<p> Calcium (Ca2+) is an important signaling molecule and hence its movement across cell membranes must be tightly regulated. The intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle and endothelium controls the coronary tone. After stimulation, decreasing the [Ca2+]i back to resting levels is achieved mainly by the sodium calcium exchanger (NCX), the plasma membrane calcium pump (PMCA) or the sarcoendoplasmic reticulum calcium pump (SERCA). The present study will focus on NCX and its interactions with SERCA in the smooth muscle and endothelium of pig coronary artery.</p> <p> Aim 1 of my thesis is determination of activity levels of NCX in smooth muscle cells (SMC) and endothelial cells (EC). The NCX activity in cultured cells was approximately 5 times greater in EC than in SMC. The NCX inhibitors KB-R7943 and SEA 0400 blocked the NCX mediated Ca2+ entry, as did collapsing the Na+ gradient with monensin. NCX1 is the isoform largely responsible for NCX activity in SMC and EC. NCX activity was also assayed as the Ca2+ efflux in cultured cells and as Ca2+ uptake in plasma membrane vesicles isolated from freshly isolated smooth muscle.</p> <p> Aim 2 is to assess the existence of a functional NCX mediated Ca2+ entry linked to SERCA in SMC. In the absence of thapsigargin, BAPTA loading SMC increased the NCX mediated uptake. Thapsigargin did not affect the Ca2+ uptake in BAPTA loaded cells but it inhibited the Ca2+ uptake in cells that were not loaded with BAPTA. These data are consistent with a model in which SER acts as a sink for the NCX mediated Ca2+ entry. However, with BAPTA chelation and the resulting lower intracellular Ca2+, the need for SER to act as a sink is eliminated, and NCX is driven in full force. EC did not demonstrate a NCX-SERCA linkage.</p> <p> Arterial SMC and EC differ in their structure and function. The function of SMC is the generation of tone which is achieved by the Ca2+ dependent contractile filaments. Since these filaments are distributed throughout the cell, Ca2+ must be transported to and removed from deep within the cell. As a result, the SER may play a large role in Ca2+ regulation in the SMC. Furthermore, SMC also contain higher levels of high affinity Ca2+ pumps (SERCA and PMCA) and thus Ca2+ is more tightly regulated. Endothelial cells release nitric oxide in response to an increase in [Ca2+]i, which relaxes the smooth muscle. The endothelial nitric oxide sythase produces nitric oxide and is located adjacent to the PM in EC. The SER that removes Ca2+ from deep within the cell cytosol may play a small role in Ca2+ dependent modulation of the endothelial nitric oxide synthase activity. Based on the Western blot data, EC contain a greater amount of the high capacity NCX, thus the larger quantities of Ca2+ can be removed from the cell and the vicinity of endothelial nitric oxide synthase.</p> / Thesis / Master of Science (MSc)

Elastography, which is based on applying pressure and estimating the resulting deformation, involves the forward problem to obtain the strain distributions and inverse problem to construct the elastic distributions consistent with the obtained strains on observation points. This thesis focuses on the former problem whose solution is used as an input to the latter problem. The aim is to provide the inverse problem community with accurate strain estimates of a coronary artery vessel wall. In doing so, a new ultrasonic image-based elastography approach is developed. Because the accuracy and quality of the estimated strain fields depend on the resolution level of the ultrasound image and to date best resolution levels obtained in the literature are not enough to clearly see all boundaries of the artery, one of the main goals is to acquire high-resolution coronary vessel wall ultrasound images at different pressures. For this purpose, first an experimental setup is designed to collect radio frequency (RF) signals, and then image formation algorithm is developed to obtain ultrasound images from the collected signals. To segment the noisy ultrasound images formed, a geodesic active contour-based segmentation algorithm with a novel stopping function that includes local phase of the image is developed. Then, region-based information is added to make the segmentation more robust to noise. Finally, elliptical deformable template is applied so that a priori information regarding the shape of the arteries could be taken into account, resulting in more stable and accurate results. The use of this template also implicitly provides boundary point correspondences from which high-resolution, size-independent, non-rigid and local strain fields of the coronary vessel wall are obtained.