INTRODUÇÃO: Existem várias doenças que evoluem com hipertensão pulmonar (HP), entre elas a Hipertensão Arterial Pulmonar Idiopática (HAPI) e a Esquistossomose. Acredita-se que um dos principais fatores desencadeantes da HP esteja relacionado com a disfunção endotelial. OBJETIVOS: 1. Avaliar a disfunção endotelial de pacientes com HAPI e esquistossomóticos com e sem HAP usando os marcadores plasmáticos Endotelina-1, Selectina E, VEGF, PDGFAB e PDGF-BB; 2.Avaliar se pacientes com HAP associada à esquistossomose possuem o mesmo grau de disfunção endotelial que pacientes com esquistossomose sem HAP. METODOLOGIA: Foram formados 4 grupos distintos: Controle (n=13), HAPI (n=11), pacientes com esquistossomose e HP (ESQ+HP) (n=13) e pacientes com esquistossomose sem HP (ESQ)(n=13). Os pacientes foram submetidos a avaliação clínica (caracterizados quanto a gravidade), funcional (realizaram ecocardiograma com medida de pressão sistólica de ventrículo direito, ultrassonografia abdominal quando indicada e exames para excluir outras doenças) e laboratorial (entre eles, contagem de leucócitos, plaquetas e dosagem de BNP). A avaliação hemodinâmica foi realizada nos pacientes com HP. Para a análise da disfunção endotelial, foram coletados 40 mL de sangue de todos os indivíduos para a dosagem de Endotelina-1, Selectina E, VEGF, PDGF-AB e PDGF-BB. RESULTADOS: Observou-se que os grupos não se diferiram quanto a idade, houve um predomínio do sexo feminino e os grupos controle e ESQ apresentaram valores de PSVD menores do que os grupos com HP (controles: 23,4±4,6, ESQ: 29,5±8,5, HAPI: 79,8±26,4 e ESQ+HP: 75,2±15,3 mmHg). As medidas hemodinâmicas foram semelhantes em ambos os grupos com HAP. Quanto aos marcadores da função endotelial, o grupo controle apresentou valores séricos de PDGF-BB mais aumentados (8,9±4,8x 103 pg/mL, p<0,001) que os grupos HAPI, ESQ+HP, ESQ (3,7±2,1; 5,2±3 ; 2,4±1,7 x 103 pg/mL, respectivamente). O grupo HAPI apresentou valores mais elevados de Selectina E (61,5±24,2 x 103 pg/mL) que os grupos controle, ESQ+HP e ESQ (14,5±12,2; 23,9±15,3; 21,4±18 x 103 pg/mL, respectivamente, p=0,005). Os valores séricos de PDGF-AB do grupo controle foram mais elevados que no grupo ESQ (p=0,006). Não foram encontradas diferenças significantes nos valores séricos de Endotelina-1 entre os grupos (p=0,281). Em relação ao VEGF, os pacientes com HAPI apresentaram valores séricos similares ao grupo ESQ+HP e mais elevados que o grupo controle e ESQ (p=0,002). O ponto de corte da dosagem da selectina E (43.806 pg/mL) para diferenciar pacientes com HAPI dos pacientes com ESQ+HP apresentou uma sensibilidade de 91% e a especificidade de 89%. O PDGF-BB apresentou uma boa acurácia para distinguir o grupo controle dos demais, com uma sensibilidade de 77% e uma especificidade de 83%. Além disso, a Selectina E apresentou uma forte correlação com o níveis séricos de BNP (r=0,74, p=0,006). O número de leucócitos e de plaquetas foram diferentes entre os três grupos do estudo. Pacientes com HAPI tinham maior número de leucócitos e plaquetas quando comparados com esquistossomóticos. CONCLUSÕES: 1.Pacientes com HAPI apresentaram valores séricos mais elevados de Selectina E do que pacientes com esquistossomose e controles; 2.Pacientes portadores de esquistossomose com e sem HP apresentaram os mesmos valores séricos dos marcadores de disfunção endotelial / INTRODUCTION: There are several diseases that cause Pulmonary hypertension (PH), such as Idiopathic Pulmonary Arterial Hypertension and Schistosomiasis. The mechanisms that lead to PH are thought to be related to endothelial dysfunction. OBJECTIVES: To evaluate endothelial dysfunction, using plasma markers such as Endothelin-1(ET-1), E-Selectin, VEGF, PDGF-AB and -BB, in patients with idiopathic pulmonary arterial hypertension (IPAH) and schistosomiasis patients with or without PH; and to evaluate if schistosomiasis groups have endothelial dysfunction in the same degree. METHODOLOGY: Patients were divided in 4 different groups: Patients with IPAH (n=11), Patients with PH associated to Schistosomiasis (SchPH)(n=13), Patients with Schistosomiasis without PH (Sch)(n=13) and Controls(n=13). PAH patients were classified according to severity. All groups were submitted to echocardiography and right ventricule systolic pressure(RVSP) was measured. Abdominal ultrassonography was used to rule in or rule out schistosomiasis diagnosis. PH patients went through haemodynamics evaluation and all patients had laboratorial assessment (leucocytes and platelet