Seta, FrancescaNg, Alexandra2025-10-062024https://hdl.handle.net/2144/514102024Sirtuin-1 (SirT1) is an NAD+-dependent deacetylase that regulates many cellular processes including inflammation, lipid homeostasis, apoptosis, and cell proliferation. In the vasculature, SirT1 has been shown to have a vasoprotective role against aortic disease when upregulated. Therefore, SirT1 upregulation has been a major interest in drug development and research. SirT1 is found to be upregulated in response to exercise, polyphenols (like resveratrol), and calorie restriction. Current polyphenolic SirT1 activators like resveratrol are not specific or efficient and several other small molecules SirT1 activators have failed to reach the clinic. Therefore, novel approaches to increase SirT1 activity like those proposed in this thesis are necessary. To test these novel therapeutics, a reliable and high-throughput assay is necessary to test a variety of molecules’ effects on SirT1 activity. Commercially available assays like the Fluor de Lys assay have been shown to be dependent on the specific fluorophore covalently attached to the substrate. The assay that our lab has been developing circumvents many of the challenges in commercially available assays. First, we optimized a standard curve to test our assay’s ability to detect the total and acetylated fraction of a SirT1 peptide substrate (p53). Next, we began testing whether SirT1 activity could be measured in vitro with our assay. In these experiments, we determined that the optimal range of the standard curve is 0 to 12 ng of the ac-p53 peptide. When testing the assay with human recombinant SirT1, we encountered a high amount of variability. Concomitantly to optimizing the SirT1 activity assay, we have concurrently optimized western blot and in-gel zymography protocols to measure SirT1 activity indirectly, by measuring the levels of SirT1 downstream targets (acetylated histone 3 (ac-H3) and matrix metalloproteinases, (MMP) activity). The western blots done using vascular smooth muscle cells (VSMCs) showed that SirT1 activity could be measured through the levels of its deacetylation target ac-H3. Our experiments showed that with increasing amounts of resveratrol, SirT1 activity increased as acetylated histone 3 levels decreased in a dose-dependent manner. The in-gel zymography experiments showed that when wild-type (WT) VSMCs and VSMCs with SirT1 deletion (SMKO) are treated with TGFβ1and H2O2, an inflammatory and oxidant stimulus the Seta laboratory previously showed inhibits SirT1 activity, MMP2 activity levels increase. Our next steps will be to continue optimizing the SirT1 activity assay and test novel small molecule activators using western blot and in-gel zymography.en-USAttribution-NonCommercial 4.0 Internationalhttp://creativecommons.org/licenses/by-nc/4.0/MedicineBiologyAortic aneurysmAortic dissectionSirt1Vascular diseasesThesis/Dissertation2025-10-05