Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 106-135).

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Dissulfeto isomerase protéica (PDIA1 ou PDI) é uma chaperona e ditiol-dissulfeto oxido-redutase residente do reticulo endoplasmático (RE). PDI é essencial à regulação da proteostase por ter função no enovelamento oxidativo de proteínas e na via de degradação associada ao RE (ERAD). Além disso, PDI interage fisicamente e regula a atividade de NADPH oxidases, e fora da célula é um regulador redox essencial à atividade de proteínas extracelulares. Este pool epi/pericelular da PDI (pecPDI) regula função de proteínas de membrana/secretadas, como integrinas, glicoproteínas gp120 do virus HIV e outras, com múltiplas funções que incluem: trombose, ativação plaquetária, adesão celular, infecção viral e remodelamento vascular. A rota de externalização da PDI permanece obscura, e seu conhecimento pode indicar mecanismos dos efeitos (fisio)patológicos da PDI. A secreção da PDI pela rota RE-Golgi foi sugerida em células endoteliais infectadas pelo vírus da dengue, células pancreáticas e tireoideanas. No entanto, uma varredura sistemática das possíveis rotas de externalização da PDI não foi previamente realizada. Neste estudo, mostramos que células endoteliais (EC) externalizam constitutivamente, por rotas distintas, dois pools de PDI, de superfície celular e solúvel, enquanto na EC não estimulada PDI não foi detectada significativamente em micropartículas. PDI externalizada corresponde a ca.1,4% do pool total de PDI celular. Tanto a PDI de superfície celular como a solúvel foram majoritariamente secretadas pela via de secreção não-convencional do tipo IV independente de GRASP. Contudo, a via de secreção clássica também contribui para externalização basal da PDI de superfície celular, mas não da solúvel basal ou estimulada por PMA, ATP e trombina indicando que todas envolvem escape do Golgi. Além disso, a externalização constitutiva da PDI de superfície em célula muscular lisa vascular também ocorre por via independente de Golgi. Externalização da PDI não foi detectavelmente mediada pela secreção não-convencional do tipo I, II, III, lisossomos secretórios, endossoma de reciclagem e transporte ativo (dependente de ATP) em EC. Considerando que chaperonas são vias essenciais de resposta a estresses, investigamos o efeito de estresse do RE e choque térmico na pecPDI. Estresse do RE não altera a PDI de superfície celular, mas aumenta PDI solúvel. Ambos os pools de PDI não foram alterados por choque térmico, embora a recuperação desse estresse diminua a secreção de PDI. Estes dados sugerem que a liberação de PDI é um processo regulado, dependente da natureza do estresse. Bloqueio da síntese de proteínas com cicloheximida não altera pecPDI, indicando que PDI recém-sintetizada não é preferencialmente externalizada e que o tráfego da PDI independe de outras proteínas recém-sintetizadas. Um aspecto importante do estudo foi indicar uma resiliência da pecPDI à modulação individual de distintas vias secretoras, consistente com uma estrita auto-regulação e possibilidade de vias sinérgicas e complementares. Estes resultados indicam que a externalização da PDI de superfície e PDI secretada possam ser externalizadas por mecanismos independentes. Estes processos compõem um processo regulado estritamente, consistente com papel homeostático da pecPDI / Protein disulfide isomerase (PDIA1 or PDI) is dithiol-disulfide oxireductase chaperone resident in the endoplasmic reticulum (ER). PDI is essential for proteostasis, due to its support of oxidative protein folding and ER-associated protein degradation (ERAD). In addition, PDI associates with NADPH oxidase(s) and regulate its activity, while outside of the cell, PDI redox-dependently modulates extracellular proteins. This