School of Chemistry - Theses
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ItemAn AFM study of nanomechanical properties of mammalian nerve and cardiac cells( 2016)The study of nano-mechanical properties of cells (under physiological conditions and/or in presence of pathological or pharmacological agents) by atomic force microscopy (AFM) is of immense interest due to the versatility of the technique. This thesis attempts to understand the nano-mechanical properties of mammalian nerve and cardiac cells under near physiological conditions using AFM. Hertz contact mechanics were employed to obtain elasticity (Young’s modulus) data. Alzheimer’s disease affects different cell types in various regions of the brain. The abnormal extracellular deposition of amyloid peptides is a hallmark of the disease. The effect of the amyloid peptide on two types of nerve cells, transformed (N2A) and primary (cortical) cells, was investigated. Initially preliminary contact mode images followed by 30x30 force grids were obtained for the whole body of N2A cells,. However, due to experimental time limitations after cell removal from the incubator, imaging was compromised and, instead, 10x10 force grids over flat regions of the nerve cells were obtained. This approach was used to obtain force grid data of untreated N2As on Day 5 and 6 from the initial seeding day. Secondly, time-lapse studies were carried out at 12, 36 and 54 hours on both untreated and Aβ1-42 treated N2As and corticals. Force measurements were taken of two regions on each cell type, which showed statistically significant differences in stiffness and adhesion properties for both untreated and Aβ1-42 treated cells. Sub-toxic amounts (5 μM) of Aβ1-42 peptide had a cell specific and time dependent effect. The effects are discussed in terms of Aβ1-42 membrane association, incorporation and internalization and probable affect on the microtubule and actin cytoskeleton network, which is characteristic for each cell type. Various pathological conditions of the heart are attributed to abnormal mechanical properties associated with the left ventricle. Such abnormalities have been imputed to variations in the mechanical properties of sarcomeres (the structural/functional units of cardiomyocytes). Therefore, studies of left ventricular cardiomyocytes were performed. Firstly, live cardiac cells were imaged in near physiological conditions to visualize topographical aspects and specifically the sarcomere regions. T-tubules at the sarcolemmal level were established. Sarcomere features were more prominent at later stages of the experiment. The cardiac cells exhibited what appeared to be highly non-linear topographical properties, that may contribute to non-linear elastic properties. Hence, imaging was followed by force measurements. Simultaneous imaging and force measurements were performed on cardiac cells in the absence and presence of a pharmacological agent, 2,3-butanedionemonomoxime (BDM),, which is an intracellular Ca2+ ion blocking agent. Results were obtained on multiple cells under both untreated and BDM treated conditions. The untreated cells demonstrated higher elasticity and adhesion values compared to BDM treated cells. Finally, in order to ascertain the effect of BDM on the z-band regions (at the sarcolemmal level), a comparison was made between two individual cardiac cells under respective conditions over the same period of 2-12 hours. The mean values of adhesion and elasticity for single cell experiments fell well within the range for that of multiple cells. Overall, the mean Young’s modulus vs. time curves show that stiffness increased with time, for both untreated and BDM treated cells and suggest that the cells continuously alternate between rigor and relax states. BDM appears to produce the relaxation effect and together with the calcium-induced calcium release process has a cumulative effect on the two states over extended time periods. BDM treatment also influences the cell surface adhesion properties.