother cells as they age, and thus accumulate exponentially in older cells. Daughter cells from young mothers benefit from asymmetric aging, but as mothers age, they produce daughters that prematurely senesce. Studies suggested that ERCs may be a cytoplasmic senescence factor that is passed from mother to daughter as the mother ages, possibly due to the mother's inability to maintain cellular pathways responsible for asymmetric processes as she ages. ASH1 is a gene that encodes an asymmetrically-distributed protein that halts expression of HO endonuclease--an enzyme critical to mating-type switch--in daughter cells. Previous studies in our lab showed that deleting ASH1 led to a decrease in daughter lifespan compared to wild-type strains. In this thesis, I present evidence of a possible connection between ASH1 and cell cycle regulation. Furthermore, the detection of ERC accumulation via Southern blotting in the mutant ASH1 strain, but not the wild-type strain, provides support that ERCs may be a senescence factor in yeast. Lastly, preliminary microarray analysis reveals several genes related to cell cycle regulation being affected by the deletion of ASH1.

Shortest-path network analysis was employed to identify novel genes that modulate longevity in the baker’s yeast Saccharomyces cerevisiae. Based upon a set of previously reported genes associated with increased life span, a shortest path network algorithm was applied to a pre-existing protein-protein interaction dataset in order to construct a shortest-path longevity network. To validate this network, the replicative aging potential of 88 single gene deletion strains corresponding to predicted components of the shortest path longevity network was determined. The 88 single-gene deletion strains identified by a network approach are significantly enriched for mutation conferring both increased and decreased replicative life span when compared to a randomly selected set of 564 single-gene deletion strains or to the current data set available for the entire haploid deletion collection. In addition, previously unknown longevity genes were identified, several of which function in a longevity pathway believed to mediate life span extension in response to dietary restriction. This study represents the first biologically validated application of a network construct to the study of aging and rigorously demonstrates, also for the first time, that shortest path network analysis is a potentially powerful tool for predicting genes that function as potential modulators of aging.

Depuis une vingtaine d’années, il est possible d’allonger la durée de vie génétiquement. Nombre d’études réalisées sur des espèces allant de la levure aux primates, ont permis d’identifier des cascades de signaux intracellulaires ayant un impact sur la longévité et la qualité du vieillissement. Il est important de noter que certaines de ces interventions réduisent considérablement l’incidence de cancers et de maladies liées au vieillissement chez les mammifères. Ceci témoigne des liens existant entre vieillissement et carcinogénèse et il probable que le développement de stratégies pharmacologiques ayant pour cible le vieillissement se révèlent efficaces contre les maladies du vieillissement comme le cancer, la maladie d’Alzheimer ou de Parkinson. Nous avons criblé la banque de mutants de Saccharomyces cerevisiae pour identifier des mutations génétiques qui augmentent la durée de vie. La plupart des gènes identifiés se sont révélés conservés puisqu’ils influencent aussi la longévité chez C. elegans. La protéine de liaison à l’acyl-CoA (ACBP) est une petite protéine (10 kDa) qui se lie avec une haute affinité aux chaîne d’acyl-CoA esters (moyennes et longues) et les transporte vers les sites de consommation de l'acyl-CoA. ACBP est hautement conservée parmi les espèces eucaryotes et joue un rôle important dans la biosynthèse des lipides et le trafic vésiculaire. Chez Saccharomyces cerevisiae, la délétion d’ACBP (ACB1) entraîne une augmentation de la longévité et favorise la résistance au stress. Pour tester si l’impact d’ACBP sur la longévité s'étend aux eucaryotes supérieurs, nous avons exploré le lien entre les gènes codant pour des ACBPs chez Caenorhabditis elegans et la longévité en utilisant l’ARN interfèrent. Chez C. elegans, sept paralogues ACBP ont été identifiés, qui sont exprimés dans différents tissus. Nous avons constaté que la réduction de l'expression de maa-1 (codant une ACBP associée aux membranes) prolonge la durée de vie des vers sauvages. Nos résultats démontrent que: 1) une perte de fonction de maa-1 entraîne une résistance au superoxyde, 2) et aux événements protéotoxiques telle que l'agrégation protéique associées aux maladies neurodégénératives comme la maladie de Huntington. Enfin, nous avons montré que l'activité du facteur de transcription HIF-1 (hypoxia inducible factor-1) contribue à la longévité causée par la mutation maa-1. En effet, la délétion du gène hif-1 annule complètement l’augmentation de la longévité causée par maa-1
Understanding the aging process, its regulation, and how to delay it has become a priority for an increasing number of scientists worldwide. The principal reason for this is that it is becoming more and more evident that anti-aging interventions may be effective against age-related diseases such as cancer, cardiovascular, and neurodegenerative diseases. Simple model organisms such as Caenorhabditis elegans and Saccharomyces cerevisiae have been instrumental to identify the principal genes implicated in aging whose role has turned out to be conserved in mammals. The project presented here has originated from a genome-wide screen performed in S. cerevisiae that has led to discover several novel life span-regulatory genes whose deletion prevents aging. One of these genes encodes for Acyl-CoA binding protein (ACBP). ACBP is a small (10 kDa) protein that binds medium- and long-chain fatty acyl-CoA esters with high affinity and transports them to acyl-CoA consuming processes. ACBP is highly conserved among eukaryotic species and plays important roles in lipid biosynthesis and vesicle trafficking. In S. cerevisiae, lack of ACBP (Acb1) extends longevity and promotes stress resistance. To test whether the life span-regulatory role of ACBP extends to higher eukaryotes, we explored the link between the C. elegans ACBP genes and longevity by RNAi screening. In C. elegans, seven ACBP paralogs have been identified, which are expressed in different tissues. We found that reducing the expression of maa-1 (encoding a membrane associated ACBP) extended the longevity of wild-type worms. Our results show that 1) a loss of function maa-1 mutant is resistant to the superoxide-generating agent paraquat and 2) reduction of maa-1 expression increases resistance to the proteotoxicity associated with the aggregation of the Huntington's disease-associated polyQ peptide. The activity of the anti-aging transcription factor HIF-1 (hypoxia inducible factor-1) contributes to the extended longevity caused by lack of maa-1. The effect of MAA-1 loss on longevity was fully reverted by the deletion of the hif-1 gene

