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Bovine Lactoferrin Regions Promoting Corneal Epithelial Healing

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Bovine Lactoferrin Regions Promoting Corneal Epithelial Healing Bovine Lactoferrin Regions Promoting Corneal Epithelial Healing Benjamin David Ashby BOptom (Hons) Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Optometry and Vision Science The University of New South Wales, Sydney, Australia and The Brien Holden Vision Institute Sydney, Australia March 2014 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: ASHBY First name: BENJAMIN Other name/s: DAVID Abbreviation for degree as given in the University calendar: PhD School: SCHOOL OF OPTOMETRY AND VISION SCIENCE Faculty: SCIENCE Title: BOVINE LACTOFERRIN REGIONS PROMOTING CORNEAL EPITHELIAL WOUND HEALING Abstract 350 words maximum: (PLEASE TYPE) Background. Corneal abrasions are a common eye injury. Fortunately they typically resolve quickly and without consequence. However until they heal they remain painful and if healing is delayed infection and scaring that may permanently impair vision is possible. The number one eye injury causing delayed healing is the alkali burn, usually from a household cleaning product or workplace accident. There are currently no medications proven to accelerate re-epithelialisation of these wounds. Purpose. Isolate the region of bovine lactoferrin (BLF) that promotes corneal epithelial wound healing, determine the mechanism and assess its efficacy. Methods. An alkali burn was applied in vitro to human corneal epithelial cells that were then incubated with BLF fragments or structural variants while wound closure was monitored. The mechanism promoting healing was determined using assays for migration, proliferation and measuring interleukin-6 and platelet-derived-growth-factor-BB (PDGF) levels. Efficacy of the regions identified in vitro were tested in vivo using Guinea pig and rabbit models of debridement and alkali burn wounds treated with BLF, fragments of BLF, or the vehicle. Wound size was monitored. Results. The C-lobe of BLF promoted greater wound healing in vitro than equimolar BLF (P<0.05) or the N-lobe (P<0.001). Further fragmentation or significant disruption to the conformation of BLF resulted in no promotion of healing compared to the control. C-lobe promoted healing was associated with corneal epithelial up-regulation of PDGF and IL-6 over the control (P<0.001), increased cell migration rates compared to BLF (P<0.05) and a small increase in proliferation rates (P<0.05) over the control. In vivo the C-lobe accelerated early wound healing in the rabbit model. For Guinea pigs with debridement wounds by 24 hours complete re-epithelialisation was observed in 67% treated with C lobe (P=0.03) and 22% with BLF (P=0.67) compared to 0% for vehicle. In the Guinea pig alkali burn model C-lobe (P=0.007) and BLF (P=0.001) promoted faster closure than the vehicle but were not significantly different from each other. Conclusions. The C-lobe of BLF promotes corneal epithelial wound healing. For abrasion-like wounds it is more effective that whole BLF. The BLF C-lobe may be a novel therapeutic for the treatment of corneal lesions. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). 2/6/14 ……………………………………………………… ……………………………………..……… ……….……………………... Signature Witness 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 must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS Bovine Lactoferrin Regions Promoting Corneal Epithelial Healing Page iii COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' 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. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date ……………………………………………........................... Certificate of Originality ‘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 …………2/6/14………………………................. Bovine Lactoferrin Regions Promoting Corneal Epithelial Healing Page ii Acknowledgements I would firstly like to thank my supervisors, Professor Mark Willcox and Dr Qian Garrett for their time, interest and experience. The combination of guidance and freedom you have given me has inspired a love of research and made the challenge of this process far less daunting. Mark, I was always helped by your ability to cut through to what really mattered and appreciated the honesty and directness of your advice. Qian, your belief in me and encouragement were always a reassurance when uncertainty entered my thoughts. Thank you also Dr Nerida Cole for always being ready to help with experiments, scientific writing and contacts. Denise and Robyn Lawler I thank for caring for all of my animals and being so willing to pass on your skills. Scott Minns, thank you for the assistance with chromatography and bringing mirth into the lab. Elaine Chew, I am indebted to you for your help and passion for quality histology. These studies were made possible by funds provided by the Australia Government through the Australian Postgraduate Award and further subsidised by additional contributions from the Brien Holden Vision Institute (BHVI). The University of New South Wales (UNSW) Graduate Research School also provided financial assistance to present this research internationally. While the bulk this research was conducted within the BHVI a great deal was achieved with the assistance from the following dedicated teams and access to their laboratories; the UNSW Recombinant Products Facility, Australian Proteome Analysis Facility, UNSW Bioanalytical Mass Spectrometry Facility, University of Sydney Faculty of Veterinary Science Veterinary Pathology Diagnostic Service, Minomic, and the UNSW Solid State & Elemental Analysis Unit. I am grateful for the support offered by each of these institutions and could not have completed this research without them. Bovine Lactoferrin Regions Promoting Corneal Epithelial Healing Page 4 The following people were always there for me and made everything worthwhile. Emma, thank you for your lifelong friendship and support, without which I may have never started this journey. Sharon, thank you for the many shared sleepless nights spent handling Guinea pigs and for trying to hold back your laughter during my first days in the lab. Kalika, thank you for never holding back your laughter. Maria M, thank you for your unwavering support and tolerating my ways, mostly. Maria B, thank you for getting me to the end. Finally, thank you to my family who have given me the opportunity to be where I am today. I would like to thank my grandfather, my role model, who has given me more than he will ever know. I am equally, and forever, grateful for the always unquestioning support and love of my parents. I will return it one day, I promise. Bovine
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