Thesis (Ph. D.)-University of Washington, 1998. / Vita. Includes bibliographical references (leaves [67]-81).

Thesis (Ph. D.)--Cornell University, August, 2006. / Vita. Includes bibliographical references (leaves 157-170.

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

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Two separate experiments were designed to measure urinary cyclic AMP and renal papillary cyclic AMP, respectively, Results suggest that urinary cyclic AMP excretion rate is unchanged and cannot be used as an index of tubular sensitivity to either vasopressin or fluoride. However, renal papillary tissue cyclic AMP increased significantly (p<0.05) at plasma fluoride concentrations which result in polyuric renal failure. Further, it appears that fluoride independently stimulates cyclic AMP in the papilla, demonstrated by the additive effect with vasopressin. It was postulated that the defect in water reabsorption induced by fluoride must be at a step subsequent to the generation of cyclic AMP, because one would expect to see an antidiuresis, not a diuresis with increased tissue cyclic AMP.

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

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) act as second messengers in intracellular signaling cascades to influence neuronal responses. Hippocampal cAMP signaling is thought to underlie the pathophysiology of major depressive disorder (MDD) and antidepressant action; however, little is known about the possible role of cGMP signaling. Furthermore, circadian rhythm disturbances can occur as part of the clinical symptoms of MDD and resolve with antidepressant therapy. The pineal gland is relevant to circadian rhythms as it secretes the hormone melatonin following activation of cAMP signaling and the rate-limiting enzyme for its synthesis, arylalkylamine N-acetyltransferase (AA-NAT). Little is known about the contribution of the phosphodiesterase (PDE) system to antidepressant-induced alterations in pineal cAMP signaling and melatonin synthesis. There is a need to clarify the trajectory of cAMP and cGMP concentrations, their synthesis by cyclases, and degradation by PDEs to understand the role of cyclic mononucleotide signaling in the effect of chronic antidepressant therapy. Using quantitative real-time PCR and enzyme immunoassay, we systematically studied elements of intracellular signaling in the hippocampus of rats chronically treated with imipramine, fluoxetine, and amitriptyline and in the pineal gland of rats treated chronically with fluoxetine. In the hippocampus, we found chronic imipramine downregulated cAMP signaling with decreased cAMP, increased PDEs and decreased adenylate cyclase mRNA. In contrast, repeated fluoxetine and amitriptyline increased hippocampal cGMP signaling, with increased cGMP and decreased PDE mRNA. We conclude that in contrast to the assumption of antidepressant-mediated increases in cAMP levels, increased hippocampal cGMP signaling might underlie the efficacy of chronic antidepressant treatment. A follow up study using cultured embryonic rat hippocampal cells in vitro treated with the PDE type 5 inhibitor, sildenafil, demonstrated increased cAMP content following acute and chronic treatment, indicating either crosstalk between cAMP and cGMP pathways or a non-specific inhibitory effect of sildenafil on other PDEs. In the pineal gland, we found elevated melatonin synthesis with increased pineal AA-NAT mRNA and daytime plasma melatonin and downregulated cAMP signaling with increased PDE and unchanged AC pineal mRNA, and decreased pineal cAMP. We conclude that chronic fluoxetine increases daytime plasma melatonin and pineal AA-NAT mRNA despite downregulated pineal cAMP signaling.

Autophagy is an evolutionarily conserved degradative eukaryotic cell pathway that plays a role in multiple cellular processes. One important function is as a key component of the cellular immune response to invading microbes. Autophagy has been found to directly target and degrade multiple intracellular bacterial species. In this thesis, I identify and characterize two distinct regulatory mechanisms for this pathway involving the second messengers: diacylglycerol and cyclic adenosine monophosphate (cAMP). Salmonella enteric serovar Typhimurium (S. Typhimurium) is a Gram-negative bacterial species that has been shown to be intracellularly targeted for degradation by autophagy. While targeting of this species has been previously shown to involve ubiquitination, this pathway accounts for only half of targeted bacteria. Here I show that ubiquitin-independent autophagy of S. Typhimurium requires the lipid second messenger diacylglycerol. Diacylglycerol localization to the bacteria precedes autophagy and functions as a signal to recruit the delta isoform of protein kinase C (PKC) in order to promote the specific autophagy of tagged bacteria. Furthermore, I have found that the role of diacylglycerol and PKC delta is not limited to antibacterial autophagy but also functions in rapamycin-induced autophagy indicating a general role for these components in this process. Multiple bacterial species have been found to be targeted by autophagy and while some have developed strategies that allow them to avoid targeting, no bacterial factor has yet been identified that is able to inhibit the initiation of this process. Here I show that two bacterial species, Bacillus anthracis and Vibrio cholera inhibit autophagy through the elevation of intracellular cAMP and activation of protein kinase A. Using two different bacterial cAMP-elevating toxins, I show that multiple types of autophagy are inhibited in the presence of these toxins. This is indicative of a general inhibitory function for these toxins and identifies a novel bacterial defence strategy. This work characterizes both a novel regulatory signal for the induction of autophagy and identifies a novel bacterial tactic to inhibit this process. Together the data presented in this thesis provide novel insight into the regulation of autophagy and offer potential targets for modulation of this process.

Autophagy is an evolutionarily conserved degradative eukaryotic cell pathway that plays a role in multiple cellular processes. One important function is as a key component of the cellular immune response to invading microbes. Autophagy has been found to directly target and degrade multiple intracellular bacterial species. In this thesis, I identify and characterize two distinct regulatory mechanisms for this pathway involving the second messengers: diacylglycerol and cyclic adenosine monophosphate (cAMP). Salmonella enteric serovar Typhimurium (S. Typhimurium) is a Gram-negative bacterial species that has been shown to be intracellularly targeted for degradation by autophagy. While targeting of this species has been previously shown to involve ubiquitination, this pathway accounts for only half of targeted bacteria. Here I show that ubiquitin-independent autophagy of S. Typhimurium requires the lipid second messenger diacylglycerol. Diacylglycerol localization to the bacteria precedes autophagy and functions as a signal to recruit the delta isoform of protein kinase C (PKC) in order to promote the specific autophagy of tagged bacteria. Furthermore, I have found that the role of diacylglycerol and PKC delta is not limited to antibacterial autophagy but also functions in rapamycin-induced autophagy indicating a general role for these components in this process. Multiple bacterial species have been found to be targeted by autophagy and while some have developed strategies that allow them to avoid targeting, no bacterial factor has yet been identified that is able to inhibit the initiation of this process. Here I show that two bacterial species, Bacillus anthracis and Vibrio cholera inhibit autophagy through the elevation of intracellular cAMP and activation of protein kinase A. Using two different bacterial cAMP-elevating toxins, I show that multiple types of autophagy are inhibited in the presence of these toxins. This is indicative of a general inhibitory function for these toxins and identifies a novel bacterial defence strategy. This work characterizes both a novel regulatory signal for the induction of autophagy and identifies a novel bacterial tactic to inhibit this process. Together the data presented in this thesis provide novel insight into the regulation of autophagy and offer potential targets for modulation of this process.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 66-80). Also available on the Internet.