Ph. D. Thesis Abstracts

Pier Morin, Ph.D. Chemistry, 2006

Transcriptional regulation in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.

 

Abstract:  

Many small mammals enter hibernation to survive the winter. Metabolic rate during torpor can drop to just 1-5 % of the euthermic rate providing energy savings of ~90% compared to the costs of remaining euthermic. Hibernation consists cycles of torpor bouts interspersed by short arousal periods. Regulation of torpor-arousal requires tight overall control of energy-consuming metabolic processes as well as selective expression of genes to accomplish these transitions and readjust metabolism for long term viability in a hypometabolic, hypothermic state. Hence, despite overall metabolic rate depression in hibernation, certain processes must be activated to ensure survival. The present studies examined transcriptional control in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus. Regulation of gene expression by the hypoxia-inducible transcription factor (HIF-1) pathway was shown to be important in hibernation; HIF-1α subunit levels rose by 60-70 % in skeletal muscle and BAT during hibernation and HIF-1 DNA-binding activity increased 6-fold in hibernating BAT.  By contrast, the overall transcriptional state in muscle was strongly suppressed during torpor.  Both activity and protein levels of histonedeacetylases, enzymes involved in transcriptional repression, were elevated during torpor whereas the activity of RNA Polymerase II, a key enzyme of gene transcription, was strongly reduced by 43 %.  To evaluate the involvement of oxidative stress and antioxidant defenses in hibernation, the responses of the Nrf2 transcription factor, involved in the oxidative stress response pathway, were evaluated as well as antioxidant genes/proteins under Nrf2 control. Nrf2 protein levels were elevated (by 1.4-fold) in all heterothermic parts of the hibernation cycle whereas the protein contents of three downstream gene targets of Nrf2 were only elevated during entrance into hibernation. This suggests aNrf2-mediated anticipatory enhancement of antioxidant defenses to deal with oxidative stress during torpor and/or arousal. Other antioxidant enzymes, the 2-Cys peroxiredoxins, also showed enhanced protein levels in torpor and increased enzyme activity (1.5-fold in heart and 3.4-fold in BAT) indicating a potential role in ROS detoxification during hibernation.  The data reported in this thesis provides new insights on the roles of selected transcription factors and on the importance of their tight regulation during all stages of hibernation.

Christopher J. Ramnanan, Ph.D. Biology, 2006

Regulation of metabolism in estivating land snails: role of reversible protein phosphorylation.

 

Abstract:

Estivation is a state of aerobic dormancy used by animals such as the desert land snail, Otala lactea, to endure harsh environmental conditions. Metabolic rate depression is key to survival during estivation and requires coordinated suppression of ATP generating and ATP-consuming cellular functions by stable regulatory mechanisms. Current studies examined the role of reversible protein phosphorylation in metabolic arrest in snail organs focusing on a major ATP-consuming process, the Na+/K+-ATPase; a key enzyme that produces NADPH for antioxidant defense, glucose-6-phosphate dehydrogenase (G6PDH); and selected protein kinases and protein phosphatases that could regulate the process. Studies documented a decrease in foot muscle and hepatopancreas Na+/K+-ATPase activity, and an increase in hepatopancreas G6PDH activity during estivation, as indicated by changes in kinetic parameters (e.g. maximal velocity, substrate affinity, Arrhenius activation energy). Furthermore, in vitro incubations stimulating specific endogenous kinases and phosphatases implicated roles for PKG and PP1 in estivation-dependent changes in Na+/K+-ATPase and G6PDH. Ion exchange chromatography of G6PDH revealed two enzyme forms – a high phosphate, high activity form and a low phosphate, low activity form – whose proportions changed in dormant snails. The peak profiles of G6PDH from active and estivating snails were also interconverted after incubations promoting PKG or PP1 activities. Profiles of protein phosphatases in O. lacteatissues revealed a general suppression of activity during estivation. For PP1 and PP2A the differential activity in estivation was linked to altered enzyme elution profiles from gel filtration chromatography, indicating that differential association into phosphatase holoenzymes is partly responsible for reduced phosphatases activities in estivation. Type-1 and type-2 phosphatases were purified and analyzed; the data generally indicated that the mammalian phosphatase classification system was applicable to O. lacteaphosphatases. Examination of several protein kinases, utilizing a relatively novel assay method with P81 paper array/phosphor imaging, revealed increased activities of AMPK, PKB, and PKG in estivating O. lactea. Increased activities of AMPK and PKB were related to changes in their phosphorylation state and confirmed by changes in activities and/or phosphorylation status of downstream targets. Overall, these studies confirm the integral role of reversible protein phosphorylation in the suppression and reorganization of metabolism during estivation.

