Sam Williamson, M.Sc. Biology 2015

Epigenetic underpinnings of freeze tolerance in the goldenrod gall fly Eurosta solidaginis and the goldenrod gall moth Epiblema scudderiana


The goldenrod gall fly Eurosta solidaginis and gall moth Epiblema scudderiana are cold hardy insects that inhabit stem galls on goldenrod plants and survive subzero temperatures during the winter. Eurosta uses a freeze tolerance strategy where it restricts the formation of ice to extracellular spaces and preserves vital intracellular functions. Epiblema supercools its bodily fluids to remain unfrozen below 0°C. This thesis examined the role of epigenetic enzymes in insect cold hardiness strategies. Cold and subzero temperature exposure in Eurosta resulted in upregulation of several DNA Methyltransferase (DNMT) enzymes, increases in DNMT and Histone Acetyltransferase (HAT) activities, and decreases in Histone Deacetylase (HDAC) and Ten-Eleven Translocation (TET) enzyme activities. Epiblema showed upregulation of several DNMT enzymes with concurrent decreases in DNMT, HAT, and HDAC activities and no change in TET activity. These findings suggest that epigenetic regulation of genes and histones underpins the winter survival strategies of these insects.

Liam Hawkins, M.Sc. Biology 2016

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


The wood frog, Rana sylvatica, has developed numerous adaptations to survive days with up to 65% of its body fluid frozen. One such adaptation is to reduce their metabolic rate, employing only those processes needed to survive until temperatures rise. The establishment of this hypometabolic state is mediated by transcriptional regulation that is elicited in part by histone methylation, however this has yet to be explored in the context of metabolic rate depression and freeze tolerance. This thesis provides the first characterization of histone methyltransferases (HMTs) and the histone and non-histone proteins they methylate in the wood frog. Transcriptionally permissive histone residues (H3K4me1 and H3K27me1) were found to decrease during freezing in skeletal muscle while those that silence transcription (H3K9me3 and H3K36me2) were maintained, whereas differential levels of histone residues were seen in liver. These findings suggest a novel role for HMTs in freeze tolerance.

Tony (Yichi) Zhang, M.Sc. Biology 2016

The molecular mechanisms underlying skeletal and cardiac muscle remodeling in the hibernating thirteen-lined ground squirrel


The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) survives winters by hibernating, whereby body temperature (Tb) cycles between 4ºC during torpor and 37ºC during arousal. Each organ/tissue of the hibernator must make specific adjustments that allow the ground squirrel to maintain or readjust physiological function during hibernation. The remodeling that occurs in skeletal and cardiac muscle is unique to hibernators, and it is fascinating as a natural means of avoiding physiological dysfunction in these tissues. The purpose of this thesis is to evaluate the molecular mechanisms underlying muscle remodeling in both tissues. It was identified that calcium signaling activates the NFAT-calcineurin pathway, leading to increased expression of hypertrophy-promoting targets in both skeletal and cardiac muscle during torpor. In addition, we found that there is differential expression and activity of transcription factors (Foxo, MyoG) and ubiquitin ligases (MAFbx and MURF1) that promote muscle atrophy in the two tissues being studied.

Rasha Al-attar, M.Sc. Biology 2016

Regulation of the nuclear factor activated T cell (NFAT) family of transcription factors in the freeze tolerant wood frog, Rana sylvatica


During winter, wood frogs (Rana sylvatica) can endure whole body freezing with 65-70% of total body water converted to extracellular ice. As a result, cells experience extensive dehydration when water exits as well as anoxia due to interruption of blood flow. Adapting to such challenges requires metabolic rearrangement, partially mediated by transcription factor control over gene expression. Here, involvement of the nuclear factor of activated T-cells (NFAT) transcription factors, isoforms c1-c4, was analyzed in liver and skeletal muscle over freeze/thaw and anoxia/re-oxygenation cycles. Freezing activated NFATc3 in liver, leading to increased osteopontin expression and glycogen synthase kinase 3β repression (the latter potentially linked with glucose production as a cryoprotectant). Anoxia activated NFATc4 in liver, leading to increased atrial natriuretic peptide levels. Neither freezing nor anoxia significantly affected NFATs in skeletal muscle. Overall, the study indicates that NFATs have a crucial role to play in the natural cryoprotection of liver.

