Therapeutic inefficacy of conventional cancer treatment is a particular dilemma associated with metastatic triple negative breast cancer (TNBC), with patients still facing poor prognosis. The design and development of novel anticancer agents specifically targeted to cancer-associated pathways is of therapeutic interest. The rationale is twofold: firstly, targeted therapy overcomes widespread toxicity and adverse effects of conventional chemotherapy due to the selectivity of the treatment modality. Secondly, synergistic combinations of different classes of highly targeted therapies could hold therapeutic promise to overcome resistance by simultaneously circumventing multiple cancer hallmarks. This study evaluates the in vitro antiproliferative activity of six compounds using breast cancer cell lines as experimental model. Five of these compounds are novel, agents designed in silico to selectively target cancer hallmarks via inhibition of specific cancer-associated proteins. The compounds include an antimitotic (STX1972), three variants of bromodomain 4 (BRD4) inhibitors (Bzt-W41, Bzt-W49 and Bzt-W52), an inhibitor of both sirtuin (SIRT) 1 and 2 (W137) and an inhibitor of janus kinases 1 and 2 (Ruxolitinib). The synergism between paired combinations was also explored. Two breast cancer cell lines, MDA-MB-231 and MCF-7 were used as experimental models. The MDA-MB-231 cell line is oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) negative and is therefore commonly used to model triple negative breast cancer with invasive and metastatic properties. MCF-7 cells are ER and PR positive and represent the hormone-dependent breast cancer model. The endothelial EA.hy926 cell line was used to represent non-cancerous cells. A crystal violet assay was used to determine the half maximal inhibitory concentration (IC50) of the six compounds on the tested cell lines after 48 h exposure. Drug combination studies based on the Chou-Talalay method of paired drug combinations were performed. Effects of treatment on cell morphology was assessed by means of confocal-microscopy. Flow cytometry was used to study the effects on cell cycle progression, apoptosis, autophagy/lysosomal activity, reactive oxygen species (ROS) production, changes in mitochondrial membrane potential (ΔΨm) and the serine 70 phosphorylation status of Bcl-2. Real-time quantitative PCR was used to analyse the effects of the compounds on the mRNA expression levels of p53, c-myc and bcl-2. Quantitative protein expression of c-MYC was analysed by means of enzyme-linked immunosorbent assay. In vitro screening for antiproliferative activity revealed that the compounds showed cancer-selective cytotoxic effects when compared to the EA.hy926 control cell line. The initial screening identified three compounds for further investigative inclusion, namely the antimitotic (STX1972), the BRD4i (Bzt-W41) and the SIRTi (W137). STX1972 was found to inhibit cell growth in the nanomolar concentration range, whilst the rest of the compounds showed growth inhibition in micromolar concentration ranges. Bzt-W41 showed significant preferential selectivity for the TNBC MDA-MB-231 cell line versus the hormone-dependent MCF-7 cell line, while STX1972 and W137 exhibited only slight differential selectivity. Two combinations (STX + Bzt-W41 and Bzt-W41 + W137) exhibited synergism, whilst the STX + W137 combination exhibited antagonistic interaction. Cell cycle and apoptosis analysis revealed that STX1972 and Bzt-W41, alone and in combination, selectively induced cell cycle arrest and apoptosis in cancer cells. However, the W137 +Bzt-W137 combination did not show preferential targeting of breast cancer cell lines, with apoptosis induced equally or even more so in the control EA.hy926 cell line. STX1972 and Bzt-W41, as well as their paired combination, was further probed in aim of deciphering their individual and combined mode of action. STX1972, Bzt-W41 as well as the paired combination proved to selectively inhibit cancer targets resulting in several molecular changes, leading to downstream pathway activation which culminates in both apoptotic and autophagy-related cellular demise. The study contributed towards deducing possible hypotheses regarding the mechanistic behaviours of the individual compounds and elucidated their combined effect during dual treatment. Results warrant future studies to further probe the intricate interaction of pathways involved in the synergistic combination of antimitotics and epigenetic regulators as a novel anticancer therapeutic modality. / Thesis (PhD)--University of Pretoria, 2020. / Physiology / PhD / Unrestricted