Rtg2p é uma proteína que participa da sinalização retrógrada, uma via de comunicação da mitocôndria para o núcleo; também tem sido associada com a longevidade em S. cerevisiae. O objetivo deste trabalho foi identificar os determinantes estruturais de Rtg2p, envolvidos na sinalização retrógrada e no envelhecimento. Para isto foram produzidos treze mutantes pontuais a partir do desenho racional por decomposição de redes de correlação de aminoácidos (DRCN). Analisaram-se as cepas mutantes por ensaio de auxotrofia para glutamato, expressão do gene CIT2 e ensaio de longevidade replicativa. Em sua grande maioria as mutações realizadas causaram perturbações nas funções de Rtg2p, com destaque para as cepas E106A, R109E, E137A, T138A e D158A, que apresentaram longevidade igual à da cepa rtg2Δ, com apenas uma mutação pontual. Em conclusão, os resultados obtidos demonstram que o domínio N-terminal é muito importante para a função de Rtg2p, e indicam que existem determinantes estruturais que controlam a longevidade de forma dependente ou independente da resposta retrógrada.
Rtg2p is a protein involved in the retrograde signaling, a pathway of communcation from mitochondria to nucleus; also has been associated with longevity in S. cerevisiae. The goal of this study was to identify the structural determinants of Rtg2p, controlling the function of this protein in retrograde response and aging. For this purpose thirteen point mutants were produced by site-directed mutagenesis, using rational design by decomposition of residues correlation networks (DRCN). The strains was analyzed by glutamate auxotrophy, CIT2 gene expression and replicative life span assays. For the most of performed mutations, generated inactivation to Rtg2p functions, highlighting to R109E, E137A, T138A, and D158A showed longevity equal to rtg2Δ strain, even with a single amino acid change. In conclusion, our results demonstrate that the N-terminal domain is very important to the function of Rtg2p and also show there are structural determinants in Rtg2p that control longevity in both dependent or independent manner of the communication between mitochondria and nucleus.

O envelhecimento envolve um progressivo declínio na eficiência metabólica dos sistemas biológicos ao longo do tempo. Embora não possa ser evitado, o envelhecimento pode ter seus fenótipos típicos mitigados em organismos submetidos à restrição calórica, um regime dietético que consiste em uma oferta diminuída de calorias. Ao longo do tempo, a levedura Saccharomyces cerevisiae mostrou-se um importante organismo modelo para o estudo de importantes marcas relacionadas ao envelhecimento, sobretudo por ser responsiva à restrição calórica. Através de uma abordagem do metabolismo energético e do estado de óxido-redução celular, nós temos buscado identificar quais são os fatores imprescindíveis para a exibição do aumento do tempo de vida cronológico dessa levedura. Nós verificamos que defeitos específicos na síntese de nicotinamida adenina dinucleotídeo aumentam a geração mitocondrial de espécies reativas de oxigênio pela enzima dihidrolipoil desidrogenase, porém não suprimem o aumento da do tempo de vida cronológico de S. cerevisiae. Por outro lado, os mutantes dessa levedura irreponsíveis à restrição calórica são aqueles que possuem defeitos no metabolismo aeróbico, mais especificamente na montagem da cadeia de transporte de elétrons. Também verificamos que diferentes mutações em enzimas do ciclo dos ácidos tricarboxílicos alteram a taxa de perda do DNA mitocondrial de S. cerevisiae numa forma dependente da concentração inicial de glicose nos meios de cultura e também do tempo de cultivo. Também observamos que a eficiência energética em S. cerevisiae cultivada sob restrição calórica é aumentada em relação à levedura cultivada em condição controle. Finalmente, também observamos que a morfologia mitocondrial é alterada pelo estado metabólico celular e se correlaciona com a geração de espécies reativas de oxigênio nesse organismo. Assim sendo, em conjunto, esses dados revelam importantes modificações metabólicas e no estado de óxido redução proporcionadas pela restrição calórica e como os fenótipos típicos do envelhecimento podem ser mitigados em S. cerevisiae, assim como quais são os fatores imprescindíveis para a resposta dessa levedura à restrição calórica.
Aging involves a progressive decline in metabolic efficiency of biological systems over time. Although it cannot be avoided, aging phenotypes are delayed in organisms undergoing caloric restriction, a dietary regimen consisting of a reduced availability of calories. The yeast Saccharomyces cerevisiae has proved to be an important model organism for studying important characteristics related to aging, and is responsive to caloric restriction. We sought to identify factors essential for increased chronological lifespan in yeast by investigating changes in energy metabolism and redox state. We found that defects in the synthesis of nicotinamide adenine dinucleotide increased mitochondrial generation of reactive oxygen species by the enzyme dihidrolipoil dehydrogenase, but did not suppress the increase in chronological life span. On the other hand, mutants of this yeast which do not respond to caloric restriction are those that have defects in aerobic metabolism, specifically in the assembly of the electron transport chain. We also found that different mutations in enzymes of the citric acid cycle alter the rate of loss of mitochondrial in a manner dependent on the initial concentration of glucose in culture media and culture time. We also observed that energy efficiency in S. cerevisiae grown under caloric restriction is increased compared to yeast grown under control conditions. Finally, we also observed that mitochondrial morphology is altered by the cellular metabolic state and correlates with the generation of reactive oxygen species in this organism. Thus, altogether, these data reveal significant changes in metabolism and redox state promoted by caloric restriction, how phenotypes typical of aging can be prevented in S. cerevisiae, as well as what factors are required for the response of yeast to caloric restriction.

