Nicotinamide adenine dinucleotide (NAD) is a key regulator of several metabolic and signaling pathways that are relevant in cancer cell survival. Cancer cells have an increased energy demand associated with an increased NAD turnover. Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme of the NAD salvage pathway, plays a crucial role in maintaining the intracellular NAD levels and in regulating the activity of NAD-dependent enzymes, such as sirtuins (SIRTs). The inhibition of NAMPT activity and the use of phytochemicals, such as resveratrol, represent novel therapeutic approaches in cancer therapy. Based on these facts, this thesis aimed to investigate (1) the chemotherapeutic potential and molecular mechanisms of FK866, a specific NAMPT inhibitor, and resveratrol on hepatocarcinoma cells and to find out whether there are differences compared to primary human hepatocytes; (2) to address the impact of NAMPT inhibition on the energy metabolism in cancer cells; and (3) to investigate the roles of NAMPT and SIRT1 in resveratrol´s mode of action and chemotherapeutic effects. This work demonstrates that FK866 and resveratrol possess potent chemotherapeutic effects in hepatocarcinoma cells which were absent in human hepatocytes. Hepatocarcinoma cells display a dysregulation in the AMP-activated kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling as well as in the NAMPT-mediated NAD salvage pathway compared to human hepatocytes. FK866-induced NAMPT inhibition induces ATP depletion associated with AMPK activation and mTOR inhibition whereas resveratrol induces caspase3-mediated apoptosis that is not dependent on NAMPT and SIRT1 function. NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and human hepatocytes. This work also reveals that resveratrol activates p53-induced cell cycle arrest in hepatocarcinoma cells which is partly mediated by SIRT1 inhibition. In summary, this thesis provides new insight into the role of the NAMPT-mediated NAD salvage pathway in energy metabolism and characterized FK866 and resveratrol as promising potential chemotherapeutic agents for treatment of hepatocellular carcinoma.

Phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. We hypothesized that SIL-induced protection may be mediated through activation of SIRT1, an enzyme which deacetylates proteins involved in cellular stress response. Adult male ICR mice were treated with SIL (0.7mg/kg ip), Resveratrol (RSV) (5mg/kg ip) (positive control), or saline (0.2 ml ip). The hearts were harvested 24 h later and homogenized for SIRT1 activity analysis. Both SIL and RSV increased cardiac SIRT1 activity (P<0.001) as compared to Saline. Adult mouse ventricular cardiomyocytes pre-treated with either SIL or RSV (1µM) in vitro also upregulated SIRT1 activity (P<0.05). SIL also reduced infarct size following 30 min. ischemia and 24 h reperfusion in vivo. Sirtinol (5mg/kg in 10% DMSO, ip), a SIRT1 inhibitor abolished the infarct-limiting effect of SIL and RSV (P<0.001). In conclusion, activation of SIRT1 by SIL plays an essential role in cardioprotection against I-R injury.

Osteoarthritis (OA) is the most common form of arthritis worldwide and is characterised by the progressive degradation of articular cartilage. Ageing is the primary risk factor associated with OA. However, the roles of ageing-related mechanisms in cartilage homeostasis are poorly understood. The class three histone deacetylase, Silent mating type information regulation 2 homolog (SIRT1) has been extensively shown to regulate lifespan in lower organisms and signalling pathways linked to mammalian ageing. My thesis explores the role of Sirtuin 1 in cartilage homeostasis and OA. I used in vitro experiments with chondrocyte cell lines, human clinical samples, novel genetically modified cartilage specific and whole body SIRT1 deficient mice alongside molecular biological tools to investigate my research questions. Human OA cartilage showed decreased SIRT1 compared to healthy cartilage. Mice with cartilage-specific SIRT1 deletion showed greater cartilage degradation during ageing and in an experimental OA model. In vitro and in vivo studies showed SIRT1 to directly regulate autophagy in chondrocytes. More importantly, the activation of autophagy using spermidine protected against experimental OA in wild-type mice but not in cartilage-specific SIRT1 deficient mice. In addition, my data revealed whole body SIRT1 deficient mice had increased early joint inflammation in repose to injury but displayed less cartilage loss over time in an experimental OA model. Together I have shown that SIRT1 declines with age and contributes to OA due to dysregulated autophagy. However, chronic low grade inflammation caused by SIRT1 loss was protective. My data suggest these pathways can be targeted to treat OA.

No description available.

Les protéines SREBP-1 sont des facteurs de transcription connus pour leur rôle dans la régulation du métabolisme lipidique. Plus récemment des études faites in vitro (myotubes humains en culture primaire) et in vivo (muscle tibial de souris) ont montré que la surexpression de SREBP-1a ou SREBP-1c induit une atrophie musculaire et bloque la différenciation musculaire, en inhibant notamment l'expression des protéines structurales du muscle squelettique et des facteurs de la différenciation musculaire (MRFs). Les travaux de thèse présentés dans ce manuscrit ont eu pour but de décrypter le mécanisme de l'atrophie induite par SREBP-1 et de déterminer comment les protéines SIRT1 pourraient réguler ce facteur de transcription. L'atrophie musculaire résulte d'un déséquilibre entre la quantité de protéines synthétisées et dégradées. Dans nos études, nous montrons que SREBP-1 régule la synthèse protéique et la dégradation protéique, respectivement via le contrôle négatif de l'expression des MRFs et via le contrôle de l'expression des atrogènes, MuRF1 et Atrogin-1. Dans le muscle squelettique, nous démontrons que la désacétylase SIRT1 régule l'activité transcriptionnelle de SREBP-1. Les protéines SREBP-1 et SIRT1 étant toutes deux impliquées dans la régulation du métabolisme lipidique, nous mettons en évidence une nouvelle voie de signalisation reliant le métabolisme énergétique et nutritionnel avec l'activité transcriptionnelle de SREBP-1 dans le muscle. Étant donné le rôle de SIRT1 et SREBP-1 dans le métabolisme lipidique et musculaire, nous nous sommes intéressés au rôle des phospholipides et plus particulièrement des céramides dans la régulation de la masse musculaire.Nos études montrent que la régulation de la quantité de céramides par la cytokine TNFα régule la masse musculaire. Ainsi, nos travaux mettent en évidence de nouveaux liens entre le métabolisme lipidique et la régulation de la masse et du métabolisme musculaire.

