Healthspan describes the length of time an individual lives without disability or chronic disease. Characteristic to aging is the risk for the onset of both through a progressive accumulation of deficits in normal physiologic function. In the past, the declines associated with aging were simply accepted as inevitable. Today, longevity research has undergone a meteoric rise in popularity. This is due to several landmark studies demonstrating that what was once thought of as inevitable has potential to be delayed. This thesis aims to consolidate current theories of biochemical processes believed to underlie aging, and explore their interconnections. Furthermore, current pharmaceutical and lifestyle interventions being studied to promote longevity and target these specific pathways will be analyzed for safety and practicality for use in a primary care setting. Through a shift from symptom-based care to personalized preventative care, the goal is to maximize function into older age and empower individuals to live life to the fullest well beyond what was previously imagined.

Abiotic stresses are constantly rising and pose a very high risk to global agricultural productivity and food security. Some plants have evolved several innate pathways for defense against these stresses. Hence, understanding stress signaling pathways can help develop crop plants with higher stress tolerance. The salicylic acid-mediated signaling pathway is important in plants experiencing biotic and abiotic stresses. In previous studies, SABP2-Interacting Protein (SIP-428) has been shown to be a negative regular of plant growth under abiotic stress. This study aimed to investigate the roles of SIP-428 in the ROS signaling of tobacco plants. We investigated transgenic RNAi-silenced lines of SIP-428 and wild-type tobacco plants for the activities of guaiacol peroxidase and catalase enzymes in Mannitol and NaCl-stressed plants for 7 and 14 days. Our results showed that SIP-428 plays a significant role in ROS signaling in Mannitol and NaCl-stressed plants via the activities of guaiacol peroxidase.

The sirtuins are a family of NAD⁺-dependent deacetylase enzymes which are implicated in various illnesses including cancer and neurodegenerative diseases. Part I of this thesis describes the synthesis and biological evaluation of inhibitors of the SIRT1 and SIRT2 isoforms of this important family of enzymes. Chapter 1 gives an overview of sirtuin biology and the physiological roles of these enzymes. In particular the link between SIRT1 and cancer and SIRT2 and its role in the onset of neurodegenerative diseases is discussed. A review of the most potent and selective inhibitors of SIRT1 and SIRT2 is given including an introduction to the tenovin and cambinol classes of inhibitor. Chapter 2 describes various issues relating to the structure of the important chemical tool tenovin-6. The synthesis of analogues to improve the solubility, determine the preferred conformation and verify the products of metabolism of tenovin-6 is presented including their evaluation by in vitro and in cell methods. Part II of this chapter reports the design and use of a ¹H NMR method used to monitor the sirtuin-mediated deacetylation reaction. This was particularly relevant due to concerns raised about the possibility of false positive results obtained with the commercially available assay kit commonly used by the sirtuin community. This new ¹H NMR method was used to validate the inhibition of SIRT2 by tenovin-6. Chapter 3 describes the parallel synthesis and evaluation of tenovin analogues as inhibitors of SIRT1 and SIRT2. This study identified that replacement of the t-butyl substituent of tenovin-6 with the 3,5-dihalogen-4-alkoxy substitution pattern led to a variety of analogues having SIRT2 selectivity. As well as the collection of valuable SAR data, in cell data is also presented for the analogues. Chapter 4 provides attempts to rationalise the SAR data collected in Chapters 2 and 3 through a computational study. The molecular docking software GOLD was used to predict the binding site of the tenovin scaffold and hence rationalise the observed potencies of various analogues. Chapter 5 reports the synthesis and biological evaluation of triazole and cambinol analogues as SIRT1 and SIRT2 inhibitors. Part I details the synthesis and in vitro testing of a series of ring constrained tenovin analogues based on the 1,4-disubstituted triazole using click chemistry. A series of 1,5-disubstituted analogues were also synthesised. Part II describes the synthesis of S-alkylated cambinol analogues and the effect of N3-methylation upon activity and selectivity towards SIRT1. Part II of this thesis details the synthesis and biological testing of novel potent allosteric inhibitors of RmlA. RmlA is the first enzyme in the L-rhamnose biosynthetic pathway in bacteria. L-rhamnose is an important component of the bacterial cell wall and as such RmlA is therefore an important target in the discovery of novel anti-bacterial compounds. Chapter 7 provides an overview of the RmlA enzyme including its role in L-rhamnose biosynthesis and why it is an attractive target for anti-bacterial drug discovery. No small molecule inhibitors of RmlA have been reported previously. Chapter 8 describes the design and synthesis of pyrimidine-2,4-dione analogues as novel allosteric inhibitors of RmlA. SAR data is generated and rationalised by X-ray crystallographic techniques to study the structures of complexes of RmlA with various analogues. Analogues were also tested for their ability to inhibit the growth of the important human pathogen Mycobacterium tuberculosis.

