In recent decades research into compartmentalization made a significant progress. Membrane-less structures that are not enclosed by a lipid bilayer were discovered, opening doors to new research...Show moreIn recent decades research into compartmentalization made a significant progress. Membrane-less structures that are not enclosed by a lipid bilayer were discovered, opening doors to new research fields. In this thesis one type of these assemblages is studied: Nuclear speckles. In this study the clusters of estrogen and glucocorticoid receptors, which belong to the family of steroid receptors are studied. An algorithm which not only identifies but also determines the properties of speckles is developed. Some parameters such as the density, width and signal inside the speckles are examined. Their dependences on the expression level of the nucleus showed interesting properties that help have a deeper insight in their nature.Show less
Micropillar arrays are used to measure traction forces of individual cells and of cells in tissue. For this technique single cells are placed on poly-di-methyl siloxane (PDMS) micropillar arrays...Show moreMicropillar arrays are used to measure traction forces of individual cells and of cells in tissue. For this technique single cells are placed on poly-di-methyl siloxane (PDMS) micropillar arrays and observed on an inverted high-resolution optical microscope. As of the requirements for a high-resolution inverted microscopy, the images of the micropillar array are taken through the cell.A refractive index change between the cell and cell medium results in refraction of light at their interface. Here we introduce a method to characterize astigmatism caused by this varying refractive index. We found that that astigmatism can lead to aberrations up to 400 nm. Further, based on the same method we were able to estimate the refractive index of the nucleus to be $1.366\pm0.004$. Our results demonstrate that astigmatism should be taken into account during traction force measurements, especially when measuring forces close to the nucleus.Show less
In recent decades research into the role of mechanical cues in cell biology has made a significant progress. The importance of understanding how cells react on changes in mechanical properties of...Show moreIn recent decades research into the role of mechanical cues in cell biology has made a significant progress. The importance of understanding how cells react on changes in mechanical properties of their local microenvironment and whether they can apply forces themselves has led to the development of a number of different methods. These methods allow changing substrate stiffness and simultaneous measuring of the cell traction forces. Hydrogels and arrays of flexible microposts are currently the most used techniques to measure cell traction forces (TF) in response to the changes in substrate stiffness. Forces are isolated by detecting displacements of beads inside hydrogels or microposts bending, caused by cells cultured on them. Further this information is combined with the known bulk substrate stiffness to calculate cellular forces needed to cause these displacements. Both techniques have certain advantages and disadvantages imposing limitations on the research opportunities. We developed a new approach by combining advantages of both techniques in order to broaden the range of possible applications. This thesis describes the generation of new substrates with a unique functional layer design to which cells can adhere. Validation was performed by using three cell types: mouse fibroblasts (3T3), human fibroblasts (SV80) and human aorta smooth muscle (HASM) cell lines. Computer analysis of the image data collected on the high-resolution confocal spinning disk microscope is used in order to obtain substrate displacement field for the cell TF calculation. We found fibronectin-based stamping on soft hydroxy-PAAm hydrogels to be most efficient.Show less
Pericytes, the mural cells of blood capillaries, have an important role in the regulation of the blood flow through the capillaries. In many pathological conditions in the vascular network,...Show morePericytes, the mural cells of blood capillaries, have an important role in the regulation of the blood flow through the capillaries. In many pathological conditions in the vascular network, pericytes are the main cause of a disease. They contain contractile proteins that are attached to fibronectin patches in a shared basement membrane with the capillary. Therefore, the pericyte is able to exert forces and regulate the vessel diameter. In our work, the main question was how the forces of pericytes relate in hypoxia and ischemia. We showed that pericytes in simulated hypoxia have a decrease in force application and an increase in cell spreading area compared to normal conditions. In simulated ischemia pericytes decrease their cell spreading area and have a slightly decreased force application. A second question we discussed is how the substrate stiffness plays a role in the force application in normal conditions. The gradient of force application for a range of substrate stiffnesses of 11.6 to 137 kPa is dependent of the cell type. For fibroblasts the force and the cell spreading area increased with increasing substrate stiffness. However, pericytes show a high force application on low and high substrate stiffnesses and have a low force application on intermediate stiffnesses. In order to find the force that a cell exert, we used a model that mimiced the fibronectin patches in the basement membrane. Cells were seeded on micropillar arrays made of polydimethylsiloxane (PDMS) that were functionalized with fibronectin. The deflection of the pillars was used to calculate the force applied by the cell.Show less
