PROJECT SUMMARY/ABSTRACT Liver dysfunction is a major global health concern. In the U.S. alone, liver disease is the 10th leading cause of death and afflicts nearly 30 million Americans. Managing and treating liver diseases is difficult and costly. In cases of end-stage liver disease, liver transplantation is the most effective therapy, but it is severely limited by donor organ supply, necessitating the development of therapeutic alternatives to whole organ replacement. Extensive necrosis and poor regeneration observed in acute liver injuries can result in liver failure. Acetaminophen (APAP), a common analgesic, can result in liver failure and death when taken in excess. A better understanding of hepatocyte biology is required to improve existing approaches and innovate therapies for liver disease treatment, including APAP-induced liver failure. Most somatic cells are diploid and contain single pairs of homologous chromosomes, while hepatocytes are characterized by polyploidy, an increase in entire chromosome sets. Polyploid hepatocytes comprise >90% of hepatocytes in adult mice and 25-50% in adult humans. The cellular and molecular mechanisms that regulate polyploidization have been well characterized; however, the functions of diploid and polyploid hepatocytes are poorly understood. In the context of liver regeneration, diploid hepatocytes begin proliferating earlier and complete the cell cycle faster than polyploids, suggesting that diploid hepatocytes drive an initial wave of proliferation. The overall goal of this project is to understand how diploid and polyploid hepatocytes function during compensatory proliferation following APAP- induced injury and the molecular mechanisms that contribute to these differences. The central hypothesis tested in the proposal is that diploid and polyploid hepatocytes respond differently to APAP overdose, where diploids resist APAP damage and drive critical, early-stage compensatory proliferation. This hypothesis will be studied with 2 aims. Aim 1 will determine ploidy-specific effects on hepatocyte regeneration during APAP overdose. Experiments will evaluate APAP-induced damage, compensatory regeneration, and mechanisms regulating oxidative stress, specifically sirtuin signaling, in polyploid knockout models and cultured hepatocytes. Aim 2 will investigate proliferation by human diploid and polyploid hepatocytes. Experiments will test the regenerative and injury responses of diploid and polyploid cells isolated from human livers. Overall, this project will expand our understanding of hepatocyte ploidy populations and their roles in acute liver injuries. These findings may lead to the development of strategies or therapeutics to improve liver disease treatments.