Many studies have demonstrated that there is a strong relationship between endothelial dysfunction and oxidative stress and have demonstrated also that hyperglycemia is associated with increased generation of oxidative stress and atherosclerotic vascular diseases, but we do not know how hydrogen peroxide (H2O2) and high glucose (HG) could affect calcium regulatory proteins of human vascular endothelial cells (HUVECs) in vitro. In the present study, we have examined the acute effect of H2O2 (100 M) and the effect of chronic exposure to HG concentration (35 mM) on the calcium regulatory system of human vascular endothelial cells using fluorescence imaging microscopy (fura-2). In this study, we tested the hypothesis that calcium regulatory proteins (SERCA-ATPase and PMCA-ATPase pumps and the NCX exchanger) of ECs have different sensitivities to H2O2 and high glucose concentration. We also tested the hypothesis that calcium regulatory proteins could be potential targets of ROS at the early stage of vascular disease. The results of this study showed that both H2O2 and high glucose induced significant delay in calcium removal time (CRT). The study of H2O2 showed that the delay in CRT was due to partial inhibition of SERCA-ATPase and the sodium calcium exchanger (NCX) activity and the effect of H2O2 on CRT was reversible. In contrast, the PMCA-ATPase pump was resistant to inhibition by H2O2. Furthermore, H2O2 induced a 40 ± 6.5 % reduction in endoplasmic reticulum refilling. The second part of the study showed that exposure of ECs to HG concentration for 10 days induced a significant delay in CRT and this delay was due to partial blockade of the SERCA-ATPase pump. Blockade of PMCA-ATPase pump with vanadate showed a further delay in CRT. We conclude that: 1- Both H2O2 and HG affected components of the calcium removal system with different sensitivities; 2- H2O2 and HG did not show any inhibitory effects on the PMCA-ATPase pump; 3- The effect of H2O2 on CRT was reversible; 4- The effect of HG on CRT could be due to increased production of H2O2; 5- The calcium regulatory proteins of ECs could be potential targets for ROS during the early stage of a cardio-vascular disease such as diabetes mellitus.

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23828号 / 人健博第99号 / 新制||人健||7(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 椎名 毅, 教授 杉本 直三, 教授 辻川 明孝 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-27T19:15:36Z No. of bitstreams: 1 eltonsoaresdelimafilho mestrado.pdf: 10470752 bytes, checksum: b2ec80f3017bdb5b70e7d1e60f734b92 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T21:06:43Z (GMT) No. of bitstreams: 1 eltonsoaresdelimafilho mestrado.pdf: 10470752 bytes, checksum: b2ec80f3017bdb5b70e7d1e60f734b92 (MD5) / Made available in DSpace on 2017-08-07T21:06:43Z (GMT). No. of bitstreams: 1 eltonsoaresdelimafilho mestrado.pdf: 10470752 bytes, checksum: b2ec80f3017bdb5b70e7d1e60f734b92 (MD5) Previous issue date: 2009-08-07 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho apresenta-se os princípios, a construção e o funcionamento de um aparato que realiza medidas interferométricas de atividade metabólica de microorganismos, em tempo real e de várias amostras simultaneamente. A construção do MAI1 compreende o desenvolvimento e implementação de técnicas de posicionamento das amostras, controle preciso de temperatura, bem como a aquisição e interpretação de padrões de sinais modulados de interferência óptica. Ao longo deste trabalho serão apresentados os modelos teóricos utilizados, os códigos de computador, os desenhos dos circuitos eletrônicos e da mecânica do aparato, bem como medidas realizadas com o mesmo. / This work presents the principles, construction and functioning of an apparatus wich measures the metabolic activity of microorganisms, in real time and from different samples simultaneously, using optical interferometry. The construction of MAI' required the development and implementation of techniques for positioning of samples, precision control of temperature, as well as the acquisition and interpretation of patterns of modulated optical interference signals. Along this work the theoretical models used, the computer codes, the electronic circuit drawings, and the mechanics of the apparatus, as well as measurements carried out with it, will be presented.

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