epi/pericellular PDI (pecPDI) pool is known to regulate membrane/secreted proteins such as integrins, HIV glycoprotein gp120 and others, with functions that involve thrombosis, platelet function, cell adhesion, viral infection and vascular remodeling. PDI externalization route remains enigmatic and its elucidation can help understand some (patho)physiological PDI effects. An ER-Golgi route for PDI secretion has been as described on dengue virus-infected endothelial cells pancreatic and thyroid) cells. However, none of these papers addressed PDI secretion routes in a systematic fashion. Here, we show that endothelial cells (EC) constitutively externalize, through different routes, two PDI pools, a cell-surface and a secreted one, while in nonstimulated ECs PDI was not significantly detected in microparticles. Externalized PDI corresponds to < 2% of total cellular PDI pool. Both cell-surface and soluble PDI were predominantly externalized through unconventional type IV GRASP-independent pathway(s). However, the classical secretory pathway also contributes to basal cell-surface, but not soluble, PDI externalization, as PMA, ATP or thrombin-stimulated secretion also involve Golgi bypass. Furthermore, constitutive cell-surface PDI externalization in vascular smooth muscle cells also occurs in a Golgi-independent way. PDI externalization was not detectably mediated by non-conventional type I, II and III secretion routes, secretory lysosomes, recycling endosomes and ATP dependent active transport in EC. Since chaperones are essential for cellular stress response, we assessed the effects of ER stress and heat-shock on pecPDI. ER stress did not affect cell-surface PDI but increased the soluble pool. Both PDI pools were unaltered by heat shock, while stress recovery decreased PDI secretion. These data suggest that PDI release is finely tuned and dependent on the type of stress. Blockade of protein synthesis with cycloheximide did not change pecPDI levels, suggesting that newly-synthesized PDI is not preferentially externalized and that PDI traffic does not require newly-synthesized proteins. An important aspect of the study was the evidence for pecPDI resilience to individual modulation of distinct secretion routes, consistent with strict auto-regulation and possible synergic or complementary pathways. Overall, our data suggest that cell-surface and secreted PDI pool externalization are regulated through independent mechanisms, which in both cases involve Type IV non-conventional routes, with some minor contribution of Golgi-dependent secretory pathway. These patterns compose a strictly regulated process, consistent with an important homeostatic role for pecPDI

cAMP-elevating agents such as adenosine and epinephrine (after binding to beta-adrenergic receptor) contribute to local vascular dilation and some of these dilations are endothelium-dependent. Previous intracellular Ca 2+ imaging studies in mouse microvessel endothelial cells reported that addition of adenosine or epinephrine induced a Ca2+ influx which is blocked by CNG channel blockers such as L-cis-diltiazem or LY83583. Inside-out patch clamp studies confirmed the existence of a cAMP-activated current in endothelial cells, strongly suggesting a functional role of CNG, in particular CNGA2, channels in endothelial cells. The current study went further to show that similar Ca2+ influx in response to adenosine or epinephrine occurred in endothelial cells in freshly isolated mouse aortic strips and was again blocked by L-cis-diltiazem. By measuring the isometric force developed in mouse aortic strips, we showed that CNGA2 channel-mediated Ca2+ influx in endothelial cells contributed to the endothelium-dependent vascular dilatation in response to adenosine and epinephrine. / In conclusion, cyclic