Ryan Girgrah, M.Sc. Chemistry 2015

SMAD transcription factor expression in anoxia-tolerant Trachemys scripta elegans and dehydration-tolerant Xenopus laevis: A comparative study 



The TGF-β signaling pathway with its associated transcription factors (SMADs) was studied in the red-eared slider (Trachemys scripta elegans; liver, heart, white skeletal muscle, red skeletal muscle, and kidney) as a model for anoxia tolerance and the African clawed frog (Xenopus laevis; liver and muscle) as a model for dehydration resistance. In addition, protein expression levels of precursor cytokines were measured, as well as known genes and proteins downstream of the SMAD signaling pathway, such as c-Myc, and AP-1 proteins. The data showed that selective control of different SMAD isoforms occurred in response to environmental stress, the mechanism being reversible protein phosphorylation. Cellular responses and signaling profiles were observed to be highly tissue-specific and stress-specific. SMADs were shown to play a role in the hypometabolic response of both animal models through the regulation of cell cycle arrest, angiogenic processes, and oxidative injury management strategies.

Anthony Ruberto, M.Sc. Biology 2015

Regulation of lactate dehydrogenase and glycerol-3-phosphate dehydrogenase in mammalian hibernation



Hibernation is a winter survival strategy for many small mammals. Animals sink into deep torpor, body temperature falls to near 0°C and physiological functions are strongly suppressed. Enzymes are the catalysts of metabolic pathways in cells and their appropriate control is critical to hibernation success. This thesis explores the properties and regulation of two key enzymes of carbohydrate metabolism (lactate dehydrogenase, LDH) and lipid metabolism (glycerol-3-phosphate dehydrogenase, G3PDH) purified from liver and skeletal muscle of ground squirrels (Urocitellus richardsonii). The studies showed that changes in pH, temperature, and inhibitors play roles in differentially regulating these enzymes between euthermic and torpid states. Furthermore, reversible protein phosphorylation proved to be a significant regulatory mechanism, producing a reduced activity state of skeletal muscle LDH and increased activity state of G3PDH in both skeletal muscle and liver during torpor. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly protein phosphorylation, contribute to reorganizing fuel metabolism during hibernation.

Keith B. Male, M.Sc. Biology, 1982

Metabolic regulation of glutamate dehydrogenase



Glutamate dehydrogenase (GDH), a key control enzyme interrelating carbohydrate and amino acid metabolism, was investigated with respect to coenzyme utilization and regulatory control via allosteric effectors. GDH from gall fly larva, Eurosta solidaginis, was purified and kinetic studies suggested that it was preferentially NADP-linked. Temperature effects on the regulatory properties of this GDH, including ATP/GTP interactions, may be responsible for the increase in proline seen at low temperature in the overwintering larva. GDH from the intertidal sea anemone, Anthopleura xanthogrammica, was purified and found to be NADP(H)-specific. This GDH was not regulated by nucleotide effectors but was strongly affected by ions. The dual coenzyme specific GDH from bovine liver was investigated with respect to its coenzyme preference. The data suggested that under increasing glutamate concentrations the preferred coenzyme was NADP, a preference which was promoted by the action of metabolic modulators.

Kama Szereszewski, M.Sc. Biology 2014

Less is mTOR: Regulation of protein synthesis via the insulin signalling pathway in the anoxia-tolerant red-painted slider Trachemys scripta elegans



The red-eared slider turtle, Trachemys scripta elegans, can survive 3-4 months of anoxic submergence in cold water during the winter. The effect of hypoxia/anoxia on protein synthesis in red-eared sliders was investigated with a focus on the insulin-signaling pathway and analysis of the mammalian target of rapamycin (mTOR) and its upstream and downstream effectors in liver and white muscle. Expression of mTORC1 did not change in muscle but increased significantly in liver after 5 and 20 hours of anoxic submergence. Upstream effectors, AKT and RAPTOR, were also elevated in liver but suppressed in muscle. PRAS40 and TSC2 inhibitors of mTOR were differentially regulated in both tissues but generally suppressed. Downstream targets of mTOR signaling (eIF4E, 4E-BP1, P70S6K, S6) as well as the poly(A) binding protein also showed differential responses to anoxia. Overall, the data indicate that the early response to anoxia by turtles is maintenance of protein synthesis in liver but suppression in white muscle.

Andrew Rouble, M.Sc. Biology 2014

Reversible protein acetylation in the regulation of mammalian hibernation



To survive the winter, many small mammals use hibernation. Employing a remarkable strategy of metabolic rate depression these animals accrue profound energy savings by remaining in a torpid state over most of the winter. Global metabolic suppression is mediated by intricate molecular mechanisms, including the post-translational modification of cellular proteins. One such modification – reversible protein acetylation – is an important regulator of metabolism, but little is known about its relevance to hibernation. This thesis provides an initial characterization of possible functions of reversible protein acetylation, and several enzymes that mediate the process (protein lysine acetyltransferases (KATs) and deacetylases (SIRTs)), in the context of a rodent model of mammalian hibernation, the thirteen-lined ground squirrel, Ictidomys tridecemlineatus. Notably, SIRT and KAT protein expression and activities increased in skeletal muscle and brown adipose tissue, respectively, during torpor, in correlation with fluctuations in downstream target acetylation. Such changes identify roles for protein acetylation during hibernation.

