Complement Function and Expression in the Red-Tailed Phascogale (Phascogale calura) Oselyne Tsuey Wei Ong B. Med. Sc. (Biomedical Sciences), M. Sc. (Conservation Biology) Submitted for the completion of a Doctor of Philosophy degree at the Western Sydney University October 2016 TABLE OF CONTENTS Table of Figures............................................................................................................. i Table of Tables ............................................................................................................ iv Acknowledgements ...................................................................................................... v Statement of Authentication .................................................................................... vii Preface ....................................................................................................................... viii Publications ................................................................................................................. ix Conference and Seminar Presentations ..................................................................... x Abstract ......................................................................................................................... 1 Introduction .................................................................................................................. 5 1.1 Marsupials as Mammals ......................................................................................... 6 1.1.2 Red-Tailed Phascogale As A Study Species ..................................................... 8 1.2 Marsupial Immunity ............................................................................................. 11 1.2.1 Marsupial Young Immunity ............................................................................ 13 1.2.2 Adult Marsupial Immunity .............................................................................. 18 1.3 Marsupial Complement System ........................................................................... 22 1.4 Aims ........................................................................................................................ 25 1.5 Thesis outline ......................................................................................................... 27 1.6 Chapter 1 References ............................................................................................ 28 Preliminary Genomic Survey and Sequence Analysis of the Complement System in Non-Eutherian Mammals ..................................................................................... 41 2.1. Chapter Outline And Authorship ........................................................................ 42 2.2. Introduction ........................................................................................................... 43 2.3. Methods .................................................................................................................. 48 2.3.1. Sequence Identity ............................................................................................ 48 2.3.2. Sequence Alignments and Motif Analysis ...................................................... 51 2.3.3. The Central Complement Component, C3 ...................................................... 53 2.4. Results ..................................................................................................................... 54 2.4.1. Sequence Identity, Alignments and Motif Identification ................................ 54 2.5. Discussion ............................................................................................................... 57 2.5.1. The Complement Component C3 .................................................................... 58 2.5.2. Classical Complement Pathway ...................................................................... 58 2.5.3. Lectin Complement Pathway........................................................................... 60 2.5.4. Alternative Complement Pathway ................................................................... 60 2.5.5. Inhibitors of Complement Pathways ............................................................... 62 2.6. Chapter 2 References ............................................................................................ 65 2.7 Chapter 2 Supplementary Materials ................................................................... 74 Evaluation of Reference Genes for Gene Expression in Red-Tailed Phascogale Liver, Lung, Small Intestine and Spleen .................................................................. 79 3.1. Chapter Outline And Authorship ........................................................................ 80 3.2. Introduction ........................................................................................................... 81 3.3. Materials And Methods ........................................................................................ 82 3.3.1. Animal and Tissue Collection ......................................................................... 82 3.3.2. RNA Extraction and cDNA Synthesis ............................................................. 83 3.3.3. Primers and Real-Time PCR ........................................................................... 83 3.3.4. Data Analysis................................................................................................... 85 3.4. Results ..................................................................................................................... 86 3.5. Discussion ............................................................................................................... 90 3.6. Conclusions ............................................................................................................ 93 3.7. Chapter 3 References ............................................................................................ 95 The Expression of Specific Complement Proteins in Developing Red-Tailed Phascogales (Phascogale calura) ............................................................................... 99 4.1. Chapter Outline And Authorship ...................................................................... 100 4.2 Introduction ......................................................................................................... 101 4.3 Materials and Methods ....................................................................................... 103 4.3.1 Animals And Tissue Collection ..................................................................... 103 4.3.2 RNA Extraction And cDNA Synthesis ......................................................... 103 4.3.3 Primers ........................................................................................................... 104 4.3.4 Sequence Identification of Complement Components .................................. 105 4.3.5 Quantitative Real-Time PCR (qPCR) ............................................................ 108 4.3.6 Statistics ......................................................................................................... 109 4.4 Results ................................................................................................................... 109 4.4.1 Sequence Identification of Complement Components .................................. 109 4.4.2 Complement Expression in Red-tailed Phascogales...................................... 111 4.5 Discussion ............................................................................................................. 112 4.6 Chapter 4 References .......................................................................................... 119 4.7 Chapter 4 Supplementary Materials ................................................................. 123 Detection of an Active Complement System in Red-Tailed Phascogales (Phascogale calura) .................................................................................................. 126 5.1 Chapter Outline And Authorship ...................................................................... 127 5.2 Introduction ......................................................................................................... 128 5.3 Materials And Methods ...................................................................................... 130 5.3.1 Animals ......................................................................................................... 130 5.3.2 Alternative Complement Pathway Assay ...................................................... 131 5.3.3 Classical Complement Pathway Assay .......................................................... 131 5.3.4 Controls, Statistics and Calculations ............................................................. 133 5.4 Results ................................................................................................................... 134 5.4.1 Alternative Complement Pathway Assay ...................................................... 134 5.4.2 Classical Complement Pathway Assay .......................................................... 135 5.5 Discussion ............................................................................................................. 137 5.6 Chapter 5 References .......................................................................................... 143 Antimicrobial Activity of Red-Tailed Phascogale (Phascogale calura) Serum .. 145 6.1 Chapter Outline and Authorship ...................................................................