Alex Watts, M.Sc. Biology 2016

Involvement of reversible protein methylation in 13-lined ground squirrel hibernation


During winter hibernation, body temperature falls to near ambient levels, metabolism is shifted to favor lipid oxidation and transcriptional and translational activity is minimized in the face of limited resources and increased heat generation costs. In order to regulate such profound changes, mammals require control at least partly brought about by protein post-translational modifications. Protein lysine methylation provides a mechanism by which enzymes may alter the activity, stability and modification states of proteins relevant to hibernator physiology. Protein abundance of SMYD2, SUV39H1, SET8, SET7/9, G9a, ASH2L and RBBP5 in 13-lined ground squirrel (Ictidomys tridecemlineatus) skeletal muscle and liver was characterized. Tissue-specific regulation was seen and enzymes changed during either torpor, arousal, or transitory periods. Methylation of H3, HSP90, and p53 proteins were also quantified and typically followed patterns of modifying enzymes. Overall, these experiments show protein lysine methylation is differentially regulated during 13-lined ground squirrel hibernation.

Altaf Mahmud, M.Sc. Biology 2012

An investigation of the relationship between dietary fiber, fecal bacterial composition, and colon cancer


Colon cancer (CC) is the second leading cause of all cancer-related deaths in North America. Dietary fiber (DF) may be an important risk factor in the aetiology and pathogenesis of CC. The anticancer effects of dietary fiber were investigated with a focus on fecal bacterial diversity and toxicity of bacterial metabolites in the aqueous phase of feces (fecal water: FW) that contains bile acids, short chain fatty acid, lactate, succinate, etc. Briefly, male Fischer-344 rats were randomized to one of 3 diets: alphacel (control), fructooligosaccharides (FOS) or wheat bran (WB) with a total fermentability level of 3% (wt/wt). Rats were injected with saline or azoxymethane (AOM) to induce tumors. FW toxicity was tested on HCT-116 cells. Rats fed alphacel and FOS diets had significantly more colon tumors than those fed WB. FW from both FOS and alphacel significantly increased apoptosis and DNA damage, and induced cell cycle arrest in HCT-116 cells after a 48 hr treatment whereas FW of WB had no effect on those cell parameters. Lower pH of FW was associated with more tumors incidence and higher cell toxicity. FOS diet was significantly associated with more Allobaculum sp. whereas Lactobacillus sp. and Clostridium XI sp. were associated with WB diet. These results suggest that dietary fiber can be an influence in CC development. This seems to be related to changes in bacterial population and bacterial metabolic activities.

Julie Wu, M.Sc. Biology 2013

Effect of fermentation rate of dietary fibre on short-term satiety, long-term food intake and gut hormone response in male rats


As obesity rates increased worldwide, nutritional strategies to reduce food intake as a weight management tool have gained much attention. Studies showed that dietary fibre can be protective against weight gain through fermentation that influences gut hormone levels to increase satiety and reduce food intake. Macronutrient-induced satiety to reduce meal size has also received a great interest. This study aimed to investigate the effects of macronutrients (carbohydrate, protein, fat) and fibres (different fermentation rates) on satiety, corresponding hormone responses (insulin, ghrelin, glucagon-likepeptide-1, peptide YY), and their relationships with food intake and body weight in rats. I found that diet containing fructooligosaccharides led to reduced long term food intake, weight gain and fat mass whereas wheat bran promoted food intake with unaffected weight gain. Both fructooligosaccharides and oil were associated with significantly lower food consumption and higher circulating PYY. My data suggested that satiety regulation is complex and can be strain-dependent.

Shahriar Saeedi, M.Sc. Biology 2000

Targeting of calcium calmodulin-dependent protein kinase II to membranes


Communication between neurons is mediated by the controlled release of neurotransmitters from small synaptic vesicles. Several proteins are involved in the regulation of neurotransmitter release. One such family of proteins is the calcium/calmodulin-dependent protein kinases. Calcium/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant members of this protein family. and is found in many tissues with specific isoforms usually predominating in certain tissues. CaMKII is found CO- purified with synaptic vesicles. The question of how CaMKII is attached to the synaptic vesicle is addressed in this thesis. It was hypothesized that CaMKII may interact with the phospholipid component of the vesicle membrane, which could mediate binding.

C-terminal deletion mutants of murine α-CaMKII were produced by PCR directional cloning and liposome-binding assays were performed on the purified proteins. Binding of murine α-CaMKII to several types of liposomes was not observed. Recent studies into targeting of this enzyme suggest a role for anchoring proteins. Murine α-CaMKII like the β isoform from rat muscle may interact with membranes through a putative non-kinase translation product (a putative anchoring protein), which encompasses amino acids 230-270 at N-terminal of the regulatory domain rather than through direct binding with phospholipids.