Cette these decrit une methode originale de comparaison automatique d'experiences de transcriptome appliquee a la determination des causes du vieillissement de l'organisme eucaryote modele: Saccharomyces cerevisiae. Les experiences de transcriptome, qui peuvent etre realisees a l'aide de microarrays, permettent au biologiste d'etudier simultanement les variations globales d'expression de milliers de genes dans de nombreuses conditions experimentales. Ces experiences a grande echelle realisees a haut-debit generent une quantite importante de donnees. En consequence, les biologistes ont besoin d'outils informatiques pour les interpreter. L'utilisation de microarrays pour l'etude des causes du vieillissement chez la levure nous a permis d'identifier les outils informatiques necessaires a l'interpretation des donnees de transcriptome. Independemment de son interet biologique, le vieillissement est un probleme particulierement bien adapte a une double approche biologique et informatique. Nous avons analyse le transcriptome des cellules de levure au cours de leur vieillissement. Deux souches ont ete etudiees: l'une d'elles est une souche sauvage et l'autre un mutant, dna2-1, vieillissant prematurement, et dont la replication de l'ADN est deficiente. Plusieurs outils existants d'analyse de donnees microarrays nous ont permis d'interpreter nos resultats. Nous avons constate un stockage d'energie associe au vieillissement: les genes impliques dans la gluconeogenese, le cycle du glyoxylate, le metabolisme lipidique, et la production de glycogene sont actives dans les vieilles cellules. Nous avons egalement observe une reponse generalisee au stress connue sous le nom ESR (Reponse Environnementale au Stress) decrite auparavant dans d'autres etudes. De plus, Nous avons observe l'induction d'un ensemble de genes qui reparent l'ADN et connus sous le nom de “signature” de degradation de l'ADN. Plusieurs genes ayant un role dans la production d'energie sont actives dans la souche mutante dna2-1. La reponse au vieillissement observee chez dna2-1 est similaire a celle decrite dans un mutant dont la telomerase est disfonctionnelle (TDR). Elle est constituee par un ensemble de genes dont l'expression change durant la senescence cellulaire. Ces resultats suggerent indirectement que les cellules agees repondent a une instabilite du genome. Le travail d'interpretation de ces donnees nous a permis d'identifier des besoins informatiques specifiques parmis lesquels la necessite de comparer des experiences microarray de facon automatique. Pour cette raison, nous avons developpe, dans la deuxieme partie de cette these, une methode permettant de comparer des experiences microarray de facon automatique. Cette methode originale repose sur une ontologie pour experiences microarrays associee a un modele de cout evaluant le degre de compatibilite de deux experiences. La validation de cette methode a conduit au developpement d'une plateforme de test, Malako, qui associe les outils informatiques classiques d'analyses de donnees microarray a un systeme de comparaison automatique d'experiences microarray.

Nuolat kintanti aplinka yra pagrindinis veiksnys, kontroliuojantis mikroorganizmų augimą ir vystymąsi. Evoliucijos eigoje organizmuose išsivystė signalinės sistemos, gebančios sujungti aplinkos signalus su ląstelės transkripcijos, transliacijos ir kt. procesais. Viena iš tokių universalių signalinių sistemų yra Ras/PKA signalinis kelias. Ši sistema leidžia mielių ląstelėms reaguoti į aplinkoje esančius maisto medžiagų šaltinius ir įvairius stresinius veiksnius. Vienas iš pagrindinių aplinkos signalų, įtakojančių ląstelių fiziologiją, yra aplinkos pH. Mielių ląstelėse į aplinkos pH reaguoja Rim101 signalinė sistema, kuri kartu su Ras/PKA reguliuoja tokius procesus kaip sporuliacija ir pseudohifinis augimas. Ekstraląstelinės terpės rūgštėjimas lemia viduląstelinį rūgštėjimą, o tai sukelia mielių ląstelėms stresą ir taip aktyvina Ras/PKA signalinį kelią. Streso poveikio pasekmė yra ląstelių augimo sulėtėjimas, gyvenimo trukmės trumpėjimas, augimo sustabdymas ar netgi žūtis. Pastaraisiais metais parodyta, kad būtent Ras/PKA signalinis kelias reguliuoja ląstelės senėjimo procesą kaip atsaką į aplinkos pH. Taigi mielių, kaip modelinio organizmo, panaudojimas žmogaus genų, reguliuojančių ląstelės senėjimą ir žūtį, tyrimuose vis plečiasi. Disertacinio darbo metu buvo įvertintas izogeninių mielių kamienų turinčių Ras/PKA signalinio kelio komponentų mutacijas augimas bei mitybinės terpės pH kitimas. Nustatyta, kad mutacijos Ras/PKA signalinio kelio komponentuose lemia skirtingą... [toliau žr. visą tekstą]
Constantly changing environment is the major factor controlling the growth and development of the microorganisms. For quick generation of the cell response, information about changes in the cell environment is rapidly transmitted to the inner molecules of the cell. During the course of evolution the cells have developed signaling systems that are able to combine extracellular signals with the inner processes, as transcription, translation etc. One of the universal signaling systems is Ras/PKA signal transduction pathway. This system helps cells recognize the nutrient sources present in the growth medium.Environmental pH is one of the main factors influencing the growth, physiology and differentiation of yeast. In Saccharomyces cerevisiae, response to pH is determined by the RIM101 pathway. Recently, it was shown, that Ras/PKA signal transduction pathway regulates cell aging as response to environmental pH. Many aging and apoptosis features are conserved between yeast and multicellular microorganisms, and this makes them perfect model organisms. Yeasts are also suitable also for acidosis related disease studies. Evaluation of the cell growth and medium acidification of isogenic strains containing mutation in the members of Ras/PKA signal transduction pathway was performed. Mutations in these genes cause changes in metabolic activity of the cell.Members of the Ras/PKA signal transduction pathway participate in regulation of cell viability and lifespan during the natural... [to full text]

