Waking the sleeping dragon: molecular insights into the hibernation of the central bearded dragon by Alexander Capraro A thesis in the fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Biochemistry and Molecular Genetics School of Biochemistry and Biomolecular Sciences Faculty of Science University of New South Wales February 2020 Thesis/Dissertation Sheet Surname/Family Name : CAPRARO Given Name/s : Alexander Abbreviation for degree as give in the University calendar : PhD Faculty : Science School : Biotechnology and Biomolecular Sciences Waking the sleeping dragon: molecular insight into the hibernation of Thesis Title : the central bearded dragon Abstract: One of the most profound adaptive strategies employed by animals in response to changes in environmental conditions is hibernation. Hibernation is an extended state of hypometabolism and dormancy induced to survive the low food availability and high physiological stress associated with winter. During the long periods of dormancy, hibernators experience changes in core biological processes, including drastic reductions in basal metabolic rate, core body temperature, oxygen consumption, heart rate and brain activity. Stress response pathways are also modulated to mitigate physiological challenges that would otherwise prove lethal to the animal. Modulation of these responses requires tightly controlled regulatory processes. While hibernation is a common response utilised by diverse lineages of animals, molecular investigations into this phenomenon have primarily focused on mammalian systems. In this thesis, I investigated the molecular mechanisms involved in the hibernation of the Australian central bearded dragon (Pogona vitticeps) using high-throughput sequencing technologies. The transcriptomic and proteomic changes in three tissues; brain, heart and skeletal muscle, during hibernation and at two time points post-arousal were examined. Hibernation was associated with the induction of common and tissue-specific stress response pathways in addition to the modulation of regulatory processes. I studied the role of microRNAs (miRNA) in mediating the changes in gene expression that are characteristic of the three tissues during hibernation and four days post-arousal. Cellular metabolism and neuroprotection of the brain emerged as key pathways under miRNA-mediated regulation. The DNA methylation dynamics in brain and skeletal muscle during hibernation and at two time points post-arousal were also examined. Alterations in DNA methylation were associated with changes in gene expression that lead to increased neuroprotection of the brain and a reduced potential for atrophy in skeletal muscle. Collectively, the data presented in this thesis shows that the precise interplay of multiple gene regulatory mechanisms is crucial in modulating the large-scale changes in cellular physiology and function observed in hibernating bearded dragons. Furthermore, the data presented bridges the gap between mammalian and reptilian hibernation and suggests they are more similar than previously thought. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents a non-exclusive licence to archive and to make available (including to members of the public) my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known. I acknowledge that I retain all intellectual property rights which subsist in my thesis or dissertation, such as copyright and patent rights, subject to applicable law. I also retain the right to use all or part of my thesis or dissertation in future works (such as articles or books). ……………………………………………………… ……………………………………………………… Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years can be made when submitting the final copies of your thesis to the UNSW Library. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. i ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………………………………………….............. ii COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents a non-exclusive licence to archive and to make available (including to members of the public) my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known. I acknowledge that I retain all intellectual property rights which subsist in my thesis or dissertation, such as copyright and patent rights, subject to applicable law. I also retain the right to use all or part of my thesis or dissertation in future works (such as articles or books).’ ‘For any substantial portions of copyright material used in this thesis, written permission for use has been obtained, or the copyright material is removed from the final public version of the thesis.’ Signed ……………………………………………........................... Date …………………………………………….............................. AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis.’ Signed ……………………………………………........................... Date …………………………………………….............................. iii Inclusion of publications statement UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure. Publications can be used in their thesis in lieu of a Chapter if: • The candidate contributed greater than 50% of the content in the publication and is the “primary author”, i.e. the candidate was responsible primarily for the planning, execution and preparation of the work for publication • The candidate has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis Please indicate whether this thesis contains published material or not: ☐ This thesis contains no publications, either published or submitted for publication Some of the work described in this thesis has been published and it has been documented ☐ in the relevant Chapters with acknowledgement This thesis has publications (either published or submitted for publication) incorporated ☒ into it in lieu of a chapter and the details are presented below CANDIDATE’S DECLARATION I declare that: • I have complied with the UNSW Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Candidate’s Name Signature Date (dd/mm/yy) POSTGRADUATE COORDINATOR’S DECLARATION I declare that: • the information below is accurate • where listed publication(s) have been used in lieu of Chapter(s), their use complies with the UNSW Thesis Examination Procedure • the minimum requirements for the format of the thesis have been met. PGC’s Name PGC’s Signature Date (dd/mm/yy) iv Details of publication #1: Full title: Waking the sleeping dragon: gene expression profiling reveals adaptive strategies of the hibernating reptile Pogona vitticeps Authors: Alexander Capraro, Denis O’Meally, Shafagh A. Waters, Hardip R. Patel, Arthur Georges and Paul D. Waters Journal or book name: BMC Genomics Volume/page numbers: 20:460 Date published: 6/6/2019 Status Published X Accepted and in In progress press (submitted) The Candidate’s Contribution to the Work Alexander Capraro contributed to the design of the study, performed the experiments and computational analysis of sequencing and mass spectrometry data, and wrote the paper. Location of the work in the thesis and/or how the work is incorporated in the thesis: This publication will be used in lieu of chapter 2. PRIMARY SUPERVISOR’S DECLARATION I declare that: • the information above is accurate • this has been discussed with the PGC and it is agreed that this publication can be included in this thesis in lieu of a Chapter • All of the co-authors of the publication have reviewed the above information and have agreed to its veracity by signing a ‘Co-Author Authorisation’ form. Primary Supervisor’s name Primary Supervisor’s signature Date (dd/mm/yy) v Details of publication #2: Full title: The role of microRNAs in regulating neuroprotection in the brain of hibernating central bearded dragons Authors: Alexander Capraro, Denis O’Meally, Shafagh A. Waters, Hardip R. Patel, Arthur Georges and Paul D. Waters Journal or book name: BMC Biology/BMC Genome