In the last decades lot of attention was conferred to mechanobiology, a multidisciplinary field bringing together concepts from biology, biochemistry, material science, engineering and biophysics. The main aim of this discipline is the understanding of the mechanisms of mechanotransduction in cells. This involves molecular events occurring at the nanoscale; therefore, nanoscience and nanotechnology are exploited for these types of studies. For these purposes, many tools and techniques have been developed for the visualization of cells and their structures enclosing: Optical Microscopy (Brightfield and Fluorescence), Digital Holographic Microscopy (DHM), and Atomic Force Microscopy (AFM). However, along with imaging setups, also mechanical stimulation instrumentations have been required and developed to perform mechanical probing. Among these there are: Quartz Crystal Microbalance (QCM), Micropipette Aspiration (MA), Atomic Force Microscopy (AFM) and Optical Tweezers (OT). Since the investigation options are enhanced when more techniques are combined together, the application of multimodal imaging, as well as multimodal mechanical stimulation, and the combination of both, is deeply investigated within this Thesis. The purposes of these techniques space from cell morphological, dynamical and viscoelastic characterization to force-induced calcium gating and mechanosensitive Piezo1 channel investigation. First, a new combination of QCM and DHM techniques was adopted for the achievement of synergic results about morphological and viscoelastic properties of cardiac fibroblasts treated with cytoskeletal drugs. The sensitivity of QCM allowed the investigation of cell-surface interaction within a nanometric depth above the quartz sensor. Subsequently, mechanical stimuli within a range of forces/pressures required for force-induced calcium gating through mechanosensitive ion channels were investigated in cardiac fibroblasts. Forces from piconewton up to nanonewton were employed by using OT and AFM respectively while cell response was followed by fluorescent calcium imaging. This multimodal investigation revealed important features about microenvironment-mediated mechanosensitive adaptation of fibroblasts which are of relevant importance for cardiac fibrotic mechanisms understanding. A new multimodal methodology to investigate red blood cells (RBCs) mechanics was also proposed. In this work, MA was coupled to multimodal imaging enclosing both bright field and fluorescent calcium recording for accessing information on mechanical properties as well as Piezo1 channel functionality at the same time, for individual cells. Finally, the application of DHM jointly with microfluidics in a novel measurement setup led to the validation of a new diagnostic method and instrument for fast bacteria analysis and urine label-free samples. The technique was compared to clinical gold standards (culture plate test and flow cytometry) showing faster analysis operation at lower cost for similar results. This fruitful project was developed in collaboration with Alifax srl. In conclusion, the Thesis aims both to consolidate and expand already existing knowledge about cell mechanobiology proposing novel interesting results and suggestions about fibroblasts fibrotic mechanisms and stimulus-induced Piezo1 channel functionality both in fibroblasts and RBCs. The achieved results reported here, together with proposed innovative experimental approaches, may open new pursuable research ways in the mechanobiology field, and impact also the clinical landscape in terms of future diagnostic tools and targeted therapies.
Nell’ultima decade una consistente attenzione è stata conferita alla meccanobiologia, un campo multidisciplinare che riunisce competenze derivanti dalla biologia, biochimica, scienza dei materiali, ingegneria e biofisica. Il principale obiettivo di questa disciplina è la comprensione dei meccanismi di meccanotrasduzione nelle cellule. Questo processo coinvolge numerosi eventi che avvengono a livello nano; perciò sia la nano-scienza che le nanotecnologie sono coinvolte in questo tipo di studi. Per questi scopi, numerosi strumenti e tecniche sono stati sviluppati per la visualizzazione delle cellule e delle loro strutture tra le quali: la microscopia ottica (in campo chiaro e di fluorescenza), la microscopia a olografia digitale (DHM), il microscopio a forza atomica (AFM). Inoltre, affianco agli strumenti per l’imaging, è stato necessario sviluppare anche strumenti per l’investigazione meccanica. Tra questi ci sono: la microbilancia ai cristalli di quarzo (QCM), l’aspirazione con micro-pipetta (MA), il microscopio a forza atomica (AFM) e le pinzette ottiche (OT). Dal momento che i punti di forza di ogni singola tecnica sono enfatizzati dall’utilizzo congiunto di più tecniche, l’applicazione multimodale di imaging e di stimolazione meccanica, e la combinazione dei due, è considerevolmente esplorata in questa Tesi. L’obiettivo di queste tecniche spazia dall’investigazione morfologica, dinamica e viscoelastica delle cellule alla caratterizzazione della mobilizzazione del calcio, a seguito di applicazione delle forze, mediata dal canale meccanosensibile Piezo1. In primis viene proposta una nuova combinazione di QCM e DHM. Questa è stata utilizzata per ottenere risultati sinergici relativi alle proprietà morfologiche e viscoelastiche di fibroblasti cardiaci. La sensibilità della QCM ha permesso di investigare l’interazione cellula-substrato focalizzando lo studio in uno spessore nanometrico al di sopra del sensore al quarzo. Successivamente, stimolazioni meccaniche in un range di forze/pressioni necessarie per indurre l’influsso si calcio attraverso i canali meccanosensibili sono state esplorate in fibroblasti cardiaci. Forze da