PROGRAMMED FUNCTION OF THE TAMMAR MAMMARY GLAND; The role of milk proteins in gut development by Sanjana Kuruppath Master of Science (MSc) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Deakin University February, 2014 Acknowledgements Thank You Almighty! I thank my family, friends and colleagues for their support without which completion of this thesis would not have been possible. First and most of all, I would like to express my sincere and deepest gratitude to my PhD supervisor Professor Kevin R Nicholas, for his constant guidance and support. I would not have been in a position to write this if he had not selected me as a student, given an opportunity to come to Australia and pursue my career in the field of scientific research. I assure, I had the best supervisor for my PhD. Under him, I had the freedom to manage and execute my project with his continuous support and timely guidance because of which I gradually grew as a confident researcher. I was always surprised to see him welcoming his students with a smiling and encouraging face, anytime they knock at his door! I greatly appreciate his commitment and passion towards research and the magical ability to continuously and convincingly promote his students to grow independently. I also thank him, from the bottom of my heart, for all the support and courage he gave me in my personal life too. I thank my co-supervisors, Dr Christophe Lefevre and Dr Julie A Sharp. I thank Christophe for his advice and assistance, mainly in the field of bioinformatics. Without him this would have been totally impossible. And above all, for always reminding to hold on to the smile, whatever happens in life! I also thank Julie for all 4 the technical advice she gave during my PhD. I thank both of them for proof-reading my research work, and for all the useful comments and remarks which helped to improve my thesis. I thank Dr Helen E Abud for welcoming me to her lab in Monash University. The collaboration with her group gave guidance and assistance for a major part of my research. I also extend my deepest gratitude to Dr Kenneth McColl for the much needed advice and assistance in histology which was a new area for me. For introducing him, I thank Tamsyn Crowley. I would like to thank Professor Norman Saunders lab of Melbourne University for assisting in getting samples for my project. I thank all my lab mates (Stephen, Laurine, Alicia, Sugeetha, Ashalyn, Ashwanth, Swathi, Amit, Akihiko, Gunveen, Venky, Meagan) for all the help and support. Their presence always motivated me to come back to the lab and work. I have gathered a lot of nice memories, especially during our lab lunches and during the wallaby “hunts”! I thank Liz for all the support. Her energy and smile has always been a motivation for me. I would also like to thank my lab mates in Monash University (Thierry, Helen, Lisa, Katja and Sivan) who guided me in different aspects. My friends…Without whom, this whole PhD and thesis would have been impossible. I thank all of them, for their presence, tolerance, patience and support. I thank Suneel and Nagu, for their constant support, even when they were far away from me. I personally thank Laurine, Adarsh, Swathi, Rajesh, Raju, Swapna, Mohan, Madhu and Ananthi. I thank “Almond family”, “Terracall family’ and “DeWaard 5 family”. Their presence never made me feel that I was away from my near and dear ones. I thank GATES foundation for the funding provided for a significant part of my PhD project. I thank Deakin University for providing scholarship during my PhD. And last but not the least, my family - Thanks for everything! 6 Communications Publications under submission Kuruppath, S., Sharp, J., Lefevre, C., Murphy, R. M. and Nicholas, K. R. (2014).Comparative analysis of Caveolins of mouse and tammar wallaby: Role in regulating mammary gland function. List of publications Kuruppath, S., Bisana, S., Sharp, J. A., Lefevre, C., Kumar, S. and Nicholas, K. R. (2012). Monotremes and marsupials: comparative models to better understand the function of milk. Journal of Biosciences 37(4): 581-588. Kuruppath, S., Kumar, A., Modepalli, V. N.,Ngo, K. P., Gras, S. L. and Lefevre, C. Buffalo Milk Transcriptomics. Submitted to the 10th WORLD Buffalo Congress 2013, Phuket Thailand Book Chapters Published Kumar, A., Buscara, L., Kuruppath, S., Ngo, K. P., Nicholas, K. R. and Lefèvre, C. The emerging role of miRNAs in the lactation process. Lactation: Natural Processes, Physiological Responses and Role in Maternity, Eds: Lisa M. Reyes Cruz and Douglas C. Ortiz Gutierrez, Nova Science Publishers. Poster presentation at conferences Sanjana Kuruppath, Julie A Sharp, Christophe M Lefevre and Kevin R Nicholas, A comparative study of the potential role of Caveolins in mouse and