• Electrospinning
• nanofibers substrates
• STORM microscopy
• Super-resolution microscopy

Living cells respond to mechanical cues from their local microenvironment by altering their morphology and modulating their gene expression accordingly. It has been demonstrated that a pivotal role in the regulation of gene expression is played by chromatin, the condensed form of DNA. Nevertheless, its spatiotemporal re-arrangement in response to mechanical cues is not fully understood, mainly due to the resolution limit of traditional microscopy. Recent studies have begun using super-resolution (SR) microscopy techniques to study chromatin, particularly observing changes in its organization in correlation with substrate stiffness.
In this thesis work, stochastic optical reconstruction microscopy (STORM), a particular SR technique, has been used to study chromatin reorganization in response to mechanical stimuli induced to cells by nanofibrous materials. Poly-lactide-co-glycolide (PLGA) nanofibers were produced by using electrospinning technique, whose parameters were optimized to produce randomly oriented nanofibers with controlled diameters, mimicking the extracellular matrix. Then, the materials have been characterized by using scanning electron microscopy, profilometer, water contact angle and weight loss measurements. Subsequently, glioblastoma cells were cultured on nanofiber mats, fixed at different time points, and immunostained for H3K4me3, a chromatin marker implicated in activation of gene transcription.
Using SR microscopy, labeled structures were localized, and clustering algorithms quantified chromatin domain reorganization for each time point. This research provides a protocol for quantifying chromatin reorganization in response to mechanical stimuli, enhancing the understanding of cell-material interactions at the molecular level, with promising implications for regenerative medicine and tissue engineering.