La levure du boulanger a une durée de vie réplicative limitée : chaque cellule mère produit un nombre fini de filles avant de mourir. Cette entrée en sénescence est supposée notamment résulter de l’accumulation d’agrégats de protéines endommagées au sein de la cellule mère. La rétention des agrégats au sein des mères permettrait de produire des filles rajeunies. Cependant, ce mécanisme de ségrégation asymétrique reste controversé, en partie car il est complexe de suivre la dynamique des agrégats protéiques in vivo. Nous avons développé une méthodologie pour suivre la formation des agrégats protéiques au cours d’un choc thermique au sein de cellules uniques. Associées à un modèle simple d’agrégation, nos données montrent que l’asymétrie peut être expliquée quantitativement par la croissance polarisée du bourgeon. De plus, des expériences de vieillissement réplicatif réalisées au sein d’une puce microfluidique suggérèrent que l’accumulation d’agrégats dans les mères pourrait induire la mort, mais seulement lorsque les cellules subissent un stress protéotoxique. Cette étude apporte donc un éclairage nouveau sur les mécanismes d’héritabilité des agrégats protéiques et leur contribution au vieillissement réplicatif chez la levure du boulanger
Budding yeast cells have a limited replicative life span: a mother cell can produce a limited number of daughter cells before it dies. It has been proposed that the progressive accumulation of aggregates of damaged proteins within mother cells drives entry into senescence. The retention of such damages by mothers is thought to permit the daughter lineage rejuvenation. Yet the mechanism of their asymmetrical segregation is still controversial, in part because of difficulties inherent to the tracking of the dynamics of protein aggregates in vivo. In this context, we have developed a single cell methodology to track the formation of protein aggregates upon heat shock in single cells. In combination with a simple computational model of aggregation, our data reveal that the asymmetry can be explained quantitatively by the polarized growth of the bud. In addition, replicative aging experiments performed in a microfluidic device suggest that the accumulation and retention of protein aggregates in mothers may be responsible for cell death, but only under particular proteotoxic stress. Therefore, this study sheds new light on the mechanism of inheritance of protein aggregates and its contribution to replicative aging in budding yeast

La biodiversité fongique interspécifique (Illumina Mi-Seq) et la dynamique des espèces Saccharomyces cerevisiae et Brettanomyces bruxellensis ont été étudiées au sein d’une nouvelle cuverie et/ou dans 3 caves d’élevage, plus particulièrement sur le sol, les murs, le matériel vinaire et l’extérieur des fûts. Dans la nouvelle cuverie, un consortium fongique (levures et moisissures) de départ est déjà présent sur tous les environnments étudiés avant l’arrivée de la première vendange. Ce consortium est constitué de genres tels que Aureobasidium, Alternaria, Didymella et Filobasidium. Ces genres qui persistent pendant deux millésimes, ne sont pas spécifiques de l’environnement de la cuverie et semblent être adaptés à tous les environnements naturels ou anthropiques au regard de leur caractère ubiquiste. Le consortium de départ est enrichi par des genres œnologiques (exemple : Hanseniaspora, Saccharomyces) qui sont introduits dans la cuverie soit par les vendanges, soit par des transferts potentiels entre les différents environnements de la cuverie. Cependant, ces genres ne semblent pas persister ou s’implanter probablement dû à leur faible adaptation aux conditions stressantes de l’environnement de la cuverie. La dynamique de la flore indigène S. cerevisiae dans la nouvelle cuverie a été également étudiée. Aucun isolat appartenant à cette espèce n’a été retrouvé avant l'arrivée de la première vendange confirmant que cette espèce n’est pas spécifique de l'environnement de la cuverie et que sa présence est en lien avec l'activité des fermentations alcooliques. Cependant, les résultats obtenus suggèrent une colonisation potentielle de l’environnement de la nouvelle cuverie par certaines souches de S. cerevisiae. Ces souches dites « colonisatrices » ont présenté une capacité plus élevée à former des biofilms comparée à celle de souches non implantées. Cette étude met en évidence l’importance de l’environnement de la cuverie qui constitue une véritable niche écologique pour les populations fongiques capables de s’implanter au cours du processus de vinification. Dans l’environnement des 3 caves d’élevage, le matériel vinaire et l’extérieur des fûts (en contact direct avec le vin) sont les environnements qui semblent favorables au développement et à l’installation des populations microbiennes cultivables (levures totales et bactéries lactiques) et des microorganismes d’altération (bactéries acétiques et B. bruxellensis), contrairement au sol et aux murs où des populations faibles ont été trouvées. Des souches récurrentes de B. bruxellensis ont été retrouvées sur le matériel et sur l’extérieur des fûts et pourraient être à l’origine de la contamination de vins au cours de l’élevage. Ces souches récurrentes présentent des capacités de formation de biofilms et de résistance plus importantes qui pourraient expliquer la persistance de B. bruxellensis dans des caves d’élevage. Ces résultats soulignent l’importance du nettoyage du matériel vinaire et du suivi microbien régulier des vins au cours de l’élevage afin de limiter les contaminations
The interspecific fungal biodiversity (Illumina Mi-Seq) and the dynamics of Saccharomyces cerevisiae and Brettanomyces bruxellensis species were studied in a new winery and/or in 3 aging cellars, more specifically on the floor, the walls, the equipment and the outside of the barrels. In the new winery, an initial fungal consortium (yeasts and molds) is already present on all the winery environments before the arrival of the first harvest. This consortium consists of fungal genera such as Aureobasidium, Alternaria, Didymella and Filobasidium. These genera, that persist during two vintages, are not specific to the winery environment and seem to be adapted to natural or anthropic environments due to their ubiquitous character. The initial consortium is enriched by oenological genera (e.g. Hanseniaspora, Saccharomyces) that are introduced into the winery either by the harvest or by potential transfers between the different environments of the winery. However, these genera do not seem to persist or establish due to their low adaptation to the stressful conditions of the winery environment. The dynamics of wild S. cerevisiae in the new winery was also studied. No isolates belonging to this species were found before the arrival of the first harvest, confirming that this species is not specific to the winery environment and its presence is related to the alcoholic fermentation activity. However, the results obtained suggest a potential colonization of the new winery environment by certain S. cerevisiae strains. These so-called « colonizing » strains showed a higher capacity to form biofilms compared to non-implanted strains. This study highlights the importance of the winery environment that constitutes a true ecological niche for fungal populations capable of implantation during the winemaking process. In the environment of the 3 aging cellars, the equipment and the outside of the barrels (in direct contact with wine) are the environments that seem to be favorable for the development and installation of cultivable microbial populations (yeasts and lactic acid bacteria) and spoilage microorganisms (acetic acid bacteria and B. bruxellensis), unlike the floor and the walls where low microbial populations were found. Recurrent strains of B. bruxellensis have been found on both the equipment and the outside of the barrels and could be the cause of wine contamination during aging. These recurrent strains showed greater biofilm formation and resistance capacities which could explain the persistence of B. bruxellensis in aging cellars. These results highlight the importance of equipment cleaning and the regular monitoring of wines during aging in order to limit the contaminations

