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Thesis Rests with Its Author University of Bath PHD An investigation of platelet-activating factor metabolism during normal and pre- eclamptic pregnancies Khan, Nighat Murad Award date: 1993 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 (i) A r c INVESTIGATION OF A 3sr o R METABOLISM DURING NORMAL AND PRE-ECLAMPTIC PREGNANCIES. Submitted by Nighat Murad Khan for the degree of Ph.D of the University of Bath 1992 Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without written consent of the author. This thesis may be made available for consultation within the University library and may be photocopied or lent to other libraries for the purposes of consultation. NIGHAT M.KHAN UMI Number: U555465 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U555465 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 (ii) This work is dedicated to Myra and Murad. Myra, for being a wonderful daughter, who trod along her milestones delightfully and Murad for his patience. (iii) ACKNOWLEDGEMENTS: First of all, I would like to thank Dr K I Williams for his supervision. I am indebted to Prof John Westwick, who inspite of his busy schedule found time for extremely useful discussions regarding my results and liis encouragements during my stay in Bath. I wish to thank Dr C. Page and C.O'Neill for provision of references which I could never have dug out. I also would like to thank Dr D. Bamford for being extremely helpful in recovery of precious amniotic fluid and other samples. Finally, I wish to thank Prof Amin Suria for his encouragement and support for my Ph.D and for the critical review of my write-up and Dr S.A.Saeed for his supervision. This research was supported by a Faculty Development Award from the Aga Khan Foundation. (iv) TABLE OF CONTENTS: TITLE..................................................................(i) DEDICATIONS...........................................................(ii) ACKNOWLEDGEMENTS................................................... (iii) CONTENTS.............................................................. (iv) LIST OF ABBREVIATIONS...............................................(ix)~ ABSTRACT............................................................... (x) SUMMARY.............................................................. (xii) INTRODUCTION............................................................1 AIMS AND OBJECTIVES..................................................21 MATERIALS AND METHODS.................................................23 Reagents and chemicals.................................................23 Buffers and solvents................................................... 23 Radiochemicals......................................................... 24 Samples collection: a .Blood............................. 25 b. Placentae and fetal membranes...................................... 26 c .Amniotic fluid........................................................ 27 Amnion cell line........................................................ 27 ^C PAF uptake and disposition by amnion cells inculture............................................................... 28 Incubations with the drugs......................................... 29 Incubations with the hormones...................... 30 (V) Chromatographic analysis of total membrane phospholipids............................................................. 31 Chromatographic analysis of phosphatidylcholines..................31 Platelet aggregation.................................................... 32 PAF acetylhvdrolase enzyme assay..................................... 33 Protein estimation.......................................................35 Statistical analysis.................................................... 36 RESULTS.....................................................................46 Section I-IV........................................................ 46-59 Figures & tables.........................................................60 Gestational age-related changes in PAF acetylhydrolase activity PAF acetylhydrolase in maternal plasma............................. 61 PAF acetylhydrolase in human plasma.................................62 PAF acetylhydrolase in amniotic fluid at term.................... 63 PAF acetylhydrolase in amniotic fluid throughout gestation 64 PAF acetylhydrolase in various cellular fractions............... 66 PAF acetylhydrolase in intrauterine tissues at term............. 67 Characterization of PAF acetylhydrolase enzyme. Effects of pre-incubation on PAF acetylhydrolase with PAF.......68 Effects of human amnion cytosol on platelet aggregation induced by various aggregating agents.............................................. 69 PAF acetylhydrolase in human amnion cytosol......................... 70 Effects of temperature on PAF acetylhydrolase enzyme activity... 71 (vi) Effects of pH on PAF acetylhydrolase enzyme activity............. 72 Effects of dialysis on PAF acetylhydrolase enzyme activity...... 73 Effects of column chromatography & amicon cell ultrafilteration on PAF acetylhydrolase enzyme activity...................................74 Effects of organic solvents on PAF acetylhydrolase enzyme activity.................................................................. 75 Effects of enzyme inhibitors on PAF acetylhydrolase enzyme activity.................................................................. 76 Effects of freezing^thawing on enzyme activity.....................77 Effects of Prostaglandins on PAF acetylhydrolase enzyme..........78 t Effects of Oxytocin on PAF acetylhydrolase enzyme activity... 79,80 Effects of Oestradiol on PAF acetylhydrolase enzyme activity.81,82 Effects of Progesterone on PAF acetylhydrolase enzyme activity.................................................................. 83 1 i ■*C PAF uptake and disposition by amnion cells in culture Uptake of **C PAF by amnion cells................................. 84, 85 Distribution of radioactivity in culture medium..................... 86 Distribution of radioactivity in cell membrane phospholipids.............................................................87 Distribution of radioactivity in phosphatidyl cholines............................................................... 88,89 Effects of treatment with phospholipase A2 on cellular radioactivity.................................................90 Effects of Bromophenacylbromide on PAF uptake..................................................................... 91 Effects of extracellular calcium on PAF uptake..................................................................... 92 Effects of Merthiolate on PAF uptake.............................93 (vii) Effects of N-ethylmaleimide on ^CPAF uptake....................... 94 Effects of WEB-2086 on i4C PAF uptake.................................95 Effects of WEB-2086 on **C PAF metabolism by amnion cells....96,97 Effects of Indomethacin on "'C PAF uptake........................... 98 Effects of NDGA on j*C PAF uptake......................................99 Effects of NDGA on PAF metabolism by amnion cells.....100,101 Effects of Indomethacin on PAF metabolism by amnion cells..103 Effects of Ouabain on PAF uptake................................ 104 Effects of Oestrogen {E2 ) on PAF uptake and metabolism by human amnion cells in culture. Effects of Oestradiol on uptake...................................... 105 Effects of Oestradiol on PAF metabolism........... 106 Effects of Oestradiol on release of PAF metabolites...............107 Effects of Progesterone on ]*C PAF uptake. Effects of Progesterone on total uptake............................ 108 Effects of Progesterone on PAF metabolism..........................109 Effects of Progesterone on release of PAF metabolites...........110 Effects of Oestradiol and Progesterone
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