Development and Application of Analytical Tools for Calcium Signaling Dynamics in MDCK-II Cells

Abstract: Calcium, a pivotal ion in various human signaling pathways, holds significant relevance in cancer research and treatment. Investigating the transmission of spontaneous calcium signals between cells and understanding how they are influenced by different treatments is therefore an interesting area of study. This master's thesis presents a newly developed tool designed for analyzing intracellular calcium concentration [Ca2+] fluctuations. The tool encompasses a cell segmentation model, tracking algorithms, nearest neighbor identification, mean intensity measurement, and calculations of dominant frequency of [Ca2+] oscillations and signal correlation among neighboring cells. The tool was applied to fluorescence microscopy images of Madin-Darby Canine Kidney II cells expressing GCaMP6m and either treated with the cardiac glycoside ouabain, a combination of ouabain and the gap junction blocker heptanol, or left untreated for comparison. Additionally, an exploration into the impact of connexin 43 (Cx43) transfection on [Ca2+] fluctuations was undertaken. The transfection rate for Cx43 did not reach a sufficient level, impeding a thorough analysis of its influence on [Ca2+] fluctuations. Furthermore, the investigation into dominant frequencies and signal correlation did not yield conclusive findings regarding the effects of ouabain and heptanol. Nevertheless, a foundational tool for analysis has been developed, with the potential for expansion to analyze more aspects calcium signaling and application to additional cell types and treatments.