Indiana University-Purdue University Indianapolis (IUPUI) / Hepatic fibrosis is a cellular repair mechanism that is initiated upon prolonged damage to the liver, resulting in an accumulation of excess extracellular matrix. This eventually leads to the formation of scar tissue, which disrupts the hepatic architecture and causes liver dysfunction. Hepatic stellate cells (HSCs) play a major role in hepatic fibrosis. However, the molecular mechanisms remain incompletely understood. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ or WWTR1), key players of the Hippo pathway, have been implicated in the liver fibrosis, but the HSC-specific functions of YAP and TAZ are largely unclear. Here we have identified Sirtuin 6 (SIRT6), an NAD+ dependent deacetylase, as a key epigenetic regulator in the protection against hepatic fibrosis by suppressing the YAP/TAZ activity. SIRT6 has been previously implicated in the regulation of the canonical transforming growth factor β (TGFβ)-SMAD3 pathway. This study has revealed the significant contribution of the non-canonical pathways including the Hippo pathway to the development of hepatic fibrosis. HSC-specific Sirt6 deficient mice developed severe fibrosis when fed a high-fat-cholesterol-cholate diet compared to their wild-type counterparts. YAP became more active in the SIRT6-deficient HSCs. Expression of the YAP/TAZ downstream genes like CTGF, CYR61 and ANKRD1 were elevated in the SIRT6-deficient HSCs. Biochemical and mutagenic analyses have revealed that SIRT6 deacetylates YAP and TAZ at key lysine residues and reprograms the composition of the TEA domain transcription factor complex to suppress the YAP/TAZ function in the hepatic fibrogenesis.

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.

Nicotinamide delivered in drinking water at about 2 g/kg/day significantly prolonged survival and showed a suggestive improvement on memory in the Npc1 (nih) / Npc1 (nih) mouse model of infantile NPC1 disease. It is likely that this role is due to its function as a histone deacetylase (HDAC) inhibitor although another HDAC inhibitor, valproic acid, was without effect. Nicotinamide could also work by preventing/reversing oxidative stress.

La schistosomiase représente actuellement la seconde endémie parasitaire mondiale après le paludisme. Annuellement, cette pathologie est responsable de 280 000 décès et 700 millions d’individus y sont exposés dans 74 pays à travers le monde. Actuellement, le traitement de la schistosomiase repose sur l’utilisation d’un seul médicament, le Praziquantel®. Ainsi, le développement de nouveaux médicaments est devenu une priorité absolue pour l’OMS. Dans cette étude, notre objectif a été d’identifier de nouvelles cibles thérapeutiques afin de développer de nouveaux précurseurs de médicaments. Au cours de ce projet, nous avons focalisé nos recherches sur les enzymes impliquées dans la modification des histones et plus particulièrement sur les sirtuines, qui sont des lysines désacétylases NAD+ dépendantes.Dans une première partie, nous avons caractérisé 5 orthologues de sirtuines de mammifères chez Schistosoma mansoni (SmSirt1, 2, 5, 6 et 7). De plus, nous avons étudié le potentiel des sirtuines comme cibles thérapeutiques pour le traitement de la schistosomiase en évaluant la toxicité d’inhibiteurs génériques de sirtuines humaines sur des parasites maintenus en culture. Ainsi, nous avons montré que les inhibiteurs de sirtuines humaines affectent in vitro la viabilité des schistosomules ainsi que la stabilité de l’accouplement et la production d’oeufs des vers adultes. De plus, ces inhibiteurs induisent des changements morphologiques de l’appareil génital du ver femelle.Dans une seconde partie, nous avons entrepris d’étudier plus spécifiquement le rôle de SmSirt2 en tant que cible thérapeutique. Ainsi, l’expression de la protéine recombinante en bactérie E. coli (collaboration: C. Romier, IGBMC, Illkirch) ainsi que l’optimisation d’un dosage fluorimétrique nous ont permis de montrer que SmSirt2 présente une activité lysine désacétylase in vitro (collaboration: M. Jung, Université Albert-Ludwigs, Freibourg). De plus, l’utilisation de ce dosage nous a permis de mettre en place le criblage à haut débit d’une chimiothèque de plus de 80 000 composés afin d’identifier de nouvelles molécules inhibitrices de l’enzyme SmSirt2 (collaboration: J. Schultz, Kancera AB, Stockholm). Les composés les plus prometteurs, ont été testés in vitro sur des parasites en culture. Les résultats obtenus démontrent que les inhibiteurs de SmSirt2 affectent également la viabilité des schistosomules ainsi que la stabilité de l’accouplement et la production d’oeufs des vers adultes.Dans une dernière partie, nous avons mis en place un criblage d’une banque d’ADNc de vers adultes par la technique du double hybride en levure dans le but d’identifier