Hapsatou Mamady, Ph.D. Biology, 2006

Mammalian hibernation: gene expression and transcription factor regulation of the unfolded protein response, apoptosis and atrophy in ground squirrels.

 

Abstract:

Various mammalian species hibernate as a way to survive extended winter periods of food scarcity and cold environmental conditions. Hibernation is an energy-conserving strategy,characterized by periods of torpor with extreme decreases in core body temperature and strong metabolic rate depression, interrupted by brief periods of arousal to euthermia. To endure the conditions of cold torpor, as well as the wide fluctuations over cycles of torpor-arousal, differential expression of genes and their tight regulation is required. The present studies examined changes in the expression and regulation of selected genes involved in the unfolded protein response, muscle atrophy and anti-apoptotic defense during hibernation in thirteen-lined ground squirrels,Spermophilus tridecemlineatus. Despite overall suppression of transcription and translation during torpor, selected genes and their products were up-regulated. Themolecular chaperone GRP78 increased in BAT and brain of torpid animals, indicating endoplasmic reticulum stress and a role for GRP78 in alleviating stresses that cause protein misfoldingduring hibernation. Regulation of thegrp78 gene by the activating transcription factor ATF4 via the PERK/eIF2a/ATF4 pathway was shown to be important in hibernation; ATF4 protein expression increased in BAT, brain and skeletal muscle of hibernating squirrels and ATF4 DNA-binding activity increased in hibernating brain. Subcellular localization of ATF4 showed that this transcription factor and its cofactor, pCREB-1, weretranslocated into the nucleus during hibernation where they could activate downstream genes. Another transcription factor, FoxO1a, and the downstream genes that it controls via the PI3K/AKT/FOXO pathway can induce muscle atrophy. Hibernators appear to counteract this by phosphorylating and inactivating FoxO1a in heart and skeletal muscle and strongly suppressing FoxO1a DNA binding activity by 76% in muscle during torpor. Finally, the anti-apoptotic proteins, Bcl-XL and Bcl-2, showed enhanced expression in tissues of ground squirrels whereas the pro-apoptotic protein, BAD, was suppressed viaphosphorylation during torpor. These results show that anti-apoptotic defense is also important to cell survival in hibernation. The data in this thesis enhance our knowledge of the molecular mechanisms that govern hibernation and the role played by selected transcription factors in regulating subsets of genes that are physiologically relevant to the hibernation phenotype.

 

Khalil Abnous. Ph.D. Chemistry 2007

Regulation of metabolic enzymes during hibernation in ground squirrels

 

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 cells and their appropriate control is critical to hibernation success. This thesis explores the properties and regulation of key enzymes of carbohydrate metabolism (hexokinase, HK), energy metabolism (creatine kinase, CK; AMP deaminase, AMPD) and signal transduction (Akt; MAPKAP-K2), highlighting skeletal muscle ground squirrels (Spermophilusrichardsonii). The studies showed that changes in pH, temperature, inhibitor and activator concentrations, mRNA transcript and protein levels, and binding to myofibrils are involved in regulating these enzymes during hibernation. Moreover, reversible protein phosphorylation proved to be a key regulatory mechanism, reducing the activity of all these enzymes during hibernation. Analysis of total protein content by Western blotting found decreased HKII, CK and P-Akt protein during hibernation but no change in Akt and MAPKAP-K2 content. Analysis of temperature effects on enzymes, via Arrhenius plots, showed that CK, AMPD and MAPKAP-K2 had significantly higher activation energies in hibernating animals Urea denaturation and pulse proteolysis showed that HKII from hibernators had greater resistance to chemical denaturation than the euthermic enzyme but studies on CK and MAPKAP-K2 found no stability differences. Affinity of CK and AMPD for their substrates decreased during hibernation. HK, Akt and MAPKAP-K2 showed reduced ATP affinity in hibernation but HK affinity for glucose remained stable, and Akt and MAPKAP-K2 showed higher affinity for their substrate peptides. Protein kinases (PKA, PKC, PKG) increased AMPD activity from both euthermic and hibernating animals but decreased CK activity; AMPK elevated HK activity in euthermic muscle. Protein phosphatases generally reversed these actions. Changes in enzyme phosphorylation state during hibernation were confirmed by elution profiles of the enzymes off DEAE Sephadex, patterns that were interconverted after incubations that stimulated protein kinases and phosphatases. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly protein phosphorylation, contribute to reorganizing enzymatic function and stability during hibernation.

