by Hing Kee Wong. / Title also in Chinese. / Thesis (M.Phil.)--Chinese University of Hong Kong. / Bibliography: leaves 67-74.

No description available.

published_or_final_version / Anatomy / Master / Master of Philosophy

Previous in vitro studies showed that exchange protein directly activated by cyclic AMP 1 (Epac1), which is a cAMP mediator, plays an important role in maintenance of endothelial barrier function. Diabetic retinopathy is characterized by impairment of retinal blood vessel integrity leading to breakdown of blood retinal barrier, retinal hypoxia, and neuronal damage. Here, we hypothesize that Epac1 regulates endothelial permeability and protects retina from the retinal damage associated with diabetes. To test such hypothesis, we first demonstrated that human retinal microvascular endothelial cells (HRMECs) exposed to high glucose concentration at 25 mM or 35 mM showed the decreased Epac1 expression level. Our preliminary data also showed that Epac1-downstream activator, Rap1, a member of Ras GTPase, was also altered by different glucose levels. In addition, retina from type 2 diabetic, db/db, mice also showed the decreased Epac1 expression compared to that of non-diabetic, db/m, mice. To further determine the role of Epac1 in diabetic retinopathy, we made use of Epac1-deficient mice. The pathogenesis of diabetic retinopathy share similar characteristics to that of ischemic retinopathy, such as neuronal cell death, glial reactivity, and glutamate toxicity. Therefore, we used our previous retinal ischemic model, i.e., transient middle cerebral artery occlusion (tMCAO). Firstly, we determined the retinal morphology of Epac1-/- mice under normal condition at 3wks. At 3 wks old, the Epac1-/- retinae showed a significantly decreased thickness of outer plexiform layer (OPL) with a trend of increase in inner nuclear layer (INL) thickness. Interestingly, there were obviously more glutamine synthetase (GS)-positive M?ller cells and protein kinase C (PKC)-α positive rod bipolar cells in INL. In addition, there were more IgG-positive blood vessels in OPL. To further determine whether these phenotypes will lead to more severe retinal damage, Epac1-/- mice were exposed to 2 hours of MCAO followed by 22 hours of reperfusion, which we have previously shown to induce retinal ischemia. There was no obvious difference in retinal thickness and expressions of glial fibrillary acidic protein (GFAP) and GS in the contralateral sides of Epac1+/+ and Epac1-/- retina after tMCAO suggesting that the Epac1-deficiency may be compensated by either protein kinase A (PKA) or Epac2. However, Epac2 level was not altered by Epac1-deficiency by Western blot analysis. The ipsilateral sides of the retina of Epac1+/+ and Epac1-/- after tMCAO also did not show obvious difference in swelling and cell death in inner retina, GFAP, glutamate, GS, nitrotyrosine (NT), and peroxiredoxin 6 (Prx6), suggesting that Epac1-deficiency may have been compensated by other cAMP mediators, such as Epac2. However, Epac2 expression in the ipsilateral side of Epac1+/+ and Epac1-/- retinae was not significantly different, although the activities of Epac and PKA were not determined. Taken together, the Epac1-deficient mice would serve as a useful model to determine the role of Epac1 in retinal development, and to determine the detail mechanisms of pathogenesis of diabetic and ischemic retinopathy. / published_or_final_version / Anatomy / Master / Master of Philosophy

The slime mould Dictyostelium discoideum contains a novel cAMP-binding protein, CABPl, that is composed of two subunits. Anti-CABPl monoclonal antibodies were used to isolate a cDNA from a $ lambda$gtll expression library. In hybrid selection experiments, this cDNA was found to be complementary to mRNAs encoding both CABPl subunits and also to share some homology with two polypeptides, p34 and p31, that copurify with CABPl. Further analysis showed that the two CABPl polypeptides are identical except for an additional 37 amino acids present in the larger polypeptide. Both polypeptides are encoded by a single gene and the production of the two CABPl mRNAs is accomplished by an unusual splicing event. The p34 and p31 polypeptides are smaller but highly homologous to the two CABPl polypeptides and are probably produced in a similar fashion. The carboxyl halves of the four polypeptides show a 50% similarity to two polypeptides of a tellurium anion resistance determinant encoded on a bacterial plasmid.

Thesis (Ph.D.)--York University, 2007. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR32052

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

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2007. / Includes bibliographical references (p. 46-54).

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