les partenaires protéiques de Sirt1 chez S. mansoni. L’analyse partielle des résultats nous a permis de mettre en évidence que SmSirt1 interagit avec plusieurs protéines impliquées dans la régulation des gènes chez le schistosome. Au cours de ce projet, nous avons également développé et optimisé un protocole permettant d’étudier l’activité enzymatique de SmSirt1 par injection d’ARNm dans des ovocytes de Xénope. Ainsi, nous avons pu montrer que le sirtinol et la salermide, deux inhibiteurs de Sirt1 humaine, présentent également une activité inhibitrice sur l’enzyme du parasite (collaboration: K. Cailliau, Université des Sciences et Technologies, Lille).L’ensemble des résultats obtenus au cours de ce projet de thèse suggère que les sirtuines sont des cibles thérapeutiques potentielles dans le traitement de la schistosomiase. Parmi les 5 orthologues identifiés chez S. mansoni, SmSirt2 semble une cible prometteuse. De plus, le criblage à haut débit que nous avons réalisé sur l’enzyme recombinante a permis d’identifier des molécules qui, après bio-optimisation, pourront être des candidats médicaments. Pour finir, ces résultats participent à une meilleure compréhension du rôle biologique des sirtuines chez S. mansoni et plus particulièrement sur leur implication dans la survie et la reproduction du parasite. / Schistosomiasis is the second most important parasitic disease worldwide after malaria. It is responsible for about 280 000 deaths annually and 700 million people in 74 countries are exposed to infection. Treatment of schistosomiasis currently depends on the use of the only available drug, praziquantel, and for this reason the development of new drugs is a strategic priority of the W.H.O. In this study, our objective was to identify novel therapeutic targets in order to develop new lead molecules for drug development. During this project we have focused our research on enzymes involved in histone modification, and more particularly on sirtuines, which are NAD+-dependent lysine deacetylases.In the first part of the project, we have identified 5 homologues of mammalian sirtuins in Schistosoma mansoni (SmSirt1, 2, 5, 6 and 7). Moreover, we studied the potential of sirtuins as therapeutic targets for the treatment of schistosomiasis by evaluating the toxicity for parasites maintained in culture of generic inhibitors of human sirtuins. In this way we showed that these inhibitors affect the viability of schistosomula and the stability of pairing and egg production of adult worms. Moreover, these inhibitors caused major morphological changes, particularly to the female worm genital apparatus.A second part of our work was devoted to the more detailed study of SmSirt2 as a therapeutic target. Immunisation of mice with the recombinant protein allowed us to obtain specific antibodies and to show that SmSirt2 protein is expressed at all parasite developmental stages. Furthermore, the use of the recombinant SmSirt2 expressed in E. coli (collaboration: C. Romier, IGBMC, Illkirch) and the optimization of a fluorimetric assay allowed us to show that SmSirt2 possesses a lysine deacetylase activity (collaboration: M. Jung, University Albert-Ludwigs, Freibourg). Moreover, the use of this assay allowed the setting up of a high-throughput screen (collaboration: J. Schultz, Kancera AB, Stockholm) of more than 80 000 compounds in order to identify novel inhibitors. The most promising candidates were tested on parasites in culture and the results obtained showed that SmSirt2 inhibitors also affect the viability of schistosomula, as well as the stability of pairing and egg production of adult worms.In parallel, we have carried out screening of a yeast two-hybrid cDNA library in order to identify protein partners of Sirt1 in S. mansoni. The partial analysis of the results obtained shows that SmSirt1 interacts with several proteins involved in gene regulation. In the course of this project, using the enzyme expressed in Xenopus oocytes we were able to show that both sirtinol and salermide, inhibitors of human Sirt1, also inhibit the schistosome enzyme (Collaboration: K. Cailliau, University of Sciences and Technologies, Lille).Taken together, the results of this thesis project suggest that sirtuins are potential therapeutic targets for the treatment of schistosomiasis. Of the five orthologues of human sirtuins identified in S. mansoni, SmSirt2 seems to be a promising target. Moreover, high-throughput screening using the recombinant enzyme identified inhibitors that, after bio-guided optimization, could be drug candidates. Finally, these results contribute to a better understanding of the biological role of S. mansoni sirtuins and in particular their importance in parasite survival and reproduction.

