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2013/2014 Gli oscillatori micro e nanomeccanici stanno trovando crescente applicazione come biosensori, grazie alla loro elevata sensibilità, alla possibilità di effettuare analisi con un ridotto volume di campione biologico e senza molecole di marcatura secondarie e alla loro integrabilità come sensori in dispositivi di analisi portatili a basso costo. Questo progetto ha come scopo l’utilizzo di oscillatori micromeccanici a geometria verticale (micropillars) come sensori biomolecolari, rispetto ai più comuni oscillatori orizzontali (cantilevers). La struttura dei micropillars, infatti, permette di confinare l’adsorbimento molecolare alla parte superiore dell’oscillatore, consentendo una facile quantificazione della massa depositata attraverso la variazione della frequenza di risonanza; inoltre, i tempi di risposta del sensore risultano ridotti, grazie alla più veloce diffusione delle molecole verso l’area sensibile, rispetto ad altri sensori basati su effetti di superficie e di più grandi dimensioni. Dense matrici di micropillars idrofobici permettono anche la formazione dello stato superidrofobico di Cassie-Baxter, riducendo l’interazione del liquido alla sola parte superiore dell’oscillatore. Durante questo progetto è stato sviluppato un opportuno trattamento idrofobico delle matrici, basato sulla deposizione di un alcanosilano idrofobico, e caratterizzato rispetto alla bagnabilità della matrice e agli effetti sulla risposta meccanica e sulla risoluzione dell’oscillatore come sensore di massa. È stata inoltre verificata la compatibilità di questo trattamento con la formazione di uno strato di oro sulla parte superiore dell’oscillatore, usato come substrato per l’adsorbimento di biorecettori per il riconoscimento e la cattura della biomolecola di interesse. Il risultato di questa ottimizzazione è stato applicato alla rilevazione di un biomarcatore per il tumore alla prostata (PSMA) a concentrazioni utilizzate nella pratica clinica (nanomolari), sia in soluzione fisiologica che in plasma diluito. Infine, è stata dimostrata la misura della frequenza di risonanza dei micropillars a contatto con il liquido nello stato superidrofobico di Cassie-Baxter, come alternativa al problematico utilizzo dei cantilever completamente immersi in liquido, aprendo la strada alla rilevazione in tempo reale di biomolecole da campioni biologici. Micro and nanomechanical resonators are playing a growing role in biosensing due to their high sensitivity, the possibility of label-free biomolecular detection with a reduced amount of biological sample and their potential integration as sensing tool on low cost, point of care devices. This project focuses on the application of arrays of micropillars resonators for biomolecular sensing, exploiting the advantages coming from the vertical geometry respect to the most common micro and nanocantilever horizontal sensors. Indeed, the biomolecular adsorption can be confined to the micron sized top area of pillars, allowing an easy quantification of the deposited mass and reducing the response time of the resonator due to the faster diffusion on the sensitive area respect to larger, surface based sensors. In addition, the superhydrophobic Cassie-Baxter state can be obtained on dense, hydrophobic micropillars arrays, limiting the interaction with the analyte solution to the top area of pillars. A proper hydrophobization treatment of the arrays, based on the deposition of a hydrophobic alkanosilane coating, has been developed and characterized in terms of wettability of the arrays and of the effects on the mechanical performance and on the mass resolution of the resonators. The compatibility of this treatment with the formation of stable gold layer on top of pillars as a substrate for bioreceptor adsorption has been also verified. The optimized micropillars arrays have been used for the detection of PSMA (Prostate Specific Membrane Antigen) at diagnostically relevant concentrations (nM level) both from physiological solution and from diluted serum. Finally, by exploiting the superhydrophobic Cassie-Baxter state, the direct measurement of the resonance frequency of micropillars in a liquid environment has been demonstrated, as an alternative to the cumbersome application of micro and nanocantilevers on the same conditions, paving the way toward real-time, biomolecular detection from biological samples. XXVII Ciclo 1983

Given the clinical effect of progeria syndrome, understanding the cell mechanical behavior of this pathology could benefit the patient's treatment. Progeria patients show a point mutation in the lamin A/C gene (LMNA), which could change the cell's biomechanical properties. This paper reports a mechano-dynamic analysis of a progeria mutation (c.1824 C > T, p.Gly608Gly) in neonatal rat ventricular myocytes (NRVMs) using cell indentation by atomic force microscopy to measure alterations in beating force, frequency, and contractile amplitude of selected cells within cell clusters. Furthermore, we examined the beating rate variability using a time-domain method that produces a Poincaré plot because beat-to-beat changes can shed light on the causes of arrhythmias. Our data have been further related to our cell phenotype findings, using immunofluorescence and calcium transient analysis, showing that mutant NRVMs display changes in both beating force and frequency. These changes were associated with a decreased gap junction localization (Connexin 43) in the mutant NRVMs even in the presence of a stable cytoskeletal structure (microtubules and actin filaments) when compared with controls (wild type and non-treated cells). These data emphasize the kindred between nucleoskeleton (LMNA), cytoskeleton, and the sarcolemmal structures in NRVM with the progeria Gly608Gly mutation, prompting future mechanistic and therapeutic investigations.