count and BNP levels) Soluble adhesion molecules such as E-Selectin, VEGF, PDGF-AB, PDGF-BB e ET-1 were determined by ELISA. Leucocytes and platelet counts as well as BNP levels were also evaluated. Results: Subjects did not differ according to age and there was a higher proportion of female patients. Controls and Sch subjects had lower RVSP compared to PH groups (Sch: 23.4±4.6, controls: 29.5±8.5, IPAH: 79.8±26.4 and Sch+HP: 75.2±15.3 mmHg). Haemodynamics data did not differ in PH patients. In IPAH group, E-selectin was elevated (61.5±24,2x103pg/mL) compared to controls, Sch+HP and Sch (14.5±12.2; 23.9±15.3; 21.4±18 x103pg/mL, respectively, p=0,005). PDGF-BB was decreased in IPAH, Sch+HP, Sch (3.7±2.1; 5.2±3; 2.4±1.7x103pg/mL, respectively) compared to controls (8.9±4.8x 103 pg/mL, p<0.001). PDGF-AB was elevated in controls (25.6±8.6x103pg/mL) when compared to Sch (11.4±8.6 x103pg/mL)(p=0.006). There were no differences in ET-1 levels within groups. In relation to VEGF, IPAH group had higher levels compared to controls and Sch (96,6±68,2, 38,4±28, 37,±19,2 pg/mL, respectively) (p=0,002). Based on ROC curve, E-selectin cutoff value of 43.806 pg/mL showed a sensitivity of 91% and a specificity of 89% to distinguish IPAH patients from other groups and PDGF-BB had a good accuracy to differentiate controls with a sensitivity of 77% and a specificity of 83%. Furthermore, E-selectin had a strong correlation with BNP levels (r=0,74, p=0,006). The number of leucocytes and platelets were different within groups. IPAH patients had the highest, and Sch group had the lowest blood cells and platelets count. Conclusions: 1. IPAH patients had higher levels of serum E-selectin and VEGF and controls had higher levels of PDGF-BB and AB; 2. Schistosomiasis patients with or without PH had the same levels of endothelial dysfunction serum markers

INTRODUÇÃO: Existem várias doenças que evoluem com hipertensão pulmonar (HP), entre elas a Hipertensão Arterial Pulmonar Idiopática (HAPI) e a Esquistossomose. Acredita-se que um dos principais fatores desencadeantes da HP esteja relacionado com a disfunção endotelial. OBJETIVOS: 1. Avaliar a disfunção endotelial de pacientes com HAPI e esquistossomóticos com e sem HAP usando os marcadores plasmáticos Endotelina-1, Selectina E, VEGF, PDGFAB e PDGF-BB; 2.Avaliar se pacientes com HAP associada à esquistossomose possuem o mesmo grau de disfunção endotelial que pacientes com esquistossomose sem HAP. METODOLOGIA: Foram formados 4 grupos distintos: Controle (n=13), HAPI (n=11), pacientes com esquistossomose e HP (ESQ+HP) (n=13) e pacientes com esquistossomose sem HP (ESQ)(n=13). Os pacientes foram submetidos a avaliação clínica (caracterizados quanto a gravidade), funcional (realizaram ecocardiograma com medida de pressão sistólica de ventrículo direito, ultrassonografia abdominal quando indicada e exames para excluir outras doenças) e laboratorial (entre eles, contagem de leucócitos, plaquetas e dosagem de BNP). A avaliação hemodinâmica foi realizada nos pacientes com HP. Para a análise da disfunção endotelial, foram coletados 40 mL de sangue de todos os indivíduos para a dosagem de Endotelina-1, Selectina E, VEGF, PDGF-AB e PDGF-BB. RESULTADOS: Observou-se que os grupos não se diferiram quanto a idade, houve um predomínio do sexo feminino e os grupos controle e ESQ apresentaram valores de PSVD menores do que os grupos com HP (controles: 23,4±4,6, ESQ: 29,5±8,5, HAPI: 79,8±26,4 e ESQ+HP: 75,2±15,3 mmHg). As medidas hemodinâmicas foram semelhantes em ambos os grupos com HAP. Quanto aos marcadores da função endotelial, o grupo controle apresentou valores séricos de PDGF-BB mais aumentados (8,9±4,8x 103 pg/mL, p<0,001) que os grupos HAPI, ESQ+HP, ESQ (3,7±2,1; 5,2±3 ; 2,4±1,7 x 103 pg/mL, respectivamente). O grupo HAPI apresentou valores mais elevados de Selectina E (61,5±24,2 x 103 pg/mL) que os grupos controle, ESQ+HP e ESQ (14,5±12,2; 23,9±15,3; 21,4±18 x 103 pg/mL, respectivamente, p=0,005). Os valores séricos de PDGF-AB do grupo controle foram mais elevados que no grupo ESQ (p=0,006). Não foram encontradas diferenças significantes nos valores séricos de Endotelina-1 entre os grupos (p=0,281). Em relação ao VEGF, os pacientes com HAPI apresentaram valores séricos similares ao grupo ESQ+HP e mais elevados que o grupo controle e ESQ (p=0,002). O ponto de corte da dosagem da selectina E (43.806 pg/mL) para diferenciar pacientes com HAPI dos pacientes com ESQ+HP apresentou uma sensibilidade de 91% e a especificidade de 89%. O PDGF-BB