Tighe Bloskie, M.Sc. Biology 2021

Methyl epigenetic mechanisms in the freeze-tolerance response of Rana sylvatica nervous tissue


Wood frog freeze tolerance is a classic example of metabolic rate depression (MRD), which facilitates reprioritization of minimal anaerobic resources to pro-survival pathways. Global gene expression is consequentially suppressed due, in part, to transcriptional controls, but to date, specific mechanisms have received little attention. Methylation of DNA and histone lysine residues are common epigenetic mechanisms that are tightly associated with control of transcription and thus have been implicated in MRD. However, preliminary findings appeared tissue- and species-specific, and considering research into nervous tissues was lacking, further investigation is required. This thesis tracks the expression and activity of some key methyl epigenetic modifiers like lysine/DNA methyltransferases and DNA demethylases, as well as selected putative targets across the wood frog freeze-thaw cycle and associated sub-stresses. This thesis provides strong evidence in favour of roles for H3K9 and DNA hypomethylation during freeze recovery, which are largely correlated with changes in expression of catalyzing enzymes. Some non-histone target roles are also suggested. Alleviation of repressive epigenetic controls likely contribute to the resumption of a permissive transcriptional state and may induce the activity of essential repair pathways during thawing.

Sarah Breedon, M.Sc. Biology 2021

MicroRNA biogenesis and expression in the anoxia-tolerant red-eared slider turtle, Trachemys scripta elegans


The red-eared slide turtle (Trachemys scripta elegans) is able to survive prolonged episodes of anoxia without suffering any apparent damage. This feat is underscored by a complex set of regulatory mechanisms to achieve metabolic rate depression (MRD) including microRNA (miRNA)-mediated gene silencing. Immunoblotting of relative protein abundance was used to analyze miRNA biogenesis under anoxic and reoxygenated conditions in liver and muscle, finding tissue-specific regulation of miRNA production. Cytoplasmic granule proteins were also assessed to inspect mRNA fate, the results of which indicated that mRNA sequestration to stress granules or processing bodies was also tissue specific. Next, the miRNAome was analyzed using RNA sequencing and bioinformatic analyses, revealing anoxia-induced suppression of processes associated with cell cycle progression and protein turnover. Altogether, these results indicate that miRNA targeting of mRNA transcripts occurs in a tissue specific manner with a particular focus on the suppression of energetically expensive processes.

Aline Ingelson-Filpula, M.Sc. Biology 2021

A winter’s tale: microRNA biogenesis and regulation in the freeze-tolerant grey tree frog, Hyla versicolor


Grey tree frogs (Hyla versicolor) can endure full-body freezing over the winter, with survival aided by metabolic rate depression. Post-transcriptional controls on gene expression include microRNA regulation of gene transcripts that can aid implementation of protein changes required for freezing survival. Western immunoblotting was used to examine protein expression levels of the miRNA biogenesis pathway in three tissues. During freezing, four proteins were upregulated in liver, whereas four proteins in muscle and five proteins in kidney were downregulated. Small RNA-sequencing and bioinformatic analysis of liver showed that seven miRNAs were freeze-upregulated and four were freeze-downregulated. Functions for these miRNAs may involve inhibition of signaling pathways, apoptosis, and nuclear processes. Interestingly, miRNAs may enhance ribosomal biogenesis. Overall, the data show miRNA biosynthesis is altered during freezing and differentially regulated across tissues, with computational predictions highlighting specific functions and processes that may be disproportionally altered during freezing.

Ranim Saleem, M.Sc. Biology 2020

Enzymatic regulation in the liver of the freeze-tolerant wood frog in metabolically depressed states


The wood frog, Rana sylvatica, can tolerate high degrees of freeze-tolerance, a stress that also requires anoxia, dehydration and hyperglycemia tolerance. Frozen wood frogs show no heartbeat, brain activity, muscle movement or breathing, but phenomenally return to normal once thawed. Control of enzymatic activity is crucial for regulating metabolism and it is imperative for wood frog survival. This thesis investigates the properties of two key enzymes metabolic enzymes, glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase in liver of the wood frogs exposed to freezing, dehydration and anoxia. Current data show that changes in activity, substrate affinity and stability of the enzymes play a major role in their regulation to support the survival of the wood frog during stress, and these regulations are partly controlled by post-translational modifications. Therefore, these enzymes undergo regulation at the level of posttranslational modification to contribute to the overall readjustment of energy production in the wood frog.

Zephanie Lung, M.Sc. Biology 2019

Regulation of DNA damage repair in response to freezing and anoxia in the wood frog, Rana sylvatica


The wood frog, Rana sylvatica, can survive freezing up to 65% of its body water in subzero temperatures and endures anoxia due to the cessation of vital functions while frozen. Fluctuating oxygen levels, particularly upon reperfusion and reoxygenation, increase vulnerability to reactive oxygen species and oxidative damage to macromolecules, including DNA. This thesis assesses DNA damage and responses by antioxidant capacity and DNA damage repair pathways to freezing or anoxia in wood frog liver and skeletal muscle. DNA oxidation remained largely constant, with observed increased antioxidant capacity in anoxia but not freezing. Although many observed DNA repair proteins displayed constant expression through stress and recovery, the MRN complex, Ku heterodimer, and ligation complexes, displayed changes in expression that vary based on tissue and stress. Overall, the data indicate that DNA damage is minimized through tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.