Stuart Green, Ph.D. Biology 2021

Regulation of citric acid cycle enzymes and related pathways in the skeletal muscle of hibernating Richardson’s ground squirrels, Urocitellus Richardsonii


Richardson’s ground squirrels (Urocitellus richardsonii) are small rodents inhabiting western Canada that spend a large portion of their life in hibernation. Hibernation is accompanied by a profound drop in body temperature to a minimum of 2-3 °C and a notable shift from carbohydrate to lipid consumption that involves large-scale rearrangements of central metabolic processes. This thesis investigated the regulation of key enzymatic checkpoints in the citric acid cycle (CAC) as well as enzymes that shuttle substrates into the CAC in skeletal muscle of ground squirrels during hibernation. Initial work investigated regulation of the pyruvate dehydrogenase complex (PDC) that bridges glycolysis and the CAC. Muscle PDC showed few changes in properties in terms of activity and inhibitory phosphorylation of the enzyme. This was in stark contrast to liver where strong suppression of PDC activity occurred during hibernation correlated with increased inhibitory phosphorylation on serine-300. This then led to investigation of two crucial irreversible regulatory steps of the CAC in the muscle: citrate synthase (CS) and the α-ketoglutarate dehydrogenase complex (KGDC). CS activity decreased significantly during hibernation. This correlated with decreased lysine succinylation of CS that reflected increased SIRT5 levels, the enzyme responsible for desuccinylase activity in mitochondria. KGDC also showed decreased affinity for coenzyme A in hibernating squirrels and marked differences in posttranslational modifications including increased tyrosine phosphorylation on all three enzyme subunits and increased serine phosphorylation on E2 subunit. Stimulating the action of endogenous protein kinases demonstrated decreased affinity for coenzyme A. Finally, regulation of muscle glutamate dehydrogenase (GDH) was analyzed to ascertain how GDH regulation mediated the flow of α-ketoglutarate into the CAC from amino acid catabolism. Most GDH kinetic parameters were unaffected between hibernating and euthermic states, except that glutamate affinity was substantially lower at 8 °C (a physiologically relevant temperature) for the enzyme from hibernating squirrels. GDH from hibernating animals also exhibited significantly higher ADP-ribosylation, suggesting a regulatory mechanism for modulating GDH. Taken together these findings suggest that enzymatic regulation in Richardson’s ground squirrel muscle is actively mediated by a variety of posttranslational mechanisms of the CAC and related enzymes to coordinate metabolic suppression during hibernation.

Hanane Hadj-Moussa, Ph.D. Biology 2021

Molecular adaptations of mammalian hypoxia tolerance: Regulation of oxidative damage, neuroprotection, and microRNA


Prolonged exposure to limited oxygen can be lethal. Investigating the biological consequences of oxygen-deprivation in a hypoxia tolerant mammalian model can provide us with novel insights that could be applied to alleviate the ischemic insults experienced during stroke, or to better tolerate the hypoxia of high-altitude. Naked mole-rats (Heterocephalus glaber) represent nature’s solution to the problem of both acute and chronic oxygen limitation among mammals, solutions that have developed over evolutionary time. In this thesis I investigate their unique adaptations. The data I collected paints a picture of intricate signalling mechanisms in place to facilitate metabolic reorganization and protection during hypoxia. I determine that naked mole-rats are not as vulnerable to hypoxia-induced oxidative damage, as compared to hypoxia intolerant animals, and that brains appear to be the most resilient. The cell-survival proteins I profile implicate the induction of mechanisms responsible for conserving energy and maintaining neural integrity under low oxygen levels. Next, I perform the first microRNA-sequencing analysis in naked mole-rats, focusing on the hypoxic brain. Hypoxia-induced microRNAs suppress ATP-expensive processes, activate central signalling pathways, and coordinate a shift to non-fructose based anaerobic glycolysis. I then examine global metabolic reorganization and characterize a microRNA-mediated, AMPK-driven shift to carbohydrate metabolism in hypoxic skeletal muscles that may support tissue-specific prioritization of energy for more essential organs. Taken together, these findings advance our understanding of mammalian hypoxia tolerance and highlight the molecular mechanisms and complex layered regulatory controls required to endure frequent hypoxia exposures, as well as provide directions for future studies.