nucleotides playa vital role in the regulation of intracellular Ca2+ concentration in vascular cells and Jurket T cells. / In Jurkat T cells, cyclic nucleotides regulated Ca2+ mobilization in a different way. Fluorescence-imaging studies showed that cGMP inhibited store-operated Ca2+ influx and histamine-induced Ca 2+ rise in Jurkat T cells through activation of PKG. / Thromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can partly be attributed to TxA2-induced Ca2+ influx, which activates the Ca2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca2+ influx in vascular smooth muscle cells. Application of U-46619, a thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relied on the presence of extracellular Ca2+, because removal of extracellular Ca2+ abolished the constriction. This constriction was partially inhibited by a L-type Ca2+ channel inhibitor nifedipine (0.5-1 muM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner, Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca2+, which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence Of CNGA2 protein in vascular smooth muscle cells, These data suggest a functional role of CNG channels in U-46619-induced Ca 2+ influx and contraction of smooth muscle cells. / Leung, Yuk Ki. / "August 2010." / Adviser: Yao Xiaoxiang. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Han, Jianguo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 66-76). / Abstracts in English and Chinese. / Declaration --- p.i / Acknowledgement --- p.□ / Publication --- p.□ / Abstract (English) --- p.□xi / Abstract (Chinese) --- p.□ / Abbreviations --- p.ix / List of figures / tables --- p.x / Chapter Chapter 1. --- General Introduction / Chapter 1.1 --- Role of vascular smooth muscle cells in the control of coronary circulation --- p.1 / Chapter 1.1.1 --- Potassium channels in the coronary smooth muscle cells --- p.2 / Chapter 1.1.1.1 --- Voltage -dependent potassium (Kv) channels --- p.3 / Chapter 1.1.1.2 --- Inward rectifier K+ (Kir) channels --- p.4 / Chapter 1.1.1.3 --- ATP-sensitive potassium (Katp) channels --- p.4 / Chapter 1.1.2 --- BKCa channels in the regulation of vascular function --- p.6 / Chapter 1.1.2.1 --- The structure of BKCa channels --- p.6 / Chapter 1.1.2.2 --- Role of BKCa channels in the regulation of vascular function --- p.6 / Chapter 1.2 --- Functional alteration of the coronary SMCs during cardiac surgery --- p.7 / Chapter 1.2.1 --- Effect of ischemia on the function of SMCs in the coronary circulation --- p.8 / Chapter 1.2.2 --- Effect of cardioplegic/organ preservation solutions on the function of SMCs in the coronary circulation --- p.11 / Chapter Chapter 2. --- Materials and Methods --- p.14 / Chapter 2.1 --- Isometric force study in small coronary arteries --- p.14 / Chapter 2.1.1 --- Preparation of porcine small coronary arteries --- p.14 / Chapter 2.1.2 --- Experiment procedure --- p.15 / Chapter 2.1.2.1 --- Mounting of small coronary arteries --- p.15 / Chapter 2.1.2.2 --- Normalization procedure for small coronary arteries --- p.16 / Chapter 2.1.2.3 --- Precontraction and relaxation --- p.17 / Chapter 2.1.3 --- Data acquisition and analysis --- p.17 / Chapter 2.2 --- Patch-clamp electrophysiology --- p.18 / Chapter 2.2.1 --- Preparation of porcine coronary arteries --- p.18 / Chapter 2.2.2 --- Enzymatic dissociation of coronary arterial SMCs --- p.18 / Chapter 2.2.3 --- Primary cell culture --- p.19 / Chapter 2.2.4 --- Recording of BKca channel currents --- p.19 / Chapter 2.3 --- Statistical analysis --- p.21 / Chapter 2.4 --- Chemicals --- p.21 / Chapter Chapter 3. --- The Effect of Ischemia on BKCa channels in the Isolated SMCs of Coronary Arteries --- p.22 / Chapter 3.1 --- Abstract --- p.22 / Chapter 3.2 --- Introduction --- p.23 / Chapter 3.3 --- Experimental