CORE Metadata, citation and similar papers at core.ac.uk Provided by ScholarWorks@UA INDUCTION OF HEAT SHOCK PROTEINS IN COLD- ADAPTED AND COLD- ACCLIMATED FISHES By Laura Elizabeth Teigen Dr. Kristin O'Brien Advisory Committee Chair Dr. Diane Wagner Chair, Department of Biology and Wildlife APPROVED: ;t.-/ INDUCTION OF HEAT SHOCK PROTEINS IN COLD- ADAPTED AND COLD- ACCLIMATED FISHES A THESIS Presented to the Faculty of the University of Alaska Fairbanks in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE By Laura Elizabeth Teigen, B.A. Fairbanks, Alaska May 2014 v Abstract I examined the effects of oxidative stress and changes in temperature on heat shock protein (Hsp) levels in cold-adapted and cold-acclimated fishes. Adaptation of Antarctic notothenioids to cold temperature is correlated with high levels of Hsps, thought to minimize cold-induced protein denaturation. Hsp70 levels were measured in red- and white-blooded Antarctic notothenioid fishes exposed to their critical thermal maximum (CTMax), 4C warm acclimated, and notothenioids from different latitudes. I determined the effect of cold acclimation on Hsp levels and the role of sirtuins in regulating Hsp expression and changes in metabolism in threespine stickleback, Gasterosteus aculeatus, cold-acclimated to 8C. Levels of Hsps do not increase in Antarctic notothenioids exposed to their CTMax, and warm acclimation reduced levels of Hsp70. Hsp70 levels were higher in Antarctic notothenioids compared to a temperate notothenioid and higher in white-blooded notothenioids compared to red-blooded notothenioids, despite higher oxidative stress levels in red-blooded fish, suggesting Hsp70 does not mitigate oxidative stress. Cold acclimation of stickleback resulted in tissue-specific increases in some Hsps and sirtuins. My research indicates that cold acclimation increases Hsp levels, and moderate increases in temperature reduce Hsp levels in cold-adapted fishes. Together, these data lend support to the hypothesis that cold denatures proteins. vii Table of Contents Page Signature Page .................................................................................................................... i Title Page ........................................................................................................................... iii Abstract ................................................................................................................................v Table of Contents .............................................................................................................. vii List of Figures .................................................................................................................... xi List of Tables ................................................................................................................... xiii List of Appendices ........................................................................................................... xiii Acknowledgements ............................................................................................................xv Introduction ..........................................................................................................................1 References ............................................................................................................................7 Chapter 1: Heat shock proteins in hearts of red- and white-blooded notothenioids ..........13 1.1 Abstract ..................................................................................................................14 1.2 Introduction ............................................................................................................16 1.3 Materials and Methods ...........................................................................................20 1.3.1 Tissue collection ....................................................................................20 1.3.2 Experimental design...............................................................................21 1.3.3 Hsp70/Hsc70 protein levels ...................................................................22 1.3.4 RNA extraction ......................................................................................23 1.3.5 Cloning and sequencing of HSP70 and EF-1α ......................................24 1.3.6 Quantitative real-time PCR ....................................................................25 1.3.7 Statistical analyses .................................................................................26 1.4 Results ....................................................................................................................27 1.4.1 Hsp70 levels in fishes exposed to their CTMax ....................................27 1.4.2 Hsp70 levels after 1 week acclimation to 4°C .......................................27 1.4.3 Hsp70 levels in icefishes and red-blooded notothenioids ......................28 1.5 Discussion ..............................................................................................................28 1.5.1 Levels of Hsps in hearts of red- blooded notothenioids and icefishes ....29 viii Page 1.5.2 Hsp protein levels do not differ among notothenioids from different regions ............................................................................................30 1.5.3 HSP70 mRNA levels decline in response to moderate warming ..........31 1.6 Figures....................................................................................................................35 1.7 Tables .....................................................................................................................38 1.8 Acknowledgements ................................................................................................39 1.9 Appendix A ............................................................................................................40 1.10 References ............................................................................................................53 Chapter 2: Cold acclimation induces expression of heat shock proteins and sirtuins in threespine stickleback ..................................................................................65 2.1 Abstract ..................................................................................................................66 2.2 Introduction ............................................................................................................68 2.3 Methods..................................................................................................................72 2.3.1 Animal collection and experimental procedures....................................72 2.3.2 RNA extraction ......................................................................................73 2.3.3 Quantitative real-time PCR ....................................................................74 2.3.4 Hsp70/Hsc70 protein levels ...................................................................75 2.3.5 Statistical analyses .................................................................................77 2.4 Results ....................................................................................................................77 2.4.1 Levels of heat shock proteins .................................................................77 2.4.2 Expression of sirtuins .............................................................................79 2.5 Discussion ..............................................................................................................80 2.5.1 Hsps may increase during cold acclimation in response protein denaturation....................................................................................81 2.5.2 Increases in Hsps in pectoral muscle may assist with mitochondrial biogenesis .......................................................................................83 2.5.3 Transcript levels of HSP70 mRNA do not change with cold acclimation .....................................................................................84 ix Page 2.5.4 Expression of some sirtuins change with cold acclimation ...................85 2.5.5 Mitochondrial SIRT3 and SIRT4 do not change during cold acclimation .....................................................................................86 2.6 Conclusion .............................................................................................................87 2.7 Figures....................................................................................................................89 2.8 Tables .....................................................................................................................92 2.9 Acknowledgements ................................................................................................92 2.10 Appendix A ..........................................................................................................93 2.11 References ..........................................................................................................103 Conclusion .......................................................................................................................115 References ........................................................................................................................121 xi List of Figures Page Fig. 1.1: HSP70 transcript levels in ventricle of heat stressed Antarctic