Mackenzie Parent, M.Sc. Biology 2023

Epigenetic adaptations for survival of extreme dehydration in the estivating African clawed frog, Xenopus laevis


The African clawed frog, Xenopus laevis, is a model organism for different fields of scientific study and has a remarkable ability to tolerate dehydration. RNA (m6A) and histone arginine methylation are known to contribute to the regulation of gene expression and have not been previously studied in relation to severe dehydration. This thesis presents an introduction to the potential involvement of these pathways in the survival of severe dehydration in X. laevis liver and kidney tissues. 35 ± 0.93% dehydration of X. laevis led to differential expression of many proteins/marks in both of these pathways. There were many notable changes observed in the levels of many key proteins and marks. These results indicate that both m6A and histone arginine methylation may have a role in supporting the metabolic reorganization that takes place to during dehydration of X. laevis.

Kurtis Douglas, M.Sc. Biology 2023

Response of DNA and histone lysine methylation regulators
during anoxia-reoxygenation and dehydration-rehydration
in wood frog (Rana sylvatica) skeletal muscle


The ability of wood frogs, Rana sylvatica¸ to survive freezing and clinical death during harsh northern winters necessitates well-grounded mechanisms for managing extreme anoxia, dehydration, and reperfusion damage. These stresses are major instigators of pathogenesis yet to be overcome by humans. Contemporary efforts in the field are focused on interrogating stress-specific mediators that play a cytoprotective role in vital tissues such as skeletal muscle, providing valuable information to the biomedical community. Herein, the potential role of DNA and histone lysine methylation enzymes are examined in wood frog skeletal muscle in response to 24 h anoxia and 40% dehydration independently, and recovery from both stresses. This thesis demonstrates a condition-specific response of many epigenetic methylation regulators, highlighting some conserved similarities in comparison to prior freeze-thaw models. These findings support an integral role of epigenetic regulators in survival of hypometabolic stresses, most prominently during recovery stages. These cytoprotective effects are likely attributed to functional roles in transcriptional suppression during hypometabolism and activation during recovery, but also alternative roles based on known interactions with regulators of the cell cycle and repair pathways.

Mairelys Naranjo Vera, M.Sc. Biology 2023

Histone arginine methylation in the freeze-tolerant wood frog, Rana sylvatica


The wood frog, Rana sylvatica, is well known for its freeze tolerance ability. To endure winter, frozen frogs switch to a hypometabolic state via transcriptional regulation. Histone methylation is known to play a crucial role in regulating gene transcription. However, histone arginine methylation or demethylation has not previously been studied in the context of freeze tolerance. This thesis presents the first characterization of arginine methylation in a freeze tolerant vertebrate. Overall, levels of protein arginine methyltransferases (PRMTs) and methylated histone residues showed differential regulation over the freeze/thaw-cycle in wood frog liver. All PRMTs and downstream targets showed no changes during freezing, but protein levels of targets associated with transcription activation were elevated during thaw in skeletal muscle. Differential levels of histone demethylases were found in both tissues among the experimental conditions. These results indicate a role for histone methylation in supporting metabolic rate depression and tissue homeostasis during freezing.

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.