In diversi organismi, dal lievito ai mammiferi, è stato osservato che la riduzione dell'introito calorico aumenta la sopravvivenza e protegge dall'insorgenza di numerose patologie associate all'età. Il meccanismo molecolare tramite cui questo effetto si realizza non è ancora chiaro. Questa ricerca mette in evidenza il ruolo di singoli nutrienti nella sensibilizzazione agli stress e nella promozione dell'invecchiamento, identifica in particolare tre specifici aminoacidi che agiscono da segnalatori pro-aging e descrive le vie molecolari attraverso cui tali molecole agiscono. Poiché i pathway di segnalazione coinvolti nell'invecchiamento e nella risposta ai nutrienti sono significativamente conservati in tutti gli eucarioti, i risultati ottenuti nel sistema modello Saccharomyces cerevisiae suggeriscono un possibile intervento per la modulazione dell'invecchiamento anche nei mammiferi.
Calorie restriction (CR), but also the restriction of specific components of the diet, has been known to affect longevity. However, the understanding of how each component of the macronutrients affects longevity and stress resistance is poorly understood. This study describes how each amino acid and glucose cooperate to activate cell sensitizing pathways and promote aging, identifies specific amino acids that affect cellular protection and aging and shows how different pathways mediate these pro-aging effects. Because of the conserved pro-aging role of orthologs of many of the genes in the signaling network involved, these results are likely to serve as a foundation for the elucidation of similar nutrient-dependent pro-aging mechanisms in mammals.

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.
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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.

Proteins in Escherichia coli were compared in terms of essentiality, centrality, and conservation. The hypotheses of this study are: for proteins in Escherichia coli, (1) there is a positive, measureable correlation between protein conservation and essentiality, (2) there is a positive relationship between conservation and degree centrality, and (3) essentiality and centrality also have a positive correlation. The third hypothesis was supported by a moderate correlation, the first with a weak correlation, and the second hypotheis was not supported. When proteins that did not map to orthologous groups and proteins that had no interactions were removed, the relationship between essentality and conservation increased to a strong relationship. This was due to the effect of proteins that did not map to orthologus groups and suggests that protein orthology represented by clusters of orthologus groups does not accurately dipict protein conservation among the species studied.

La recherche présentée dans cette thèse concerne différents sujets dans le domaine de la biomathématique. J’aborde diverses questions en biologie (et liées aux systèmes complexes) avec des méthodes mathématiques et numériques. Ces questions sont les suivantes: (i) Les processus passifs sont-ils suffisants pour justifier la distribution asymétrique des protéines endommagées pendant et après la cytokinèse de la levure? (ii) Quels processus sont à l’origine des schémas complexes d’expansion de l’amyloïde bêta dans le cerveau des patients atteints de la maladie d’Alzheimer? (iii) Qu’y a-t-il derrière la dichotomie de ‘clusters’ vs. ‘cline-like’ dans les modèles d’évolution le long de gradients environnementaux? (iv) Comment cette dichotomie affecte-t-elle la dynamique spatiale des invasions? (v) Comment la multi-stabilité se manifeste-t-elle dans ces modèles? Ces questions sont abordées (à différentes échelles, certaines totalement et certaines partiellement) avec différentes méthodes théoriques. Les résultats devraient permettre de mieux comprendre les processus biologiques analysés et de motiver la poursuite des travaux expérimentaux et empiriques susceptibles de contribuer à résoudre les incertitudes persistantes
The research presented in this thesis concerns different topics in the field of Biomathematics. I address diverse questions arising in biology (and related to complex systems) with mathematical and numerical methods. These questions are: (i) Are passive-processes enough to justify the asymmetric distribution of damaged proteins during and after yeast cytokinesis? (ii) What processes are behind the complex patterns of expansion of Amyloid beta in the brains of patients with Alzheimer’s disease? (iii) What is behind the clustering and cline-like dichotomy in models of evolution along environmental gradients? (iv) How does this dichotomy affect the spatial dynamics of invasions and range expansions? (v) How does multi-stability manifest in these models? These questions are approached (at different scales, some fully and some partially) with different theoretical methods. Results are expected to shed light on the biological processes analyzed and to motivate further experimental and empirical work which can help solve lingering uncertainties

