Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xxii, 260 p.; also includes graphics (some col.). Includes bibliographical references (p. 232-260). Available online via OhioLINK's ETD Center

The Gilman's cyclic AMP binding assay was used to examine the possibility of adopting this method for adenylate cyclase determinations. Cyclic AMP determinations were not invalidated by the reagents used in the adenylate cyclase reaction. Cyclic AMP measured by the binding assay was directly proportional to adenylate cyclase activity. Although variability in recovery of cyclic AMP was obtained, it could be reduced by performing triplicate assays. Thus, the cyclic AMP binding assay, with some reservations, would appear applicable for measuring adenylate cyclase activity. Adenylate cyclase in rat brain was studied by using the cyclic AMP binding method for determination of product formed. Rat brain cortex was fractionated by the method of Whittaker. The highest adenylate cyclase activity was found in the fraction containing the highest acetylcholinesterase activity, and this fraction was shown by electronmicroscopic studies to be rich in synaptosomes. A modified sucrose gradient was used for isolating satisfactory synaptosomal fractions (the layer between 1.0 M and 1.1 M sucrose). Properties of synaptosomal adenylate cyclase were examined. The enzyme was dependent on the concentrations of ATP and Mg²⁺ or Mn²⁺ ion. The enzyme was stimulated by fluoride and inhibited by calcium ion. Synaptosomal adenylate cyclase was not sensitive to catecholamines or adenosine. No hormonal stimulation was obtained in the presence of GTP. In experiments where the effects of endogenous catecholamines were reduced by the addition of α and β adrenergic blocking agents or by prior treatment of the animals with reserpine, hormonal stimulation of adenylate cyclase in particulate preparations could not be demonstrated. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Intracerebral injections of the neurotoxin 6-OHDA into the dorsal bundle (DB) causes selective depletion of cortical noradrenaline (NA) stores. The cortical neurons may then develop supersensitivity to NA and this may be measurable by the level of cAMP accumulation. Seven days was chosen as a period of time from injection to the development of the supersensitive response, and ten weeks was taken as the long-term period to measure permanent effects of this treatment. At seven days there was a significant increase in maximal stimulation and a slight, but not significant, shift of the dose-response curve. The baseline values of cAMP remained unchanged. The effect of the cAMP system after ten weeks post-injection consisted of a significant shift of the dose-response curve to the left, corresponding to a lowering of K[sub D], and a significant increase in both baseline and maximal stimulation levels, or V[sub max], of cAMP. The very high responsiveness of the adenylate cyclase system during the end of the second post-natal week was characterized by higher baseline levels of cAMP and greater cAMP accumulation in response to all NA concentrations tested. However, there was no significant shift of the dose-response curve. Kindling had no effect on the NA-stimulated cAMP response, showing unchanged basal and maximal stimulation levels in both anterior and posterior cortical slices. These results are discussed in terms of our present knowledge of the role of cAMP as a component of the post-synaptic receptor complex. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Lai, Ka Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 114-121). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.x / Chapter Chapter1 --- General introduction --- p.1 / Chapter 1.1 --- Cell differentiation --- p.1 / Chapter 1.1.1 --- S tem cell treatments --- p.2 / Chapter 1.1.2 --- Differentiation therapy for cancer --- p.3 / Chapter 1.2 --- Cyclic adenosine monophosphate (cAMP) signaling involved in cell differentiation --- p.4 / Chapter 1.2.1 --- cAMP -signaling pathways leading to transcription activities --- p.4 / Chapter 1.2.1 --- Regulation of cell differentiation by cAMP/PKA signal --- p.5 / Chapter 1.3 --- Aim of thesis --- p.5 / Chapter Chapter2 --- "Materials, media, buffers and solutions" --- p.7 / Chapter 2.1 --- Mate rials --- p.7 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.12 / Chapter 2.2.1 --- General culture buffers --- p.12 / Chapter 2.2.2 --- Culture medium --- p.12 / Chapter 2.2.3 --- Assay buffers and solutions --- p.13 / Chapter 2.2.3.1 --- Buffers and solutions for RT-PCR --- p.13 / Chapter 2.2.3.2 --- Buffers and solutions for assay of [3H]cAMP production --- p.13 / Chapter 2.2.3.3 --- Buffers and solutions for Western blotting --- p.14 / Chapter 2.2.3.4 --- Buffers and solutions for histamine assay --- p.16 / Chapter 2.2.3.5 --- Buffers and solutions for flow cytometry --- p.17 / Chapter Chapter3 --- Methods --- p.18 / Chapter 3.1 --- Maintenance of rat pheochromocytoma (PC12) cells --- p.18 / Chapter 3.2 --- Dete rmination of AC isoforms expression in PC12 cells by RT-PCR analysis --- p.19 / Chapter 3.2.1 --- RNA isolation --- p.19 / Chapter 3.2.2 --- cDNA synthesis by reverse transcription (RT) --- p.20 / Chapter 3.2.3 --- Semi-quantitative PCR --- p.21 / Chapter 3.3 --- Maintenance of human erythroleukemia (HEL) cells --- p.23 / Chapter 3.4 --- Dete rmination of [3H]cAMP Production in HEL cells --- p.23 / Chapter 3.4.1 --- Principle of assay --- p.23 / Chapter 3.4.2 --- Column preparation --- p.24 / Chapter 3.4.3 --- Measurem ent of [3H]cAMP production in HEL cells --- p.24 / Chapter 3.4.4 --- Data analysis --- p.25 / Chapter 3.5 --- Im munodetection of STAT3 and pTyr705STAT3 by western blotting --- p.25 / Chapter 3.6 --- Harvesting of HE L cells after differentiation treatment --- p.27 / Chapter 3.7 --- Flow cyto metry analysis of HEL cells --- p.27 / Chapter 3.7.1 --- F ITC-conjugated CD41 -antibody staining --- p.28 / Chapter 3.7.2 --- P I staining --- p.28 / Chapter 3.8 --- Determination of extracellular and intracellular histamine of HEL cells --- p.29 / Chapter 3.8.1 --- Sample preparation --- p.29 / Chapter 3.8.2 --- Automated assay of histamine content --- p.30 / Chapter 3.9 --- siRNA mediated knockdown of STAT3 in HEK293 cells --- p.30 / Chapter 3.9.1 --- Culture human embryonic kidney (HEK293) cells --- p.30 / Chapter 3.9.1 --- siRNA transfection --- p.31 / Chapter Chapter4 --- mRNA expression of adenylyl cyclase isoforms during early stage of NGF-induced differentiation of PC12 cells --- p.33 / Chapter 4.1 --- Introduction --- p.33 / Chapter 4.1.1 --- Dif ferentiation of PC12 cells --- p.33 / Chapter 4.1.1.1 --- Induction of neurite outgrowth by NGF in PC12 cells --- p.33 / Chapter 4.1.1.2 --- Effect of cAMP on NGF-induced neurite outgrowth in PC12 cells --- p.34 / Chapter 4.1.1.3 --- Effect of cAMP on NGF-induced neurite outgrowth in PC12 cells --- p.35 / Chapter 4.1.2 --- Enhanced forskolin-stimulated [3H]cAMP productionin NGF-difFerentiated PC12 cells --- p.36 / Chapter 4.1.3 --- Classification of adenylyl cyclases --- p.38 / Chapter 4.1.4 --- Aims of study --- p.39 / Chapter 4.2 --- Results and discussion --- p.40 / Chapter Chapter5 --- Effect of cicaprost on PMA-mediated differentiation of human erythroleukemia (HEL) cells --- p.48 / Chapter 5.1 --- Introduction --- p.48 / Chapter 5.1.1 --- Differentiation of HEL cells --- p.48 / Chapter 5.1.2 --- Prostac yclin (PGI2) and human IP receptors --- p.49 / Chapter 5.1.3 --- Agonists and antagonists of IP receptors --- p.50 / Chapter 5.1.4 --- IP signaling in HEL cells --- p.52 / Chapter 5.1.5 --- Effect of cAMP on megakaryocytic differentiation --- p.52 / Chapter 5.1.6 --- Aims of study --- p.54 / Chapter 5.2 --- Results and discussion --- p.56 / Chapter 5.2.1 --- Preliminar y studies --- p.56 / Chapter 5.2.1.1 --- PMA induced cell adhesion and morphological change --- p.56 / Chapter 5.2.1.2 --- Cell proliferation and protein content --- p.57 / Chapter 5.2.1.3 --- IP signaling in HEL cells --- p.57 / Chapter 5.2.1.4 --- Presence of histaminase in FBS --- p.60 / Chapter 5.2.1.5 --- Summary of preliminary studies --- p.61 / Chapter 5.2.2 --- PMA -induced cell spreading of HEL cells --- p.63 / Chapter 5.2.3 --- PMA -induced DNA synthesis of HEL cells --- p.65 / Chapter 5.2.4 --- PMA -induced cell size and cell complexity of HEL cells --- p.67 / Chapter 5.2.5 --- PMA -induced CD41/CD61 expression of HEL cells --- p.69 / Chapter 5.2.6 --- PMA -induced histamine production of HEL cells --- p.72 / Chapter 5.2.7 --- IP receptor-dependent and IP receptor-independent actions of cicaprost --- p.74 / Chapter 5.2.8 --- STAT3 knockdown by siRNA --- p.75 / Chapter 5.3 --- Role of STAT3 in MK differentiation --- p.76 / Chapter 5.4 --- Summary --- p.78 / Chapter Chapter6 --- General discussions and future study --- p.105 / Chapter 6.1 --- General discussions --- p.105 / Chapter 6.2 --- Future study --- p.111 / References --- p.114

