During placental development, human trophoblasts differentiate along two pathways leading to the formation of the extravillous cytotrophoblasts (EVCT) and the villous cytotrophoblasts. EVCTis responsible for trophoblast invasion, which is an important process for successfulplacentation in early pregnancy.Dysregulation of the process is associated with a wide range of pregnancy complications, including intrauterine growth restriction, preeclampsia and choriocarcinoma. EVCTsproducematrix metalloproteinase and urokinase plasminogen activator(uPA)that degrade the extracellular matrix of the endometrium for the invasion process. Adrenomedullin is a 52-amino acid polypeptide belonging to the calcitonin/calcitoningene-related peptide/amylin peptide family. The expression of adrenomedullin in trophoblasts is most abundant in the first-trimester human placentas, consistent with the involvement of the molecule in early placentation. Most of the studies on adrenomedullin concentrate on its vasodilatory activity. Studies on the action of adrenomedullin in human trophoblast functions are limited. Therefore the role of adrenomedullin in early placentation is not known. The hypothesis of this project is that adrenomedullin regulates the invasion of human EVCT. Two human EVCT cell lines, JEG-3 and TEV-1 were used as study models in this project. The functions of adrenomedullin aremediated through a receptor system composedof two transmembrane components, the calcitonin receptor-likereceptor and receptor activity-modifying protein(RAMP). Adrenomedullin binds to calcitoninreceptor-likereceptorwhen coupled with RAMP2 or RAMP3. Immunostaining, western blotting and RT-PCR demonstrated the presence of these components on EVCT cell lines. Adrenomedullin treatment significantly enhanced invasiveness, migration, but not proliferation in EVCT as demonstrated by transwell invasion assay, transwell migration assay and cell proliferation assayrespectively. The adrenomedullin antagonist, adrenomedullin22-52, blocked the effects of adrenomedullin on EVCT cell lines, confirming adrenomedullin exerted its biological effects through its classical receptors. RT-PCR results further demonstrated that while adrenomedullin treatment had no effect on matrix metalloproteinase-2 expression, it up-regulated uPA expression and activity. Silencingof uPA by siRNA transfection abolished the stimulatory effect of adrenomedullin, suggesting uPA is the key mediator for adrenomedullin-induced invasion. Adrenomedullin increased nitric oxide synthase expression and nitric oxide (NO) production in JEG-3 cells. The involvement of NO in adrenomedullin-induced EVCT invasion and biosynthesis of uPAwas confirmed bypharmacologicalstudy using nitric oxide synthase inhibitorsor NO donors.Protein kinase G was shown to be one of the downstream regulator of NO that mediated the adrenomedullin-induced EVCT invasion. By using a biotin-switch based method to purify theS-nitrosylated proteins, adrenomedullin was further demonstrate to up-regulate the NO-dependent S-nitrosylation in EVCTs.Interestingly, S-nitrosylation of uPA in vitro induced a higher proteinaseactivitywhich may be responsiblefor the stimulatory action of adrenomedullin. In conclusion, thisstudyhas provided evidence that adrenomedullin modulates EVCT invasion by regulating the uPA expression andactivity through NO signaling pathway. Abnormal trophoblast invasion can lead to a range of major pregnancy complications. The outcome of this project enhanced our understanding of the mechanisms that regulate trophoblast invasion. These data could constitute the basis for new therapeutic strategies of the diseases in the future. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Bibliography: leaves 192-269. / xi, 269 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the impacts of development and acute and chronic stress upon the gene expression of two key catecholamine synthetic enzymes, tryosine hydroxylase (TH) and pheylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of the fetal sheep. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2000

Thesis (Ph.D.) -- University of Adelaide, Dept. of Physiology, 2000. / Bibliography: leaves 192-269.

ADM is a 52-amino acid peptide which carries out multiple biological functions in cardiovascular system such as vasodilation and hypotension, and is a prognostic marker for cardiovascular diseases. Recent studies show that its plasma level is elevated in diabetes, however the reason and significance for such an increase has not been understood. Recent research has proposed that inflammation and oxidative stress both contribute to the pathogenesis of diabetes. If ADM is a marker in diabetes, it is possible that ADM is regulated by these two mechanisms, and so this project aims to investigate how these mechanisms could affect ADM expression. Recent studies have demonstrated that advanced glycation endproducts (AGEs) could lead to development of various diabetic complications. AGEs are formed as intermediate product in the non-enzymatic glycation of reducing sugars. Formation of these products is stimulated by hyperglycemia and oxidative stress, which could also induce ADM expression. Hence one of the studies investigated the direct effect of AGEs on ADM expression in an in vitro model. A rat macrophage cell line NR8383 was used to investigate the dose-response and time-point responses of macrophage cells in expressing ADM stimulated by AGEs. 6 hours of AGEs treatment resulted in no significant effect on ADM gene expression. The gene expression increased in all time points in which the change was at maximum after 1 hour of AGE treatment compared with other time points (P<0.05). However the time-dependent effect on ADM gene expression was insignificant compared with controls. How oxidative stress could lead to increased ADM expression deserves further investigation. ADM plays a role in inflammation that it could influence IL-6 and adiponectin expressions. This project also investigated whether IL-6 and adiponectin could affect ADM levels on the opposite. The associations between IL6 and adiponectin single nucleotide polymorphisms (SNPs) with plasma ADM levels were studied using a cohort of 476 subjects from the Cardiovascular Risk Factor Prevalence Study (CRISPS). Specific tagging SNPs were genotyped for the 476 subjects. Significant associations were identified for the IL6 SNP rs17147230 and adiponectin SNP rs182052 with plasma ADM levels in additive model (β=-0.096, P=0.034, and β=0.104, P=0.023 respectively adjusting for age and sex). The associations remained significant after adjusting for various covariates (P<0.05). Genotypic model shows that the minor alleles of rs17147230 and rs182052 resulted in 12.8% decrease and 17.7% increase in plasma ADM levels. These findings show that ADM level could be regulated by IL-6 which is an inflammatory cytokine, and adiponectin which is a protective peptide in inflammation. Reducing inflammation could lower ADM level and adiponectin might be a therapeutic candidate. / published_or_final_version / Medicine / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

published_or_final_version / Physiology / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 77-97).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences