Vliv mechanotransdukce na nádorové jaterní buňky kultivované v 3D kolagenovém nosiči

Title: Mechanotransduction of Hepatic Cancer Cells cultured in a 3D Collagen Scaffold Author: Mgr. Adam Frtús Department: Department of Optical and Biophysical Systems, Institute of Physics of the Czech Academy of Sciences Supervisor: PharmDr. Šárka Kubinová, Ph.D., Department of Optical and Biophysical Systems, Institute of Physics of the Czech Academy of Sciences Abstract: Cell culture models transformed over time from a simple monolayer culture in 2D Petri dish to advanced 3D platforms, that provide conditions and features for cellular growth in all directions, similarly to in vivo. Moreover, cells experience distinct microenvironmental features within the extracellular matrix in tissues, that 3D cell culture might replicate. In fact, majority of studies indicate that physical cues generated from extracellular microenvironment drive the cellular behavior and functions. In this dissertation thesis, we attempt to provide the mechanistic explanation behind the changes in cellular metabolism and molecular signaling pathways induced by physical cues of 3D cell culture. We have used the advanced biomaterial-based 3D cell culture, biochemical assays, genetic manipulation and photobiomodulation in order to reveal the molecular mechanisms of mechanotransduction in hepatic cancer cells under physical cues of 3D...Název práce: Vliv mechanotransdukce na nádorové jaterní buňky kultivované v 3D kolagenu Autor: Mgr. Adam Frtús Katedra: Oddělení optických a biofyzikálních systémů, Fyzikální ústav AV ČR Školitel: PharmDr. Šárka Kubinová, Ph.D., Oddělení optických a biofyzikálních systémů, Fyzikální ústav AV ČR Abstrakt: Modely buněčných kultur se v průběhu času transformovaly z jednoduchých monovrstvých kultur v Petriho miskách do pokročilých 3D platforem, které poskytují podmínky a vlastnosti pro růst buněk ve všech směrech, podobně jako in vivo. Buňky tkání ovlivňuje mikroprostředí okolní extracelulární matrix, které 3D buněčná kultura může replikovat. Výsledky výzkumu naznačují, že mechanické signály generované extracelulárním mikroprostředím ovlivňují buněčné chování a funkce. V této disertační práci jsme se zaměřili na odhalení molekulárních mechanismů mechanotransdukce, změn buněčného metabolismu a molekulárních signálních drah v 3D kultuře jaterních nádorových buněk v kolagenovém nosiči. Využili jsme celou řadu biochemických testů sledujících proliferaci, expresi signálních proteinů, imunofluorescenční barvení a konfokální mikroskopii, genetickou manipulaci a fotobiomodulaci. Náš výzkum si klade za cíl nejen získat základní znalosti o plasticitě nádorových buněk a molekulárních signálních drah v 3D prostředí, ale...Faculty of Mathematics and PhysicsMatematicko-fyzikální fakult

Iron Oxide Nanoparticle-Induced Autophagic Flux Is Regulated by Interplay between p53-mTOR Axis and Bcl-2 Signaling in Hepatic Cells

Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies

Sensitivity of endogenous autofluorescence in HeLa cells to the application of external magnetic fields

Abstract Dramatically increased levels of electromagnetic radiation in the environment have raised concerns over the potential health hazards of electromagnetic fields. Various biological effects of magnetic fields have been proposed. Despite decades of intensive research, the molecular mechanisms procuring cellular responses remain largely unknown. The current literature is conflicting with regards to evidence that magnetic fields affect functionality directly at the cellular level. Therefore, a search for potential direct cellular effects of magnetic fields represents a cornerstone that may propose an explanation for potential health hazards associated with magnetic fields. It has been proposed that autofluorescence of HeLa cells is magnetic field sensitive, relying on single-cell imaging kinetic measurements. Here, we investigate the magnetic field sensitivity of an endogenous autofluorescence in HeLa cells. Under the experimental conditions used, magnetic field sensitivity of an endogenous autofluorescence was not observed in HeLa cells. We present a number of arguments indicating why this is the case in the analysis of magnetic field effects based on the imaging of cellular autofluorescence decay. Our work indicates that new methods are required to elucidate the effects of magnetic fields at the cellular level

Hepatic Tumor Cell Morphology Plasticity under Physical Constraints in 3D Cultures Driven by YAP–mTOR Axis

Recent studies undoubtedly show that the mammalian target of rapamycin (mTOR) and the Hippo–Yes-associated protein 1 (YAP) pathways are important mediators of mechanical cues. The crosstalk between these pathways as well as de-regulation of their signaling has been implicated in multiple tumor types, including liver tumors. Additionally, physical cues from 3D microenvironments have been identified to alter gene expression and differentiation of different cell lineages. However, it remains incompletely understood how physical constraints originated in 3D cultures affect cell plasticity and what the key mediators are of such process. In this work, we use collagen scaffolds as a model of a soft 3D microenvironment to alter cellular size and study the mechanotransduction that regulates that process. We show that the YAP-mTOR axis is a downstream effector of 3D cellular culture-driven mechanotransduction. Indeed, we found that cell mechanics, dictated by the physical constraints of 3D collagen scaffolds, profoundly affect cellular proliferation in a YAP–mTOR-mediated manner. Functionally, the YAP–mTOR connection is key to mediate cell plasticity in hepatic tumor cell lines. These findings expand the role of YAP–mTOR-driven mechanotransduction to the control hepatic tumor cellular responses under physical constraints in 3D cultures. We suggest a tentative mechanism, which coordinates signaling rewiring with cytoplasmic restructuring during cell growth in 3D microenvironments