 

Christopher Dieni.  Ph.D. Chemistry 2008

Regulation of enzyme function in freeze tolerance

 

Abstract:

The wood frog (Rana sylvatica) is one of the few vertebrate species that can survive whole-body freezing during the cold winter months. The frog endures the freezing of 65-70% of total body water as extracellular ice and, while frozen, shows no respiration, heart beat, or brain activity. Consequently, the frogs experience anoxia and ischemia throughout the freeze followed by oxidative stress when oxygen is reperfused. Enzymes, the biochemical catalysts of cells, must be appropriately controlled to ensure survival. This thesis explores the properties and regulation of key enzymes of adenylate metabolism (AMP-deaminase, AMPD; creatine kinase, CK) and glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH; hexokinase, HK). The studies showed that changes in pH, temperature, inhibitor and activator concentrations, and binding to myofibrils are involved in regulating these enzymes in the transition to the frozen state. Moreover, reversible protein phosphorylation appears to be a key regulatory mechanism, altering enzyme activity and substrate affinity to suit physiological needs during freezing. Analysis of kinetic parameters showed an increase in enzyme activity for CK and decreased activity for HK. Affinity of CK for one of its substrates, creatine, increased, whereas HK, G6PDH, and myofibril-bound AMPD showed reduced substrate affinity in the transition to the frozen state. These changes in kinetic parameters were the result reversible protein phosphorylation; bound AMPD and CK both increased in phosphorylation state in frozen frogs, whereas G6PDH and HK both decreased in phosphorylation state. Changes in enzyme activity as a result of reversible phosphorylation were analyzed by in vitro stimulation of endogenous protein kinase and protein phosphatase activities. Native phosphorylation states of these enzymes, and changes between control and frozen frogs were further confirmed by elution profiles off DEAE-Sephadex ion-exchange columns that were interconverted between the two physiological states, as well as SDS-PAGE studies that compared phosphoprotein levels to total protein levels. Though phosphorylation states of these enzymes changed, protein levels remained constant in the transition to the frozen state. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly reversible protein phosphorylation, control enzyme function and the reorganization of metabolic pathways in freeze-tolerance.

Amal I. Malik, Ph.D. Biology, 2009

Cellular adaptation to dehydration stress: Molecular adaptations for dehydration tolerance in the African clawed frog, Xenopus laevis

 

Abstract:

The thesis addressed multiple questions about the signal transduction mechanisms that trigger gene expression responses to dehydration signals, and about the role of antioxidant defenses in combating dehydration stress in the African clawed frog, Xenopus laevis. In the first part of the thesis the responses to dehydration stress by the three main MAPK cascades were traced by measuring both total protein levels, and the relative amounts of active phosphorylated proteins for multiple intermediates in the p38 MAPK, stress-activated protein kinase (SAPK), and extracellular signal-regulated kinase (ERK) cascades. The data documented a major activation of the ERK pathway in most organs of X. laevis during dehydration. Selected upstream activator and downstream targets of the ERK pathway also showed pronounced tissue specific regulation in response to dehydration. The SAPK was activated in skeletal muscle, lung, and skin whereas the p38 MAPK was activated in the lung and kidney of X. laevis.  The second part of the thesis focused on antioxidant defenses that are known to be contributors to cell preservation under various types of stress. Two main transcription factors that regulate antioxidant genes were activated in response to dehydration in X. laevis organs:  NF-E2 related factor (Nrf2) and forkhead box, class O (FoxO). Immunoblottingshowed a significant increase in their nuclear translocation, and enzyme-linked immunosorbant assays showed increased DNA binding activity by FoxO1 under dehydration stress. Expression of downstream target genes controlled by these transcription factors was enhanced during dehydration. Six family members of the glutathione S-transferase (GST) and three family members of the aldo-keto reductase (AKR) showed tissue specific expression, correlated with Nrf2 activation. Manganese-dependent superoxide dismutase (MnSOD) and catalase expression were also elevated under FoxO1 control. Improved antioxidant defenses may be critical to dealing with variations in tissue oxygenation and reactive oxygen species generation that are one consequence of large changes in body hydration that affect oxygen delivery to tissues. This thesis showed for the first time that the MAPKs family members are selectively activated in response to two levels of dehydration stress in X. laevis. Also, antioxidant defenses play a critical protective role during dehydration stress in these frogs.

