The presence of acetic acid during industrial alcohol fermentation reduces the yield of fermentation by imposing additional stress on the yeast cells. The biology of cellular responses to stress has been a subject of vigorous investigations. Although much has been learned, details of some of these responses remain poorly understood. Members of heat shock chaperone HSP proteins have been linked to acetic acid and heat shock stress responses in yeast. Both acetic acid and heat shock have been identified to trigger different cellular responses including reduction of global protein synthesis and induction of programmed cell death. Yeast HSC82 and HSP82 code for two important heat shock proteins that together account for 1–2% of total cellular proteins. Both proteins have been linked to responses to acetic acid and heat shock. In contrast to the overall rate of protein synthesis which is reduced, the expression of HSC82 and HSP82 is induced in response to acetic acid stress. In the current study we identified two yeast genes DOM34 and RPL36A that are linked to acetic acid and heat shock sensitivity. We investigated the influence of these genes on the expression of HSP proteins. Our observations suggest that Dom34 and RPL36A influence translation in a CAP-independent manner.

Mole-rats are champions of self-preservation, with increased longevity compared with other rodents their size, strong antioxidant capabilities and specialized defenses against endogenous oxidative stress. However, how the brains of these subterranean mammals handle acute in vivo hypoxia is poorly understood. This study is the first to examine the molecular response to low oxygen in six different species of hypoxia-tolerant mole-rats from sub-Saharan Africa. Protein carbonylation, a known marker of DNA damage (hydroxy-2′-deoxyguanosine), and antioxidant capacity did not change following hypoxia but HIF-1 protein levels increased significantly in the brains of two species. Nearly 30 miRNAs known to play roles in hypoxia tolerance were differentially regulated in a species-specific manner. The miRNAs exhibiting the strongest response to low oxygen stress inhibit apoptosis and regulate neuroinflammation, likely providing neuroprotection. A principal component analysis (PCA) using a subset of the molecular targets assessed herein revealed differences between control and hypoxic groups for two solitary species (Georychus capensis and Bathyergus suillus), which are ecologically adapted to a normoxic environment, suggesting a heightened sensitivity to hypoxia relative to species that may experience hypoxia more regularly in nature. By contrast, all molecular data were included in the PCA to detect a difference between control and hypoxic populations of eusocial Heterocephalus glaber, indicating they may require many lower-fold changes in signaling pathways to adapt to low oxygen settings. Finally, none of the Cryptomys hottentotus subspecies showed a statistical difference between control and hypoxic groups, presumably due to hypoxia tolerance derived from environmental pressures associated with a subterranean and social lifestyle.

Hibernation is a highly regulated stress response that is utilized by some mammals to survive harsh winter conditions and involves a complex metabolic reprogramming at the cellular level to maintain tissue protections at low temperature. In this study, we profiled the expression of 117 conserved microRNAs in the heart, muscle, and liver of the 13-lined ground squirrel (Ictidomys tridecemlineatus) across four stages of the torpor-arousal cycle (euthermia, early torpor, late torpor, and interbout arousal) by real-time PCR. We found significant differential regulation of numerous microRNAs that were both tissue specific and torpor stage specific. Among the most significant regulated microRNAs was miR-208b, a positive regulator of muscle development that was found to be upregulated by fivefold in the heart during late torpor (13-fold during arousal), while decreased by 3.7-fold in the skeletal muscle, implicating a potential regulatory role in the development of cardiac hypertrophy and skeletal muscle atrophy in the ground squirrels during torpor. In addition, the insulin resistance marker miR-181a was upregulated by 5.7-fold in the liver during early torpor, which supports previous suggestions of hyperinsulinemia in hibernators during the early stages of the hibernation cycle. Although microRNA expression profiles were largely unique between the three tissues, GO annotation analysis revealed that the putative targets of upregulated microRNAs tend to enrich toward suppression of progrowth-related processes in all three tissues. These findings implicate microRNAs in the regulation of both tissue-specific processes and general suppression of cell growth during hibernation.

Hibernation requires the intricate regulation of physiological and biochemical adaptations to facilitate the decrease in metabolic rate and activation of prosurvival factors needed for winter survival. Mitochondria play important roles in eliciting these responses and in coordinating the required energy shifts. Herein, we report the presence of a novel mitochondrial peptide, s‐humanin, in the hibernating 13‐lined ground squirrel, Ictidomys tridecemlineatus. S‐humanin was shown to have strong structural and sequence similarities to its human analogue, humanin—a powerful neuroprotective mitochondrial peptide. An assessment of the protein and gene expression levels of this peptide in ground squirrels revealed stark tissue‐specific regulatory responses whereby transcript levels increased in brain cortex, skeletal muscle, and adipose tissues during hibernation, suggesting a protective torpor‐induced activation. Accompanying peptide measurements found that s‐humanin levels were suppressed in liver of torpid squirrels but enhanced in brain cortex. The enhanced transcript and protein levels of s‐humanin in brain cortex suggest that it is actively involved in protecting delicate brain tissues and neuronal connections from hibernation‐associated stresses. We propose that this squirrel‐specific peptide is involved in modulating tissue‐specific cytoprotective functions, expanding its role from human‐specific neuroprotection to environmental stress protection.Significance of the studyUnderstanding the molecular mechanisms, which protect against oxidative stress in a model hibernator such as the ground squirrel, could be pivotal to the regulation of cytoprotection. This study expands on our knowledge of metabolic rate depression and could suggest a potential role for humanin therapy in neurodegenerative diseases.