apresentou uma boa acurácia para distinguir o grupo controle dos demais, com uma sensibilidade de 77% e uma especificidade de 83%. Além disso, a Selectina E apresentou uma forte correlação com o níveis séricos de BNP (r=0,74, p=0,006). O número de leucócitos e de plaquetas foram diferentes entre os três grupos do estudo. Pacientes com HAPI tinham maior número de leucócitos e plaquetas quando comparados com esquistossomóticos. CONCLUSÕES: 1.Pacientes com HAPI apresentaram valores séricos mais elevados de Selectina E do que pacientes com esquistossomose e controles; 2.Pacientes portadores de esquistossomose com e sem HP apresentaram os mesmos valores séricos dos marcadores de disfunção endotelial / INTRODUCTION: There are several diseases that cause Pulmonary hypertension (PH), such as Idiopathic Pulmonary Arterial Hypertension and Schistosomiasis. The mechanisms that lead to PH are thought to be related to endothelial dysfunction. OBJECTIVES: To evaluate endothelial dysfunction, using plasma markers such as Endothelin-1(ET-1), E-Selectin, VEGF, PDGF-AB and -BB, in patients with idiopathic pulmonary arterial hypertension (IPAH) and schistosomiasis patients with or without PH; and to evaluate if schistosomiasis groups have endothelial dysfunction in the same degree. METHODOLOGY: Patients were divided in 4 different groups: Patients with IPAH (n=11), Patients with PH associated to Schistosomiasis (SchPH)(n=13), Patients with Schistosomiasis without PH (Sch)(n=13) and Controls(n=13). PAH patients were classified according to severity. All groups were submitted to echocardiography and right ventricule systolic pressure(RVSP) was measured. Abdominal ultrassonography was used to rule in or rule out schistosomiasis diagnosis. PH patients went through haemodynamics evaluation and all patients had laboratorial assessment (leucocytes and platelet count and BNP levels) Soluble adhesion molecules such as E-Selectin, VEGF, PDGF-AB, PDGF-BB e ET-1 were determined by ELISA. Leucocytes and platelet counts as well as BNP levels were also evaluated. Results: Subjects did not differ according to age and there was a higher proportion of female patients. Controls and Sch subjects had lower RVSP compared to PH groups (Sch: 23.4±4.6, controls: 29.5±8.5, IPAH: 79.8±26.4 and Sch+HP: 75.2±15.3 mmHg). Haemodynamics data did not differ in PH patients. In IPAH group, E-selectin was elevated (61.5±24,2x103pg/mL) compared to controls, Sch+HP and Sch (14.5±12.2; 23.9±15.3; 21.4±18 x103pg/mL, respectively, p=0,005). PDGF-BB was decreased in IPAH, Sch+HP, Sch (3.7±2.1; 5.2±3; 2.4±1.7x103pg/mL, respectively) compared to controls (8.9±4.8x 103 pg/mL, p<0.001). PDGF-AB was elevated in controls (25.6±8.6x103pg/mL) when compared to Sch (11.4±8.6 x103pg/mL)(p=0.006). There were no differences in ET-1 levels within groups. In relation to VEGF, IPAH group had higher levels compared to controls and Sch (96,6±68,2, 38,4±28, 37,±19,2 pg/mL, respectively) (p=0,002). Based on ROC curve, E-selectin cutoff value of 43.806 pg/mL showed a sensitivity of 91% and a specificity of 89% to distinguish IPAH patients from other groups and PDGF-BB had a good accuracy to differentiate controls with a sensitivity of 77% and a specificity of 83%. Furthermore, E-selectin had a strong correlation with BNP levels (r=0,74, p=0,006). The number of leucocytes and platelets were different within groups. IPAH patients had the highest, and Sch group had the lowest blood cells and platelets count. Conclusions: 1. IPAH patients had higher levels of serum E-selectin and VEGF and controls had higher levels of PDGF-BB and AB; 2. Schistosomiasis patients with or without PH had the same levels of endothelial dysfunction serum markers

Local neuronal activity in the brain results in increased blood flow and is called neurovascular coupling. Such blood flow changes result in the blood-oxygen level dependent (BOLD) fluctuations detectable by functional magnetic resonance imaging (fMRI). The hemodynamic response is also an essential component of brain health and is impaired in various models of cognitive dysfunction. However, we still do not understand why functional hyperemia in the brain is important. To understand this question, various groups have studied brain metabolic activity as well as the mechanisms underlying neurovascular coupling. Over the years, several cell types have been proposed to contribute to functional hyperemia in the brain, including neurons, astrocytes and pericytes. However, the picture remains incomplete – controversies abound regarding the exact role of astrocytes, and pericytes in neurovascular coupling. Our lab has studies the mechanisms of neurovascular coupling from a mesoscopic perspective, as vasodilation in the rodent cortex involves capillaries and diving arterioles in the brain parenchyma as well as surface vasculature in the brain. We proposed that the vascular endothelium itself might provide a continuous conduit for transmitting vasodilatory signals initiated at the capillary level due to local neuronal activity. Given that systemic endothelial dysfunction could contribute to decreased neurovascular function, this hypothesis raised important concerns regarding endothelial vulnerabilities in common diseases like hypertension and diabetes and its role in diminished cognitive function and neurodegeneration. Based on findings from vascular research in other organ systems, we hypothesized that two distinct mechanisms of endothelium-derived vasodilation significantly contribute to neurovascular coupling the brain. These two mechanisms were expected to consist of fast long-range endothelium-derived hyperpolarization (EDH) dependent vasodilation (conducted vasodilation) and slower, more localized endothelium calcium-wave dependent vasodilation (propagated vasodilation). Together, we expected these mechanisms to shape the spatio-temporal evolution of hemodynamic responses in the brain. This dual mechanism of endothelial control of the hyperemic response in the brain might explain the complex spatiotemporal properties and non-linearities of the fMRI blood oxygen level dependent (BOLD) signal. My initial experiments were conducted in anesthetized rats, where I pharmacologically inhibited endothelial dependent vasodilation during functional hyperemia in the somatosensory cortex under a hind-paw electrical stimulus paradigm. While the results gleaned from these experiments were very revealing, it was important to consider the effect of the pharmacological manipulations on neuronal activity in the brain. In addition, neurovascular coupling and overall brain blood flow in anesthetized animals is dramatically altered when compared to awake animals. In order to accomplish these goals, I built a wide-field optical imaging system that could simultaneously measure fluorescence-based neuronal activity and reflectance-based hemodynamic activity in awake head-restrained mice. I then used non-blood brain barrier permeable pharmacology to study endothelial mechanisms of neurovascular coupling in awake Thy1-GCaMP6f mice, which express the calcium fluorophore in a subset of excitatory neurons in the cortex. I found that using this pharmacology I could dissect out the hypothesized two spatiotemporally distinct components of whisker-stimulus evoked neurovascular coupling in awake mice. With simultaneous recording of the neuronal activity driving this blood flow, I was able to build a mathematical model for neurovascular coupling that accounted for these two mechanisms by allowing for the superposition of a time-invariant, constant hemodynamic response with a hemodynamic response obtained by convolving the underlying neuronal response with a hemodynamic response function (HRF). I was able to linearize these apparent non-linearities in the hemodynamic response by studying the properties of deconvolved HRFs for stimuli of different durations before and after pharmacological manipulation of endothelial activity. Two important considerations remain. Firstly, our wide-field, mesoscopic view of the brain prevents observations of endothelial function (hyperpolarization and calcium activity) and the propagation dynamics of dilation best observed at the microscopic level. To accomplish this task, ongoing experiments currently use our high-speed volumetric imaging technology (SCAPE – Swept Confocally Aligned Planar Excitation microscopy) to study stimulus-evoked vascular dynamics in mouse lines expressing GFP and GCaMP8 in endothelial cells. Secondly, our longitudinal imaging of these animals is ideal for studying the acute and long-term effects of endothelial dysfunction on cognitive function. This requires adequate study of changes in mouse behavior during manipulations of endothelial function longitudinally in awake mice. Future experiments should involve the development and implementation of appropriate task-based behavior experiments, and analysis methods for more carefully exploring changes in neuronal activity in the mouse brain during stimulus and non-stimulus dependent activity.