published_or_final_version / Medicine / Master / Master of Research in Medicine

Cyclic adenosine monophosphate (cAMP) is an important intracellular secondary messenger. The major target of cAMP was traditionally considered as protein kinase (PK) A. This belief has been challenged by the discovery of exchange protein activated by cAMP 1 (Epac1), a cAMP-dependent guanine-nucleotide-exchange factor (GEF). Epac1 is ubiquitously expressed in all tissues and plays important roles particularly in the cardiovascular system. As cAMP activates both PKA and Epac1, the development of 8-pCPT-2'-O-Me-cAMP (8-pCPT), which has 107-fold higher affinity to bind and activate Epac1 than PKA, aids the researches on Epac1-mediated responses. In the present study, the protein expressions of Epac1 in the porcine coronary arteries and rat aortas were confirmed by Western blot analysis. In organ chambers, 8-pCPT induced acute relaxations in isolated porcine coronary arteries contracted to thromboxane receptor (TP-receptor) antagonists, and the relaxation was endothelium-independent. The 8-pCPT-induced Epac1 activation selectively altered the vasoactive responses to the TP-receptor agonists. The Epac1-mediated relaxation was found not related to PKA, PKG and the opening of ATP-sensitive potassium channels. Although Epac1 was first cloned as a Rap-linked GEF, in the porcine coronary artery, small GTPase Rac1 is the downstream target of Epac1 instead of Rap1 for relaxation. Activation of TP-receptors stimulates Rho-kinase to cause contraction, and the 8-pCPT-induced relaxation was Rho-kinase dependent, probably through pathway that is distinct from Rac1. Activation of Epac1 also inhibited the contraction to PKC, which is also downstream of TP-receptor but independent to Rho-kinase activity. On the contrary, in the aorta from male Sprague-Dawley rats aged 10-12 weeks, 8-pCPT induced relaxation in rings contracted to phenylephrine (PE) and the relaxation was endothelium-dependent. The relaxation depended mainly on endothelial nitric oxide synthase (eNOS) and partly on cyclooxygenase (COX). Western blot analysis found that 8-pCPT did not enhance eNOS phosphorylation, which is one of the mechanisms for eNOS activation. Activation of Epac1 also did not alter the phosphorylation of Akt and ERK1/2 which play important roles in cAMP-dependent eNOS. More experiments are needed to examine whether or not Epac1 alters nitric oxide (NO) and prostanoids synthesis, which are the major endothelium-derived mediators responsible for vascular tone regulation. In summary, the selective Epac activator 8-pCPT induced significant relaxations by distinct mechanisms in porcine coronary arteries and rat aortas. It is most likely that the relaxing effects of Epac1 activator are tissue and/or species specific. Owing to the effects of 8-pCPT on vascular relaxation, Epac1 might be an alternative therapeutic target for the treatment of vasospasm and hypertension. Further studies are necessary to explore the detailed mechanisms of Epac1 and its in vivo effects and in diseased models. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

The effects of dibutyryl 3'; 5' cyclic monophosphate (dbcAMP) on several parameters of transformation were studied using a Kirsten sarcoma virus (Ki-MSV)-transformed mouse cell line (K-A31). Treated cells showed changes in morphology, decreased motility, saturation density and growth rate, and lost the capacity for anchorage-independent growth. In contrast to many other transformed cell lines, fibronectin and an elaborate cytoskeleton were present in K-A31 cells. The transcription of the proviral genome was examined using both reverse-transcribed and nick-translated ('3)H-DNA probes, and certain viral-specific RNAs were found restricted to the nucleus of dbcAMP-treated cells. Additive hybridization experiments suggested these RNAs were transcribed from rat-derived sequences located in the 5'-half of the proviral genome. These results are discussed with respect to the properties most closely associated with cellular malignancy, and the possible mechanism of dbcAMP-mediated reverse-transformation in K-A31 cells.

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

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 107-121).

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 61-73). Also available via the Internet.