No description available.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Sirtuins (SIRT) regulate the transcription of various genes involved in the development of diet-induced obesity and chronic disease; however, it is unknown how they change acutely following a high-fat meal. The purpose of this study was to determine the effect of a high-fat meal (65% kcals/d; 85% fat recommendation), on SIRT1-7 mRNA expression in blood leukocytes at 1, 3, and 5-h post-prandial. Men and women (N=24) reported to the lab following an overnight fast (>12H). Total RNA was isolated and reverse transcribed prior to using a Taqman qPCR technique with 18S rRNA as a normalizer to determine SIRT1-7 mRNA expression. An additional aliquot of serum was used to measure triglycerides. Data was analyzed using a RM ANOVA with P<0.05. Triglycerides (P<0.001; 124%) peaked at 3-h. SIRT 1 (P=0.004; 70%), and SIRT 6 (P=0.017; 53%) decreased expression at 3-h. SIRT4 (P=0.024) peaked at 5H relative to baseline (70%) and 3-h (68%). To our knowledge, this is the first study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent in a pattern that mirrors changes in serum triglycerides. Decrease in expression of SIRT1 and SIRT6 combined with an increased SIRT4 would be consistent with an increase in metabolic disease risk if maintained on a chronic basis.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 4, 2010). Includes bibliographical references.

Dietary restriction (DR), or the reduction of food consumption without malnutrition, is the most conserved method of preventing or reversing age associated diseases. It is also the most conserved method of increasing lifespan, across model organisms. We developed a robust liquid DR method for C. elegans, to investigate requirements for stress defense and NAD\(^+\)-associated mechanisms in mediating DR induced longevity. We found that DR lifespan extension depended upon stress defense regulators that act downstream of TORC1 and growth pathways, as well as SIR-2.1/SIRT1 and the NAD\(^+\) salvage pathway enzyme PNC-1. Surprisingly, PNC-1 was not required for improvement in two measures of healthspan, or the period of life spent free from disease. This suggests that the genetic regulation of DR effects can be uncoupled from one another, and that increased healthspan does not always indicate increased lifespan.