Yeung Lam. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-150). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGMENT --- p.V / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General review of angiogenesis --- p.1 / Chapter 1.2 --- Vascular endothelial growth factors (VEGFs) --- p.2 / Chapter 1.2.1 --- VEGF-A --- p.2 / Chapter 1.2.2 --- P1GF --- p.4 / Chapter 1.2.3 --- VEGF-B --- p.5 / Chapter 1.2.4 --- VEGF-C and VEGF-D --- p.6 / Chapter 1.3 --- VEGF receptors (VEGFRs) --- p.9 / Chapter 1.3.1 --- VEGFR-1 (or flt-1) --- p.9 / Chapter 1.3.2 --- VEGFR-2 ( or flk-1) --- p.10 / Chapter 1.3.3 --- VEGFR-3 ( or flt-4) --- p.11 / Chapter 1.4 --- Hormonal regulation of VEGFs by LH/hCG --- p.14 / Chapter 1.5 --- General review of the testis --- p.17 / Chapter 1.5.1 --- Structure and function of the testis --- p.17 / Chapter 1.5.2 --- Testicular vasculature --- p.18 / Chapter 1.5.3 --- Testicular angiogenesis --- p.19 / Chapter 1.6 --- Localization of VEGF and VEGF receptors in the testis --- p.20 / Chapter 1.7 --- Aims of the present study --- p.21 / Chapter 2. --- Materials and methods --- p.23 / Chapter 2.1 --- Animals --- p.23 / Chapter 2.1.1 --- Depletion of Leydig cell --- p.23 / Chapter 2.1.2 --- Suppression of Leydig cell and stimulation by hCG --- p.24 / Chapter 2.1.3 --- Collection of tissue --- p.25 / Chapter 2.2 --- Preparation of primary cells from rat testes --- p.27 / Chapter 2.2.1 --- Sertoli cell preparation --- p.27 / Chapter 2.2.2 --- Germ cell preparation --- p.29 / Chapter 2.2.3 --- Interstitial cell and Leydig cell preparation --- p.30 / Chapter 2.3 --- Cell cultures --- p.32 / Chapter 2.3.1 --- Reagents and cell lines --- p.32 / Chapter 2.3.2 --- "Mouse Leydig cell line, TM3 and Sertoli cell line, TM4" --- p.33 / Chapter 2.3.3 --- "Mouse tumor Leydig cell line, MLTC-1" --- p.34 / Chapter 2.3.4 --- "Rat tumor Leydig cell line, R2C" --- p.34 / Chapter 2.3.5 --- "Rat tumor Leydig cell line, LC540" --- p.35 / Chapter 2.4 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.35 / Chapter 2.4.1 --- Extraction of total RNA --- p.35 / Chapter 2.4.2 --- Quantitation of total RNA --- p.37 / Chapter 2.4.3 --- RT-PCR --- p.37 / Chapter 2.4.4 --- Purification and authentication of PCR products --- p.47 / Chapter 2.5 --- Immunohistochemical staining --- p.48 / Chapter 2.5.1 --- Perfusion and processing of testes for histological sections --- p.48 / Chapter 2.5.2 --- Immunohistochemical staining of tissue sections --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.52 / Chapter 2.6.1 --- Extraction and quantitation of total protein --- p.52 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Immunoblotting --- p.55 / Chapter 2.7 --- Statistical analyses --- p.57 / Chapter 3. --- Results --- p.58 / Chapter 3.1 --- Expression and localization of VEGFs in the rat testis --- p.58 / Chapter 3.1.1 --- VEGF-A --- p.58 / Chapter 3.1.2 --- VEGF-B --- p.64 / Chapter 3.1.3 --- VEGF-C --- p.69 / Chapter 3.1.4 --- VEGF-D --- p.73 / Chapter 3.1.5 --- P1GF --- p.77 / Chapter 3.2 --- Effect of Leydig cell depletion on VEGFs expression in the rat testis --- p.81 / Chapter 3.2.1 --- Effect on VEGF-A --- p.81 / Chapter 3.2.2 --- Effect on VEGF-B --- p.82 / Chapter 3.2.3 --- Effect on VEGF-C --- p.88 / Chapter 3.2.4 --- Effect on VEGF-D --- p.91 / Chapter 3.2.5 --- Effect on P1GF --- p.94 / Chapter 3.3 --- Effect of Leydig cell suppression and hCG stimulation on VEGFs expression in the rat testis --- p.97 / Chapter 3.3.1 --- Effect on VEGF-A --- p.97 / Chapter 3.3.2 --- Effect on VEGF-B --- p.107 / Chapter 3.3.3 --- Effect on VEGF-C --- p.113 / Chapter 3.3.4 --- Effect on VEGF-D --- p.119 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- References --- p.134

