Most diseases are being found to have a genetic component, which most likely triggers a cascade of events and eventually affects the overall cell mechanical properties. Over the years, the mechanical characteristics of cells have raised a great interest in the scientific community, since cells have been shown to play a key structural role in building complex structures like tissues and organs, and are able to sense, transduce and exert forces on their surroundings. Among the numerous techniques developed to study cell mechanics, atomic force microscopy (AFM) has often proven to be effective in discerning cell mechanical properties in health and disease. This work aimed to show the power of AFM in the study of cell mechanics under the effect of genetic mutations. To prove the versatility of AFM technique, this thesis contemplated three genetic diseases, and likewise genes. Specifically, the present study focused on arrhythmogenic cardiomyopathy (AC) and PKP2 gene, dilated cardiomyopathy (DCM) and FLNC gene, Hutchinson-Gilford progeria syndrome (HGPS) and LMNA gene. Despite being generally different, all the aforementioned diseases affect the heart, which was therefore chosen as the target of this work. Either gene suppression or mutation were induced in cardiac cells, which were then probed by AFM to assess their mechanical properties. PKP2 was knocked down in HL-1 cells by shRNA targeting; CRISPR/Cas9 was applied to knock out FLNC in human induced pluripotent stem cells (hiPSC), then differentiated into cardiomyocytes; and a mutant form of LMNA, known to cause HGPS, was expressed in primary rat cardiac fibroblasts using an adenoviral vector. It is worth mentioning that rat cardiac fibroblasts were isolated from neonatal, juvenile, and adult animals. PKP2-deficient HL-1 cells and mutant juvenile rat cardiac fibroblasts exhibited altered mechanical properties compared to controls, whereas no variation in the mechanical behaviour was detected in all the other samples. Remarkably, a prospective relationship between variation of cell stiffness and alteration of the distance at which the maximum adhesion force occurs was discovered. These results undeniably demonstrated that AFM is a powerful toolbox to study certain mechanical aspects in in vitro models of genetic diseases. Although, consideration may want to be given to some critical issues that had emerged, like the choice of an appropriate experimental setup and the cellular region to be investigated. In conclusion, this study should encourage more researchers to address biological questions from a mechanical point of view, since biomechanical properties can be identified as potential targets for novel diagnostic and therapeutic approaches.

Al giorno d’oggi, è ormai noto che la maggior parte delle malattie possiede una componente genetica, la quale può potenzialmente innescare una cascata di eventi ed avere come risultato finale un’alterazione delle proprietà meccaniche cellulari. Queste ultime hanno considerevolmente attratto l’attenzione della comunità scientifica, la quale ha preso coscienza del ruolo strutturale delle cellule in organi e tessuti e della capacità delle cellule stesse di captare, trasdurre ed esercitare forze sull’ambiente circostante. Tra i metodi sviluppati per lo studio della meccanica cellulare, la microscopia a forza atomica (AFM) si è ripetutamente dimostrata efficace nel discernere il comportamento meccanico delle cellule in condizioni fisiologiche e patologiche. Il presente lavoro di tesi si propone di illustrare le potenzialità dell’AFM nello studio delle proprietà meccaniche di cellule in modelli in vitro di malattie genetiche. Per avvalorare la versatilità della tecnica, sono state prese in considerazione tre malattie genetiche, ed altrettanti geni ad esse associati: nello specifico, la cardiomiopatia aritmogena ed il gene PKP2, la cardiomiopatia dilatativa ed il gene FLNC, e la sindrome di Hutchinson-Gilford (detta anche progeria) ed il gene LMNA. Nonostante siano complessivamente diverse, le suddette malattie condividono un aspetto comune: tutte e tre, infatti, hanno effetti di variabile intensità sul cuore, il quale è stato pertanto scelto come oggetto di studio della tesi. In alcuni tipi cellulari cardiaci sono state indotte la soppressione o la mutazione dei geni sopra citati: ad esempio, nelle cellule HL-1, ovvero una linea cellulare rappresentativa del muscolo cardiaco, è stato introdotto un meccanismo shRNA per silenziare PKP2. Il gene FLNC, invece, è stato modificato tramite CRISPR/Cas9 in cellule staminali (hiPSC) che sono state successivamente differenziate in cardiomiociti. Per lo studio della progeria, infine, fibroblasti cardiaci primari di ratto in età neonatale, giovane ed adulta sono stati infettati con un vettore adenovirale che esprimeva la proteina LMNA in forma wild-type o mutata. Ogni tipo cellulare, con la rispettiva mutazione genetica, è stato poi studiato con l’AFM. Nelle cellule HL-1 con PKP2 soppresso e nei fibroblasti da ratto giovane con LMNA mutata sono state riscontrate variazioni delle proprietà meccaniche, mentre negli altri campioni non è stato rilevato alcun cambiamento. In particolare, laddove sono state misurate differenze nelle proprietà meccaniche di controlli e mutanti, i parametri soggetti a variazione erano sempre gli stessi, ovvero il modulo di Young e la distanza a cui si manifestava la massima forza di adesione. Essi parrebbero dunque legati da una relazione inversa, ovvero all’aumentare di uno, l’altro diminuisce, e viceversa. I risultati ottenuti dimostrano inequivocabilmente che l’AFM può essere considerata una tecnica di elezione anche nello studio delle caratteristiche meccaniche di modelli in vitro per malattie genetiche. Nonostante ciò, bisogna sempre tenere in considerazione le potenziali criticità che questo lavoro ha messo in luce, come ad esempio la necessità di scegliere in modo appropriato il setup sperimentale, nonché la regione della cellula da sottoporre ad indagine. In conclusione, questa tesi dovrebbe incoraggiare sempre più ricercatori ad intraprendere lo studio delle proprietà meccaniche di una cellula, in modo tale che in un futuro, sperabilmente non lontano, esse possano diventare il target di approcci diagnostici e terapeutici innovativi.

