Rationale and hypothesis: Diabetic sensory neuropathy involves a distal dying-back of nerve fibers. Neuronal mitochondrial function is impaired in diabetes and Sirtuin 2 (SIRT2) is a sensor of redox state that regulates cellular bioenergetics. The role of SIRT2 in regulating the phenotype of adult sensory neurons derived from both control and diabetic rats or wild type and SIRT2 knockout (KO) mice was studied. It was hypothesized that sensory neurons under a hyperglycemic state would have a lowered NAD+/NADH ratio thus deactivating the SIRT2 pathway. It was further hypothesized that the down regulation of SIRT2 would diminish the activity of the AMP-activated protein kinase (AMPK) pathway resulting in mitochondrial dysfunction. This defect would contribute to distal dying-back of axons observed in diabetes. Methodology: Type 1 diabetes was induced in rodents by streptozotocin (STZ). Adult sensory neurons derived from control or STZ-diabetic rats or control and SIRT2 knockout (KO) mice were cultured in defined media with varying doses of neurotrophic factors and D-glucose. Protein levels were determined by quantitative Western blotting and neurite outgrowth quantified by immunocytochemistry. Plasmid transfection was initiated for overexpression of SIRT2 constructs and Seahorse XF24 analyzer was utilized to measure mitochondrial function of cultured neurons. Results: Overexpression of SIRT2 elevated total neurite outgrowth in cultures derived from control and STZ-diabetic rats. Cultures derived from SIRT2 KO mice exhibited diminished neurite outgrowth. The AMPK pathway was inhibited under high glucose treatment through activation of the polyol pathway. Pharmacological inhibition of the polyol pathway improved mitochondrial bioenergetics and neurite outgrowth in sensory neurons. Augmented expression of electron transport proteins and increased mitochondrial mass was associated with enhanced bioenergetic function. Conclusion: SIRT2 is a key component driving mitochondrial function and axon regeneration through the activation of AMPK pathway. In diabetes this pathway is suppressed via elevated polyol pathway activity. / October 2016

No description available.

No description available.

Neurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets. The disease is caused by mutations in the NF2 gene that encodes a tumor suppressor called merlin. Loss of merlin function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrates for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin, an actin severing and depolymerizing agent. LIMKs also translocate into the nucleus and regulate cell cycle progression. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2ΔEx2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2ΔEx2 MSC reduced LIMK1 and LIMK2 levels. Pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of iv control MSCs. Similarly, LIMK knockdown decreased viability of Nf2ΔEx2 MSCs. The decreased viability of Nf2ΔEx2 MSCs was due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation and cofilin phosphorylation. To increase the search for NF2 therapeutics, we applied an alternative approach to drug discovery with an unbiased pilot high-throughput screen of the Library of Pharmacologically Active Compounds. We assayed for compounds capable of reducing viability of Nf2ΔEx2 MSC as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2, a mammalian sirtuin, is a NAD+ -dependent protein deacetylase. We show that Nf2ΔEx2 MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases Nf2ΔEx2 MSC viability in a dose dependent manner without substantially reducing wildtype MSC viability. Inhibition of SIRT2 activity in Nf2ΔEx2 MSC causes cell death accompanied by release of the necrotic markers lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. Overall this work uncovered two novel potential therapeutic targets, LIMK and SIRT2 for NF2 and tumors associated with merlin deficiency.

NAMPT (Nicotinamide phosphoribosyltransferase) catalyses the rate-limiting step in the NAD biosynthesis from nicotinamide and thereby regulates the activity of NAD-dependent enzymes. Cancer cells are highly dependent on NAD for energy and DNA repair processes and are assumed to be more susceptible to an inhibition of NAD synthesis than non-transformed cells. We aimed to investigate whether or not inhibition of NAMPT with its specific inhibitor FK866 can sensitize leukemia cells for chemotherapeutic agents. NAMPT protein abundance, enzymatic activity and NAD concentrations were significantly higher in Jurkat and Molt-4 leukemia cell lines compared to normal peripheral blood mononuclear cells. Combination of etoposide and FK866 caused increased cell death in leukemia cell lines compared to etoposide alone. Etoposide decreased protein abundance of NAD-dependent deacetylases SIRTUIN1. After combining etoposide and FK866 treatment SIRTUIN2 was further decreased and accumulation and acetylation of the downstream target p53 was further enhanced in MOLT4 cells. Concomitantly, protein abundance of p21 and cleaved BAX was increased. Targeting NAMPT could be a novel therapeutic strategy to enhance the efficacy of chemotherapeutic agents such as etoposide against leukemia.

SIRT1 and SIRT2 are two NAD⁺-dependent deacetylases which negatively modulate the activity of p53, a protein which is involved in cell cycle arrest, senescence and apoptosis following genotoxic stress. Part I of the thesis describes the exploration of the chemical space around a reported unselective and modest inhibitor of SIRT1 and SIRT2 with the aim of improving the selectivity and potency of the inhibitor against the two isoforms. Particular emphasis is placed upon understanding the mode of binding of the novel analogues within the active site of the enzymes. Chapter 1 reviews the physiological roles of class III NAD⁺-dependent deacetylases, also known as sirtuins. In particular, the application of SIRT1 and SIRT2 inhibitors as potential anticancer agents is described. Amongst these, only cambinol and the tenovins showed in vivo activity in a mouse xenograft model. Previously only one analogue of cambinol had been reported in the literature. Chapter 2 describes the development of a small collection of novel cambinol analogues (First Generation Studies). The role played by different substituents at the phenyl group and at the N-1 of the thiouracil core is discussed. Along with the synthesis and structure activity relationship (SAR) associated with the core structure, in-cell experiments intended to confirm the activity of the most active compounds are reported. Chapter 3 provides a rationalisation for the SAR discussed in Chapter 2. Based on computational molecular modelling studies (GOLD), the activity of the most potent and selective SIRT2 inhibitors is explained. Two series of novel cambinol analogues were designed (Second and Third Generation Analogues) in order to assess further the proposed binding mode. Chapter 4 focuses on the development of the “Second Generation” analogues, characterised by the presence of lipophilic substituents at the sulfur atom and at the N-3 position of the thiouracil core. The synthesis, biological evaluation and SAR are discussed in detail. Chapter 5 reports the development of the “Third Generation” analogues, characterised by either a benzyl group or para-alkoxy-substituted benzyl group at the N-1 position of cambinol. Once again, the synthesis, biological evaluation and SAR data are presented. An improved understanding of the mode of binding of the novel compounds is proposed based on molecular dynamics (MD) studies. Indole-based alkaloids, such as Vincristine and Vinblastine, are well known for their anticancer activity. Recently, the anticancer activity of members of the calycanthaceous family of alkaloids has been discovered. Part II of the thesis focuses on model studies aimed at developing the total synthesis of one of these compounds, perophoramidine. Chapter 7 provides an overview of the calycanthaceous alkaloid family of natural products, including their biological properties. The structural features of perophoramidine, along with the previously reported synthetic studies are outlined. Chapter 8 describes the synthesis of an advanced intermediate in the total synthesis of dehaloperophoramidine, a structural analogue of perophoramidine Problems encountered, optimisation studies and the synthesis of a re-designed intermediate are also reported in this chapter.

Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte. / Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.