The red-eared slider turtle (Trachemys scripta elegans), has developed remarkable adaptive mechanisms for coping with decreased oxygen availability during winter when lakes and ponds become covered with ice. Strategies for enduring anoxia tolerance include an increase in fermentable fuel reserves to support anaerobic glycolysis, the buffering of end products to minimize acidosis, altered expression in crucial survival genes, and strong metabolic rate suppression to minimize ATP-expensive metabolic processes such as protein synthesis. The mammalian target of rapamycin (mTOR) is at the center of the insulin-signaling pathway that regulates protein translation. The present study analyzed the responses of the mTOR signaling pathway to 5 (5H) or 20 h (20H) of anoxic submergence in liver and skeletal muscle of T. scripta elegans with a particular focus on regulatory changes in the phosphorylation states of targets. The data showed that phosphorylation of multiple mTOR targets was suppressed in skeletal muscle, but activated in the liver. Phosphorylated mTOR showed no change in skeletal muscle but had increased by approximately 4.5-fold in the liver after 20H of anoxia. The phosphorylation states of upstream positive regulators of mTOR (p-PDK-1, p-AKT, and protein levels of GβL), the relative levels of dephosphorylated active PTEN, as well as phosphorylation state of negative regulators (TSC2, p-PRAS40) were generally found to be differentially regulated in skeletal muscle and in liver. Downstream targets of mTOR (p-p70 S6K, p-S6, PABP, p-4E-BP1, and p-eIF4E) were generally unchanged in skeletal muscle but upregulated in most targets in liver. These findings indicate that protein synthesis is enhanced in the liver and suggests an increase in the synthesis of crucial proteins required for anoxic survival.

Hibernation is a fascinating survival adaptation that allows animals to transition into a torpid state to survive the winter by coordinating a strong suppression of metabolic rate, conservation of fuel/energy, and reduction of body temperature. This strategy permits thirteen-lined ground squirrels (Ictidomys tridecemlineatus) and other hibernating mammals to endure the harsh winter season when there is little access to food. Many energy-expensive cellular processes are suppressed, including gene transcription and protein synthesis/turnover, but are reactivated rapidly when animals arouse back to euthermia. Both torpor and arousal can have damaging consequences; for example, during arousal, reactive oxygen species flood the cell causing oxidative damage to numerous cellular components. Therefore, hibernation requires many pro-survival mechanisms to mitigate multiple types of damage: e.g. from oxidative damage, DNA damage, and pathogen attack, among others. The research reported in this thesis on damage control processes in hibernators shows that antioxidant enzymes such as PRDXs are upregulated in key tissues but in an isoform-specific and time-specific manner over the torpor-arousal cycle. PRDX2, 3, 4 and 6 were found to be significantly upregulated in specific tissues.Similarly, DNA damage repair is initiated during torpor and is characterized by the binding of repair proteins such as Ku80 and the MRN complex to the site of breaks, but ligation (with XLF) reactions to fully repair DNA do not appear to occur until the arousal period.Pro-inflammatory mechanisms are also used to deal with pathogens; these remain active at basal levels in a tissue-specific manner during torpor, but are up-regulated in the final stages just before arousal or only during arousal depending on the tissue, such as the induction of CCL5, a recruiter of monocytes. A cyto/neuro-protective mitochondrial peptide, shumanin, was also identified that is induced in a tissue-specific manner, helping to protect key organs such as the brain cortex and adipose tissues. The results show that hibernation is a complex, multi-faceted process that employs specific adaptations of damage prevention/repair pathways to protect squirrel tissues from damage not only during prolonged torpor but over the transitional states to/from torpor and does so expertly while conserving energy until such a time that repair mechanisms may be fully initiated.iv

No abstract

The red-eared slider turtle, Trachemys scripta elegans, can survive 3-4 months of anoxic submergence in cold water during the winter. The effect of hypoxia/anoxia on protein synthesis in red-eared sliders was investigated with a focus on the insulin-signaling pathway and analysis of the mammalian target of rapamycin (mTOR) and its upstream and downstream effectors in liver and white muscle. Expression of mTORC1 did not change in muscle but increased significantly in liver after 5 and 20 hours of anoxic submergence. Upstream effectors, AKT and RAPTOR, were also elevated in liver but suppressed in muscle. PRAS40 and TSC2 inhibitors of mTOR were differentially regulated in both tissues but generally suppressed. Downstream targets of mTOR signaling (eIF4E, 4E-BP1, P70S6K, S6) as well as the poly(A) binding protein also showed differential responses to anoxia. Overall, the data indicate that the early response to anoxia by turtles is maintenance of protein synthesis in liver but suppression in white muscle. iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my thesis supervisor, Dr. Kenneth Storey for accepting me into his lab. I am eternally grateful for his advice and persistence in pushing me to work harder than I ever have in order to become the molecular biologist I am today. I have learned so much more than new molecular techniques from Dr. Storey. He has helped me develop on a personal level and pursue higher levels of achievement. I must also thank Janet Storey for her extensive knowledge and hours of work put into helping me write this thesis. Furthermore, the continued amount of work put into keeping the lab running. We would be lost without you. I would like to thank my family and friends without whom I would not be where I am today. To my parents for causing me to push myself towards higher academic achievement, to my lab mates: Dr. Barb Katzenback and Dr. Shannon Tessier for teaching me everything I know, Sanoji Wijenayake for her endless guidance and support, as well as Kyle Biggar, Mike Wu, and Bryan Luu for their countless hours of training and tutelage. Finally, I would like to thank the rest of the lab for guiding me along the way and for our endless scientific discussions. Most importantly, I would like to thank Avery, without you, I would not have gotten where I am today. I thank you for your endless support, wisdom and guidance. You have been there for me from the beginning and continuously throughout this journey. v