Preservation of biological tissues by freezing has application in fields ranging from reproductive technology to conservation of endangered species. However, while scientists can successfully cryopreserve small living structures like eggs and embryos, application of those strategies to larger structures remains challenging. The natural world can provide insights into overcoming those challenges. Many "cold-blooded" animals encounter sub-freezing temperatures in their natural environment and, among the vertebrates, a few species have evolved physiological and biochemical strategies to survive freezing. This project will study one of those species, Cope?s gray treefrog. Freezing imposes daunting challenges for an animal, including potential physical damage from ice, fluctuating tissue oxygenation as the blood freezes and thaws, and disruption of molecular and cellular structures as liquid water freezes and body fluid concentrations change. A key to surviving these challenges is regulation of the distribution of water and dissolved solutes. Ice formation must be restricted to fluids outside of cells, and so water must be able to leave cells rapidly when freezing begins. In addition, some solutes, such as glycerol are cryoprotective and help to preserve molecular and physiological integrity during freezing. Gray treefrogs present a natural model for unraveling the mechanisms behind these capabilities. During the cooling autumn months they accumulate glycerol. They also regulate the expression of proteins ("aquaporins") that facilitate movement of water and glycerol across cell membranes. This project will evaluate how these molecular mechanisms are regulated, what is the resulting impact on cellular properties, and how this contributes to freeze tolerance of the whole animal. This integrative approach provides an excellent framework for training students from high school through PhD level in methods and ideas of molecular physiological regulation, and it will yield novel insights into the challenge of keeping tissues frozen alive.