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Ischemic preconditioning is an endogenous cardioprotective mechanism in which short periods of ischemia and reperfusion provide protection when given before a subsequent ischemic event. Early mechanistic studies showed ATP-sensitive potassium (KATP) channels to play an important role in ischemic preconditioning. KATP channels link intracellular energy metabolism to membrane excitability and contractility. It is thought that KATP channels provide a cardioprotective role during ischemia by inducing action potential shortening, reducing an excessive Ca^2+ influx, and by preventing arrhythmias. However, the mechanisms by which KATP channels protect during ischemic preconditioning are not known. In this study, we investigated a novel potential mechanism in which alterations in subcellular KATP channel trafficking during ischemia and ischemic preconditioning may result in altered levels of surface channel density, and therefore, a greater degree of cardioprotection. In the optimization of our experiments, we compared various antibodies for their specificity and sensitivity for channel subunit detection in immunoblotting. In addition, we examined the effects of varying salt concentrations during tissue homogenization in order to determine the optimal conditions for protein isolation. Furthermore, we examined the effect of heating the samples prior to SDS-PAGE for improved detection of channel proteins by immunoblotting. The subcellular trafficking of some membrane proteins is altered by ischemia. For example, the glucose transporter, Glut4, translocates from endosomal compartments to the sarcolemma (Sun, Nguyen, DeGrado, Schwaiger, & Brosius, 1994). Conflicting data exists regarding the effects of ischemia on KATP channel subcellular trafficking and the regulation of KATP channel surface density (Edwards et al., 2009 and Bao, Hadjiolova, Coetzee, & Rindler, 2011). We therefore, sought to test our hypothesis that KATP channels are internalized from the surface of cardiomyocytes to endosomal compartments during ischemia, and this internalization can be reduced and/or reversed by ischemic preconditioning. We subjected isolated Langendorff-perfused mouse hearts to ischemia, reperfusion, or ischemic preconditioning events and measured the density of KATP channels in the sarcolemmal and endosomal compartments. We also determined the degree of injury by staining heart slices with triphenyltetrazolium chloride and compared infarct sizes between hearts subjected to ischemia and ischemic preconditioning. Our data demonstrated that KATP channels are, in fact, internalized during ischemia and that reperfusion led to a slow recovery of surface KATP channel density. Interestingly, ischemic preconditioning reduced the size of infarcts induced by ischemia and also prevented the ischemia-induced decrease of KATP channel surface density, thereby, contributing to cardioprotection.

There has been an increased interest in optimization for the analysis of large-scale data sets which require gigabytes or terabytes of data to be stored. A variety of applications originate from the fields of signal processing, machine learning and statistics. Seven representative applications are described below. - Magnetic Resonance Imaging (MRI): A medical imaging tool used to scan the anatomy and the physiology of a body. - Image inpainting: A technique for reconstructing degraded parts of an image. - Image deblurring: Image processing tool for removing the blurriness of a photo caused by natural phenomena, such as motion. - Radar pulse reconstruction. - Genome-Wide Association study (GWA): DNA comparison between two groups of people (with/without a disease) in order to investigate factors that a disease depends on. - Recommendation systems: Classification of data (i.e., music or video) based on user preferences. - Data fitting: Sampled data are used to simulate the behaviour of observed quantities. For example estimation of global temperature based on historic data. Large-scale problems impose restrictions on methods that have been so far employed. The new methods have to be memory efficient and ideally, within seconds they should offer noticeable progress towards a solution. First-order methods meet some of these requirements. They avoid matrix factorizations, they have low memory requirements, additionally, they sometimes offer fast progress in the initial stages of optimization. Unfortunately, as demonstrated by numerical experiments in this thesis, first-order methods miss essential information about the conditioning of the problems, which might result in slow practical convergence. The main advantage of first-order methods which is to rely only on simple gradient or coordinate updates becomes their essential weakness. We do not think this inherent weakness of first-order methods can be remedied. For this reason, the present thesis aims at the development and implementation of inexpensive higher-order methods for large-scale problems.

Statins can act as preconditioning agents against ischemia reperfusion (IR)-injury through a mechanism involving cyclooxygenase (COX)-2 and the upregulation of prostaglandin synthesis. The following study investigated the effect of selective and non-selective COX inhibition on rosuvastatin-mediated protection against IR-induced endothelial dysfunction in the human forearm vasculature. Healthy volunteers were randomized to drugs with different COX-inhibiting properties: 81mg aspirin (OD), 325mg aspirin (OD), 400mg ibuprofen (QID), 200mg celecoxib (BID) or placebo. A single dose of 40mg rosuvastatin was also administered 24-hours prior to IR. Endothelial function before and after IR was assessed by measuring flow-mediated dilation of the radial artery. Our results show that 81mg and 325mg aspirin (more COX-1 selective), 400mg ibuprofen (similar selectivity for COX-1/2) and 200mg celecoxib (COX-2 selective) all effectively abolished statin-mediated protection against IR-induced endothelial dysfunction in the forearm (2-way ANOVA, p<0.05). These findings indicate that even partial COX-2 inhibition is sufficient to attenuate statin-induced preconditioning.