Yang Qin. / "June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 168-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lau Kin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-103). / Abstracts in English and Chinese. / Contents --- p.1 / Chapter Chapter 1 --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling in Endothelial Cells --- p.5 / Chapter 1.1.1 --- Calcium and its functions --- p.5 / Chapter 1.1.2 --- "Second Messengers: Inositol-1,4,5-Triphosphate and Diacylglycerol" --- p.6 / Chapter 1.1.3 --- Propagation of Ca2+ Signals --- p.8 / Chapter 1.1.4 --- Ca2+-ATPases --- p.9 / Chapter 1.1.5 --- Regulation of Sarcoplasmic Reticulum --- p.10 / Chapter 1.1.6 --- Agonist-induced Ca2+ Entry --- p.11 / Chapter 1.2 --- Mechanism of Store-Operated Ca2+ Entry --- p.14 / Chapter 1.2.1 --- Signaling Mechanisms of SOC --- p.14 / Chapter 1.2.1.1 --- A Diffusible Messenger --- p.14 / Chapter 1.2.1.2 --- Conformational Coupling --- p.15 / Chapter 1.2.1.3 --- Vesicle Secretion --- p.16 / Chapter 1.3 --- Regulation of Ca2+ Entry by cGMP --- p.20 / Chapter 1.4 --- Molecular Structres of Store-operated Channels --- p.22 / Chapter 1.4.1 --- Drosophila Transient Receptor Potential (trp) Gene --- p.22 / Chapter 1.4.2 --- Trpl Gene --- p.23 / Chapter Chapter 2 --- Methods and Materials --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Phosphate-buffered saline --- p.27 / Chapter 2.1.2 --- Culture Media and Materials --- p.27 / Chapter 2.2 --- Preparations and Culture of Cells --- p.28 / Chapter 2.2.1 --- Culture of Rat Aortic Endothelial Cells --- p.28 / Chapter 2.2.2 --- Culture of Human Bladder Epithelial Cell Line --- p.29 / Chapter 2.2.3 --- Culture of Human Embryonic Kidney Epithelial Cell Line --- p.29 / Chapter 2.3 --- Cell. Subculture and Marvest --- p.29 / Chapter 2.4 --- Intracellular Free Calcium Ions ([Ca2+]i) measurment --- p.30 / Chapter 2.4.1 --- Chemicals --- p.30 / Chapter 2.4.2 --- Bathing solutions --- p.31 / Chapter 2.4.3 --- Preparations of Cells for [Ca2+]i Measurement --- p.31 / Chapter 2.4.3.1 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with PTI RatioMaster Fluorescence System --- p.31 / Chapter 2.4.3.2 --- Plating cells on Glass Cover Slips for [Ca2+]i Measurement with Confocal Imaging System and Confocal Laser Scanning Microscopy --- p.32 / Chapter 2.4.4 --- PTI RatioMaster Fluorescence System --- p.35 / Chapter 2.4.4.1 --- Experimental Setup --- p.35 / Chapter 2.4.4.2 --- Fura-2/AM Dye loading --- p.35 / Chapter 2.4.4.3 --- Background Fluorescence and [Ca ]i Measurement --- p.37 / Chapter 2.4.5 --- Confocal Imaging System --- p.37 / Chapter 2.4.5.1 --- Experimental Setup --- p.37 / Chapter 2.4.5.2 --- Fluo-3/AM Dye Loading --- p.39 / Chapter 2.4.5.3 --- [Ca2+]i Measurement --- p.39 / Chapter 2.4.6 --- Confocal Laser Scanning Microscopy --- p.40 / Chapter 2.4.6.1 --- Principles --- p.40 / Chapter 2.5 --- Cloning and expression of Trpl in HEK293 cell line --- p.43 / Chapter 2.5.1 --- Cloning of Htrpl Gene into pcDNA3 Vector --- p.43 / Chapter 2.5.1.1 --- Enzyme Digestion --- p.43 / Chapter 2.5.1.2 --- Gel electrophoresis and Isolation of Htrpl by GeneCIean II Kit --- p.44 / Chapter 2.5.1.3 --- Ligation of Trpl and pcDNA3 Vector --- p.44 / Chapter 2.5.1.4 --- Transformation --- p.47 / Chapter 2.5.1.5 --- Purification of cloned Trpl-pcDNA3 by QIAprep Spin Miniprep Kit --- p.47 / Chapter 2.5.2 --- Transfection of HEK293 Cells with Htrpl and pEGFP-Nl Vector --- p.48 / Chapter 2.5.2.1 --- Cell Preparation for Transfection --- p.48 / Chapter 2.5.2.2 --- Transfection --- p.48 / Chapter 2.5.3 --- Fluorescence Labeling of Expressed Htrpl Channel in HEK293 Cells --- p.49 / Chapter 2.5.3.1 --- Immunostaining with Anti-TRPCl Antibody --- p.49 / Chapter 2.5.3.2 --- Labeling with FITC2° Antibody --- p.50 / Chapter Chapter 3 --- Results --- p.51 / Chapter 3.1 --- Propagation of Ca2+ Signaling --- p.51 / Chapter 3.2. --- Effect of cGMP on SERCA --- p.55 / Chapter 3.2.1 --- ATP stimulated Ca2+ release from internal stores --- p.55 / Chapter 3.2.2 --- Effect of cGMP on the falling phase of [Ca2+]i --- p.55 / Chapter 3.2.3 --- Effect of CPA on the falling phase of [Ca2+]i --- p.58 / Chapter 3.2.4 --- Effect of KT5823 on cGMP --- p.63 / Chapter 3.3. --- Effect of cGMP on bradykinin-activated capacitative Ca2+ entry --- p.65 / Chapter 3.3.1 --- Bradykinin induced capacitative Ca2+ entry --- p.65 / Chapter 3.3.2 --- Effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.3 --- Effect of KT5823 on the inhibitory effect of cGMP on Ca2+ entry activated by bradykinin --- p.67 / Chapter 3.3.4. --- Effect of cGMP and KT5823 on capacitative Ca2+ entry activated by a combination of different agonists. --- p.71 / Chapter 3.4 --- Cloning and expression of htrpl in HEK 293 cell line --- p.75 / Chapter 3.4.1 --- Optimizing transfection conditions using pEGFP-Nl --- p.78 / Chapter 3.4.2 --- Transient transfection of htrpl channel in HEK293 cells --- p.81 / Chapter 3.4.3 --- Channel properties of expressed htrpl channel --- p.84 / Chapter Chapter 4 --- Discussion --- p.88 / Chapter 4.1 --- Ptopagation of Ca2+ Signaling --- p.88 / Chapter 4.2 --- Effect of cGMP on[Ca2+]i of Vascular Endothelial Cells --- p.89 / Chapter 4.2.1 --- Effect of cGMP on SERCA --- p.89 / Chapter 4.2.2 --- Effect of cGMP on Regulation of Agonist-Activated Capacitative Ca2+ Entry --- p.92 / Chapter 4.2.3 --- Physiological Property of Expressed Htrpl in HEK293 cells --- p.95 / References --- p.97

Aumento no risco de tromboembolismo venoso têm sido descrito em usuárias de anticoncepcionais hormonais combinados orais de terceira geração. Como a disfunção endotelial é reconhecida como o primeiro passo para o desenvolvimento da doença cardiovascular e de trombose, nosso objetivo foi analisar a função endotelial venosa em mulheres saudáveis em uso do anticoncepcional hormonal combinado oral contendo 15 ug etinilestradiol associados a 60 ug gestodeno. Avaliou-se a função endotelial venosa em 21 mulheres saudáveis pela técnica da complacência venosa da veia do dorso da mão, em estudo prospectivo caso-controle. Em 12 delas estudou-se a função endotelial venosa no momento basal e ao final do 4º mês do uso do anticoncepcional (grupo-caso). Em outras nove, usuárias de dispositivo intra-uterino T de cobre (grupo-controle), a função endotelial venosa foi avaliada no mesmo intervalo. Construíram-se curvas doses-resposta para acetilcolina e nitroprussiato de sódio após a pré-constrição da veia com fenilefrina. No grupo de usuárias de anticoncepcional hormonal houve diminuição da venodilatação máxima em resposta à acetilcolina e ao nitroprussiato de sódio, porém, essa mudança não foi estatisticamente significante (p > 0,05). No grupo-controle não se detectaram mudanças significantes na venodilatação máxima entre o momento basal e o 4° mês. Assim, nosso estudo não mostrou mudanças significantes na função endotelial venosa em mulheres saudáveis em uso de anticoncepcional hormonal combinado oral em relação às não usuárias, avaliadas pela técnica da complacência venosa de uma veia do dorso da mão. / An increase in the incidence of venous thromboembolic events has been reported among users of third generation oral contraceptives. As endothelial dysfunction is recognized as an initial step in the development of cardiovascular disease and venous thromboembolism, our goal was to evaluate the effect of an oral contraceptive (15 ug ethinyl estradiol/ 60 ug gestodene) on the venous endothelial function of healthy women. Twenty one women were evaluated by the dorsal hand vein technique. In twelve women venous endothelial function was studied at baseline and after 4 months of oral contraceptives use. Nine users of intrauterine device (control group) had their venous endothelial function evaluated twice, in a similar time interval. After preconstriction of the vein with phenylephrine, dose-response curves for acetylcholine and sodium nitroprusside were constructed. In the oral contraceptive group, the maximum venodilation response to acetylcholine and sodium nitroprusside decreased after a four months but this change was not statistically significant (p > 0,05). In the control group no significant changes were detected in maximum venodilation responses in a 4-month study interval. Therefore in our study, the use of oral contraceptives was not associated with significant changes in venous endothelial function accessed by the dorsal hand vein technique.