Jessica Mattice, M.Sc. Biology 2018

Regulation of glutathione-based antioxidant defenses in response to dehydration stress in the African Clawed frog, Xenopus laevis


The African clawed frog (Xenopus laevis) needs efficient antioxidant defenses to mitigate oxidative damage and endure dehydration stress under arid conditions in its natural environment. Two enzymes that aid glutathione-based antioxidant defenses, glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), were purified from liver of control and dehydrated (~35% total body water lost) frogs. Kinetic analysis revealed that GR was positively regulated in response to dehydration to regenerate GSH, particularly when physiological urea concentrations were high. G6PDH from dehydrated frogs showed lower affinity for glucose-6-phosphate than control but was strongly activated in the presence of high ATP. Both enzymes showed regulatory modification by reversible protein phosphorylation that affected substrate affinities. Dehydration also induced other PTMs including protein sumoylation, cys-nitrosylation, and acetylation that could affect kinetic properties and protein stability. This thesis provides novel insights into the regulation of glutathione-based enzymatic defenses against dehydration-induced oxidative stress.

Trong Nguyen, M.Sc. Biology 2017

Regulation of glutamate dehydrogenase and lactate dehydrogenase in the freeze tolerant wood frog, Rana sylvatica


Freeze tolerance is a survival strategy used by the wood frog, Rana sylvatica, for winter survival. Drastic changes to physiology and biochemistry are required to enter a state of metabolic rate depression in order to reestablish homeostasis during whole-body freezing. Enzymes are biocatalysts that mediate these metabolic functions and regulate survival of this environmental stress. This thesis explores the properties and regulation of two key enzymes of carbohydrate metabolism (lactate dehydrogenase, LDH) from liver and amino acid metabolism (glutamate dehydrogenase, GDH) from skeletal muscle. The studies showed that allosteric effectors play a role in differentially regulating these enzymes between freezing and control conditions. Furthermore, reversible protein phosphorylation appears to be a common regulatory mechanism reducing activity of both LDH and GDH in the frozen state. Altogether, these studies support theories that multiple mechanisms of enzyme regulation, particularly protein phosphorylation, contribute to the reorganization of metabolism during freeze tolerance.

Hanane Hadj-Moussa, M.Sc. Biology 2017

Micromanaging freeze tolerance: The biogenesis and regulation of microRNAs in frozen frogs


When temperatures plummet below 0°C, the wood frog (Rana sylvatica) freezes up to 65% of its body water in extracellular ice masses, displaying no measurable brain activity, no breathing, and a flat-lined heart. Various molecular mechanisms including microRNAs, a multifunctional group of short non-coding RNAs, are in place to facilitate freeze tolerance. This thesis provides the first large-scale investigation of microRNA in a freeze tolerant vertebrate. Immunoblotting was used to investigate protein abundance of key microRNA biogenesis factors in brain and liver of control, 24 h frozen, and 8 h thawed R. sylvatica. Biogenesis capacity was reduced in brains and elevated in livers during freezing and thawing. This correlated with RT-qPCR levels of ~110 microRNAs, where the majority of differentially expressed miRNAs were downregulated in brains and upregulated in livers. Bioinformatic miRNA targeting predicted brain miRNAs to play a neuroprotective role, while hepatic miRNAs suppressed energy-expensive pro-growth processes.

Sam Logan, M.Sc. Biology 2017

Control of selective mRNA translation over the torpor-arousal cycle of thirteen-lined ground squirrels


Mammalian hibernation is an interesting adaptation that allows many capable animals like the thirteen-lined ground squirrel to endure the winter months on a low or absent food supply. Metabolic suppression during hibernation is facilitated by diverse biochemical mechanisms including the global shut-down of energy expensive processes like transcription and translation, the use of post-translational modifications to regulate protein activity, and differential gene/protein expression of essential protein products. Differential protein expression can occur during hibernation, but it remains incompletely understood how transcripts are “chosen” to be translated instead of stored or degraded. This thesis explores two mechanisms that may regulate differential gene and protein expression during hibernation, including RNA-binding protein (RBP) stabilization and transport of transcripts, and translation machinery activation. Notably, RBPs and cap-dependent translation factors were upregulated over the torpor-arousal cycle. These results suggest a possible role for proteins that regulate mRNA stability and enhance translation during metabolic suppression

Stuart Green, M.Sc. Biology 2017

Regulation of urea cycle related enzymes in response to freezing in the wood frog, Rana sylvatica


The North American wood frog, Rana sylvatica, is one of the few vertebrate species that is capable of surviving freezing. Prevention of intracellular ice formation and maintenance of osmotic balance is facilitated by the production of large concentrations of urea during freezing in the liver. The regulatory roles of three enzymes critical to production of urea; glutamate dehydrogenase (GDH), carbamoyl phosphate synthetase I (CPS1), and ornithine transcarbamylase (OTC), were investigated in the liver in response to freezing. Freeze-exposed GDH had heightened substrate affinity and higher maximal activity than the control. Freeze-exposed GDH also had decreased acetylation and ADP-ribosylation. CPS1 from frozen frogs was demonstrated to have a higher affinity for ammonium and decreased lysine glutarylation relative to the control. Freeze-exposed OTC demonstrated improved affinity for ornithine and increased serine phosphorylation. Taken together, the results suggest that urea production is sustained in the liver of the wood frog during freezing.