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Ragazzini, Gregorio, (2021)  - Meccanobiologia di sistemi biologici: da doppi strati lipidici a cellule in-vitro  - , Tesi di dottorato - (, , Universitą degli studi di Modena e Reggio Emilia ) - pagg. -

Abstract: Mechanical properties of biological systems play a crucial role for their own behavior. As an example, many potential drugs could modify mechanical properties of the biological membrane and indirectly modulate transmembrane protein functions. Similarly, many pathological conditions at the cellular level are characterized by a phenotype with altered mechanical properties, and these alterations are due to cytoskeleton reorganization. At the same time, cells continuously probe rheological properties of extracellular matrix (ECM) enabling, depending on response obtained by the substrate, different downstream signaling cascades. In many cases, cytoskeleton reorganization occurs also when cells are experiencing periodic mechanical stimuli, as it happens for example in the cardiovascular system or in lungs. All these aspects are treated by a recent branch of physic and biology sciences: “Mechano-biology”. This PhD thesis work has been devoted to study some specific aspects of mechanical properties of biological systems: from simple models of the biological-membrane, like supported-lipid-bilayer (SLB) or giant-unilamellar-vescicle (GUV), to in-vitro cells. Investigation techniques exploited in this work include: phase-contrast optical microscopy, DIC and fluorescence microscopy and atomic force microscopy (AFM). In the thesis we developed analysis-methods and devices dedicated to specific application and measurements of biological samples. It has been designed, tested and employed successfully an on-stage cell incubator for live cell imaging. From time-lapse microscopy experiments we obtained different quantitative migration parameters both for cell exposed to different drugs and for cells seeded on substrates with different mechanical rigidity. The same cell incubator has been modified to include an uniaxial stretcher, able to provide specific periodic deformation functions to the substrate on which cells are growing, and we studied the effect of the periodic stimulation on cell migration and polarization. Among the different analysis methods, a single cell migration analysis protocol has been developed, exploiting the “Persistence-Random-Walk” model. The ultimate goal was that of analyzing the cytostatic effect of a potential drug for U87MG cell line, employed as model of the glioblastoma multiforme disease. The analysis has in fact shown the efficiency of this molecule for both migration and replication of this cell line. Furthermore, possible biochemical mechanisms of action involved in these effects have been investigated. In the context of SLBs and GUVs a line tension analysis of domains recapitulating lipid-raft and a bending constant measurement have been implemented, both based on Flickering spectroscopy theory. In the former case, line tension results of ternary mixture containing different components relevant for lipid-rafts formation have been compared for different lipid compositions. In the latter case, the role of exogenous molecules (antimicrobial peptides and lipopeptides) on the bending constant has been investigated. In viscoelastic characterization of the cell cytoskeleton through AFM, a Ting model-based software has been implemented, allowing to extrapolate viscoelastic parameters from single indentation-retraction curves. Using this method, the effect of the previously mentioned potential drug has been investigated, trying to correlate rheological properties to migration capabilities of U87MG. Finally, software dedicated to Jump-Through-Force curves by AFM to identify specific events on SLB, and tether pulling events during AFM tip retraction on plasma-membrane have been developed; in order to find possible methods to highlight variations in rheological properties of membrane exposed to different drug treatments.