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.

Le proprietà meccaniche dei sistemi biologici hanno una grande importanza nel determinare il loro comportamento. Molti potenziali farmaci possono modificare le proprietà meccaniche della membrana biologica e indirettamente modulare la funzione di proteine di membrana. Analogamente, molti stati patologici a livello cellulare presentano un fenotipo con proprietà meccaniche alterate e la modifica di tali proprietà è tipicamente il risultato di una riorganizzazione del citoscheletro. Allo stesso tempo, le cellule sondano le proprietà reologiche della matrice extracellulare (ECM) attivando, a seconda della risposta ottenuta, diversi percorsi biochimici. Tali fenomeni sono spesso caratterizzati da una riorganizzazione citoscheletrica a seguito di stimoli periodici, come avviene ad esempio nel sistema cardiovascolare o nei polmoni. La scienza che tratta questi fenomeni è la meccanobiologia. Il lavoro di questa tesi di dottorato è dedicato all’analisi delle proprietà meccaniche di costituenti biologici, da semplici modelli di membrana, come doppi-strati-lipidici supportati (SLB) e vescicole giganti unilamellari (GUV), a sistemi quali colture cellulari in-vitro. Le tecniche di indagine usate hanno coinvolto: microscopia ottica in contrasto di fase, DIC e fluorescente; microscopia a forza atomica (AFM). Sono stati sviluppati, all’interno della tesi, metodi di analisi e dispositivi dedicati per specifiche applicazioni e misure di campioni. È stato progettato, testato e impiegato un incubatore per esperimenti di live-cell imaging da integrare direttamente sul tavolino (on-stage) di un microscopio ottico. Sono stati ottenuti simultaneamente parametri di migrazione di cellule esposte a diversi trattamenti, o poste su substrati aventi diversa rigidità meccanica. Lo stesso incubatore è stato ridisegnato per poter alloggiare uno stretcher uniassiale in grado di fornire al substrato specifiche funzioni periodiche di deformazione e valutare la conseguente risposta delle cellule in termini di migrazione e polarizzazione. Tra i metodi di indagine, è stata sviluppata l’analisi quantitativa di migrazione di singola cellula, ed è stato impiegato il modello “Persistence-Random-Walk”. Lo scopo era quello di analizzare l’effetto citostatico di un potenziale farmaco nelle cellule U87MG, usate come modello del glioblastoma multiforme. L’analisi effettuata ha infatti mostrato l’efficacia sia citostatica che antimitotica della molecola. Sono stati indagati inoltre i possibili meccanismi biochimici alla base di tali effetti. Nel contesto dei SLB e GUV è stata implementata rispettivamente l’analisi sulla tensione di linea di domini che simulano lipid-rafts e la costante di bending, basandosi sulla teoria delle fluttuazioni di membrana. Nel primo caso sono stati confrontati i risultati sulla misura della tensione di linea di miscele ternarie costituite da diverse componenti rilevanti nella formazione di lipid-rafts. Nel secondo caso, è stato valutato il ruolo di molecole esogene (peptidi antimicrobici e lipopeptidi) nella determinazione della costante di bending. Nella caratterizzazione visco-elastica del citoscheletro con AFM, è stato implementato un software basato sul modello di Ting, in grado di estrapolare i parametri viscoelastici dalle singole curve andata-ritorno. Si è studiato l’effetto del potenziale farmaco prima citato, sulle proprietà reologiche di cellule U87MG al fine di correlare migrazione e proprietà meccaniche cellulari. Sono stati sviluppati software dedicati alla ricerca di eventi Jump-Through-Force nell’analisi di SLB, e di eventi di estrazioni di tubi durante la retrazione della punta AFM sulla membrana plasmatica come possibile metodo per evidenziare variazioni di proprietà reologiche di membrane esposte a diversi trattamenti farmacologici.

Meccanobiologia di sistemi biologici: da doppi strati lipidici a cellule in-vitro / Gregorio Ragazzini , 2021 Jul 08. 33. ciclo, Anno Accademico 2019/2020.

Meccanobiologia di sistemi biologici: da doppi strati lipidici a cellule in-vitro

RAGAZZINI, GREGORIO
2021

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.
Mechanobiology of biosystems: from lipid bilayers to in-vitro cells
8-lug-2021
ALESSANDRINI, Andrea
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1250708
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