Lin Hui Yao, M.Sc. Chemistry, 2007

Profile of FoxO proteins and MnSOD in two cold-hardy insect species exposed to low temperature.



Expression of FoxO genes and proteins in two insects with different overwintering strategies were studied with Western blotting and RT-PCR. Epiblema scudderiana, a freeze-avoiding gall moth, and Eurosta solidaginis, a freeze-tolerant gall fly were used as model organisms to assess the role of FoxO transcription factors and one of their downstream proteins, manganese superoxide dismutase (MnSOD) in insect cold hardiness. FoxO3a gene and protein levels, as well as MnSOD protein, were analyzed inEpiblema scudderiana over time courses of exposure to 5°C or ‑15°C. Significant changes in total Foxo3a protein, the amount of phosphorylated inactive FoxO3a, FoxO3a mRNA levels, and MnSOD content were identified under different conditions. For example, reduced phospho-FoxO3a (Ser 253) content in larvae exposed to -15°C suggested enhanced transcriptional activation of genes under FoxO3a control at subzero temperatures whereas MnSOD content increased in larvae exposed to 5°C. Sequencing of FoxO3a mRNA from Epiblema scudderiana showed that the transcription factor was highly conserved as compared with mammals. FoxO3a, FoxO1 and MnSOD protein levels were also measured in the freeze tolerant insect Eurosta solidaginis subjected to 5 and ‑15°C exposures as well as a time course of thawing after freezing at -15°C for 1 week. The results implied that FoxO3a and FoxO1 functioned oppositely during cold, freezing and thawing of the larvae. Expression of MnSOD increased with 5°C exposure, remained elevated when larvae were frozen at -15°C, and decreased after thawing. Differential expression of FoxOs and MnSOD in both species suggest that MnSOD may not be transcriptionallycontrolled by FoxO3a or FoxO1 in cold hardy insects.


Melanie Bouffard, M.Sc. Chemistry, 2007

Involvement of FOXO transcription factors and glycogen synthase kinase 3 in the freeze tolerance capability of the wood frog, Rana sylvatica.  



Animals cope with the subzero temperatures of winter in different ways. The wood frog, Rana sylvatica, endures whole body freezing and is able to survive weeks completely frozen. Organisms that endure extreme environmental stress on a periodic or seasonal basis have developed ways to strongly suppress their metabolic rate and enter a hypometabolic state to survive. Forkhead box ‘other’ (FOXO) transcription factors have important roles in various cellular processes such as metabolism, cellular proliferation, stress tolerance and lifespan. Immunoblotting was used to assess total and phosphorylated amounts of FOXO proteins in wood frog organs. Active FOXO1 increased in brain during freezing and thawing, possibly due to a need for gluconeogenesis during this stress. The levels of active FOXO3 increased in frog brain, kidney and liver during freezing and thawing and also during anoxia and aerobic recovery after anoxia, which could be due to the need to maintain or enhance antioxidant defenses under these stresses. Glycogen synthase kinase-3 (GSK3) is a protein kinase known to inhibit glycogen synthesis, cell growth and differentiation and protein translation. The amount of active GSK3 increased in the frozen state in brain, heart, kidney, liver and muscle of wood frogs. Furthermore, kinetic analysis of GSK3 showed that the skeletal muscle of frozen frogs appears to have a higher affinity for its substrate when compared to control GSK3. Allosteric effectors of GSK3 were also identified: glucose-6-phosphate activated the enzyme whereas AMP inhibited. The data expand our understanding of metabolic regulation during natural freeze tolerance.

Mani Mathialagan, MSc Biology 2008

Transcription factor regulation in mammalian hibernation: the thirteen-lined ground squirrel as a model.



The thirteen-lined ground squirrel (Spermophilus tridecemlineatus) is one of a number of mammalian species that hibernate over the winter months. Hibernation is characterized by long periods of deep torpor that are interspersed with periodic arousals. This thesis investigated three transcription factor pathways and their regulation and roles in hibernation using tools including immunoblotting of protein and phospho-protein levels, analysis of nuclear versus cytoplasmic distribution, binding to DNA, PCR to quantify mRNA transcripts, and gene sequencing. The carbohydrate response element-binding protein (ChREBP) regulates genes that are responsible for converting excess dietary carbohydrate (glucose) into triglycerides. During torpor ChREBP protein was decreased in kidney and liver and transcripts of two downstream genes (Fasn, PK-L) were differentially expressed; this was consistent with carbohydrate sparing during torpor. Growth and differentiation processes are also minimized during hibernation and ETS transcription factors are important regulators of these. Although levels of ETS increased in most organs of hibernators, ETS binding activity to DNA was greatly reduced indicating a growth suppressive function during torpor. Upstream effectors of ETS (epidermal growth factor receptor, protein kinases C and D) were also evaluated. To assess transcriptional state during hibernation, the Rb-E2F pathway was investigated. Retinoblastoma protein (Rb) associates with E2F1 and then recruits co-factors to the promoter region of target genes to suppress transcription. Rb and E2F1 proteins were elevated during hibernation and the co-factors HDAC and SUV39H1 showed parallel increases in the organs tested; oppositely, transcription of downstream genes (cMyc, Bcl2) was suppressed. These data confirm a reduced transcriptional state during torpor.