Anastasia Krivoruchko, Ph.D. Biology, 2010

Turtle anoxia – biochemistry and gene regulation in an anaerobic extremist

 

Abstract:

While the physiological responses to oxygen deprivation have been studied extensively in the anoxia-tolerant turtle, Trachemysscripta elegans, adaptations of transcriptional regulatory processes are mostly unknown. This thesis addresses this by examining the anoxia responsiveness of several important proteins and pathways in T. s. elegans tissues. The unfolded protein response (UPR) was activated in turtle heart, kidney and liver, as evidenced by increased phosphorylation of PERK and increased expression and activation of ATF4. Enhanced expression of the molecular chaperones GRP78 and GRP94, as well as other UPR-responsive proteins was also observed. These results suggest that the UPR is an important component of stress tolerance in the turtle. The transcription factor NF-kB was also anoxia-responsive in turtle liver and activated via increased expression of its component proteins, increased nuclear presence and increased DNA-binding activity. Transcript levels of NF-kB target genes involved in antioxidant defense and anti-apoptotic signaling were also upregulated under anoxia. The FoxO transcription factors, implicated in hypometabolism and stress resistance, were also anoxia-responsive. Studies of FoxO expression, phosphorylationstatus, nuclear presence and DNA-binding activity showed that FoxO1 and FoxO3 were both activated in liver, whereas FoxO3 was activated in heart and kidney. FoxO target genes involved in cell cycle arrest and stress resistance were also upregulated in liver under anoxia. Expression and activation of the transcriptional inhibitors, histone deacetylases (HDACs), was strongly elevated in white skeletal muscle during anoxia, with a lesser response by liver, results that indicated an important role for HDACs in anoxia-mediated transcriptional suppression. Finally, the metabolic transcription factor involved in control ofglycolytic enzymes, ChREBP, was activated in the liver in response to 5 h of anoxia, and its target gene, LPK, wastranscriptionaly induced, suggesting a role for this transcription factor in adjusting carbohydrate metabolism for anaerobiosis. Overall, the data in this thesis enhance understanding of the gene and protein adaptations that support cellular endurance of anoxia and document several new mechanisms that are involved in stress resistance, hypometabolism and fuel metabolism as being key to natural anoxia tolerance.

Benjamin Lant, Ph.D. Biology, 2011

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

 

Abstract

The capacity to adapt to and survive oxygen deprivation has long been an important topic of study, in both ecological and medical fields. The freshwater crayfish, Orconectes virilis, is capable of tolerating anoxia, but the metabolic mechanisms underlying this are largely unprofiled. This thesis examines the activity and regulation of a number of stress response pathways in response to anoxia in O. virilis. The model organism Caenorhabditis elegans that shows stress-induced entry into hypometabolism (the dauer stage) was used as a template for selecting stress response pathways that could be important in crayfish anaerobiosis. The Akt signaling response showed a distinct increase in activity in crayfish tail muscle and hepatopancreas under anoxia, as assessed through phosphorylation states of the kinase and its downstream targets. This implicated a pro-survival response that functions by preventing cell cycle attenuation. Despite elevated Akt activity, residualFoxO activity remained, possibly mediating a pro-survival mechanism through transactivation of antioxidant genes (includingMnSOD) in preparation for reoxygenation. Smad and STAT transcription factors, following the pattern of pro-development Akt signaling, also showed a fairly active profile (via phosphorylation status) but upregulation was not unilateral. Hepatopancreas showed a more active profile of Smads, but this did not correlate with increased DNA binding, again hinting at a preparative mechanism for the recovery period. Apoptosis (cell death) signaling was assessed through pro-apoptosis (p53) and anti-apoptosis (Bcl) targets, whereas autophagy (a cell minimization response to stress) was assessed via expression response of multiple autophagy proteins (Atg). An anoxia-triggered, tissue-specific result arose, potentially based on the importance of individual organ integrity throughout hypometabolism. Tail muscle, which showed increased expression profiles of all three target groups (p53, Bcls, Atgs), contrasted with hepatopancreas, which appeared to be not susceptible to either apoptotic orautophagic signaling during anoxia. Finally, the cell cycle, often a target for attenuation in stress states, was analyzed. Neither tissue showed strong signs of cell cycle attenuation under anoxia, although certain inhibitor profiles were enhanced under anoxia. The data provide a comprehensive overview of the responses and integration of multiple stress-responsive signaling pathways in O. virilis that provide a novel contribution to our understanding of pro-survival mechanisms supporting invertebrate anoxia tolerance.