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Saxitoxin Binding Proteins: Biological Perspectives

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ResearchOnline@JCU This file is part of the following reference: Robertson, Alison (2005) Saxitoxin binding proteins: biological perspectives. PhD thesis, James Cook University. Access to this file is available from: http://researchonline.jcu.edu.au/34822/ If you believe that this work constitutes a copyright infringement, please contact [email protected] and quote http://researchonline.jcu.edu.au/34822/ SAXITOXIN BINDING PROTEINS: BIOLOGICAL PERSPECTIVES Thesis submitted by ALISON ROBERTSON BSc (Hons) In March, 2005 For the degree of Doctor of Philosophy In the School of Pharmacy and Molecular Sciences JAMES COOK UNIVERSITY Townsville, Queensland, Australia. In collaboration with: THE AUSTRALIAN INSTUTUTE OF MARINE SCIENCE Marine Biotechnology Townsville, Queensland, Australia. Jl/O// STATEMENT OF ACCESS I, the undersigned author of this work, understand that James Cook University will make this thesis available for use within the University Library and, via the Australian Digital Theses network, for . use elsewhere. I understand that, as an unpublished work, a thesis has significant protection under the Copyright Act and; I do not wish to place any further restriction on access to this work :Z07N Ociober .?W5 Signature Date ELECTRONIC COPY I, the undersigned, the author of this work, declare that the electronic copy of this thesis provided to the James Cook University Library, is an accurate copy of the print thesis submitted, within the limits of the technology available. Signature Date STATEMENT OF SOURCES DECLARATION I certify that this thesis is my own work and has not been admitted in any other form for another degree or diploma at any university or other institution of tertiary education. Information derived from the published or unpublished works of others has been acknowledged in the text and a list ofreferences is provided. ALISON ROBERTSON iii DECLARATION ON ETHICS The research presented and reported in this thesis was conducted within the guidelines for research ethics outlined in the Joint NHMRCIAVCC Statement and Guidelines on Research Practice (1997), the James Cook University Policy on Experimentation Ethics, Standard Practices and Guidelines (2001), and the James Cook University Statement and Guidelines on Research Practice (2001). All components of the project and reporting procedures were in compliance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the Queensland Animal Care and Protection Act (2001). The research methodology received clearance from the James Cook University Experimentation Ethics Review Committee (approval number A745-02). ALISON ROBERTSON IV STATEMENT ON THE CONTRIBUTION OF OTHERS Financial I am very grateful for the considerable financial support that was provided by the following organisations over the course of my candidature: An Australian Postgraduate Award (APA), granted by James Cook University (JCU) and financed by the federal Department of Science, Education and Training (DEST), provided a generous stipend and was greatly appreciated. I am indebted to the School of Pharmacy and Molecular Sciences, JCU, who financed bench fees, transport and overheads incurred from my full­ time placement at the Australian Institute of Marine Science (AIMS), allowing me the opportunity to work in a world class marine research institution. Finally I would like to express my appreciation to AIMS for providing cutting edge research facilities, and a considerable postgraduate research award which covered research consumables and equipment purchases over the course of candidature. Collaborations I am sincerely grateful to Dr. David Stirling from the Institute of Environmental Science & Research Limited, Porirua, New Zealand for his collaboration and insights. David performed preliminary MS analysis of several octopi extracts and contributed to an accepted manuscript as detailed in Chapter 4. I am also grateful and humbled by the generosity of Dr. David Rowell from the Australian National University (ANU) who provided his time, transport, permits and experience to assist me on a field collection of log dwelling invertebrates from the Tallaganda State forest in NSW. v Bio-molecular Analysis Services • The Australian Proteome Analysis Facility (APAF) performed tryptic digests of purified saxitoxin binding protein and subsequent peptide sequencing by manual QTOF mass spectrometry, on a fee for service basis. • Likewise, nucleotide sequencing was performed by Dr. Lynn Woodward from the Genetic Analysis Facility (GAF) in the Advanced Analytical Centre (AAC) at JCU. All other personal and scientific contributions are listed as acknowledgements for each relevant thesis chapter. ALISON ROBERTSON VI ACKNOWLEDGEMENTS