AFM as a toolbox for assessing the mechanical properties of cells with genetic mutations / Pecorari, Ilaria. - (2019 Mar 28).

AFM as a toolbox for assessing the mechanical properties of cells with genetic mutations

PECORARI, ILARIA
2019-03-28

Abstract

Most diseases are being found to have a genetic component, which most likely triggers a cascade of events and eventually affects the overall cell mechanical properties. Over the years, the mechanical characteristics of cells have raised a great interest in the scientific community, since cells have been shown to play a key structural role in building complex structures like tissues and organs, and are able to sense, transduce and exert forces on their surroundings. Among the numerous techniques developed to study cell mechanics, atomic force microscopy (AFM) has often proven to be effective in discerning cell mechanical properties in health and disease. This work aimed to show the power of AFM in the study of cell mechanics under the effect of genetic mutations. To prove the versatility of AFM technique, this thesis contemplated three genetic diseases, and likewise genes. Specifically, the present study focused on arrhythmogenic cardiomyopathy (AC) and PKP2 gene, dilated cardiomyopathy (DCM) and FLNC gene, Hutchinson-Gilford progeria syndrome (HGPS) and LMNA gene. Despite being generally different, all the aforementioned diseases affect the heart, which was therefore chosen as the target of this work. Either gene suppression or mutation were induced in cardiac cells, which were then probed by AFM to assess their mechanical properties. PKP2 was knocked down in HL-1 cells by shRNA targeting; CRISPR/Cas9 was applied to knock out FLNC in human induced pluripotent stem cells (hiPSC), then differentiated into cardiomyocytes; and a mutant form of LMNA, known to cause HGPS, was expressed in primary rat cardiac fibroblasts using an adenoviral vector. It is worth mentioning that rat cardiac fibroblasts were isolated from neonatal, juvenile, and adult animals. PKP2-deficient HL-1 cells and mutant juvenile rat cardiac fibroblasts exhibited altered mechanical properties compared to controls, whereas no variation in the mechanical behaviour was detected in all the other samples. Remarkably, a prospective relationship between variation of cell stiffness and alteration of the distance at which the maximum adhesion force occurs was discovered. These results undeniably demonstrated that AFM is a powerful toolbox to study certain mechanical aspects in in vitro models of genetic diseases. Although, consideration may want to be given to some critical issues that had emerged, like the choice of an appropriate experimental setup and the cellular region to be investigated. In conclusion, this study should encourage more researchers to address biological questions from a mechanical point of view, since biomechanical properties can be identified as potential targets for novel diagnostic and therapeutic approaches.
28-mar-2019
SBAIZERO, ORFEO
31
2017/2018
Settore ING-IND/22 - Scienza e Tecnologia dei Materiali
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2991025
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