Tek hücre kümelenmesinde intersellüler kalsiyum sinyallerinin takibi

The modulation of calcium concentration and rate of elimination in the cytoplasma is a crucial intracellular mechanism that control different cellular process, including gene expression, proliferation, differentiation and energy metabolism. Spikes, waves and continuous oscillation deliver information throughout the cell by frequency and amplitude changes or may adversely affect other functions if modes are altered by external stimuli. Despite the detailed studies and an insight into the mechanism of calcium elevation, elucidating temporal patterning of signals, small number of dynamic imaging and intrinsic noise remain elusive and limits the development of reliable models. Measuring the metabolic changes at single-cell resolution plays a key role to understand the heterogeneous signal propagation in tissues. Hovewer, ensemble measurements mask the activity of single-cells; fast tracking methods are needed to resolve the dynamics at spatiotemporal resolution. Single-cell studies of calcium signaling and tracing can also decipher its association with downstream events. In this thesis, we have used calcium tracking methods to monitor real-time changes, and used statistical methods to elucidate the heterogenous signal formation. Genetically encoded reporters derived from permutated GFP were expressed in different cells and used as a probe to measure calcium waves mediated by various hydroxytrytamine compunds. To determine rare cells, calcium traces from single-cells was analyzed by a linear transformation. The coefficients of principal components with largest eigenvalues were used to replot single-cell with new orthonormal coordinates. We have determined that cells were segregated differently based on initial rate and magnitude of calcium changes, therefore isolated rare and unresponsive cells. After preprocessing, single-cells were selectively sorted by various clustering methods. We found that calcium signal modulated by serotonin and histamine can be divided into two states depending on falling and raising patterns and the length of oscillation. The coefficients were also tested to determine the state of signals that had various signal-to-noise ratios. Our study suggests that dynamic measurements of calcim signaling and fast segregation can be used to cluster calcium patterns at single-cell level. We envisioned that quantitative imaging can reveal spatiotemporal dynamics of metabolic states, therefore establish an alternative method to study calcium signal in single-cells.