碩士
國立清華大學
生物科技研究所
99
粒線體融合與分裂的動態調控影響其於細胞中的功能，而在老化所導致的神經退化性疾病中，已經有很多研究顯示與粒線體的功能喪失及形態調控有直接關聯。故而本篇論文是從細胞老化的角度切入，釐清粒線體融合、分裂之動態平衡與老化的關係。在本篇研究中，採用了釀酒酵母(Saccharomyces cerevisiae)此種被廣泛利用的模式生物來作為本研究之對象，以修飾過的生物素(Biotin)標記在細胞表面，利用其與卵白素(Streptavidin)之高度親和力，再利用其與已鍵結上卵白素的微鐵珠 (micro-magnetic beads)作用，即可使用強力磁鐵達到分離已標記之原始細胞的目的。藉由此技術，可以將出芽生長分裂代數較多的酵母菌從培養的族群中分離出來，再利用已送入含有可標記粒線體綠螢光蛋白(Green-Fluorescent Protein)之質體，便可以觀察酵母菌因粒線體融合與分裂平衡的傾向所導致的不同粒線體形態。在細胞老化的層次上，可發現生長代數較多之野生種酵母菌顯現出較多粒線體碎裂的特徵，此現象在基因剔除粒線體分裂機制上必要的FIS1 以及DNM1 菌株中即不復出現；在mRNA 的表現量而言，FIS1 以及DNM1基因在分裂代數較多之酵母菌的樣品裡表現量也明顯提高，證實粒線體的動態調控機制參與了老化細胞裡粒線體分裂多於融合的過程。我們的實驗驗證了以酵母菌為模式生物來研究細胞層次老化的過程是可行的。而我們的結果更指出粒線體動態平衡的調控機制 與細胞老化是有相關的。

碩士
國立臺灣大學
食品科技研究所
102
Replication checkpoints serve as control mechanisms that ensure the fidelity of the replicating genome in eukaryotes. It was previously reported that a mouse model of checkpoint (ATR) deficiency exhibited replicative stress during embryogenesis, which resulted in premature aging. To further understand the role of the replication checkpoint in cellular aging, we took advantage of the ATR homologous gene MEC1 and its hypostatic gene RAD53 in yeast. We determined the replicative lifespans (RLS) of the hypomorphic mec1-100 and rad53-11 mutants; the life span of these mutants was decreased significantly as compared to the wild type strain. Therefore, we examined the genes and pathways that modulate RLS to establish the relevance of checkpoint function in cellular aging. We found that calorie restriction (CR) required the checkpoint kinase function of Mec1 and Rad53 to extend life span. We further demonstrated that checkpoint function is required to protect the stability of the rDNA array, mating loci and telomeres but this is independent of the protection by sirtuins Sir2. Moreover, while a defect in chromatin assembly (the asf1Δ mutation) did not further decrease the life span of mec1-100, it did decrease that of rad53-11. Furthermore, an increased histone supply (hir3Δ) extended the lifespan of mec1-100 cells, but not those of rad53-11. Deletion of histone acetyltransferase SAS2 results in tighter packaging of telomere and life span extension; surprisingly, mec1-100 and rad53-11 mitigate the life span extension effect of sas2Δ with the decrease being greatest in mec1-100. Collectively, our results suggest that the kinase function of Mec1 and Rad53 mediates the effect of calorie restriction on replicative life span extension. The genetic analyses reveal that the checkpoint pathway may contribute to preserving chromatin integrity in both heterochromatin (rDNA and telomere) and euchromatin (active chromatin). We propose that loss of function of the checkpoint kinase may cause aging due to failure to respond replicative stress, which increases of sporadic damage to the genome when chromatin is improperly assembled.

The aging process is unforgiving, targeting a decline in cellular function. Originally, the actin cytoskeleton has not been defined as a hallmark of aging biology, however, numerous studies provide evidence that actin cytoskeleton integrity is declining with age. Mammalian cells express an aged-linked decline in their actin dynamics, consequently defecting their migratory movements, immunological synapse formation, and phagocytosis. Overall, suggesting actin integrity is specifically targeted by aging. Despite the substantial evidence, the underlying mechanism remains elusive, however, current research indicates actin stability as a possible mechanistic aging target. Therefore, our research goal is to further elucidate the mechanism for actin cytoskeleton aging biology in a streamlined model organism, budding yeast, Saccharomyces cerevisiae. Here, we use aging enrichment protocols, streptavidin affinity purification, to isolate a population of older cells to examine any changes in the actin cytoskeleton with age. With an isolated aging population, we analyzed the actin cytoskeleton by testing its stability against a destabilizing drug, Lat-A, and morphology with imaging analysis. We find significant age-associated changes in the actin cytoskeleton, which we later conclude may be a consequence of the age-linked decline in the actin stability that we identified in an aging cell. Additionally, we uncovered a perplexing finding that there is an age-linked decline in actin cable bundling. How actin stability effects actin cable bundling, remains to be determined. However, our actin stability model was further supported by our research characterizing an open reading frame, YKL075C, as a novel actin cable regulatory protein whose deletion: increased actin cable stability, abundance, and mitochondrial quality to extend the replicative lifespan. Upon further insight into YKL075C underlying mechanism, we find YKL075C effects on actin stability and morphology is dependent on alterations in branched-chain amino acid (BCAA) metabolism. Overall, our research discovered a novel actin regulatory protein, Ykl075cp, whose actin function is dependent on BCAA homeostasis, and deleting specifically YKL075C reduces BCAA levels that subsequently increases actin cable stability and abundance to enhance mitochondrial quality and extends longevity.