Cette thèse porte sur le métabolisme du parasite Schistosoma mansoni. Au cours de son cycle de vie, S. mansoni subit des changements drastiques de l'environnement qui comprennent les étapes de libre-vie dans l'eau et l’endoparasitisme en invertébrés hôtes (à savoir les escargots) et en vertébrés hôtes (à savoir les mammifères). En conséquence, ce parasite montre une plasticité métabolique étonnante caractérisée par d’importants changements métaboliques d'une étape à une autre. L'un des plus intéressants changements est le passage de la phosphorylation oxydative à la glycolyse aérobie qui se produit lorsque l’étape libre-vie, c’est-à-dire le stade cercaires, infecte l'hôte mammifère. Ce changement est dépendant du glucose de l’hôte et est totalement réversible lorsque la concentration en glucose est faible. Ici, nous étudions en détail l'évolution des transporteurs de glucose de S. mansoni, ainsi que le rôle potentiel des enzymes de modification des histones (à savoir les sirtuines) dans la régulation du changement métabolique. La thèse est organisée en trois chapitres. Le chapitre I est une introduction générale au genre Schistosoma, à sa taxonomie et à ses membres les plus importants, à leur épidémiologie et aux stratégies de contrôle actuelles. Nous présentons en outre nos connaissances actuelles sur le mécanisme épigénétique de ces parasites, ainsi que sur la famille des protéines sirtuines. Dans le chapitre II, nous présentons les résultats de la thèse et ce chapitre est séparé en deux parties: (i) l'évolution et les propriétés moléculaires des transporteurs de glucose dans S. mansoni, (ii) et le rôle de la Sirtuine 1 dans la régulation du métabolisme mitochondrial des schistosomules, le stade mammifère de S. mansoni. Nous avons constaté que les transporteurs de glucose de S. mansoni, SGTP1, SGTP2, SGTP3 et SGTP4 suivaient différentes voies évolutives. Nos résultats suggèrent que les transporteurs de glucose de classe I de S. mansoni (SGTP2 et SGTP3) ont perdu leur capacité de transport du glucose et que cette fonction a évolué indépendamment dans les transporteurs de glucose spécifiques des Plathelminthes (SGTP1 et SGTP4). En ce qui concerne le rôle des sirtuines dans la régulation du métabolisme du glucose dans S. mansoni, nous avons constaté qu’à des concentrations élevées de glucose, les Sirtuines 1 de S. mansoni (SmSirt1) stimulent l'activité mitochondriale. Des inhibiteurs de Sirtuines 1 ainsi qu’un knockdown/qu’une inactivation du gène de SmSirt1 par ARN interférence réduisent l'activité mitochondriale des schistosomules. En outre, SmSirt1 est un répresseur de la pyruvate déshydrogénase kinase 1 (PDK1), un régulateur majeur de l'activité mitochondriale. Cependant, SmSirt1 ne semble pas réprimer les transporteurs de glucose de S. mansoni. Ceci est en accord avec notre analyse de l’évolution des transporteurs du glucose, car chez les mammifères, Sirt1 régule l'expression des transporteurs de glucose de classe I et nos résultats ont montré que les transporteurs de glucose de classe I ne transportent pas de glucose chez S. mansoni. Enfin, dans le chapitre III, nous faisons une discussion générale sur les principales conclusions de la thèse. / This thesis focuses on the metabolism of the parasite Schistosoma mansoni. During its life cycle, S. mansoni experiences drastic environmental changes that include: free-living stages in water and endoparasitism in invertebrate (i.e. snails) and vertebrate (i.e. mammals) hosts. In consequence, this parasite shows an amazing metabolic plasticity characterized by drastic metabolic switches from one stage to another. One of the more interesting is the switch from oxidative phosphorylation to aerobic glycolysis that occurs when the free-living stage, cercariae, infects the mammalian host. This switch is dependent on host glucose and is totally reversible when glucose concentration is low. Here, we study in details the evolution of the glucose transporters of S. mansoni as well as the potential role of histone modifying enzymes (i.e. sirtuins) in the regulation of the metabolic switch. The thesis is organized of three chapters. Chapter I is a general introduction to genus schistosoma, its taxonomy and more prominent members, their epidemiology and current control strategies. We further introduce our current knowledge on the epigenetic machinery of these parasites as well as the sirtuin protein family. In Chapter II we present the results of the thesis and this chapter is separated in two parts: (i) the evolution and molecular properties of glucose transporters in S. mansoni, (ii) and the role of Sirtuin 1 in the regulation of the mitochondrial metabolism of schistosomula, the mammalian stage of S. mansoni. We found that the glucose transporters from S. mansoni, SGTP1, SGTP2, SGTP3 and SGTP4 followed different evolutionary paths. Our results suggested that S. mansoni class I glucose transporters (SGTP2 and SGTP3) lost their capacity to transport glucose and that this function evolved independently in the Platyhelminthes-specific glucose transporters (SGTP1 and SGTP4). Regarding the role of sirtuins in the regulation of glucose metabolism in S. mansoni, we found that at high concentrations of glucose, S. mansoni sirtuin 1 (SmSirt1) stimulate mitochondrial activity. Sirtuin 1 Inhibitors as well as SmSirt1 gene knockdown, by RNA interference, reduce the mitochondrial activity of schistosomula. In addition, SmSirt1 is a repressor of pyruvate dehydrogenase kinase 1 (PDK1), a major regulator of mitochondrial activity. However, SmSirt1 did not appear to repress the S. mansoni glucose transporters. This is in agreement with our analysis of glucose transporter evolution because in mammals, Sirt1 regulates the expression of class I glucose transporters and our results showed that class I glucose transporters in S. mansoni do not transport glucose. Finally, in Chapter III we make a general discussion of the main findings of the thesis.