I would like to thank my supervisors Dr. Lyndon Llewellyn and Dr. Andrew Negri from the Australian Institute of Marine Science and Prof. Jim Burnell from James Cook University for providing me with the opportunity to embark upon this PhD research and for believing in me. Thanks to Lyndon who's greatest attribute is his love of science. I appreciate you letting me in to the world of toxins and proteins and hope that one day we can solve this saxiphilin riddle. Thanks also to Jim for administrative assistance, encouragement and molecular biology input. Special thanks to Andrew, who was a continual source of friendship, motivation, advice and encouragement. Thanks for holding the torch at the end of the long winding tunnel, whenever I lost sight of the goal. I would also like to acknowledge the laughs, friendship, support and encouragement of everyone in the Marine Biotechnology group at AIMS. It was a wonderful opportunity to work with and learn from such a great group of people. To my office buddies, Carsten, Neil, Enrique and Carlos for the laughs and friendship. I would like to thank Dr. Cherie Motti for being a impartial sounding board to new ideas, for helping me troubleshoot new chemistry methods and equipment and for reviewing some of my early manuscripts with humility. I would also like to thank Dr. Diane Tapiolas who has been a constant source of encouragement and support and for urging me to seek out post doctoral opportunities overseas. Thanks to Dr. Rick Willis who invited me into the world of Mass Spectrometry and was always willing to have a bash at some new and interesting samples. Special thanks to Dr. Cedric Robillot for his friendship, interest and support of my project and training and assistance with newly commissioned LC and LCMS equipment. Thanks to Dr. Kate Wilson for her SUppOlt, ideas, molecular biology advice and wealth of experience which really transformed my approach to the partial sequencing of saxiphilin and to Jennie Swan for showing me that molecular vii biology could be fun. Thanks also to Lesa, Enrique, Dave and Nicole for fielding my many questions on molecular strategies. I am grateful to Jason, Lyndon and Rick who enthusiastically provided me with a steady stream of toads when I needed them and to all who assisted with field collections. Particular thanks go to Mr. Carsten Wolff (AIMS), Dr. John Collins (JCU), Dr. Mark Norman and Mr. Julian Finn who assisted with taxonomy following extensive field collections. Appreciation is extended to all staff from AIMS including members from science, IT, administration, stores, purchasing, accommodation, security, finance, marine operations, library, registry, and human resources for their kindness, laughs and assistance in day to day activities. I would particularly like to acknowledge the assistance of Gavin, Carsten and Darren who helped me resurrect my PC and sanity through four successive hard drive crashes and a multitude of computer related dramas. I deeply appreciate the support and friendship of Dr. Anna Marie Babey. Thanks for the independent advice and perspective during tough times. To my beloved family Heather, Jim and Bruce, thank you all for your unwavering love and encouragement which has made the vast distances between us disappear. Last, but certainly not least, I would like to thank Terry who has been by my side for the whole journey, through the highs and lows with his unconditional love and support. Life has hit you with some tough blows but I am constantly amazed at you inner strength and determination. There are only great things to look forward to from now on. viii PUBLICATIONS ARISING FROM THESIS Each research chapter of this thesis will stand alone as at least one peer reviewed publication as detailed below. Additional publications and conference presentations indirectly relating to my thesis research are detailed as additional outcomes in Chapter 6. Thesis Research Papers Robertson, A., Stirling, D., Robillot, C., Llewellyn, L., Negri, A.P. (2004) First report of saxitoxin in octopi. Toxicon, 44, 765- 771. Robertson, A., Llewellyn, L. E., Negri, A. P. (2005) Development of a robust radio-receptor assay for discovery and screening of novel saxitoxin binding proteins and paralytic shellfish toxins. Target Journal: Environmental Science & Technology (in preparation). Robertson, A., Negri, A.P., Llewellyn, L.E. (2005) Extending the phylogenetic distribution of saxitoxin binding proteins in search of functionality. Target Journal: Toxicon (in preparation). Robertson, A., Llewellyn, L.E., Negri, A.P. (2005) Survey of a tropical saxitoxin "hotspot": Port Hedland, Western Australia. Target Journal: Marine Biology (in preparation). Robertson, A., Swan, J., Negri, A. P., Burnell, J., Llewellyn, L., Wilson, K. (2005) Purification and partial sequencing
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