Des altérations de fonctions physiologiques du réticulum endoplasmique (RE) induisent un processus appelé stress RE. Dans le domaine cardiovasculaire, plusieurs travaux ont montré que le stress RE contribue au développement de la majorité des pathologies cardiaques. En réponse au stress RE, la réponse UPR (Unfolded Protein Response) est activée afin de restaurer l’homéostasie du RE et de permettre la survie de la cellule. Néanmoins, dans le cas d’un stress RE excessif ou prolongé, les altérations ne pouvant plus être compensées, la cellule est éliminée par apoptose contribuant au développement de la pathologie cardiaque. Une thérapie prometteuse pour lutter contre ce type de pathologie consisterait donc à moduler la réponse au stress RE afin d’inhiber l’apoptose des cardiomyocytes. Au cours de ma thèse, je me suis intéressé aux modifications induites en réponse au stress RE dans le cœur et au rôle de la sirtuine 1 (SIRT1) dans la modulation de cette réponse. SIRT1 est une déacétylase activée par différents stress cardiaques et connue pour favoriser la survie cellulaire. D’une part, j’ai mis en évidence que le stress RE induit une modification importante de l’architecture des cardiomyocytes et en particulier une augmentation des contacts RE/mitochondries associée à une altération de la fonction mitochondriale. D’autre part, en utilisant une lignée cellulaire (H9c2), des cardiomyocytes de rat adulte et des souris invalidées pour SIRT1, j’ai démontré in vitro et in vivo (i) que SIRT1 est activée et joue un rôle cardioprotecteur en réponse au stress RE, (ii) que SIRT1 limite la réponse UPR en régulant spécifiquement la voie PERK, et (iii) que SIRT1 régule la voie PERK en déacétylant le facteur d’initiation de la traduction, eIF2 sur deux résidus lysine. Ces résultats montrent donc pour la première fois que SIRT1 est impliquée dans la régulation de la réponse apoptotique au stress RE des cardiomyocytes et suggèrent que cette déacétylase serait une cible thérapeutique intéressante pour prévenir l’apoptose dans les pathologies cardiaques liées au stress RE. / Impairment of physiological functions of the endoplasmic reticulum (ER) induces the so-called ER stress. ER stress has been implicated in many cardiovascular diseases including ischemic heart, hypertrophy and heart failure. To overcome the deleterious effect of ER stress, an evolutionarily conserved adaptive response known as Unfolded Protein Response (UPR) is activated in order to restore ER homeostasis and promote cell survival. Nevertheless, in the case of prolonged or severe ER stress, apoptotic cell death is ultimately activated to eliminate stressed cells, thus contributing to the development of the pathology. The modulation of ER stress response, in order to reduce cardiomyocyte apoptosis, thus appears as a promising therapeutic strategy for such pathologies. During my Ph.D thesis, I studied the modification that occur during ER stress response in the heart and the role of the sirtuine 1 (SIRT1) in the modulation of this response. SIRT1 is a deacetylase activated in response to many cardiac stresses to promote cell survival. First, we showed that ER stress induces important structural modifications of cardiomyocytes and in particular an increase in contact sites between ER and mitochondria associated with an alteration of the mitochondrial function. Secondly, using a cell line (H9c2), freshly isolated adult rat ventricular cardiomyocytes and SIRT1-KO mice, we demonstrated in vitro and in vivo (i) that SIRT1 is activated and plays a cardioprotective role in ER stress response, (ii) that SIRT1 attenuates the UPR by specifically regulating the PERK pathway, and (iii) that SIRT1 modulates PERK axis by deacetylating the translation initiation factor, eIF2on two lysine residues. Collectively, our results provide the first evidence that SIRT1 modulates ER stress-induced apoptosis in the heart and suggest that this deacetylase may represent a therapeutic target to prevent apoptosis in cardiac pathologies associated to ER stress.