碩士
國立臺灣大學
食品科技研究所
102
Skin is the most visible organ in the aging process. The characteristics of skin aging include wrinkling, sallowness, laxity and dryness. Aging may result from both the passage of time (intrinsic aging) and from ultraviolet light exposure (extrinsic aging). Past research showed that sake (rice wine) has positive effect on skin, including prevention of UV damage, reduction of matrix metalloproteinase-1 (MMP-1) expression and increase of typeⅠprocollagen synthesis. However, rice wine production requires tedious procedures and longtime processing. In this study, we attempt to simulate the fermentation conditions of rice wine production but with much shorter time, and hope that the fermentation product also possess anti-aging effect on skin. Since both peptide and carbohydrate are all considered to possess anti-aging effect for skin, other than rice, soybean meal was also used as substrates for fermentation. The fermentation experiment was divided into two parts. First, the optimal composition and fermentation time of koji prepared by Aspergillus oryzae were determined. Then, two kind of Saccharomyces cerevisiae were added to complete the fermentation process. The human skin fibroblast (CCD-966SK) was used as in vitro model to evaluate the bioactivity of the fermentation product. Results showed that the dose below 50μg/mL would not exert cytotoxic effect on the human skin fibroblast. The samples derived from ethanol extract of fermented product which fermented by Aspergillus oryzae and Saccharomyces cerevisiae YCL1087 could significantly reduce about 20％ of the accumulation of UV-induced ROS production in skin fibroblast. But the samples with higher concentration could inhibit typeⅠprocollagen synthesis. If treated with low concentration of the product fermented with Aspergillus oryzae for a shorter time, compared with control group, there was no significant effect on typeⅠprocollagen synthesis. Therefore, this fermented extract could scavenge ROS production significantly and reduce UV-induced oxidative stresses in skin cells. But this product is not a comprehensive skin care product, because of they cannot stimulate typeⅠprocollagen synthesis.

Both an intuitive observation and maybe the most mysterious process of biology, aging describes the progressive deterioration of cellular functions with time. Asymmetric cell divisions stand at the center of ability to reset age in offspring and for stem cells to self-renew. This requires the asymmetric segregation of age-determinants, many of which have been identified in the budding yeast Saccharomyces cerevisiae. We here use budding yeast to explore fundamental aspects underlying the asymmetric inheritance of mitochondria and the concurrent rejuvenation of daughter cells. We show that in addition to the preferential inheritance of high-functioning mitochondria to daughter cells, a distinct population of high-quality organelles must also be retained within the mother cell. We find that both physical retention and qualitative maintenance of a distinct mitochondrial population at the mother cell tip depends on Mitochondrial F-box protein (Mfb1p) and that MFB1-deletion leads to premature aging. Our findings outline a critical balance between the need for daughter cell rejuvenation and the requirement to conserve replicative potential within the mother cell. The particular mechanism by which Mfb1p functions further lead us to uncover a critical role of globally maintained cellular polarity in form of an axial budding pattern in lifespan regulation, the functional significance of which thus far remained essentially unexplored. We also find that the asymmetric localization of Mfb1p depends on potentially novel structures of the actin cytoskeleton and the loss of Mfb1p-polarization with age may accurately predict remaining cellular lifespan.

碩士
國立臺灣大學
食品科技研究所
102
Mismatch repair (MMR) is a DNA repair system which is critical for the maintenance of genome stability. Defects in mismatch repair have been linked to colorectal and sporadic cancers. Calorie restriction (CR) has been shown to extend life span and increases stress resistance via TOR/SCH9 and RAS signaling pathways in various organisms. We have found that CR containing 0.5% glucose compared to normal treatment (2.0% glucose) can extend life span and promote HOM3 gene stability in MMR-defected cells during aging process in yeast previously. Here, we demonstrate tor1&;#8710; can mimic CR condition and extend life span of msh2&;#8710; and msh3&;#8710; mutants with low mutation frequency. We also found the levels of reactive oxygen species (ROS) were significantly increased during aging and tor1&;#8710; dramatically reduced the levels of ROS. However, tor1&;#8710; could not completely reverse mutation rates in msh2&;#8710;msh3&;#8710; mutants. According to these, we believe TOR1 gene can only partially regulates genome stability in MMR defected cells during aging. Whether RAS also affects the genome stability in MMR defected cells during aging needs to be further investigated.