Orientador: Maria Luiza Silveira Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T06:29:06Z (GMT). No. of bitstreams: 1 Ghiraldini_FlaviaGerelli_D.pdf: 75704253 bytes, checksum: 9d1afc3cebcffd05000b5efb25d3996d (MD5) Previous issue date: 2013 / Resumo: O diabetes mellitus do tipo I (DM1) caracteriza-se pela ocorrência de insulite com consequente hiperglicemia e poliuria. Alterações celulares estruturais e metabólicas decorrentes do aumento da glicemia podem provocar fenótipos de envelhecimento precoce. O envelhecimento celular e resultado de fatores intrínsecos e extrínsecos, que alteram a estrutura e a organização da cromatina e que, consequentemente, afetam a expressão gênica. As sirtuinas, deacetilases NAD+-dependentes, estão envolvidas na transcrição gênica, reparo de DNA, transcrição do rDNA, regulação metabólica e remodelação cromatinica. As sirtuinas nucleares, especialmente Sirt1 e Sirt6 estão envolvidas com o envelhecimento precoce, no metabolismo de glicose e na resposta a inflamação. O presente trabalho teve como objetivo geral comparar os processos de remodelação cromatinica em hepatocitos sob o efeito da hiperglicemia e do envelhecimento, usando-se modelo animal (camundongos). Um modelo de cultura celular (HepG2) foi também utilizado para estudo de efeitos da hiperglicemia, utilizando-se como metodologias analisem morfológicas e moleculares. Foi observado um aumento em conteúdo de DNA e em acessibilidade da cromatina a MNase mais acentuado em hepatocitos de animais DM1 do que de idosos. O aumento na abundancia de Sirt1 em animais hiperglicêmicos não refletiu em sua maior atividade, enquanto em idosos houve um decréscimo generalizado nesses parâmetros e aumento da aceptilação de sítios histónicos. Em animais DM1, Sirt6 apresentou abundancia semelhante à de Sirt1, possivelmente devido à alta fragmentação de DNA observada nesses animais, diferente do ocorrido em idosos. Ambos os animais DM1 e idosos apresentaram baixa relação área AgNOR+/área nuclear. Em animais diabéticos isto foi devido ao aumento na área nuclear, enquanto nos idosos, foi devido à diminuição na área AgNOR+ e aumento na área nuclear. O aumento na metilação de rDNA na porção 18S e a baixa abundancia de Sirt7 confirmam diminuição no metabolismo celular no envelhecimento. Em hepatocitos de camundongos DM1 e idosos foi observado genes diferencialmente expressos relacionados à inflamação. Admite-se que no primeiro caso este achado seja devido à natureza auto-imune da doença, enquanto no segundo possa ser um indício de inflamação naturalmente encontrada em processos de envelhecimento. Em animais DM1, a expressão diferenciada de genes envolvidos com metabolismo de lipídios poderia contribuir para com a peroxidação lipídica e produção de ROS levando a esteatose hepática. Nas células HepG2, alterações na expressão dos genes Apoe, Igfbp1 e Foxo1, ocorridas em meio de cultura hiperglicêmicas, tornaram-se revertidas quando as células foram retornadas a normoglicemia. Contudo, as abundancias das marcações epigenéticas nos promotores desses genes decresceram progressivamente, indicação de uma memória hiperglicêmica, dado não observado em modelo animal. A análise do fenótipo nuclear dessas células indicou possível indução da proliferação celular quando retornadas a normoglicemia. A inibição de sirtuinas aumentou o conteúdo Feulgen-DNA e o contraste entre cromatina condensada e não-condensada, indicativo de atuação na proliferação celular e na remodelação cromatinica. DM1 e envelhecimento, portanto, não podem ser considerados fenômenos idênticos, pois enquanto no primeiro ha um mecanismo compensatório que promove