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Moving Beyond Serovars ABSTRACT Title of Document: MOLECULAR AND BIOINFORMATICS APPROACHES TO REDEFINE OUR UNDERSTANDING OF UREAPLASMAS: MOVING BEYOND SEROVARS Vanya Paralanov, Doctor of Philosophy, 2014 Directed By: Prof. Jonathan Dinman, Cell Biology and Molecular Genetics, University of Maryland College Park Prof. John I. Glass, Synthetic Biology, J. Craig Venter Institute Ureaplasma parvum and Ureaplasma urealyticum are sexually transmitted, opportunistic pathogens of the human urogenital tract. There are 14 known serovars of the two species. For decades, it has been postulated that virulence is related to serotype specificity. Understanding of the role of ureaplasmas in human diseases has been thwarted due to two major barriers: (1) lack of suitable diagnostic tests and (2) lack of genetic manipulation tools for the creation of mutants to study the role of potential pathogenicity factors. To address the first barrier we developed real-time quantitative PCRs (RT-qPCR) for the reliable differentiation of the two species and 14 serovars. We typed 1,061 ureaplasma clinical isolates and observed about 40% of isolates to be genetic mosaics, arising from the recombination of multiple serovars. Furthermore, comparative genome analysis of the 14 serovars and 5 clinical isolates showed that the mba gene, used for serotyping ureaplasmas was part of a large, phase variable gene system, and some serovars shown to express different MBA proteins also encode mba genes associated with other serovars. Together these data suggests that differential pathogenicity and clinical outcome of an ureaplasmal infection is most likely due to the presence or absence of potential pathogenicity factors in individual ureaplasma clinical isolates and/or patient to patient differences in terms of autoimmunity and microbiome. To address the second barrier we are adapting the traditional molecular biology and novel synthetic biology tools to Ureaplasma, such as creation of oriC plasmids, use of transposons, and most prominently the engineering bacterial genomes cloned as yeast centromeric plasmids followed by genome transplantation to make ureaplasma mutants programmed by the genomes manipulated in yeast. This will allow for the creation of targeted single or multiple mutants that will greatly increase the understanding of ureaplasma pathogenicity. Efforts to transplant the genomes of bacteria, outside the mycoides group have been thwarted due to recombination between the donor and recipient cell genomes. We are exploring the use of the DNA cross-linking drug, Mitomycin C, to inactivate the recipient cell genomes and thus prevent false positive transplantation results and potentially increase the genome transplantation efficiency. MOLECULAR AND BIOINFORMATICS APPROACHES TO REDEFINE OUR UNDERSTANDING OF UREAPLASMAS: MOVING BEYOND SEROVARS By Vanya Metodieva Paralanov Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2014 Advisory Committee: Professor Jonathan Dinman, Chair Professor John I. Glass, Co-Chair Professor Daniel Stein Professor Philip Bryan Professor Rose Viscardi Dr. Elisabet Caler © Copyright by Vanya Metodieva Paralanov 2014 Dedication To all the little ones born too soon and fighting for their lives, all the little angels gone too soon and their families! “Sometimes the smallest things take up the most room in your heart!” -Winnie the Pooh Just For By Unknown Author Just for this morning, I am going to step over the laundry, And pick you up and take you to the park to play. Just for this morning, I will leave the dishes in the sink, And let you teach me how to put that puzzle. Just for this afternoon, I will unplug the telephone and keep the computer off, And sit with you in the backyard and blow bubbles. Just for this afternoon, I will not yell once, not even a tiny grumble when You scream and whine for the ice cream truck, and I will buy you one if he comes by. Just for this afternoon, I won't worry about what you are going to be When you grow up, or second guess every decision I have made Where you are concerned. ii Just for this afternoon, I will let you help me bake cookies, And I won't stand over you trying to fix them. Just for this afternoon, I will take us to McDonald's And buy us both a Happy Meal so you can have both toys. Just for this evening, I will hold you in my arms and tell you a story About how you were born and how much I love you. Just for this evening, I will let you splash in the tub and not get angry. Just for this evening, I will let you stay up late While we sit on the porch and count all the stars. Just for this evening, I will snuggle beside you for hours, And miss my favorite TV shows. Just for this