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.

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.