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Characterisation of Guanylin and Uroguanylin in the Digestive System 10.17863/CAM.38505 Dye, Florent Serge O'Rahilly, Stephen Guanylin and uroguanylin are closely related peptides originating from the intestinal mucosa with established roles in salt and water homeostasis and the regulation of gut physiology. These hormones are secreted as the propeptides, ProGuanylin and ProUroguanylin, and post- translationally processed into active peptides in the intestinal lumen. Those active peptides activate the guanylyl cyclase C (GC-C) receptor from the lumen and catalyse the production of the intracellular secondary messenger cGMP. Subsequently cGMP stimulates both cGMP- dependent protein kinase II (PKGII) and protein kinase A (PKA). Both PKGII and PKA phosphorylate the cystic fibrosis transmembrane conductance regulator (CFTR), leading to an efflux of chloride in the lumen, resulting in fluid secretion into the intestine. The importance of this hormone-receptor system in human gastrointestinal physiology is epitomised by GC-C being the receptor for diarrheagenic bacterial enterotoxins. Observed human mutations in the receptor cause its hyperactivation or inactivation, leading to familial syndromes of diarrhoea and meconium ileus, respectively. The function of guanylin and uroguanylin is not restricted to intestinal physiology, as several other studies demonstrated their role on water and salt homeostasis in the kidney as well as a potential hypothalamic satiety effect. Therefore, the control of the guanylin peptides’ secretion may serve to regulate food intake through endocrine regulation. Understanding the role of guanylin peptides in healthy & disease states, the regulation of their secretion and their localisation in the digestive system has been limited by the lack of specific antibodies against the active human peptides and their prohormone precursors. The aim of this project was to address these deficiencies. In this thesis, specific antibodies were generated and utilised in immunoassays, which provided the ability to quantify accurately the circulating levels of proforms in human samples. Furthermore, liquid chromatography–mass spectrometry (LC-MS) analysis on human and mouse biological samples challenged the current knowledge of the bioactive peptides. The newly developed immunoassays provided opportunities to investigate pharmacological mediators that modulate guanylin and uroguanylin secretion from intestinal epithelium into the gut lumen and bloodstream. Finally, using newly generated transgenic mice expressing a fluorescent protein under the control of the guanylin promoter, the localisation of guanylin- expressing cells was examined in the gastrointestinal tract. Whilst several models used in this study did not draw a consensus on the specific stimuli resulting in proforms’ secretion, the tools developed advanced the knowledge on the localisation of guanylin-expressing cells. Understanding the mechanisms of expression will provide novel insights into the physiological roles of these peptides and their possible involvement in pathophysiological states. University of Cambridge 2019-04-03 Thesis Doctoral Doctor of Philosophy (PhD) en BBSRC i-Case MedImmune https://www.repository.cam.ac.uk/handle/1810/291324 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2848fc2c-0797-4c23-9e88-f707a6dc14a3/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/85ac56a4-c19b-4043-9c22-6c210c7a162b/download d90e2a108556c8da3f68ebef9c58be8a https://www.rioxx.net/licenses/all-rights-reserved/ Guanylin Uroguanylin Guanylate-CyclaseC Guanylyl-cyclaseC Guanylate-cyclase