Loma Linda University TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works Loma Linda University Electronic Theses, Dissertations & Projects 6-2014 Effect of Maternal Hypoxia on Fetal Development: Programming of HIE Phenotype in Neonatal Rat Brain Pablo J. Gonzalez-Rodriguez Follow this and additional works at: http://scholarsrepository.llu.edu/etd Part of the Biochemistry Commons, and the Medicine and Health Sciences Commons Recommended Citation Gonzalez-Rodriguez, Pablo J., "Effect of Maternal Hypoxia on Fetal Development: Programming of HIE Phenotype in Neonatal Rat Brain" (2014). Loma Linda University Electronic Theses, Dissertations & Projects. 204. http://scholarsrepository.llu.edu/etd/204 This Dissertation is brought to you for free and open access by TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works. 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LOMA LINDA UNIVERSITY School of Medicine in conjunction with the Faculty of Graduate Studies ____________________ Effect of Maternal Hypoxia on Fetal Development: Programming of HIE Phenotype in Neonatal Rat Brain by Pablo J González-Rodríguez ____________________ A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biochemistry ____________________ June 2014 © 2014 Pablo J González-Rodríguez All Rights Reserved Each person whose signature appears below certifies that this dissertation in his/her opinion is adequate, in scope and quality, as a dissertation for the degree Doctor of Philosophy. , Chairperson Lubo Zhang, Professor of Physiology, Pharmacology and Pediatrics Carlos Casiano, Professor of Microbiology and Molecular Genetics, Biochemistry and Medicine Charles A. Ducsay, Professor of Physiology, Gynecology and Obstetrics Penelope Duerksen-Hughes, Professor of Biochemistry Dalia Xiao, Associate Research Professor of Physiology and Pharmacology iii ACKNOWLEDGEMENTS First of all, I want to thank my God for this amazing adventure that He allowed me to experience for these past years. His blessing was abundant every single day and His guidance was with me in every single step. Thank you Lord. Dr. Lubo Zhang, thanks for the opportunity you gave me to work in this wonderful lab; for your support, comprehension and knowledge. I am blessed to have a mentor like you. Dr. Marino de León, words are not enough to thank you for all you have done for me. Thanks for the opportunity to be part of the IMSD program and thank you for believing in me. Dr. Casiano, Dr. Ducsay, Dr. Duerksen-Hughes and Dr. Xiao, members of my guidance committee; thank you for your time, guidance, advice and support. Thanks to the Center of Perinatal Biology and the Center for Health Disparities and Molecular Medicine at Loma Linda University for allowing me to use their facilities, thank you. Thanks to all my colleagues, in Dr. Zhang’s lab, for their support and valuable ideas. Gracias a mis padres por su apoyo incondicional, por creer en mí y hacerme quien soy hoy en día. Los amo. Este triunfo es para ustedes, mi orgullo. A mi princesa Jeanaidyl Paulette, sé que no ha sido fácil para nosotros el no poder estar cerca por todo este tiempo, pero te aseguro que pensé en ti cada día. Gracias por tu paciencia y apoyo. Te amo. A mi hermana Karen, gracias por tu apoyo, oraciones, por estar ahí…. te amo. A mi cuñado, mis sobrinos, tías y tíos, primos y primas, abuelas y abuelos que siempre me apoyaron y oraron por mí. Les amo. iv CONTENTS Approval Page .................................................................................................................... iii Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix List of Abbreviations ......................................................................................................... xi Abstract ..............................................................................................................................xv Chapter 1. Introduction ..............................................................................................................1 Programming Diseases.......................................................................................1 Kidney Development ...................................................................................2 Brain Development ......................................................................................4 Maternal Hypoxia ..............................................................................................5 Epigenetic ..........................................................................................................6 Hypoxic Ischemia Encephalopathy ....................................................................8 Glucocorticoids ............................................................................................9 Prostaglandin..............................................................................................10 The Purpose of These Studies………………………………………………..11 References……………...…………………………………………………….13 2. Fetal Hypoxia Results in Programming of Aberrant Angiotensin II Receptor Expression Patterns and Kidney Development ......................................................20 Abstract ............................................................................................................21 Introduction ......................................................................................................22 Materials and Methods .....................................................................................23 Experimental Animals and Hypoxic Exposure ..........................................23 Immunoblotting..........................................................................................24 RT-PCR......................................................................................................25 Glomerular Counting .................................................................................26 v Statistical Analysis .....................................................................................26 Results ..............................................................................................................27 Effects of fetal hypoxia on body and kidney weight .................................27 Effects of fetal hypoxia on renal AT1R and AT2R protein and mRNA abundance ......................................................................................29 Effects of fetal hypoxia on glomerular number .........................................29 Discussion ........................................................................................................35 References ........................................................................................................42 3. Fetal Hypoxia Increases Vulnerability of Hypoxic-Ischemic Brain Injury in Neonatal Rats: Role of Glucocorticoid Receptors .................................................47 Abstract ............................................................................................................48 Introduction ......................................................................................................49 Materials and Methods .....................................................................................50 Experimental Animals ...............................................................................50 Brain HI Treatment ....................................................................................51 Measurement of Brain Infarction ...............................................................51 Western Blot ..............................................................................................52 Real Time - RT-PCR .................................................................................52 Methylated DNA Immunoprecipitation .....................................................53 Chromatin Immunoprecipitation ................................................................55 Statistical Analysis .....................................................................................55 Results ..............................................................................................................55 Maternal hypoxia induced asymmetrical growth restriction in the fetus ............................................................................................................55 Maternal hypoxia increased neonatal brain vulnerability to HI injury ..........................................................................................................56 Dexamethasone reduced HI-induced neonatal brain damage ....................56 Maternal hypoxia downregulated GR expression in the developing brain ...........................................................................................................61