Oscar A. Aguilar, M. Sc. Biology, 2009

Regulation of the MEF-2 and the SMAD family of transcription factors in the freeze tolerant wood frog, Rana sylvatica



The wood frog, Rana sylvatica is a native North America capable of withstanding full body freezing when ambient temperatures drop below 0°C. During the freeze exposure, approximately 65-70% of the extracellular fluid gets converted into ice. Anoxia, ischaemia, osmotic and oxidative stress are some of the consequences which result from a freezing cycle. The dynamic nature of cells allows them to adapt to a wide array of stress conditions at different organizational levels. Transcription factors are key regulators of gene expression responsible for adaptation. In the present study, the MEF2 and SMAD family of transcription factors are demonstrated to have importance in the wood frog during freezing. The proteins were initially associated with developmental controls, however recent studies have found them to be involved in stress responses. Western blots were used to assess the expression and phosphorylation levels of MEF2A, MEF2C, SMAD1, SMAD2, SMAD3, and SMAD4 during torpor. It was generally found that MEF2A, MEF2C, and SMAD3 were post-translationally (phosphorylated) at Thr312, thr300, ser425 sites, respectively during 24h and 8h thawing. RT-PCR analysis of MEF2 and SMAD target genes (calreticulin, glucose transporter-4, creatine kinase (brain and muscle) and serpine1, myostatin, tsc22d3, respectively) revealed a modest up-regulation during 24h freezing in wood frog brain, heart, skeletal muscle, liver and kidney in selected transcripts. These results show that the two families of transcription factors are transcriptionally active during freezing, which comes as no surprise given the signals which regulate these proteins as well as the functions of the genes they activate.

Marcus Allan, M.Sc. Biology, 2010

Nuclear factor (NF)-kappaB regulation in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.



When environmental conditions become unfavorable, such as during winter, many small mammals are able to enter into a state of dormancy known as hibernation in order to conserve energy. Energy conservation is accomplished via a drastic decline in metabolic and physiological activity in association with a decrease in body temperature, which is periodically interspersed with brief bouts of arousal back to their euthermic values. These drastic changes in oxygen consumption and concentration, perfusion of tissues and energy consumption results in an elevated susceptibility to oxidative stress which can cause severe tissue damage. Hibernators are able to mitigate this damage using antioxidants and their associated pathways in a coordinated response. In the present study, the role of the redox sensitive transcription factor NF-κB was investigated to gain insight into its regulation during hibernation. NF-κB is an essential transcription factor which is known to regulate many targets including antioxidant, antiapoptotic/pro-survival and pro-inflammatory genes. The extent and duration of the NF-κBs response depends on its interactions with its multiple upstream effectors. During hibernation it was found that NF-κB and its signaling components have different expression patterns which are tissue dependant and change along the torpor–arousal cycle. Overall, NF-κB was found to be maximally activated during entrance into torpor, with its cytoprotective downstream genes being upregulated in time for next subsequent arousal in both liver and skeletal muscle tissue. Therefore, these results suggest that antioxidant defenses are upregulated throughout torpor-arousal and that NF-κB may help mediate such protective responses.

Shannon N. Tessier, M.Sc. Biology, 2010

Molecular adaptations of skeletal muscle and cardiac muscle in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.



Many small mammals face severe problems during the winter – little or no food supply and yet huge energy costs for keeping their bodies warm. To escape these problems, they hibernate, entering states of deep torpor where metabolic rate falls to just 2-4% of normal and body temperature falls to near 0°C. Remarkably, skeletal muscle sustains cell size and strength despite extended periods of disuse during torpor whereas cardiac muscle actually promotes cell growth (hypertrophy) to support the stronger cardiac contractions needed in the cold. Despite overall suppression of transcription and translation during hibernation, the present research identified and analyzed selected muscle genes and their products that were up-regulated during torpor in striated muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus). These changes in myocyte enhancer factor-2 (MEF2a, MEF2c) transcription factor levels as well as altered expression of selected downstream targets (e.g. glucose transporter 4, myogenic differentiation protein) aid skeletal and cardiac muscle in meeting metabolic challenges associated with hibernation. MEF2 transcription factors were significantly elevated at various points in the torpor-arousal cycle suggesting a significant role for MEF2-mediated gene transcription in the selective adjustment of striated muscle proteins. Muscle plasticity in the hibernator was also evidenced by torpor-responsive changes in the levels of important contractile (troponin I, α/β-tropomyosin), sarcomeric (myomesin) and cytoskeleton proteins (desmin, andvimentin). These data provides new insights into muscle remodeling during hibernation and the role of selected genes/proteins in balancing programs of atrophy, stasis andmyogenesis.


Helen Alyx Holden, M.Sc. Biology, 2011

Anuran adaptations to climatic stress: Immune responses and the SMAD family in the wood frog, Rana sylvatica, and the African clawed frog, Xenopus laevis



The wood frog, Rana sylvatica, survives freezing over winter. The African clawed frog,Xenopus laevis, withstands substantial dehydration seasonally. The effects of environment on these frogs‟ immunity were investigated with a focus on antimicrobial peptides. Expression of brevinin-1SY was analyzed during freezing, dehydration, anoxia, and development in R. sylvatica. Brevinin-1SY responded differently to each stress, suggesting environmentally regulated expression. Upregulation of hepcidin mRNA was demonstrated during dehydration in X. laevis liver, as were hepcidin agonists, STAT 3 andcMYC. Alternatively, hepcidin antagonizing TGF-β-mediated SMADs weredownregulated and the BMP-mediated SMADs, promoters of hepcidin expression, did not change. Molecular controls of X. laevis skeletal muscle growth were also explored during dehydration. Myostatin, a muscle growth antagonizer, was downregulated during dehydration, whereas cMYC, a muscle growth agonizer, and GLUT 4, a glucose transporter, were upregulated; differential control of SMADs was documented. The data suggest that, during estivation, muscle growth signals are promoted.