Given the clinical effect of laminopathies, understanding lamin mechanical properties will benefit the treatment of heart failure. Here we report a mechano-dynamic study of LMNA mutations in neonatal rat ventricular myocytes (NRVM) using single cell spectroscopy with Atomic Force Microscopy (AFM) and measured changes in beating force, frequency and contractile amplitude of selected mutant-expressing cells within cell clusters. Furthermore, since beat-to-beat variations can provide clues on the origin of arrhythmias, we analyzed the beating rate variability using a time-domain method which provides a Poincaré plot. Data were further correlated to cell phenotypes. Immunofluorescence and calcium imaging analysis showed that mutant lamin changed NRVMs beating force and frequency. Additionally, we noted an altered microtubule network organization with shorter filament length, and defective hemichannel membrane localization (Connexin 43). These data highlight the interconnection between nucleoskeleton, cytoskeleton and sarcolemmal structures, and the transcellular consequences of mutant lamin protein in the pathogenesis of the cardiac laminopathies., Biological sciences; Cardiology; Biomechanics; Cytoskeleton; Mechanical property; Membrane; Lamin A/C; Cardiomyopathy; Atomic force microscopy; Cardiomyocytes; Beating; Cx43

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte&ndash, myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-&alpha, 1 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM.

Micro and nanomechanical resonators represent a promising platform for proteins label-free detection because of their extreme sensitivity, fast response and low cost. Micro-pillars are columnar resonators that can be easily arranged in dense arrays of several thousand sensors in a squared mm. To exploit such a large density, however, a method for tracking independently micropillars resonance frequency is required. Here we present a detection method based on CCD imaging and software image analysis, which can measure the resonance frequency of tens of pillars in parallel. Acquiring simultaneously the frequency shift of up to 40 sensors and applying a proper statistical analysis, we were able to overcome the variability of the single measures improving the device sensitivity at low analyte concentration range. As a proof of concept, this method has been tested for the detection of a tumor marker, the Prostate Specific Membrane Antigen (PSMA). Pillars have been functionalized with an antibody against PSMA. The tumor marker (PSMA) has been detected in a range of concentrations between 300 pM and 100 nM, in buffer and in diluted bovine serum. The sensitivity of our method was limited only by the affinity constant of the antigen–antibody recognition. Moreover, this detection technique demonstrated to be effective in the 1–6 nM range, which is the window of PSMA concentration of clinical interest.

Lamins are type V intermediate filaments that collectively form a meshwork underneath the inner nuclear membrane, called nuclear lamina. Furthermore, they are also present in the nucleoplasm. Lamins are experiencing a growing interest, since a wide range of diseases are induced by mutations in the gene coding for A-type lamins, globally known as laminopathies. Moreover, it has been demonstrated that lamins are involved in other pathological conditions, like cancer. The role of lamins has been studied from several perspectives, exploiting different techniques and procedures. This multidisciplinary approach has contributed to resolving the unique features of lamins and has provided a thorough insight in their role in living organisms. Yet, there are still many unanswered questions, which constantly generate research in the field. The present work is aimed to review some interesting experimental techniques performed so far to study lamins. Scientists can take advantage of this collection for their novel investigations, being aware of the already pursued and consolidated methodologies. Hopefully, advances in these research directions will provide insights to achieve better diagnostic procedures and effective therapeutic options.

Aims Given the clinical impact of LMNA cardiomyopathies, understanding lamin function will fulfill a clinical need and will lead to advancement in the treatment of heart failure. A multidisciplinary approach combining cell biology, atomic force microscopy (AFM), and molecular modeling was used to analyse the biomechanical properties of human lamin A/C gene (LMNA) mutations (E161K, D192G, N195K) using an in vitro neonatal rat ventricular myocyte model. Methods and results The severity of biomechanical defects due to the three LMNA mutations correlated with the severity of the clinical phenotype. AFM and molecular modeling identified distinctive biomechanical and structural changes, with increasing severity from E161K to N195K and D192G, respectively. Additionally, the biomechanical defects were rescued with a p38 MAPK inhibitor. Conclusions AFM and molecular modeling were able to quantify distinct biomechanical and structural defects in LMNA mutations E161K, D192G, and N195K and correlate the defects with clinical phenotypic severity. Improvements in cellular biomechanical phenotype was demonstrated and may represent a mechanism of action for p38 MAPK inhibition therapy that is now being used in human clinical trials to treat laminopathies.

It is widely accepted that the pathological state of cells is characterized by a modification of mechanical properties, affecting cellular shape and viscoelasticity as well as adhesion behaviour and motility. Thus, assessing these parameters could represent an interesting tool to monitor disease development and progression, but also the effects of drug treatments. Since biomechanical properties of cells are strongly related to cytoskeletal architecture, in this work we extensively studied the effects of selective impairments of actin microfilaments and microtubules on HeLa cells through force-deformation curves and stress relaxation tests with atomic force microscopy. Confocal microscopy was also used to display the effects of the used drugs on the cytoskeletal structure. In synergy with the aforementioned methods, stress relaxation data were used to assess the storage and loss moduli, as a complementary way to describe the influence of cytoskeletal components on cellular viscoelasticity. Our results indicate that F-actin and microtubules play a complementary role in the cell stiffness and viscoelasticity, and both are fundamental for the adhesion properties. Our data support also the application of biomechanics as a tool to study diseases and their treatments.

Dielectric barrier discharge (DBD) plasma is usually referred in literature as a form of cold plasma. Many applications of DBD plasma rely on this characteristic, which allow to treat also sensitive materials, including biological tissues, to exploit a range of different effects. Atmospheric pressure plasma treatment is regularly used on polymers to enhance surface properties such as wettability and adhesion. However, in the present work, we show that DBD plasma can also be used as an alternative for heating polymeric based materials, as an initial step for further industrial processing such as thermo-forming. In particular, the efficiency of the heating process has been measured, and a novel heating mechanism has been proposed based on the experimental results