The Role of Peptidyl arginine deiminase, type IV (PAD4) in the Pathology of Shock/Sepsis By Bethany Marie Biron B.A., Worcester State College M.S., Johns Hopkins University Dissertation Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Division of Biology and Medicine at Brown University Providence RI May 2017 © Copyright 2017 by Bethany M. Biron This dissertation by Bethany Marie Biron is accepted in its present form by the Department of Pathobiology as satisfying the dissertation requirement for the degree of Doctor of Philosophy. Date_____________ _________________________________ Alfred Ayala, Co-Advisor Date_____________ _________________________________ Jonathan Reichner, Co-Advisor Advisor Recommended to the Graduate Council Date_____________ _________________________________ Craig Lefort, Reader Date_____________ _________________________________ Daithi Heffernan, Reader Date_____________ _________________________________ Bruce Levy, Outside Reader Approved by the Graduate Council Date_____________ _________________________________ Andrew Campbell, Dean of the Graduate School i Bethany M. Biron Girard, BA, MS Pathobiology Graduate Program-Pre-doctoral Student Co-Mentored by Alfred Ayala/Jonathan Reichner Division of Surgical Research Rhode Island Hospital Brown Medical School Aldrich 239, 593 Eddy Street Providence, RI 02903 Email: [email protected] Primary Research Field My current research involves studying the citrullination of histones via Protein Arginine Deiminase 4 (PAD4) and its involvement in Neutrophil Extracellular trap formation in disease states. Acute lung injury (ALI) is a common sequela of shock/trauma and is associated with significant mortality. Trauma patients exposed to a secondary infectious challenge are considered at significant risk for inflammatory lung injury. Neutrophil extracellular traps (NETs) are capable of microbial killing essential for combating systemic infection, but are also associated with detrimental inflammation in the host. My research entails defining the role of NET formation in the protection of the host from indirect acute lung injury as a result of hemorrhage and subsequent septic challenge, as well as elucidates the importance of NETs to host defense and whether the host is benefited or harmed by NET release by using an array of basic science laboratory protocols and methods.. Education 2013-present Ph.D. Pending. Pathobiology, Brown University, Providence, RI 2011 M.S. in Biotechnology concentration Biodefense, Johns Hopkins University, Rockville MD. 2007 B.A. in Spanish and Natural Sciences, Worcester State College, Worcester, MA Professional Experience Aug 2009- Aug2012 Research Microbiologist, Center for Aerobiological Sciences, USAMRIID, Frederick, MD I participated in GLP and non-GLP infectious disease vaccine research and aerosol model development research experiments in BSL-3 laboratory environments. Under the supervision of the division chief for Aerobiological Sciences I developed, wrote, and carried out animal protocols in accordance to IACUC standards. More specifically I was in charge of developing and executing adjunct therapy studies against inhalational anthrax infection. In addition, I performed aerosolization of bacterial and viral agents in a class III biosafety cabinet, and then employed various biological techniques to determine aerosol concentrations of biological agents as well as ii for protein quantization, antibody assays, and tissue culture assays. Upon completion of studies I analyzed data and wrote scientific reports and papers for publication. Completed all background checks and obtained top secret clearance to perform duties in BSL 3-4 laboratories, as well as enrolled in Personal Reliability Program (PRP) for work with select agents. Oct 2007- Aug 2009 Research Laboratory Technician II, Charles River Labs, Shrewsbury, MA I served as lead/primary technician for multiple basic and complex in-life studies simultaneously. This included generating data with minimal supervision in the performance of studies as well as being responsible for handling and restraining animals, clinical observations, sample collections, monitoring food consumption, animal husbandry, and performing accurate data collection and reporting. Specialized in small animal procedures and worked in compliance with Good Laboratory Practice Regulations (GLP’s), study protocols, and Standard Operation Procedures (SOP’s) Awards, Honors, Fellowships, Grants: Phi Eta Sigma National Honor Society, 2004 Johns Hopkins University Research Fellowship, USAMRIID, 2009-2011 Shock Society-Travel Award to support presentation of a Poster/Abstract as a part of the 38th Annual Conference on Shock in Denver, CO, June 6-9, 2015 Society for Leukocyte Biology – Travel award to support presentation of Poster/Abstract as part of the 49th Annual SLB Meeting in Verona Italy, Sep 15-18, 2016 Societies: SHOCK Society: Student member Society of Leukocyte Biology: Student member American Society for Investigative Pathology: Graduate student member Publications: Biron BM, Beck K, Dyer D, Mattix M, Twenhafel N, Nalca A. Efficacy of ETI-204 monoclonal antibody as an adjunct therapy in a New Zealand white rabbit partial survival model for inhalational anthrax. Antimicrob. Agents Chemother. 