design and analysis --- p.25 / Chapter 3.3.1 --- Isometric force study in small coronary arteries --- p.25 / Chapter 3.3.2 --- Effect of ischemia on NS1619-induced relaxation in small coronary arteries --- p.26 / Chapter 3.3.3 --- Effect of ischemia on smooth muscle BKca channel currents --- p.27 / Chapter 3.3.3.1 --- Preparation of porcine coronary artery --- p.27 / Chapter 3.3.3.2 --- Enzymatic dissociation of coronary arterial SMCs --- p.27 / Chapter 3.3.3.3 --- Recording of BKCa channel currents --- p.27 / Chapter 3.3.4 --- Data acquisition and analysis --- p.28 / Chapter 3.4 --- Results --- p.28 / Chapter 3.4.1 --- Electrophysiological studies --- p.28 / Chapter 3.4.1.1 --- Effect of IBTX on the whole cell outward currents --- p.29 / Chapter 3.4.1.2 --- Effect of ischemia on the IBTX-sensitive BKca currents --- p.30 / Chapter 3.4.2 --- Relaxation studies --- p.30 / Chapter 3.4.2.1 --- Resting force --- p.30 / Chapter 3.4.2.2 --- U46619-induced contraction force --- p.31 / Chapter 3.4.2.3 --- Effect of IBTX on the NS1619-induced relaxation --- p.31 / Chapter 3.4.2.4 --- Effect of ischemia on the NS1619-induced relaxation --- p.31 / Chapter 3.5 --- Discussion --- p.32 / Chapter 3.5.1 --- Functional changes of the coronary smooth muscle BKca channels after ischemic exposure --- p.33 / Chapter 3.5.2 --- Role of BKca channels in SMCs during ischemia --- p.33 / Chapter 3.5.3 --- Clinical implications --- p.35 / Chapter Chapter 4. --- The Effect of Hyperkalemia on BKCa channels in the Isolated SMCs of Coronary Arteries --- p.41 / Chapter 4.1 --- Abstract --- p.41 / Chapter 4.2 --- Introduction --- p.42 / Chapter 4.3 --- Experimental design and analysis --- p.44 / Chapter 4.3.1 --- Isometric force study in small coronary arteries --- p.44 / Chapter 4.3.1.1 --- Effect of hyperkalemia on NS1619-mediated relaxation in small coronary arteries --- p.44 / Chapter 4.3.2. --- Effect of hyperkalemia on BKCa currents of SMCs --- p.45 / Chapter 4.3.2.1 --- Preparation of porcine coronary arteries --- p.45 / Chapter 4.3.2.2 --- Enzymatic dissociation of coronary arterial SMCs --- p.45 / Chapter 4.3.2.3 --- Recording of BKca channel currents --- p.46 / Chapter 4.3.3. --- Data acquisition and analysis --- p.46 / Chapter 4.4 --- Results --- p.47 / Chapter 4.4.1 --- Effect of hyperkalemia on the iberiotoxin-sensitive BKCa channel currents --- p.47 / Chapter 4.4.2 --- Relaxation studies --- p.48 / Chapter 4.4.2.1 --- Resting force --- p.48 / Chapter 4.4.2.2 --- U46619- and high K+-induced contraction force --- p.48 / Chapter 4.4.2.3 --- Effect of high K+ on the NS1619-induced relaxation --- p.48 / Chapter 4.4.2.4 --- Effect of IBTX on the NS1619-induced relaxation --- p.49 / Chapter 4.5 --- Discussion --- p.49 / Chapter 4.5.1 --- Role of BKCa channels in the isolated SMCs in hyperkalemic solution --- p.50 / Chapter 4.5.2 --- Functional changes of BKCa channels in coronary SMCs in hyperkalemia exposure --- p.51 / Chapter 4.5.3 --- Clinical implications --- p.52 / Chapter Chapter 5. --- General Discussion --- p.58 / Chapter 5.1 --- BKCa channels in porcine coronary SMCs --- p.59 / Chapter 5.2 --- Alteration of BKCa function related to ischemia in porcine coronary SMCs --- p.60 / Chapter 5.3 --- Alteration of BKCa function related to hyperkalemia in porcine coronary SMCs --- p.61 / Chapter 5.4 --- Limitation of the study --- p.62 / Chapter 5.5 --- Future investigations --- p.63 / Chapter 5.6 --- Conclusions --- p.63 / References --- p.66

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 186-210). Also available on the Internet.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xxiii, 251 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 214 pages. Includes vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 12, 2010). Includes bibliographical references.