evening when I run my finger through your hair as you pray, I will simply be grateful that God has given me the greatest gift ever given. I will think about the mothers and fathers, Who are searching for their missing children, The mothers and fathers who are visiting Their children's graves instead of their bedrooms, And mothers and fathers who are in hospital rooms Watching their children suffer senselessly, And screaming inside that they can't handle it anymore. And when I kiss you goodnight I will hold you a little tighter, a little longer. It is then, that I will thank God for you and ask him for nothing, Except one more day........... iii Acknowledgements I have come so far with the encouragement, support, and mentorship of so many important people that it only makes sense to acknowledge everyone chronologically! My dear grandmother, who I call Mami, was always sure and used to tell me: “One day you will be a Doctor of the sciences!” This is a word for word translation from what she used to often tell me in Bulgarian when I was still in elementary school and all throughout my school and university career. I am so grateful that she never doubted my abilities and her voice has always been in the back of my head when I needed a dose of encouragement! My parents, Rossitza and Metodi, who are both physicist, instilled in me curiosity and love of science and research. They nurtured my science interests and followed my lead! The most fun part for me was visiting often their laboratory and exploring with them. They have been very supportive and selflessly provided academic, moral, and financial support, including countless days of childcare for my sweet son, Christopher! I will be eternally grateful for the sacrifices they have made and continue to make! Another person, who has also always believed in me and has been proud of me, is my dear sister, Radostina! We have been through a lot together, helping each other. I am eternally grateful for all the sacrifices she has made to help me and our parents, and take care of our grandparents! I am proud to say that I have known my best friend, who is also my wonderful husband, for 15 years or half of my life! I honestly don’t remember and don’t want to know what life is like without him! He has been extremely supportive throughout my student life, even when it seemed it will never end and he was getting a bit inpatient! iv He has always been there for me, believing in me, loving, reminding me that I can do it, and telling me how proud of me he is! He was there to help me through countless sleepless nights, making sure that I don’t forget to eat, don’t drink too much caffeine, and get some rest and sleep as soon as possible. Thank you for providing for us all the necessities, love and much more, so I can concentrate on my school and research! But most of all thank you for Christopher and being a wonderful, loving and engaged father! We will always love you unconditionally! I would also like to acknowledge the support, advice, and help from my husband’s family, Mariana, Ljuben, Blagovest, and Grandma Sava or my second mom, dad, brother and grandmother, who have taken interest in my research and were proud of my every academic milestone and achievement! My passion for science, and in particular biology, was reignited when I came to the USA and met my college professors. I was amazed and inspired by their dedication, passion, and enthusiasm for research and teaching. They were approachable by their students and eager to answer student’s questions! These were all qualities that my University professors in Europe, unfortunately, failed to demonstrate. I would like to say that I am so happy and grateful I met Dr. Thomas Burkett, my Biotechnology degree advisor and professor. I owe a lot of my success today and how far I have gone to his enthusiasm and passion for science along with his guidance, advising and encouragement. Dr. Burkett introduced me to the Molecular Biology, Biochemistry and Bioinformatics (MB3) Program at Towson University and informed me about and strongly recommended that I apply to the summer internship program at the J. Craig Venter Institute. v After I graduated the Biotechnology Program, I transferred to the MB3 Program at Towson University, where I continued to meet wonderful professors, inspiring me to continue my University career. My program advisor and course professor, Dr. James Saunders was extremely supportive. His office door was always open for the students in the MB3 program and other students. He made sure that we all felt like one big family! Dr. Saunders fostered my leadership skills and further encouraged me to pursue a doctoral degree.
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