piconewton fino a nanonewton sono state applicate usando OT e AFM rispettivamente, mentre la risposta della cellula è stata seguita con imaging di fluorescenza. Questo tipo di investigazione multimodale ha rivelato l’importanza del microambiente nell’adattamento meccanosensibile dei fibroblasti. Inoltre, una nuova metodologia multimodale per indagare la meccanica dei globuli rossi (RBCs) è stata proposta. In questo lavoro MA è stata accoppiata con l’imaging multimodale che include sia il campo chiaro che la fluorescenza per accedere ad informazioni relative alle proprietà meccaniche e alla funzionalità del canale Piezo1 nello stesso momento, per cellule isolate. Infine, l’applicazione di DHM congiuntamente alla micro-fluidica in un nuovo setup di misura ha permesso la validazione di un nuovo metodo diagnostico per l’analisi di campioni di urina in condizioni label-free. La tecnica è stata comparata ai gold standards (urino-cultura e citometria a flusso) e si è dimostrata essere un’analisi più veloce ed economica per l’ottenimento di un simile risultato. Il fruttuoso progetto è stato sviluppato e condotto in collaborazione con Alifax srl. In conclusione, la Tesi ambisce a consolidare ed espandere le presenti conoscenze relativamente alla meccanobiologia, proponendo nuovi interessanti risultati relativi ai meccanismi fibrotici dei fibroblasti e alla funzionalità di Piezo1 a seguito di stimoli meccanici sia in fibroblasti che in RBCs. I risultati ottenuti e riportati qui, assieme agli innovativi approcci proposti, potrebbero aprire nuove vie di ricerca nel campo della meccanobiologia e impattare anche il panorama clinico in termini di futuri strumenti diagnostici e terapie target.
Proprietà morfologiche e meccaniche di cellule investigate attraverso l’applicazione multimodale di tecniche di imaging e di stimolazione locale / Braidotti, Nicoletta. - (2024 Mar 13).
Proprietà morfologiche e meccaniche di cellule investigate attraverso l’applicazione multimodale di tecniche di imaging e di stimolazione locale
BRAIDOTTI, NICOLETTA
2024-03-13
Abstract
In the last decades lot of attention was conferred to mechanobiology, a multidisciplinary field bringing together concepts from biology, biochemistry, material science, engineering and biophysics. The main aim of this discipline is the understanding of the mechanisms of mechanotransduction in cells. This involves molecular events occurring at the nanoscale; therefore, nanoscience and nanotechnology are exploited for these types of studies. For these purposes, many tools and techniques have been developed for the visualization of cells and their structures enclosing: Optical Microscopy (Brightfield and Fluorescence), Digital Holographic Microscopy (DHM), and Atomic Force Microscopy (AFM). However, along with imaging setups, also mechanical stimulation instrumentations have been required and developed to perform mechanical probing. Among these there are: Quartz Crystal Microbalance (QCM), Micropipette Aspiration (MA), Atomic Force Microscopy (AFM) and Optical Tweezers (OT). Since the investigation options are enhanced when more techniques are combined together, the application of multimodal imaging, as well as multimodal mechanical stimulation, and the combination of both, is deeply investigated within this Thesis. The purposes of these techniques space from cell morphological, dynamical and viscoelastic characterization to force-induced calcium gating and mechanosensitive Piezo1 channel investigation. First, a new combination of QCM and DHM techniques was adopted for the achievement of synergic results about morphological and viscoelastic properties of cardiac fibroblasts treated with cytoskeletal drugs. The sensitivity of QCM allowed the investigation of cell-surface interaction within a nanometric depth above the quartz sensor. Subsequently, mechanical stimuli within a range of forces/pressures required for force-induced calcium gating through mechanosensitive ion channels were investigated in cardiac fibroblasts. Forces from piconewton up to nanonewton were employed by using OT and AFM respectively while cell response was followed by fluorescent calcium imaging. This multimodal investigation revealed important features about microenvironment-mediated mechanosensitive adaptation of fibroblasts which are of relevant importance for cardiac fibrotic mechanisms understanding. A new multimodal methodology to investigate red blood cells (RBCs) mechanics was also proposed. In this work, MA was coupled to multimodal imaging enclosing both bright field and fluorescent calcium recording for accessing information on mechanical properties as well as Piezo1 channel functionality at the same time, for individual cells. Finally, the application of DHM jointly with microfluidics in a novel measurement setup led to the validation of a new diagnostic method and instrument for fast bacteria analysis and urine label-free samples. The technique was compared to clinical gold standards (culture plate test and flow cytometry) showing faster analysis operation at lower cost for similar results. This fruitful project was developed in collaboration with Alifax srl. In conclusion, the Thesis aims both to consolidate and expand already existing knowledge about cell mechanobiology proposing novel interesting results and suggestions about fibroblasts fibrotic mechanisms and stimulus-induced Piezo1 channel functionality both in fibroblasts and RBCs. The achieved results reported here, together with proposed innovative experimental approaches, may open new pursuable research ways in the mechanobiology field, and impact also the clinical landscape in terms of future diagnostic tools and targeted therapies.File | Dimensione | Formato | |
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