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The Role of Lipid Mediators in the Aetiology of Endometriosis

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The Role of Lipid Mediators in the Aetiology of Endometriosis The role of lipid mediators in the aetiology of endometriosis A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2018 Orsolya Kiss School of Health Science Division of Pharmacy and Optometry LIST OF CONTENTS LIST OF CONTENTS ........................................................................................................................ 2 LIST OF FIGURES .................................................................................................................... 5 LIST OF TABLES .................................................................................................................... 10 FREQUENTLY USED ABBREVIATIONS .................................................................................. 12 ABSTRACT ............................................................................................................................ 15 DECLARATION ..................................................................................................................... 17 Acknowledgement .............................................................................................................. 19 CHAPTER: Introduction and Literature Review .................................................................. 19 Overview of Endometriosis ......................................................................................... 20 Pathophysiology .......................................................................................................... 21 Histology of endometrium .................................................................................. 21 Histology of ectopic lesions ................................................................................ 21 Morphology of the lesions .................................................................................. 22 Evolution of the lesions ....................................................................................... 23 Phenotypes of lesions ......................................................................................... 24 Aetiological Theories ................................................................................................... 27 Uterine origin ...................................................................................................... 27 Embryonic origin ................................................................................................. 28 Stem cells origin .................................................................................................. 30 Lymphatic spread ................................................................................................ 30 Scope of Endometriosis .............................................................................................. 31 Risk factors .......................................................................................................... 31 Epidemiology ....................................................................................................... 35 Burden of the disease ......................................................................................... 35 Diagnosis ............................................................................................................. 36 Management ....................................................................................................... 39 Elements of Pathophysiology ...................................................................................... 41 Steroid hormones ............................................................................................... 41 Lipid mediators ................................................................................................... 43 Peritoneum as microenvironment for endometriosis ........................................ 56 Pathomechanism ........................................................................................................ 59 Peritoneal cells with impaired functions ............................................................ 60 Proliferation of endometrial lesions ................................................................... 61 PGE2 as the master regulator for the proliferation of ectopic lesions ................ 63 Role of lipid mediators in the inflammation process .......................................... 64 2 Peroxisome proliferator-activated receptor γ (PPARγ) ...................................... 66 Aims and Objectives .................................................................................................... 67 CHAPTER: Materials and Methods ..................................................................................... 69 Clinical specimens ....................................................................................................... 70 Mass Spectrometry Analysis of Lipid Mediators ......................................................... 72 Overview of mass spectrometry ......................................................................... 72 Experimental description .................................................................................... 74 Materials for LC/ESI-MS/MS ............................................................................... 75 Preparations ........................................................................................................ 77 Performance of LC/ESI-MS/MS ........................................................................... 78 Qualitative Real-Time Polymerase Chain Reaction (qRT-PCR).................................... 84 Overview of qRT-PCR .......................................................................................... 84 Materials ............................................................................................................. 85 RNA extraction .................................................................................................... 86 cDNA synthesis .................................................................................................... 88 Primer design ...................................................................................................... 88 Qualitative Real-Time Polymerase Chain Reaction ............................................. 89 Validation of primers and qRT-PCR assay ........................................................... 90 Quantification of genes of interest ................................................................... 100 Validation of the endogenous reference gene ................................................. 102 Data analysis ..................................................................................................... 103 Histological methods................................................................................................. 104 Materials ........................................................................................................... 104 Sample preparation .......................................................................................... 105 Haematoxylin and eosin staining ...................................................................... 105 Immunohistochemistry ..................................................................................... 106 Enzyme-linked Immunosorbent Assay ...................................................................... 114 Overview of Enzyme-linked Immunosorbent Assay ......................................... 114 Materials ........................................................................................................... 115 Preparation ....................................................................................................... 115 Performance of TNFα and IL-1β ELISA assay ..................................................... 116 Determination of analyte concentration .......................................................... 116 Glucose concentration determination in peritoneal washes ........................... 118 CHAPTER: Lipid Mediators in Biological Fluid Specimens ................................................ 120 Introduction .............................................................................................................. 121 Participant characteristics ......................................................................................... 121 Normalisation of peritoneal washes ......................................................................... 123 3 Results ....................................................................................................................... 125 Lipid mediators in peritoneal fluid .................................................................... 125 Lipid mediators in plasma ................................................................................. 139 Discussion .................................................................................................................. 157 Lipid mediators in peritoneal fluid .................................................................... 157 Lipid mediators in plasma ................................................................................. 166 CHAPTER: Pro-inflammatory cytokines in fluid specimens .............................................. 170 Introduction .............................................................................................................. 171 Results ......................................................................................................................
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