<div>Mesenchymal stem cells (MSCs) are responsive to mechanical stimuli that play an essential role in directing their differentiation to the chondrogenic lineage. A better</div><div>understanding of the mechanisms that allow MSCs to respond to mechanical stimuli is important to improving cartilage tissue engineering and regenerative medicine. Hydrostatic pressure (HP) in particular is known to be a primary mechanical force in joints. However, little is known about the underlying mechanisms that facilitate HP</div><div>mechanotransduction. Understanding the signaling pathways in MSCs in transducing HP to a beneficial biologic response and their interrelationship were the focus of this thesis. Studies used porcine marrow-derived MSCs seeded in agarose gel. Calcium ion Ca++ signaling, focal adhesion kinase (FAK) involvement, and sirtuin1 activity were investigated in conjunction with HP application.</div><div><br></div><div><div>Intracellular Ca++ concentration was previously shown to be changed with HP application. In our study a bioreactor was used to apply a single application of HP to the MSC-seeded gel structures and observe Ca++ signaling via live imaging of a fluorescent calcium indicator in cells. However, no fluctuations in Ca++ concentrations were observed with 10 minutes loading of HP. Additionally a problem with the biore actor design was discovered. First the gel was floating around in the bioreactor even without loading. After stabilizing the gel and stopping it from floating, there were still about 16 µm of movement and deformation in the system. The movement and deformation was analyzed for the gel structure and different parts of the bioreactor. </div><div><br></div><div>Furthermore, we investigated the role of FAK in early and late chondrogenesis and also its involvement in HP mechanotransduction. A FAK inhibitor was used on MSCs from day 1 to 21 and showed a dose-dependent suppression of chondrogenesis. However, when low doses of FAK inhibitor added to the MSC culture from day 21 to 42, chondrogenesis was not inhibited. With 4 hour cyclic HP, FAK phosphorylation increased. The beneficial effect of HP was suppressed with overnight addition of the</div></div><div><div>FAK inhibitor to MSC medium, suggesting FAK involvement in HP mechanotransducation by MSCs.</div></div><div><br></div><div>Moreover, sirtuin1 participation in MSC chondrogenesis and mechanotransduction was also explored. The results indicated that overnight sirtuin1 inhibition increased chondrogenic gene expression (Agc, Col2, and Sox9) in MSCs. Additionally, the activity of sirtuin1 was decreased with both 4 hour cyclic hydrostatic pressure and inhibitor application. These two together demonstrated that sirtuin1 inhibition enhances chondrogenesis.</div><div><br></div><div><div>In this research we have investigated the role of Ca++ signaling, FAK involvement, and sirtuin1 activity in the mechanotransduction of HP in MSCs. These understandings about the mechanisms regulating the chondrogenesis with respect to HP could have important implications for cartilage tissue engineering and regenerative studies.</div></div>

Manganese superoxide dismutase (MnSOD), is a mitochondria-localized antioxidant enzyme that scavenges superoxide anions generated in the respiratory chains, has been known to play an important role in the radioprotection, and function as a tumor suppressor gene in many types of cancer. Sirtuin 3 (Sirt3) is a mitochondrial NAD+-dependent deacetylase that regulates mitochondrial oxidative metabolism, MnSOD activity, and tumorigenesis. The current study was designed to examine the role of MnSOD and Sirt3 in regulating mitochondrial metabolism and steady-state levels of O2*- in thymocyte responses to ionizing radiation (IR) and lymphomagenesis. Loss of MnSOD in thymocytes resulted in the decreased levels of viability when mice were exposed to 0.1 or 1 Gy 137Cs radiation. In contrast, loss of Sirt3 did not affect thymocyte radiosensitivity or radiation-induced superoxide levels when mice were exposed to a single dose of 137Cs radiation (0.1 or 1 Gy). Interestingly, the Sirt3-/- thymocytes demonstrated a compromised ability to induce an adaptive response following whole body exposure to a 0.1 Gy dose of 137Cs radiation when challenged 4 h later with a 1.5 Gy dose of 137Cs radiation. Of note, both Sirt3-/- and MnSOD-/- thymocytes did not demonstrate increased T cell lymphoma in C57BL/6 mice at 15-18 months following low dose (0.1 Gy or 1 Gy) radiation exposure to low linear energy transfer (LET) 137Cs or high LET 300 MeV/n Silicon (67 kev/µM) irradiation. Interestingly, the loss of Sirt3 in Bax overexpressing transgenic thymocytes did cause significant alterations in mitochondrial oxidative metabolism including increased levels of superoxide, increased mitochondrial membrane potential, and increased oxygen consumption, as well as decreased levels of MnSOD activity and decreased steady-state levels of ATP. Sirt3 was also found to be upregulated in Lck-Bax transgenic pre-malignant thymocytes, and downregulation of Sirt3 was noted in lymphomas from Lck-Bax mice. Furthermore, a significant acceleration of thymic lymphoma development was observed in Lck-Bax transgenic animals lacking Sirt3. In conclusion, the data presented here support the hypothesis that increased levels of superoxide in thymocytes induced by loss of Sirt3 or MnSOD and/or exposure to low dose radiation was not sufficient to induce T cell lymphomas. However, loss of Sirt3 significantly accelerated thymic lymphoma formation induced in Lck-Bax overexpressing C57BL/6 mice, showing that Sirt3 could act as a tumor suppressor in this model system. These results also support the speculation that mitochondrial oxidative metabolism regulated by Sirt3 leads to increased steady-state levels of O2*- and may contribute to the promotion of carcinogenesis in the Lck-Bax model of T cell lymphoma development.