Remote ischemic preconditioning (rIPC) through transient limb ischemia induces potent cardioprotection against ischemia reperfusion (IR) injury. I examined the delayed phase of protection that appears 24 hours after the initial rIPC stimulus. The primary objective of this study was to establish a mode of sedation and control treatment for delayed rIPC experiments. I used an ex-vivo, Langendorff isolated-mouse heart preparation of IR injury to examine the delayed effects of an intra-peritoneal (IP) injection, sodium-pentobarbital (SP), halothane and nitrous oxide (N2O) anesthesia on post-ischemic cardiac function. Each anesthetic method improved left-ventricular function after IR injury. SP and halothane anesthesia also reduced LV infarct size. Delayed cardioprotection after IP injections was associated with an increase in phosphorylated-Akt levels. The present study shows that IP injections and inhalational anesthesia invoke cardioprotection and, therefore, indicates that these modes of sedation should not be used as control treatments for studies examining the delayed rIPC phenotype.

Statins can act as preconditioning agents against ischemia reperfusion (IR)-injury through a mechanism involving cyclooxygenase (COX)-2 and the upregulation of prostaglandin synthesis. The following study investigated the effect of selective and non-selective COX inhibition on rosuvastatin-mediated protection against IR-induced endothelial dysfunction in the human forearm vasculature. Healthy volunteers were randomized to drugs with different COX-inhibiting properties: 81mg aspirin (OD), 325mg aspirin (OD), 400mg ibuprofen (QID), 200mg celecoxib (BID) or placebo. A single dose of 40mg rosuvastatin was also administered 24-hours prior to IR. Endothelial function before and after IR was assessed by measuring flow-mediated dilation of the radial artery. Our results show that 81mg and 325mg aspirin (more COX-1 selective), 400mg ibuprofen (similar selectivity for COX-1/2) and 200mg celecoxib (COX-2 selective) all effectively abolished statin-mediated protection against IR-induced endothelial dysfunction in the forearm (2-way ANOVA, p<0.05). These findings indicate that even partial COX-2 inhibition is sufficient to attenuate statin-induced preconditioning.

Remote ischemic preconditioning (rIPC) through transient limb ischemia induces potent cardioprotection against ischemia reperfusion (IR) injury. I examined the delayed phase of protection that appears 24 hours after the initial rIPC stimulus. The primary objective of this study was to establish a mode of sedation and control treatment for delayed rIPC experiments. I used an ex-vivo, Langendorff isolated-mouse heart preparation of IR injury to examine the delayed effects of an intra-peritoneal (IP) injection, sodium-pentobarbital (SP), halothane and nitrous oxide (N2O) anesthesia on post-ischemic cardiac function. Each anesthetic method improved left-ventricular function after IR injury. SP and halothane anesthesia also reduced LV infarct size. Delayed cardioprotection after IP injections was associated with an increase in phosphorylated-Akt levels. The present study shows that IP injections and inhalational anesthesia invoke cardioprotection and, therefore, indicates that these modes of sedation should not be used as control treatments for studies examining the delayed rIPC phenotype.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22884号 / 医博第4678号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 羽賀 博典, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In this thesis, the different solution methods for saddle-point systems aris- ing from fluid dynamics are studied. The main emphasis is on Krylov subspace methods with effective preconditioning techniques for saddle-point systems ob- tained from finite element discretization of the Navier-Stokes equations. Two preconditioning techniques are presented: pressure-convection-diffusion precon- ditioning (PCD) and least-square commutator preconditioning (LSC). Both pre- conditioners are validated on two benchmarks: lid-driven cavity and flow around cylinder. From the computational point of view, we focus on comparing the performance of used solvers, with emphasis on our implementation of PCD pre- conditioning. All numerical simulations are performed by software Firedrake. 1

Sarcolemmal blebbing and rupture are prominent features of irreversible ischemic myocardial injury. Dystrophin and spectrin are sarcolemmal structural proteins. Dystrophin finks the transmembrane dystroglycan complex and extracellular laminin receptors to intracellular F-actin. Spectrin forms the backbone of the membrane skeleton confering an elastic modulus to the sarcolemmal membrane. An ischemic loss of membrane dystrophin and spectrin, in ischemically pelleted rabbit cardiomyocytes or in vivo 30-45 rain permanently ischemic. LAD-ligated hearts, was detected by immunofluorescence with monoclonal antibodies. Western blots of light and heavy microsomal vesicles and Triton-extracted membrane fractions from ischemic myocytes demonstrated a rapid loss of dystrophin coincident with sub-sarcolemmal bleb formation, subsequent to a hypotonic challenge. The loss of spectrin from purified sarcolemma of autolysed rabbit heart, and both isolated membrane vesicles and Triton solubilized membrane fractions of ischemic cardiomyocytes correlated linearly with the onset of osmotic fragility as assessed by membrane rupture, subsequent to a hypotonic challenge. In contrast to the ischemic loss of dystrophin and spectrin from the membrane, the dystrophin-associated proteins. α-sarcoglycan and β-dystroglycan and the integral membrane protein, sodium-calcium exchanger, were maintained in the membrane fraction of ischemic cells as compared to oxygenated cells. Preconditioning protected cells, but did not significantly alter ischemic dystrophin or spectrin translocation. This previously unrecognized loss of sarcolemmal dystrophin and spectrin may be the molecular basis for sub-sarcolemmal bleb formation and membrane fragility during the transition from reversible to irreversible ischemic myocardial injury.