Anastasia Krivoruchko, Ph.D. Biology, 2010

Turtle anoxia – biochemistry and gene regulation in an anaerobic extremist



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.