M. Sc. Thesis Abstracts

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

Abstract:

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.

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

Abstract:

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.

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

Abstract:

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.

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

Abstract:

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.

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

Abstract:

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.

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

Abstract:

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.

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

Abstract:

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.

Targeting of calcium calmodulin-dependent protein kinase II to membranes

Abstract:

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.

SMAD transcription factor expression in anoxia-tolerant Trachemys scripta elegans and dehydration-tolerant Xenopus laevis: A comparative study 

 

Abstract:

The TGF-β signaling pathway with its associated transcription factors (SMADs) was studied in the red-eared slider (Trachemys scripta elegans; liver, heart, white skeletal muscle, red skeletal muscle, and kidney) as a model for anoxia tolerance and the African clawed frog (Xenopus laevis; liver and muscle) as a model for dehydration resistance. In addition, protein expression levels of precursor cytokines were measured, as well as known genes and proteins downstream of the SMAD signaling pathway, such as c-Myc, and AP-1 proteins. The data showed that selective control of different SMAD isoforms occurred in response to environmental stress, the mechanism being reversible protein phosphorylation. Cellular responses and signaling profiles were observed to be highly tissue-specific and stress-specific. SMADs were shown to play a role in the hypometabolic response of both animal models through the regulation of cell cycle arrest, angiogenic processes, and oxidative injury management strategies.

Regulation of lactate dehydrogenase and glycerol-3-phosphate dehydrogenase in mammalian hibernation

 

Abstract:

Hibernation is a winter survival strategy for many small mammals. Animals sink into deep torpor, body temperature falls to near 0°C and physiological functions are strongly suppressed. Enzymes are the catalysts of metabolic pathways in cells and their appropriate control is critical to hibernation success. This thesis explores the properties and regulation of two key enzymes of carbohydrate metabolism (lactate dehydrogenase, LDH) and lipid metabolism (glycerol-3-phosphate dehydrogenase, G3PDH) purified from liver and skeletal muscle of ground squirrels (Urocitellus richardsonii). The studies showed that changes in pH, temperature, and inhibitors play roles in differentially regulating these enzymes between euthermic and torpid states. Furthermore, reversible protein phosphorylation proved to be a significant regulatory mechanism, producing a reduced activity state of skeletal muscle LDH and increased activity state of G3PDH in both skeletal muscle and liver during torpor. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly protein phosphorylation, contribute to reorganizing fuel metabolism during hibernation.