Corneal endothelial cells have been shown to possess a uniform polygonal and complex multipolar shape at their apical and basolateral surface respectively. We established a morphological timetable to study how this complex shape arises postnatally. Corneas were collected from mice between postnatal day 8 to postnatal day 35 and labelled with antibodies specific for ZO-1 and NCAM at apical and basolateral region, respectively. Images were collected using wide-field fluorescence microscopy and morphometrically analyzed. Results showed that apical cell sizes increase linearly over the first 3 weeks, plateauing at 4-5 weeks postnatally with increased regularity. Basolateral membrane surfaces remained relatively smooth prior to eyelid opening and thereafter begins developing showing differences in development from periphery to the center until about 4 weeks postnatally when the wavy processes get vivid. Results indicate concurrent and independent development at both poles of the corneal endothelium, with more complexity seen at the basolateral surface.

Profound vascular leakage in conjunction with elevated viremia is the hallmark of Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). Antibody (Ab)-dependent enhancement (ADE), in which pre-existing, cross-reactive Abs enhance virus infectivity, is thought to be responsible for increased viremia, while loss of endothelial cell (EC) barrier integrity is the precursor to plasma leakage. However, the relationship between viremia and vascular leak has not been established. The objective of this dissertation project was to determine the involvement of antibodies in the pathogenesis of vascular leak syndrome associated with DHF/DSS by establishing a relationship between Ab-mediated increase in viremia and changes in vascular permeability, the hallmark of DHF/DSS. Our approach focused on characterization of human monoclonal antibodies (hMAbs) from a previously dengue virus (DENV)-infected patient for their ability to both neutralize and enhance infection and increase vascular permeability in vitro. Our results revealed that the human antibody response to DENV E protein elicited by natural infection is predominantly comprised of broadly cross-reactive antibodies targeting domain II epitopes. Using a multiplex cytokine immunoassay, qRT-PCR, and plaque assay, we demonstrated an association between viral load and cytokine production in DENV-infected FcγR-bearing K562 cells, and determined that DENV infection of K562 cells in the presence of hMAb resulted in a modulated inflammatory cytokine response with an overall pro-inflammatory profile. Using human microvascular ECs (HMEC-1), we further demonstrated an association between viral load, cytokine production, and the onset of permeability changes via an indirect mechanism in which inflammatory mediators released by DENV-infected K562 cells altered HMEC-1 barrier function and observed a synergistic effect between active DENV infection and release of inflammatory mediators by both K562 and HMEC-1 that increased permeability. Collectively, our results support the multifactorial nature of the pathogenesis underlying vascular leak, involving a complex interaction between ECs and FcγR-bearing cells, and a synergistic relationship between enhanced viremia and inflammatory mediators leading to increased permeability. Our use of hMAbs provided a novel approach to understanding how Abs impact the vasculature during DENV infection and enable identification of Ab characteristics that may trigger vascular leak, a crucial concern for DENV vaccine design. / acase@tulane.edu

Von Willebrand factor (VWF) is a plasma glycoprotein that can bind collagen at a wound site as well as circulating platelets. VWF forms high molecular weight multimers (>20,000 kDa). VWF can also form VWF strings that appear to be attached to the endothelial surface and are capable of binding platelets. These strings are only observed in vitro and in vivo in the absence of the VWF-cleaving protease ADAMTS13. Deficiency in ADAMTS13 results in thrombotic thrombocytopenic purpura (TTP), a clotting disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, renal dysfunction, neurological dysfunction and fever. Patients suffering from TTP demonstrate VWF-and platelet-rich thrombi in the microvasculature of numerous organ systems, but most notably in the kidneys, heart, and brain. While VWF strings have not been directly connected to TTP, their presence in vivo was only identified with the ADAMTS13 knockout mouse (a model of TTP), suggesting a possible relationship. Recently we identified glycerol as an agent, similar to histamine, that triggers the formation of VWF strings in vitro. We found that glycerol and histamine trigger TTP in an ADAMTS13-deficient mouse model. In addition, we determined conditions in vitro that promote the formation of dense VWF networks. These networks of VWF can be greater than 70 μm thick and appear to be able to form fibers as long as several millimeters in length. These networks have not been previously identified and may underlie a possible mechanism by which VWF-rich thrombi form in TTP. These networks were formed solely from cultured endothelial cells, leading us to believe that endothelial cells alone are capable of producing more VWF than perhaps previously appreciated. These data suggest that secretion of VWF from the endothelium may play an important role in the pathophysiology of TTP.