Hello. My name is Victoria Zika and today I'll be presenting the work I did at the Brunelleschi lab at Maine Medical Center Research Institute during those last academic year. My project is about the unconventional effects of trying to exomic acid try and exomic acid is a common drug administered by emergency physicians on trauma and patients suffering from severe hemorrhage. When there's the loss of blood, it is very important to prevent Fiber Analysis, which is the break down or to lysis of fibrin in clocks, tick say is a very efficient inhibitor of the maturation of both manage into plasma and takes a, exhibits important effects besides blocking fibre analysis itself. Patients who are treated with trying to exomic acid present with less inflammation, edema, and recover faster than those who don't receive the treatment. Several studies in literature showing that the effects of tick, say, transcends its anti February analytic activity. Some of these effects include the inhibition of lung injury and inflammation, as well as the decrease of pro-inflammatory cytokines in blood contact in hemorrhagic and rarely burnt experimental animals. The goal of our lab is to answer the following question. What are cell and molecular mechanisms underlying the extensive beneficial effects of THC? We're working collaboratively with the emergency and cardiovascular Department of Maine Medical Center with doctors report, Carter and Kramer. Previously it has been shown that when take say is applied into the culture medium, it prevents the increase of the markers of cell damage. The main one is mitochondrial DNA that leaks out of the cell in case their damage, the suppressive effect of tick, say, on mitochondrial DNA release has been shown. And endothelial cells and in neutrophils. To study neutrophils, we are using an in-vitro model where neutrophils are obtained by differentiation of my LM1 acidic leukemia cell line known as H0 60, treated with retina OIC acid. Previously, our group conducted experiments using the seahorse rapid to analyze mitochondrial respiration of endothelial cells treated with DXA, it was observed that try and exomic acid is increasing the mitochondrial respiration and the synthesis of ATP. My task was to figure out what else was take, say, doing to the mitochondria. The initial experiment used endothelial cells and consisted of one control group and two concentrations of take say, 2000 micrograms per ML, which is very similar to clinical conditions. This experiment was done by treating the cells for 24 hours, 6EI, and then staining them with the mitochondrial specific fluorescent marker MitoTracker. And the cells were studied using confocal microscopy. On these confocal images, the more MitoTracker is taken by the mitochondria, the brighter they are, and therefore they are more active. Mitotracker also allows us to study the size and the morphology of the mitochondria. After takes a treatment, mitochondria are longer and more abundant. This confocal results showed that takes a stimulates mitochondrial activity and increases biogenesis. This data were confirmed by our studies using immunoblotting method. First of all, using the immunoblotting method, we determine the effects of tics a, an intracellular content of Cox for an endothelial cells. Cox for is a typical mitochondrial protein, the major component of the mitochondria of cytochrome chain, the more Cox for the more mitochondrial mass. We observed that 24 hour incubation with TSA increases significantly. Cox for invoke neutrophils and NK cells. In a separate experiment, we found that three hour incubation of endothelial cells in a serum free medium with TX a enhance the phosphorylation of AKT. Akt signaling is known to stimulate mitochondrial biogenesis. We also found out that 24 hour incubation of neutrophils with higher concentration of THC, a 100 micrograms per ml increase the expression of the transcription factor PGC one alpha, a key positive regulator of mitochondrial biogenesis. Collectively, our confocal microscopy and amino blotting results indicate that take say, stimulates mitochondrial biogenesis. The other question we asked was, What are the targets of taxa in the cells besides plasmon? To answer this question is not such an easy task. In the last ten years, a method called darks allows us to determine the protein targets for different small molecules. Barks Stanford drug affinity responsive target stability. This method consists of repairing glides, eats up the cells, in our case, endothelial cells. In treating what the small molecule we're study for us takes a, takes a molecule, binds to the target proteins and protect some molecule from proteolysis by the enzyme pronates. The following mass spectroscopy analysis showed the increase abundance of the protective proteins. In our studies, we have found several proteins for that to protected by takes a and the most consistently protection in a series of darts experiments was demonstrated for pyruvate kinase MB, glycolytic enzyme, which catalyzes the less sap of glycolysis, resulting in the production of pyruvate. Based on this, we hypothesize that try next. I'm an acid, binds to pyruvate kinase MB and inhibits its activity, which results in the increase of mitochondrial biogenesis and respiration. This hypothesis will be evaluated in the near future. In particular, we will assess the effects of tics a on pyruvate kinase MB activity. Using the available commercial kits to the following mass spectrometry analysis showed the increased abundance of the protected proteins. In our studies, we have found several proteins protected by takes a and the most consistent protection in a series of darts experiments was demonstrated for pyruvate kinase MB, a glycolytic enzyme that catalyzes the last step of glycolysis, resulting in the production of pyruvate. It has been shown that the SH RNA knocked down on pyruvate kinase and results in the enhancement of mitochondrial biogenesis. Based on this, hypothesize that trauma exomic acid binds to pyruvate kinase MB and inhibits its activity, which results in an increase of mitochondrial biogenesis and respiration. This hypothesis will be evaluated in the near future. In particular, we will assess the effects of tics a on pyruvate kinase activity using the available commercial kids. We also hypothesized that the enhancement of mitochondrial biogenesis and the activity by tick, say, could help to overcome the metabolic stress, which is typically observed in severe hemorrhagic patients. To verify this hypothesis, the animal experiments are important. First of all, I would like to thank Dr. championed list burst and Doctor Lu Si Liao, pretty amazing us. Mmm. Cri, academic research internship program. I would like to specifically thank Dr. pred off ski for the amazing mentorship and tutoring case. Her and met some risky for teaching me so many protocols and helping me throughout the last 14 months. I would also like to thank Karen Wayne, Damien Carter, monarchical, Mary Calvin Barry, Robert Kramer, and Joseph report. Thank you so much for your time.