alterações genéticas, epigenéticas e remodelação cromatinica, no segundo ha um decréscimo generalizado no metabolismo celular levando a modificações diferentes nos mesmos parâmetros / Abstract: Diabetes mellitus type I (DM1) is characterized by insulitis and consequent hyperglycemia and polyuria. Structural and metabolic changes in the cell caused by hyperglycemia might induce an early-ageing phenotype. Both intrinsic and extrinsic agents might contribute to cellular ageing thus leading to chromatin structural changes and differential gene expression. Sirtuins, NAD+-dependent deacetilases, play a role in cell metabolism, transcription, DNA repair and chromatin remodeling. Sirt1 and Sirt6, especially, are nuclear proteins related to early-ageing, glucose metabolism and inflammatory response. The general purpose of the present work was to compare processes of chromatin remodeling in hepatocytes under the effects of hyperglycemia and aging, using mouse models. A model using cells in culture (HepG2) was also used to study the effects caused by hyperglycemia. The methodology used involved morphological and molecular analysis. An increase in DNA content and chromatin accessibility to MNAse was found more pronounced in hepatocytes from DM1 than from aged mice. Despite the high abundance of Sirt1 in DM1 animals, its activity was not proportionally high, whereas in old animals there was a reduction in these parameters, increasing the acetylation of Sirt1-histonic sites. In DM1 mice, Sirt6 presented similar abundance as Sirt1, possibly due to the high DNA fragmentation, different to what was found in aged animals. Both DM1 and normoglycemic old mice presented a decrease in AgNOR+ area/nuclear area ratio. While in DM1 animals it was a result from the increase in nuclear area, in old animals it was a combination of increased nuclear areas and decreased AgNOR+ areas. The DNA methylation increase in the 18S rDNA region and the decrease in Sirt7 abundance in the hepatocytes from old mice support the hypothesis of diminished cellular metabolism. Differential expression analysis for DM1 and old mouse hepatocytes presented a high number of genes involved in the inflammatory response. While in the former it could be an autoimmune characteristic of the disease, in the latter it might be an evidence of inflammatory state naturally associated with aging. Moreover, DM1 mice also presented differential gene expression related to lipid metabolism, which could contribute to increase lipid peroxidation and ROS production leading to hepatic steatosis. HepG2 cells showed changes in Apoe, Igfbp1 and Foxo1 expression in hyperglycemic medium and they were reverted when the cells returned to normoglycemic medium. The epigenetic marks, however, presented a progressive decrease in abundance, indicative of a hyperglycemic memory, which was not observed in DM1 animals. The nuclear phenotype in HepG2 cells under these same experimental conditions indicated a possible induction in cellular proliferation when the cells were returned to the normoglycemic medium. Inhibition of sirtuins increased the contrast between condensed and non-condensed chromatin and the Feulgen-DNA content, indicating a role in cell division and chromatin remodeling. Therefore, DM1 and ageing cannot be considered as identical processes, because while in DM1 there is a compensatory mechanism that induces changes in epigenetic marks, chromatin remodeling and differential gene expression, there is a general decrease in cell metabolism under aging that leads to different changes in the same parameters / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural