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Thesis Full Text Studies on the regulatory roles for Retinoic Acid (RA) during host-microbial interaction: implications for S. aureus and C. albicans infections A thesis submitted for the degree of Doctor of Philosophy In the Faculty of Science by Preeti Yadav Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India July 2019 DECLARATION I hereby declare that the work presented in this thesis is the result of investigation carried out by me under the supervision of Prof. K. N. Balaji, at the Department of Microbiology and Cell Biology (MCB), Indian Institute of Science, Bangalore, India. This work is submitted for the award of the Ph. D. degree of the Indian Institute of Science and does not form the subject matter for any other degree, diploma, associate-ship or membership of any institute or university. Contents ACKNOWLEDGEMENTS ....................................................................................................... 1 ABBREVIATIONS ................................................................................................................... 3 Synopsis ..................................................................................................................................... 5 CHAPTER 1: INTRODUCTION ............................................................................................. 9 Retinoic acid mediated modulation of host immune signaling events during S. aureus and C. albicansinfection .............................................................................................................................. 10 1.1 RA family and their transcriptional regulation ........................................................................ 11 1.2 Role of RA in infection and immunity ....................................................................................... 13 1.3 Therapeutic role of RA ................................................................................................................ 14 S. aureus infection driven modulation of host innate immunity ................................................ 15 1.5 Development of infection induce septic arthritis ............................................................................... 15 1.6S. aureus virulence factors and clinical manifestations ..................................................................... 16 1.7 Immune evasion of host by S. aureus .................................................................................................. 18 1.8 Host directed therapies against S. aureus infection .......................................................................... 19 C. albicans infection driven modulation of host innate immunity .............................................. 21 1.9Background: .......................................................................................................................................... 21 1.10Prognosis of wound and its associated microbiome ......................................................................... 22 1.11Role of C. albicans infection in wound pathogenesis ........................................................................ 24 1.12Pathogenicity factors associated with C. albicans ............................................................................ 24 1.13Immune evasion mechanism employed by C. albicans .................................................................... 25 1.14Immunotherapies against fungal burden .......................................................................................... 26 1.15 Rationale of current study: .................................................................................................... 28 1.15.1 S. aureus activated AURKA-WNT signaling axis and its implication on development of septic arthritis ........................................................................................................................................................ 28 1.15.2 C. albicans impaired wound contraction and its effect on perturbation of Rho/Rac GTPase signaling ....................................................................................................................................................... 29 1.15.3 Regulatory effects of RA in modulating host -microbial pathogenesis ...................................... 29 1.16 Objectives of the current study: ............................................................................................ 31 CHAPTER 2: METHODS ...................................................................................................... 33 2.1 Cells, mice and bacteria ....................................................................................................................... 34 2.2 Design of in vivo models ....................................................................................................................... 35 2.3 Reagents and antibodies ...................................................................................................................... 35 2.4 Treatment with pharmacological reagents ........................................................................................ 36 2.5 RNA isolation and quantitative real-time RT-PCR .......................................................................... 36 2.6 Immunoblotting .................................................................................................................................... 37 2.7 Transient transfection .......................................................................................................................... 38 2.8 Micro computed tomography .............................................................................................................. 38 2.9 Histopathological examination ............................................................................................................ 38 2.10 Immunohistochemistry (IHC) ........................................................................................................... 39 2.11 Immunofluorescence (IF) .................................................................................................................. 39 2.12 Statistical analysis .............................................................................................................................. 40 CHAPTER 3: SELECTIVE ACTIVATION OF MST1/2 KINASES BY RETINOID AGONIST ADAPALENE ABROGATES AURKA-REGULATED SEPTIC ARTHRITIS . 41 3.1 Introduction .......................................................................................................................................... 42 3.2 Results: .................................................................................................................................................. 45 3.2.1 Establishment of arthritis by S. aureus infection ........................................................................... 45 3.2.2 AURKA responsive WNT signaling regulates S. aureus infection triggered septic arthritis ..... 48 3.2.3 ADA rescues S. aureus induced cartilage damage ......................................................................... 50 3.2.4 ADA differentially regulates WNT and HIPPOsignaling ............................................................. 52 3.2.5 HIPPO signaling potentiated by ADA regulates pro-resolving mediators .................................. 54 3.3 Discussion: ............................................................................................................................................. 57 CHAPTER 4: EXPLORATION OF REGULATORY EFFECTS OF RA DURING C. ALBICANS INDUCED DELAYED WOUND HEALING AND PATHOGENESIS .......... 60 4.1 introduction ........................................................................................................................................... 61 4.2 Results ................................................................................................................................................... 63 4.2.1 Adapalene treatment modulates C. albicans induced delayed wound closure ............................ 63 4.2.2 C. albicans infectionimpairs wound healing and modulates inflammatory transcripts profile . 65 4.2.3 RA augments phagocytic uptake of C. albicans without effecting its survival ............................ 67 4.2.4 Crosstalk of BMP signaling with downstream effectors of Rho/Rac GTPases ........................... 69 4.3 Discussion: ............................................................................................................................................. 72 PERSPECTIVE ...................................................................................................................... 74 CHAPTER 4: REFERENCES ............................................................................................... 75 PUBLICATIONS: ................................................................................................................... 89 Acknowledgements ACKNOWLEDGEMENTS I am grateful to my mentor, Prof. K N Balaji for offering me a position in his laboratory, and an exciting project to pursue. I thank him for the constant supervision and advice and immense support for my work. He has taught me the importance of being well-read, informed and diligent about my work and for others who are part of scientific community. It has been a privilege to have his guidance and be a part of KNB lab. I thank lab alumni Sahana, Ravinder, Vikas, Kasturi, Praveen, Monoranjan and Tanushree have been
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