59(4):2206-14, 2015. Biron BM, Ayala A, Lomas-Neira JL. Biomarkers for Sepsis: What Is and What Might Be? Biomark. Insights 15;10(Suppl 4):7-17, 2015 (review). Cheng T, Bai J, Chung CS, Chen, Y, Biron BM, Ayala, A. Enhanced innate inflammation induced by anti-BTLA antibody in dual insult model of hemorrhagic shock/sepsis. Shock 45:40-49, 2016 Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. Cl-amidine Prevents Histone 3 Citrullination/NET Formation and Improves Survival in a Murine Sepsis Model. J. Innate Immun. Sep 14, 2016. (Epub ahead of print) iii O’Brian X, Biron BM, Reichner J. Consequences of Extracellular Trap Formation in Sepsis. Current Opinions in Hematology. (in press), 2016 (review) Abstracts (published): Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. The role of PAD4 histone 3 citrullination and net formation in a murine polymicrobial sepsis model. Shock 43:49 (supplt), 2015. Biron BM, Chung C-S, Heffernan DS, Ayala A. 2016. Citrullinated histone modification and NET production is increased the septic patients. Shock 45: supplement 1, 2016 Bethany Biron Girard, Chun-Shiang Chung, Yaping Chen , Jonathan Reichner, Alfred Ayala. Do NETs Matter? Establishing a Role for Neutrophil Extracellular Traps in Patho- Biology of Indirect Acute Lung Injury. Inflammation, Immunity and Cancer: Neutrophils and Other Leukocytes, Verona Italy, 2016 Bethany Biron Girard, Yaping Chen, Chun-Shiang Chung, Jonathan Reichner, Alfred Ayala. PAD4 Deficiency Limits Kidney Dysregulation in a Murine Model of Shock/Sepsis. Experimental Biology, Chicago Il, 2017 Presentations (Oral & Poster): Bethany Biron Girard, Yaping Chen, Chun-Shiang Chung, Jonathan Reichner, Alfred Ayala. PAD4 Deficiency Limits Kidney Dysregulation in a Murine Model of Shock/Sepsis. Experimental Biology/ASIP, Chicago IL (2017) Bethany Biron Girard, Chun-Shiang Chung, Yaping Chen, Jonathan Reichner, Alfred Ayala. Do NETs Matter? Establishing a Role for Neutrophil Extracellular Traps in Patho- Biology of Sepsis and Indirect Acute Lung Injury. Brown University-Graduate Pathobiology Retreat, East Providence, RI (2016) Biron BM, Chen Y, Chung CS, Reichner J, Ayala A. Cl-amidine Prevents Histone 3 Citrullination and Improves Survival in a Murine Sepsis Model. 23rd Annual Hospital Research Celebration, Providence, RI (2015). Biron BM, Chen Y, Chung CS, Reichner J, Ayala A. Cl-amidine Prevents Histone 3 Citrullination and Improves Survival in a Murine Sepsis Model. Brown University- Graduate Pathobiology Retreat, West Greenwich, RI (2015). Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. The Role of Peptidylarginine Deiminase 4 Catalyzed Histone 3 Citrullination and NET formation in Murine Polymicrobial Sepsis Model. 22nd Annual Hospital Research Celebration, Providence, RI (2014). iv Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. The Role of Peptidylarginine Deiminase 4 Catalyzed Histone 3 Citrullination and NET formation in Murine Polymicrobial Sepsis Model. Brown University-Graduate Pathobiology Retreat, Bristol, RI (2014). Biron BM, Chung CS, Ayala A. Expression of Soluble PD-L1 in a Mouse Model of Polymicrobial Sepsis. Brown University-Graduate Pathobiology Retreat, Bristol, RI (2013). Biron BM, Dyer D, Pitt ML. Development of a New Zealand White Rabbit Partial Survival Model for Inhalational Anthrax to Demonstrate Added Value of Adjunct Therapies, Aerobiology in Biodefense IV, Richmond VA (2011) Biron BM, Dyer D, Pitt, ML. Indication Markers for the Treatment of Pneumonic Plague in African Green Monkeys, JHU research symposium, Rockville MD (2011); Ft Detrick Spring Research Festival, Frederick MD (2011) Biron BM, Dyer D, Pitt ML. Comparison of Inhalational Anthrax in African Green Monkeys, Rhesus Macaques, and Cynomologous Macaques. JHU research symposium , Rockville MD (2010); Ft. Detrick Spring Research festival, Frederick MD (2010) Teaching Experience Fall 2013: Teaching Assistant for Dr. Peter Shank PhD. Biology of AIDS, Brown University, Providence RI v ACKNOWLEDGEMENTS The journey to this point has been long and winding with many ups as well as downs. I could not have made it this far without the support system I have. To my mom: I feel like