Hanane

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.