Craig Brooks, M. Sc. Biology, 2009

Regulation of NF-κB and p53 in the liver and skeletal muscle of the freeze tolerant wood frog, Rana sylvatica



The wood frog, Rana sylvatica, is the primary model animal used for studying vertebrate freeze tolerance.  During Canadian winters, wood frogs can endure the freezing of about 70% of their total body water and then thaw and resume life in the spring.  Frogs have multiple ways to protect themselves against potential freezing injuries including adaptive changes to intermediary metabolism and gene expression.  One way that wood frogs deal with freezing stress is via upregulation of several freeze-responsive genes.  Previous studies provided excellent presumptive evidence for the involvement of the NF-κB and p53 transcription factors in freeze tolerance.  The studies in this thesis used Western blotting to quantify levels of NF-κB subunits p50 and p65, its inhibitor, p-IκB, and downstream targets (ferritin heavy chain, manganese superoxide dismutase) as well as protein levels of p53, post-translationally modified p53, and some p53 downstream genes in the muscle and liver of control versus frozen wood frogs. Nuclear distributions of NF-κB and p53 were also assessed. RT-PCR was used to quantify transcript levels of select targets of NF-κB and p53.  Significant increases in the expression levels of NF-κBand its downstream targets as well as in levels of p53, post-translationally modified p53, and its downstream targets were observed during freezing.  These findings suggest the activation of NF-κB antioxidant defenses in the wood frog during freezing in anticipation of reperfusion during thawing and the activation of p53 in the wood frog which would lead to cell cycle arrest in the frozen state.