Histone deacetylases (HDACs) are a family of enzymes whose functions have been overwhelmingly associated with gene expression and chromatin dynamics by modifying the histone tails. In recent years, intensive studies have demonstrated that many non-histone proteins also could serve as substrates for HDACs. And their functions and activities have been found to be regulated by posttranslational acetylation on the ε-amino group of lysines. Here, we report that two DNA repair factors including NBS1 (Nijmegen breakage syndrome 1) and ATDC (Ataxia-Telangiectasia Group D Complementing) are acetylated proteins. SIRT1 could maintain NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. And by modulating the acetylation of ATDC, HDAC9 could prevent ATDC-p53 complex formation, promoting IR-induced cell death. These data suggest HDACs play much wider roles in cells in addition to their transcriptional repression function.

SIRT1, appartenant à la famille des sirtuines, est une déacétylase NAD-dépendante, jouant un rôle essentiel dans le contrôle de l'expression génique. En plus de modifier les histones, SIRT1 peut affecter l'activité de certains facteurs de transcription et leurs gènes cibles. Une question fondamentale est de comprendre le mécanisme moléculaire par lequel SIRT1 contrôle l'expression des gènes impliqués dans la prolifération cellulaire et le métabolisme énergétique. Pour identifier les partenaires protéiques de SIRT1, nous avons utilisé la méthode de purification TAP-TAG à partir d'une fraction nucléaire soluble et d'une fraction ancrée à la chromatine de cellules Mef exprimant stablement une copie ectopique de SIRT1 (e-SIRT1). Nous avons ainsi pu identifier un complexe SIRT1 associé à la fois au facteur de prolifération cellulaire Ki67, et à la sous-unité TFIIIC, nécessaire à l'assemblage du complexe de pré-initiation de l'ARN Polymérase III. En délétant sirt1, et en inhibant spécifiquement l'expression de Ki67, nous avons montré que la machinerie de transcription de l'ARN Polymérase III et la prolifération cellulaire étaient fortement affectées. L'ensemble de mes résultats démontre très clairement que SIRT1, Ki67, et TFIIIC sont au sein d'un même complexe protéique, SIRT1 et Ki67 agissant de manière coordonnée pour réguler le niveau d'expression des SINEs et des LINEs, transcrits issus de la machinerie de transcription de l'ARN Polymérase III.

The aim of this thesis was to examine role of Sirt1 in the Notch signalling pathway, using Drosophila as a model organism. Based on in vivo and in vitro studies, we conclude that Sirt1 plays a positive role in Notch signalling. In embryonic S2N cells, Sirt1 is responsible for the protection from metabolic stress-induced down-regulation of subset of E(Spl) genes. During development, Sirt1 is responsible for proper Notch-dependent specification of SOPs and wing development. Sirt1 can regulate the Notch signalling on multiple levels via deacetylation of various substrates involved in the Notch signalling revealed by our proteomic survey.

SIRT1 histone deacetylase acts towards many epigenetic and non-epigenetic targets. The involvement of SIRT1 in oocyte maturation is assumed and the importance of ooplasmic SIRT1 pool for further destiny of matured oocyte is strongly suggested. We hypothesized that SIRT1 play role of the signal molecule in mature oocyte through selected epigenetic and non- epigenetic regulation. We observed SIRT1 re-localization in mature oocyte and the association with spindle microtubules. In matured oocyte, SIRT1 shows a spindle-like pattern and spindle- specific SIRT1 action is supported decreasing α-tubulin acetylation. Based on the observation of histone code in immature and matured oocytes, we suggest that SIRT1 is mostly predestined for epigenetic mode of action in germinal vesicle (GV) of immature oocyte. Accordingly, SIRT1- driven trimethylation of histone H3 on lysine K9 in matured oocyte is considered to be an inheritance of GV epigenetic transformation. Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocyte capable to be switched from epigenetic to the non- epigenetic mode of action readily depending on meiosis progress. Keywords: oocyte, SIRT1, histone, developmental competence, tubuline, epigenetics

This thesis investigates the connection between the Sirtuin1 protein (Sirt1) and the Notch signaling pathway in Drosophila melanogaster. We used RNAi to downregulate the Sirt1 gene expression in a tissue specific manner in the muscles, brain, fat body, and wing disc and tested the sensitivity of Notch pathway to dietary amino acids. We identified tissue specific requirements for Sirt1 protein to mediate the sensitivity of Notch pathway to amino acids.