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Anchal Varma, Ph.D. Biology 2023

Enzymatic regulation of hepatic carbohydrate metabolism in freeze-tolerant wood frog, Rana sylvatica

Abstract:

Wood frogs (Rana sylvatica) are a widely researched vertebrate species due to their ability to endure natural freeze tolerance. These frogs can survive months of sub-zero temperatures during winter, even when 65-70% of their total body water is frozen as extracellular ice. However, this freezing results in the cessation of blood circulation, heartbeat, and breathing, leading to limited oxygen supply. Wood frogs must depend on anaerobic glycolysis for energy production during this time. Two of the basic mechanisms underlying freeze tolerance in wood frogs are metabolic rate depression (MRD) and the production of high concentrations of glucose as a cryoprotectant by the liver. This thesis aimed to investigate the regulation of key enzymatic checkpoints in hepatic carbohydrate metabolism in wood frogs. The research revealed the downregulation of pyruvate kinase (PK) during freezing, leading to the inhibition of glycolysis. The study also shed light on the suppression of fructose-1,6-bisphosphate (FBPase) and citrate synthase (CS), which subsequently inhibited flux through gluconeogenesis and TCA, respectively. This suppression is likely to aid in the survival of MRD during severe winters. Moreover, it was found that glycerol-3-phosphate dehydrogenase (G3PDH)—an enzyme linking lipid and carbohydrate metabolism—is upregulated despite the hypometabolic conditions during freezing. This upregulation of G3PDH activity likely plays a vital role in supporting the metabolic survival strategies of wood frogs. Overall, this thesis uncovered an intricate yet synchronized network of enzymes that support MRD and initiate hepatoprotective mechanisms allowing wood frogs to endure prolonged freezing and maintain cellular homeostasis.

Aakriti Gupta, Ph.D. Biology 2022

Complex yet coordinated: regulation of transcriptional factors and cell signaling pathways to endure anoxia in Rana sylvatica

Abstract:

Wood frogs (Rana sylvatica) are a well-studied vertebrate model of natural freeze tolerance, surviving several months of winter subzero temperatures with 65-70% of total body water frozen as extracellular ice. Freezing halts blood circulation, heartbeat and breathing, restricting oxygen availability throughout the body and requiring a switch to anaerobic glycolysis for energy production, with its much lower ATP yield. To survive, wood frogs suppress their metabolic rate by about 90% to match ATP availability from glycolysis alone. Multiple cellular processes are regulated and suppressed, sustaining only pro-survival pathways until thawing occurs. Episodes of anoxia/reoxygenation also elevate reactive oxygen species (ROS) production that can surpass the antioxidant capacity of cells causing oxidative stress and tissue damage. This thesis examined a network of stress-responsive transcription factors (NRF2, OCT1, OCT4, YAP/TEAD, and RBPJ) and their associated pathways to determine their response and regulation over the anoxia/reoxygenation cycle. Decreased binding of transcriptional complexes to the promoter regions of target genes indicated a global reduction in transcription/translation processes. The data show also “functional switching” of OCT1, OCT4, and MAML while selectively upregulating antioxidants in a stress/organ specific manner. The present studies also shed new light on tissue repair mechanisms by demonstrating upregulation of selected pathway proteins. An increase in AHCY levels in liver also suggests maintenance of redox control, and elevated JMJD2C, TAZ, and MAML in skeletal and cardiac muscles indicates a potential increase in the expression of MyoD for muscle regeneration. Overall, the findings of this thesis document a complex yet coordinated network of transcriptional factors that support metabolic rate depression during freezing, combat oxidative stress, and initiate tissue repair mechanisms to endure prolonged anoxia and maintain cellular homeostasis in frozen wood frogs.

Gurjit Singh, Ph.D. Biology 2022

Role of glucose-induced transcription factor signalling and mitochondrial epigenetics in stress tolerant wood frog, Rana sylvatica

Abstract:

The freeze-tolerant wood frogs, Rana sylvatica are one of only a few vertebrate species in the animal kingdom, which are extensively studied to understand vertebrate freeze tolerance. They undergo whole-body freezing during winter and become ice solid with no heartbeat, brain activity and blood flow but amazingly come back to life during spring unharmed without any major changes in their body. Freeze survival is challenging, with wood frogs facing ischemia due to freezing of blood, dehydration via cell volume reductions due to loss of 60-70% of total body water into extracellular space as well as hyperglycemia, producing a huge amount of glucose as a cryoprotectant. Interestingly, wood frogs can also tolerate these stresses independent of freezing. Also, winter survival by wood frogs is associated with a metabolic reorganization to reduce their energy demands to a bare minimum by globally suppressing energy-expensive pathways and selectively regulating genes to prioritize available energy use for pro-survival pathways. This thesis examined the effects of freezing and dehydration-induced hyperglycemic response in selectively inducing transcription factor MondoA in regulating glucose-induced transcription and activating an adaptive transcriptional response to induce stress response via inflammasome activation, mitochondrial dysfunction and mitochondrial epigenetics. The current findings establish MondoA in guiding an adaptive transcriptional response to activate genes regulating glucose homeostasis and circadian rhythm in a tissue-specific manner in the liver during the freeze/thaw cycle. Also, the role of TXNIP (downstream to MondoA) and its PTMs, in activating inflammasome via NLRP-3 in stress-specific way during freezing was shown. Moreover, the higher mitochondrial presence of TXNIP did not correlate to protein expression of its downstream targets in inducing mitochondrial dysfunction in any of the stresses, which were attributed to its low/weak binding to TRX-2. Investigating the role of mitochondrial methylation suggests its tissue-specific regulation in the liver and potential role in maintaining a tight regulation of mitochondrial transcriptional and gene expression response. Altogether, findings from this thesis demonstrate that a highly synchronized and intricate control via multiple levels of regulation is present in activating mechanisms that are involved in maintaining cellular milieu during stress in wood frogs.

Mackenzie Parent, M.Sc. Biology 2023

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

Abstract:

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

Abstract:

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

Abstract:

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

Abstract:

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

Abstract:

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

Abstract:

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

Abstract:

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.