Christine Childers, M.Sc. Biology 2014

Regulation of skeletal muscle glycolysis during dehydration in the aestivating African clawed frog, Xenopus laevis



Seasonally arid conditions can trigger African clawed frogs (Xenopus laevis) to enter aestivation. This process includes whole body dehydration that at high levels can create hypoxic conditions due to impaired blood circulation and increase the need for glycolytic energy production. This thesis examines hexokinase (HK) and lactate dehydrogenase (LDH) purified from skeletal muscle of control versus dehydrated (~30% body water lost) frogs. Studies analyzed substrate affinities, urea effects, thermal stability and protein posttranslational modifications (PTM) to understand how enzyme properties are modified under dehydration stress. Muscle HK and LDH showed regulation by reversible protein phosphorylation and nitrosylation. These PTM’s correlated with reduced affinities for glucose by HK and lactate by LDH, overall lower Vmax for LDH in both directions, and altered thermal stabilities. The two enzymes responded to the same PTMs, which suggests that coordinated controls over these first and last enzymes of anaerobic glycolysis contribute to dehydration responsive pathway regulation.

Bryan Luu, M.Sc. Biology 2013

Regulation of protein translation and cell cycle processes by reversible protein phosphorylation in response to dehydration in the African clawed frog



The primarily aquatic African clawed frog, Xenopus laevis, has adapted to endure substantial dehydration, partly by entering a state of hypometabolism. This thesis focuses on two processes targeted by the central protein kinase Akt (that is inhibited during dehydration) – protein translation and the cell cycle. Results suggest that dehydration leads to mTORC1 inhibition via PRAS40 activation in both liver and skeletal muscle, thereby suppressing protein synthesis. Suppression of Akt also allows activation of p21 and p27 cell cycle inhibitors to promote cell cycle arrest in liver. Analysis of multiple protein components shows that cell cycle arrest is actively facilitated in liver, but not in muscle. Regulation of liver kinases and phosphatases led to hypophosphorylation of Rb which inhibits E2F1-induced transcription of genes required for cell cycle progression. Overall, during dehydration, frogs suppress protein translation in liver and muscle, and show regulated cell cycle arrest in liver, a proliferative tissue.

Yulia Maistrovski, M.Sc. Biology 2013

Regulation of anti-apoptotic pathways in skeletal muscle and liver of an estivating species, Xenopus laevis



This study investigated the anti-apoptotic pathways activated during dehydration and estivation of the African clawed frog, Xenopus laevis. Staying immobile for a significant period of time can cause serious tissue atrophy in non-adapted animals. To preserve skeletal muscle and liver mass, certain metabolic pathways with functions in cell preservation and protection must be activated. This study focused on two families of transcription factors, NF-κB and STAT whose target genes include those with anti-apoptotic functions. The data indicated an upregulation of the NF-ᴋB pathway in liver of dehydrated frogs along with downstream targets Bcl-xL and c-IAP. STAT3 appeared to be active in liver whereas STAT5 was active in skeletal muscle. Relative levels of the NF-κB and STAT downstream target Bcl-2 were elevated in response to dehydration. Analysis of pro- and anti-apoptotic microRNAs indicated that these contribute to post-transcriptional regulation of mRNA transcripts that encode proteins with roles in cell survival.

Jean Abboud, M.Sc. Biology 2015

Regulation of pyruvate kinase and glycerol-3-phosphate dehydrogenase in the freeze tolerant goldenrod gall fly: Role in polyol cryoprotectant production



Larvae of the goldenrod gall fly, Eurosta solidaginis, can survive winter temperatures as cold as –55oC by using the freeze tolerance strategy of cold hardiness. This study examines pyruvate kinase (PK) and glycerol-3-phosphate dehydrogenase (G3PDH) that were purified from gall fly larvae acclimated to 5oC (control) and −15oC (freeze-exposed). The roles that these enzymes play in polyol cryoprotectant synthesis and metabolic regulation were investigated. A primary focus was the potential for reversible post-translational modifications (PTMs) to regulate enzyme activity. Several PTMs were analyzed for PK and G3PDH from control and freeze-exposed gall fly larvae using dot-blot analysis. Results showed that low and high phosphate forms of each enzyme exist and these differ in kinetic properties, G3PDH also showed different levels of ubiquitination. Changes in substrate affinities and different interactions with endogenous sugars suggest that PK activity is maintained whereas G3PDH is inhibited during freezing.


Tamara English, M.Sc. Biology, 1995

Comparisons of the effects of temperature on the liver fatty acid binding proteins from hibernator and nonhibernator mammals



Hibernating mammals rely heavily on lipid metabolism to supply energy during hibernation. We wondered if the fatty acid binding protein from a hibernator responded to temperature differently than a non-hibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 mM) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37°C, while the rat liver fatty acid binding protein was affected with the Kd at 37°C being about half (0.8 mM) that found at lower temperatures. This same trend was observed when comparing the specificity of various fatty acids of differing chain length and degree of unsaturation for both proteins at 5°C and 37°C. At the lower temperature, ground squirrel protein bound long chain unsaturated fatty acids, particularly linoleate and linolenate, at least as well as at the higher temperature and matches requirements for these fatty acids in the diet. The most common long chain fatty acid, palmitate, was a more effective ligand for ground squirrel liver fatty acid binding protein at 5°C than at 37°C with the opposite occurring in the eutherm. Rat protein was clearly not adapted to function optimally at temperatures lower than the animal’s body temperature.