Judeh Lama.  M.Sc. Biology 2008

Regulation of glucose-6-phosphate dehydrogenase and hexokinase in anoxia-tolerant mollusks: role of reversible phosphorylation



Metabolic rate depression is key to animal survival without oxygen and requires coordinated suppression of ATP-generating and ATP-consuming cellular functions by stable regulatory mechanisms. This thesis examined the possible role of reversible protein phosphorylation in metabolic suppression in organs of the anoxia-tolerant intertidal marine mollusc, Littorina littorea. Studies focused on glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway (PPP), and hexokinase (HK), an important enzyme at the forefront of carbohydrate metabolism. The data show that hepatopancreas G6PDH is regulated by phosphorylation and entry into anoxia leads to a more dephosphorylated form, whose properties suggest a more active enzyme. This would favour enhanced carbon flow through the PPP to sustain NADPH production for antioxidant defense. Furthermore, the study provides one of the first demonstrations of coordinated regulation of the PPP between active and hypometabolic states and implicates specific protein kinases and phosphatases in G6PDH regulation. Studies of HK showed that it is also regulated by reversible protein phosphorylation, in a tissue-specific manner. In the hepatopancreas, it appears that HK affinity for one of its substrates is enhanced during anoxia whereas in foot muscle, HK activity is suppressed. Differences between control and anoxic HK were also observed in their susceptibility to urea denaturation, response to specific protein kinase and protein phosphatase incubations as well as their elution profiles from an ion-exchange column. Overall, these studies confirm an integral role of reversible protein phosphorylation in the suppression and reorganization of L.littorea metabolism for anoxia survival.


Craig Brooks, M. Sc. Biology, 2009

Regulation of NF-κB and p53 in the liver and skeletal muscle of the freeze tolerant wood frog, Rana sylvatica



The wood frog, Rana sylvatica, is the primary model animal used for studying vertebrate freeze tolerance.  During Canadian winters, wood frogs can endure the freezing of about 70% of their total body water and then thaw and resume life in the spring.  Frogs have multiple ways to protect themselves against potential freezing injuries including adaptive changes to intermediary metabolism and gene expression.  One way that wood frogs deal with freezing stress is via upregulation of several freeze-responsive genes.  Previous studies provided excellent presumptive evidence for the involvement of the NF-κB and p53 transcription factors in freeze tolerance.  The studies in this thesis used Western blotting to quantify levels of NF-κB subunits p50 and p65, its inhibitor, p-IκB, and downstream targets (ferritin heavy chain, manganese superoxide dismutase) as well as protein levels of p53, post-translationally modified p53, and some p53 downstream genes in the muscle and liver of control versus frozen wood frogs. Nuclear distributions of NF-κB and p53 were also assessed. RT-PCR was used to quantify transcript levels of select targets of NF-κB and p53.  Significant increases in the expression levels of NF-κBand its downstream targets as well as in levels of p53, post-translationally modified p53, and its downstream targets were observed during freezing.  These findings suggest the activation of NF-κB antioxidant defenses in the wood frog during freezing in anticipation of reperfusion during thawing and the activation of p53 in the wood frog which would lead to cell cycle arrest in the frozen state.

Rabih Roufayel, M. Sc. Biology, 2009

Regulation of the Tb-E2F pathway in the freeze tolerant wood frog, Rana sylvatica



The North American wood frog, Rana sylvatica, is a primary model animal used in the study of freeze tolerance in vertebrates. During Canadian winters, wood frogs endure the freezing of about 65-70% of their total body water. Anoxia, ischemia, oxidative stress and many other consequences are a result of freeze/thaw cycles. A variety of adaptations are known that protect the frogs against potential freezing injuries as well as regulate their intermediary metabolism and gene expression to support survival. Selected transcription factors have critical roles to play in freezing survival by regulating the expression of genes that control the adaptations needed to handle freezing stress. In my present study, the retinoblastoma (Rb) protein coupled with the E2F transcription factor family were demonstrated to have roles in controlling the cell cycle in wood frog liver and skeletal muscle during freezing and associated stresses (anoxia, dehydration). Western blotting was used to quantify total Rb, phosphorylation or acetylation at different sites on Rb, E2F members and selected downstream targets under E2F control. Other central regulators of the cell cycle were also quantified including Cyclins, Cyclin dependent kinases (Cdks), and checkpoint proteins. Nuclear distributions of Rb-E2F and Cdk:Cyclins were also assessed. RT-PCR was used to quantify mRNA transcript levels of Cyclin D1 which decreased significant during freezing as well as c-Myc, a downstream target of E2F. The data indicate that the cell cycle is under regulation during freezing through E2F upregulation and Rb phosphorylation via Cdk:Cyclin activity.