tammar wallaby lactation. Poster presented in “Gordon Research Conference on Mammary Gland Biology”, 12th-17th June 2011, Salve Regina University, Newport, RI. 7 Sanjana Kuruppath, Julie A Sharp, Christophe M Lefevre and Kevin R Nicholas, A study of the potential role of Caveolins in mouse and tammar wallaby lactation. Poster presented in “8th Annual Symposium on Milk Genomics and Human Health”, 15th-17th November 2011, Melbourne, Australia. Sanjana Kuruppath, Julie A Sharp, Christophe M Lefevre and Kevin R Nicholas, A study of the potential role of Caveolins in mouse and tammar wallaby lactation. Poster presented in “ASMR Victorian Student Research Symposium”, 4th June 2012, Bio21 Institute, Parkville, VIC, Australia. Sanjana Kuruppath, Helen E Abud, Julie A Sharp, Christophe Lefevre and Kevin R Nicholas, Tammar Wallaby: a marsupial model to understand the role of milk in regulating stomach development. Poster presented at the 10th “International Milk Genomics and Human Health Annual Symposium”, October 1-3, 2013 in Davis, California. Oral presentation at conferences Sanjana Kuruppath, Helen E Abud, Julie A Sharp, Christophe Lefevre and Kevin R Nicholas, Tammar Wallaby: a marsupial model to understand the role of milk in regulating stomach development. Presented at the 10th “International Milk Genomics and Human Health Annual Symposium”, October 1-3, 2013 in Davis, California. Student travel award. 8 Table of Contents CHAPTER 1: General Introduction and Literature Review............29 1.1 Introduction...............................................................................................30 1.2 Evolution of Mammals.............................................................................31 1.3 Mammary gland structure and lactation...................................................35 1.4 Lactation: comparison between eutherians and marsupials......................35 1.4.1 Eutherians..................................................................................35 1.4.2 Marsupials..................................................................................36 1.5 Milk and its components...........................................................................40 1.5.1 Carbohydrate..............................................................................40 1.5.2 Protein........................................................................................40 1.5.3 Fat..............................................................................................42 1.5.4 Growth factors...........................................................................43 1.5.5 MicroRNA.................................................................................43 1.6 Regulation of lactation..............................................................................44 1.6.1 Hormones...................................................................................46 1.6.1.1 Insulin.....................................................................................46 1.6.1.2 Glucocorticoid........................................................................47 1.6.1.3 Prolactin.................................................................................47 1.6.1.4 Hormonal regulation of milk protein gene expression: comparison between mouse and tammar wallaby..............................48 1.6.2 Extracellular Matrix...................................................................49 1.6.2.1 Eutherians...............................................................................50 1.6.2.2 Marsupials..............................................................................50 1.6.3 Different cell types....................................................................51 1.6.3.1 Myoepithelial cells..................................................................51 1.6.3.2 Adipocytes and stromal fibroblasts........................................52 1.6.3.3 Caveolins; potential influence on mammary function by a mechanism of cell-cell interaction......................................................52 9 1.6.4 Milk factors................................................................................53 1.7 GIT development and maturation in mammals........................................54 1.7.1 Stomach development in mammals...........................................55 1.7.1.1 Stomach development in mouse..............................................58 1.7.1.1.1 Gastric mucosa development...............................................58 1.7.1.1.2 Muscle layer development...................................................59 1.7.1.2 Stomach development in Opossum and Tammar Wallaby.....59 1.8 Role of milk in GIT development.............................................................61