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A Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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A Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MATERNAL IMMUNE CHANGES DURING BOVINE PREGNANCY: A FOCUS ON THE ENDOMETRIAL MACROPHAGE By LILIAN DE JESUS OLIVEIRA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1 © 2010 Lilian de Jesus Oliveira 2 To my parents Luiz Manoel and Maria Aparecida de Jesus, my brother and sister, Rodolfo Manoel and Luciana de Jesus Oliveira, my boyfriend Robson Fortes Giglio, and my major advisor, Dr. Peter J. Hansen 3 ACKNOWLEDGMENTS I would like to express my heartfelt thanks in having Dr. Peter J. Hansen as my major advisor. His guidance, support and challenges throughout my PhD program have made me grow not only as scientist but also as a person. His enthusiasm, knowledge and intelligence inspired me every day during these years. He is more than an advisor; he is a very good friend that I am glad to have the opportunity to meet. I am going to miss our weekly meetings. Also, I would like to thank my committee members, Dr. Nasser Chegini, Dr. William Thatcher, Dr. Daniel Sharp and Dr. Ammon Peck, for their contributions and suggestions for improving my research projects and academic training. I am also grateful to Dr. Joel Yelich his continuous support and the opportunity to teach in his class. This experience was indispensable to my formation as a teacher; his passion for teaching is contagious. I would like to thank my current lab mates, Dr. Silvia Carambula, Sarah Fields, Barbara Loureiro, Luciano and Aline Bonilla, Justin Fear, and Jim Moss, as well as my old lab mates, Dr. Jeremy Block, Dr. Maria Padua, Dr. Dean Jousan, Dr. Luiz Augusto de Castro, Dr. Katherine Hendricks, Adriane Bell (in memoriam) and Amber Brad, for their friendship and help. A special thanks to William Rembert, for his friendship and help. I am also grateful to Dr. Carlos Risco for his help in the obtention of the samples for my first chapter. Likewise, I would like to thank my friends at the Department of Animal Sciences for serving as my family all these years. Thanks are also extended to all personnel in the Department of Animal Sciences with special mention to Joann Fischer, Cora, Darlene; Dolores, Rick, Frank and Joyce for all their help and friendship. 4 I also want to thank the International Student Center staff, especially to Ms. Debra Anderson, for her kindness and assistance to me and all international students. I thank my Fulbright fellows for their support and friendship during this time. To Dr. Debora Faria from the University of Oklahoma, your friendship is one of the most valuable things that I have received. I also thank Neal Benson and my dear friend Steve McClellan from the Flow Cytometry Core Laboratory for their help during all my Ph.D. Many times they worked after hours so I could finish my experiments. Without their help, my dissertation would have been very different. I also thank Dr. Gigi Ostrow and Dr. Jason Li from the Gene Expression Core Laboratory for giving me valuable advice. I could not name all the friends I have made during these years in Gainesville, but without them, my journey here would have seemed longer. I want to thank especially Izabella and Bryan Thompson; Juliana Fernandes; Juliano Laran; Lincoln Zotarelli; Flavio Avila; Luis Symanski; Luciano Silva; Carlos Canas; Leandro Grecco; Aline Monari; Oscar Queiroz; Maria Belen Rabaglino; Beatriz Sanz; Qien Yang; Flavia Barros; Jacob Bulboz; Suzan Rodgers; and John Midolo for their friendship and the good times that we spent together. It was really fun, I really love you guys and I miss you all. You are lifetime friends. I would like to especially thank Dr. Flavio Silvestre for his help, scientific discussions and valuable friendship. I also thank my ―American girls‖ Amber Brad, Regina Estermann, Teresa Rodina, Kathleen Pennington, Michelle Twilla and Krista DeRespino for their friendship and their valuable help since the day I arrived in Gainesville. 5 Likewise, I would like to thank Krista DeRespino and Amber Brad for their unconditional friendship and love through all good and not so good times. I also would like to thank my friends that are more likely a family to me Agda Barreirinhas, Marcelo Tamassia, Vanessa Ferraz, Cassio Auada Ferrigno, Andre and Renata Shih for the continuous support and friendship. I would like to thank my family, my mom, my dad and brother and sister for their love. I would like to extend my acknowledgments to Giglio Family. Finally, I sincerely would like to thank my partner in life and my best friend Robson Giglio for his constant love and support in every moment in my life 6 TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 11 LIST OF ABBREVIATIONS ........................................................................................... 13 ABSTRACT ................................................................................................................... 19 CHAPTER 1 INTRODUCTION .................................................................................................... 22 2 LITERATURE REVIEW .......................................................................................... 25 Origin, Development, and Function of Macrophages .............................................. 25 Development .................................................................................................... 25 Macrophage Activation and Differentiation ....................................................... 29 Regulation of Immune Responses ................................................................... 33 Wound Healing and Tissue Remodeling .......................................................... 35 Angiogenesis .................................................................................................... 36 Tumor Progression ........................................................................................... 37 Macrophages in the Pregnant Endometrium ........................................................... 38 Immunology of the Uterus in the Cow ..................................................................... 44 MHC Proteins Expression by the Trophoblast ........................................................ 46 Uterine Immune System ......................................................................................... 47 Regulatory Molecules Affecting Endometrial Immune Function .............................. 50 Progesterone .................................................................................................... 51 Prostaglandins .................................................................................................. 52 Interferon-tau .................................................................................................... 52 Parturition ............................................................................................................... 53 Synopses and Objectives ....................................................................................... 54 3 DEVIATIONS IN POPULATIONS OF PERIPHERAL BLOOD MONONUCLEAR CELLS AND ENDOMETRIAL MACROPHAGES IN THE COW DURING PREGNANCY ......................................................................................................... 55 Introduction ............................................................................................................. 55 Materials and Methods............................................................................................ 57 Materials ........................................................................................................... 57 Flow Cytometric Analysis of Peripheral Blood Mononuclear Cells ................... 58 Isolation of Peripheral Blood Mononuclear Cells (PBMC) ................................ 59 Immunohistochemistry for CD68 and CD14 ..................................................... 60 7 Two-color Immunofluorescence for CD68 and CD14 ....................................... 62 Two-color Immunofluorescence for CD68 and CD14 in Adherent Cells from Endometrium ................................................................................................. 62 Statistical Analysis ............................................................................................ 63 Results .................................................................................................................... 63 Representative Flow Cytometry Patterns ......................................................... 63 Differences between Pregnant and Nonpregnant Cows at Day 33-34 of Gestation in Subpopulations of PBMC .......................................................... 65 Differences between Preparturient and Nonpregnant Cows in Subpopulations of PBMC .............................................................................. 65 Immunolocalization of CD68+ and CD14+ Cells in Endometrium as Affected by Pregnancy Status ..................................................................................... 68 Discussion .............................................................................................................. 72 4 PHENOTYPIC CHARACTERIZATION OF MACROPHAGES IN THE ENDOMETRIUM OF THE
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