Mitochondrial superoxide production during the early and late radiation response is increasingly recognized as a critical driver of oxidative damage and injury processes in mammalian cells. The role of Sirtuin 3, a key mitochondrial regulatory deacetylase, in preventing mitochondrial superoxide generation in conditions of nutrient and oxidative stress may be critical during the radiation response in mammalian liver. Because several tumor types express lower than normal levels of Sirtuin 3, the involvement of Sirtuin 3 in the radiation response may also provide clues to improving cancer radiation therapy and understanding the process of carcinogenesis. Studies of how the SIRT3 loss impacts the hepatic radiation response may also provide insight into the role of superoxide in normal liver physiology as well as in conditions of pathology. Increased superoxide production has largely been associated with disease, but oftentimes without clear demonstration of mechanism or even clear descriptions of pathogenesis. Here we identify a target of Sirtuin 3, the mitochondrial antioxidant enzyme manganese superoxide dismutase, and delineate the role that Sirtuin 3-mediated increases in manganese superoxide dismutase may be playing in the prevention of injury following biologically relevant doses of low linear energy transfer and high linear energy transfer radiation types including Cs-137 and Fe and Si particle radiation. Loss of Sirtuin 3 appears to correlate with decreases in hepatocellular carcinoma 16 months after 0.1 and 1 Gy doses of particle radiation known to increase hepatocellular carcinoma rates. These results indicate that Sirtuin 3 is a critical regulator of superoxide metabolism in the liver following whole body irradiation.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated glutamatergic ion channels that mediate most excitatory neurotransmission in the brain. Alterations in AMPAR synaptic accumulation mediate synaptic plasticity, including long-term potentiation, long-term depression and homeostatic synaptic plasticity. AMPAR abundance in neurons is determined by balanced processes of protein translation and degradation. Changes in AMPAR function and trafficking have direct impacts on synaptic transmission and cognitive functions. However, the molecular mechanisms regulating AMPAR expression and dynamics in neurons remain largely unknown. In this thesis, two molecular mechanisms that regulate AMPAR translation and protein stability through two different signaling pathways, 5' adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 2 (SIRT2), are described. It is shown that SIRT2, a NAD+-dependent protein deacetylase, directly controls AMPAR stability by regulating AMPAR acetylation. For the first time, we discovered that AMPARs are subject to lysine acetylation, a novel form of post-translational modification for glutamate receptors. Under basal conditions, AMPARs are highly acetylated at their intracellular C termini, which protects against ubiquitination to antagonize AMPAR endocytosis and degradation, leading to prolonged receptor half-life. SIRT2 is also identified as the enzyme responsible for AMPAR deacetylation. Knockdown of SIRT2 led to elevated AMPAR acetylation and reduced ubiquitination, and consequently, increased AMPAR levels and synaptic transmission. SIRT2 knockout mice displayed weakened synaptic plasticity and impaired learning and memory. Resveratrol is a phytoalexin that has been shown to increase AMPAR expression and synaptic accumulation in neurons. The resveratrol effect on AMPAR expression is independent of sirtuin 1, the conventional target of resveratrol, but rather is mediated by AMPK and its downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Application of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), to neurons mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis through the AMPK-PI3K pathway activation. These studies describe novel regulatory mechanisms responsible for the control of AMPAR protein amount and subcellular distribution in neurons, providing insights into our understanding of synaptic plasticity, brain function and neurological disorders. / 2017-12-06T00:00:00Z