Allan Letourneau, M.Sc. Biology, 2010

Phosphorylation of glycolytic enzymes in hibernation.



Glycerol-3-phosphate dehydrogenase (G3PDH) and lactate dehydrogenase (LDH) were examined for differential phosphorylation, accompanying kinetics and stability inSpermophilus richardsonii liver and skeletal muscle. Hibernator G3PDH had a higher phosphate content and differential kinetics in both tissues which could be manipulated by kinase and phosphatase incubations in the G3P utilizing direction. Arrhenius plots and activation energies (Ea) showed that the hibernator form had a lower Ea in both tissues, especially in the liver.Euthermic LDH had a higher phosphate content and differential kinetics which could be manipulated by kinase and phosphatase incubations in both directions. Euthermic forms had lower Ea values in both tissues. G3PDH and LDH had differential urea I50 values with the hibernator more susceptible to urea denaturation in both tissues. Urea I50 values could be manipulated in LDH by kinase and phosphatase incubations in the liver but not in muscle.

Katrina J. Sullivan, M.Sc. Biology, 2011

Expression pattern of the novel freeze-responsive genes li16, fr10 and fr47 in the wood frog, Rana sylvatica



The ability of the wood frog (Rana sylvatica) to freeze up to 65% of its total body water allows it to endure subzero temperatures encountered during winter.  To survive the stresses associated with freezing, frogs have evolved multiple molecular adaptations including expression of three novel genes: li16, fr10 and fr47.  All three genes were found to be freeze responsive in a tissue-dependent manner.  They also all respond to anoxia and dehydration stresses, suggesting that the transcription of these genes is triggered by the low oxygen conditions common to all three stresses.  Protein levels were elevated after freezing for Li16; however FR10 and FR47 generally showed no change or a decrease in protein after freezing.  Transcripts of all three genes were also detected during tadpole development, indicating a possible additional role for metamorphosis.  Finally,bioinformatic analysis of their sequences provided insight into functional motifs and potential regulatory sites on the proteins.

Sandra Korycan, M.Sc. Biology 1984

Organ-specific metabolism during anoxia and recovery from anoxia in the cherrystone clam, Mercenaria mercenaria



The levels of intermediary metabolites and end products were quantified in the tissues of the cherrystone clam, Mercenaria mercenaria, over a time course of 96 h of anoxia followed by 48 h of aerobic recovery. Succinate and alanine accumulated as anaerobic products while glycogen and aspartate were utilized as substrates. Succinate accumulation ranged from 12-14 micromol/g wet weight in muscle (phasic and catch adductor, foot) to 25 micromol/g in gill and mantle with 32 micromol/mL released into the mantle cavity fluid. Lesser amounts of alanine were produced, the ratio succinate:alanine varying from 1.4:1 in phasic adductor to 3.2:1 in mantle at 96 h. Aspartate reserves apparently supply the carbon for succinate synthesis over the first 6-12 h of anoxia; subsequent succinate and alanine production probably results from glycogen fermentation. The imino acids alanopine and strombine were not produced in appreciable amounts (less than 1 micromol/g) during anoxia. When returned to aerated seawater, control levels of alanine and aspartate were reestablished within 24 h; accumulated succinate was catabolized within 48 h. Glycogen content of all tissues showed a sharp decline after 6 h of recovery, perhaps due to enhanced energy demands, but levels increased later in recovery. Tissue ATP levels, which were depressed during anoxia, were restored by 24 h.

Ajoy Chakrabarti, M.Sc. Biology 1988

Immobilization of Cellulase Using Polyurethane Foam



Cellulase was covalently immobilized using a hydrophilic polyurethane foam (Hypol FHP 2002). Compared to the free enzyme immobilized cellulase showed a dramatic decrease (7.5-fold) in the Michaelis constant for carboxymethylcellulose. The immobilized enzyme also had a broader and more basic pH optimum (pH 5.5-6.0) a greater stability under heat-denaturing or liquid nitrogen-freezing conditions and was relatively more efficient in utilizing insoluble cellulose substrates. High molecular weight compounds (Blue Dextran) could move throughout the foam matrix indicating permeability to insoluble celluloses; activity could be further improved 2.4-fold after powdering foams under liquid nitrogen. The improved kinetic and stability features of the immobilized cellulase combined with advantageous properties of the polyurethane foam (resistance to enzymatic degradation plasticity of shape and size) suggest that this mechanism of cellulase immobilization has high potential for application in the industrial degradation of celluloses.