碩士
國立臺灣大學
食品科技研究所
103
Calorie restriction (CR) is able to reduce cancer progression and extend life span in various organisms. Age-related decline of DNA repair system such as mismatch repair (MMR) can reverse by CR. Defects in MMR have been linked to colorectal and sporadic cancers. Our previous results have demonstrated that CR can extend life span and maintain genome stability in MMR-defected cells during aging, but the mechanisms are poorly understood. Therefore, we suggest base excision repair (BER) which has been proven can be up-regulated by CR, is responsible to maintain genome stability in MMR-defected cells. However, we found that CR still able to extend life span and reduce mutations in MMR/BER -defected cells. And, CR has no effect on BER gene expression in MMR-defected cells during aging. To investigate the mechanisms responsible for CR to maintain genome stability in MMR-defected cells, the whole genome profile by performing RNA-seq has been analyzed. The most significant influenced genes which regulated by CR in MMR-defected cells are fatty acid metabolism, glyoxylate and dicarboxylate metabolism pathways. Besides, we demonstrate by using hydroxyurea to slow down cell cycle progression can maintain genome stability without CR in MMR-defected cells during aging, 50mM HU can reduce mutation by patching assay, however, HU also reduce life span in cells. Furthermore, we found that phosphorylation levels of H2A in CR cells are higher than non-CR cells. H2A phosphorylation is a marker of double strand break repair (DSBR). Further studies are needed to investigate whether CR would beneficial to MMR-defected cells through affecting cell cycle and DSBR pathway.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
102
Abstract – Part I There are two methods to access the lifespan of yeast. One is called replicative lifespan (RLS) defined by the number of daughter cells produced by single mother cell. The other method called chronological lifespan (CLS) defined by how long yeasts survived in liquid culture. Nowadays, it is proposed that the cellular accumulation of reactive oxygen species (ROS) might reduce the lifespan of organism. According to previous studies, under the treatment of small natural molecule like hesperidin, the lifespan of yeast was extended by reducing oxidative pressure. Genus Ganoderma belong to kingdom Fungi, and show obvious effect of antioxidant activities. However, there is still no report clearly stated that Genus Ganoderma could extend the lifespan of an organism by the antioxidant effect. In this study, we analyzed EEGC extracted from Ganoderma colossum to understand its anti-aging effect and mechanism of yeasts. At first, we established the treatment of 10 mM N-acetylcysteine (NAC) as the positive control of RLS. We found that 2 mg/ml EEGC could lower the growth rate of yeasts. In addition, it was hard to analyze effect of >1 mg/ml EEGC because it revealed turbidity. It was observed that 0.5 mg/ml EEGC could significantly extent RLS of yeasts but not CLS. Moreover, treatment of 0.2, 0.5, or 1 mg/ml EEGC all showed antioxidant effect to reduce the accumulation of ROS levels induced by H2O2. Our data also showed that EEGC could significantly decreased SOD1、SOD2、SIR2 mRNA levels in cells. The SIRT1 activity assay implied that 0.5 or 1 mg/ml EEGC may enhance Sir2p activity in yeast. Altogether, we demonstrated that EEGC may extend yeast replicative lifespan, and could act as an antioxidant in yeast. The anti-aging mechanism of EEGC and the correlation of Sir2p activity should be further investigated. Abstract – Part II Saccharomyces cerevisiae is widely distributed in nature, and has become increasingly important in biotechnology and food industry. However, numerous cases of clinical infection caused by S. cerevisiae have been reported in recent years, considering S. cerevisiae as an emerging opportunistic pathogen. Cell wall is the first surface of the cell to encounter stresses from host defenses and environmental stresses. In addition, cell wall is responsible for yeast viability and adhesion ability to host. Our previous analysis of the composition of cell wall protein between clinical isolates and laboratory strains found that cell wall proteins Scw10p, Hsp150p, and Pst1p expressed at higher levels in clinical isolates. To clarify the role of Hsp150p in clinical isolates, we constructed three hsp150 deletion strains in clinical isolate background, and analyze the impact in cell wall integrity, adhesion ability, cell surface hydrophobicity, and virulence. We found that hsp150 deletion strains grew slower than parental clinical isolates under high dosage of Calcofluor white or Congo red. Secondly, hsp150 deletion in clinical isolates did not affect the adhesion ability or cell surface hydrophobicity. Lastly, we found that hsp150 deletion strains stimulated mouse macrophage cell lines to secret higher levels of TNF-α than parental clinical isolates, implying that deletion of Hsp150p may interrupt cell wall integrity and expose unknown virulence factors to macrophages. In this study, we found that Hsp150p may be responsible for, at least in part, the integrity of cell wall, and may play a role in yeast virulence.

A variety of genes that influence aging have been identified in a broad selection of organisms including Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worms), Drosophila (fruit flies), Macaca Mulatta (rhesus monkeys), and even Homo sapiens. Many of these genes, such the TOR’s, FOXO’s, AKT’s, and S6K’s are conserved across different organisms. All of these genes participate in nutrient sensing networks. Other conserved genetic networks may similarly affect lifespan. In this thesis, I explored genes from an iron metabolism family and a heat shock protein (HSP) gene family that have been identified, but not confirmed, to influence lifespan. Yeast is a reliable model for mitotic (replicative) aging. Using yeast, I tested whether the FET-genes, encoding a family of iron importer-related genes, are required for mitotic lifespan. I also tested whether another family of genes, the yeast SSA HSP70- encoding genes, related to mammalian HSP70s, influence mitotic aging. I primarily used the replicative lifespan (RLS) assay, in which I measured the mitotic capacity of multiple FET and SSA yeast mutants. I hypothesize that aging occurs when iron transport is misregulated, which may lead to an over-reliance on HSPs for lifespan maintenance. The results presented in this thesis support the hypothesis. First, FET3 was primarily involved in lifespan maintenance under normal conditions (2% glucose), while FET5 was primarily involved in the cellular lifespan extension characteristic of caloric restriction (0.01% glucose), a known anti-aging intervention. In addition, SSA2 appeared to facilitate lifespan maintenance in the absence of FET4, while the presence of SSA1 limited lifespan length. That the aging genes identified in this study are involved in iron metabolism or heat stress suggests that protein aggregation or reactive oxidative species production are common processes through which these genes interact.

Forkhead box (Fox) transcription factors have a conserved function in regulating lifespan and onset of age related disease in organisms from worms to mammals. Key functions in this process are the regulation of the cell cycle, oxidative stress response, and apoptosis. A complex post-translational code from nutrient, growth factor, and stress induced signals regulates Fox activity, target specificity, stability, and subcellular localization; however, many of the Fox mechanisms and targets responsible for regulating lifespan remain elusive. The budding yeast, Saccharomyces cerevisiae, is a powerful model for unravelling the genetic mechanism and pathways. Yeast encodes four Fox transcription factors, Fkh1, Fkh2, Fhl1 and Hcm1, and their roles in aging are only recently being examined. In this study, we utilized the chronological lifespan and oxidative stress assays, to explore evolutionary conservation of lifespan regulation in two of the yeast Fox orthologs, FKH1 and FKH2. We observed that deletion of both FKH genes in S. cerevisiae, impedes normal lifespan and stress resistance. Furthermore, fkh1Δ fkh2Δ cells were found to be non-responsive to caloric restriction, an intervention that extends lifespan from yeast to mammals. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress resistance. Additionally, we show the Anaphase-Promoting Complex (APC) genetically interacts with the FKHs, likely functioning in a linear pathway under normal conditions, as fkh1Δ fkh2Δ post-mitotic survival defect is epistatic to that observed in apc5CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5CA fkh1Δ fkh2Δ beyond either apc5CA or fkh1Δ fkh2Δ. Finally, we observed that both the FKHs and APC genetically interact with nutrient-responsive lifespan-regulating kinase encoding genes SCH9 and TOR1. This study establishes that the yeast FKHs play a role as regulators of lifespan in yeast and identifies the APC as a novel component of this mechanism. We speculate this involves combined regulation of stress response, genomic stability, and cell cycle.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.