521 research outputs found
AFM as a toolbox for assessing the mechanical properties of cells with genetic mutations
No full textAl 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.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
A Perspective on the Experimental Techniques for Studying Lamins
No full textLamins 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
Atomic force microscopy and lamins: a review study towards future, combined investigations
No full textIn the last decades, atomic force microscopy (AFM) underwent a rapid and stunning development,
especially for studying mechanical properties of biological samples. The numerous discoveries relying
to this approach, have increased the credit of AFM as a versatile tool, and potentially eligible
as a diagnostic equipment. Meanwhile, it has become strikingly evident that lamins are involved on
the onset and development of certain diseases, including cancer, Hutchinson-Gilford progeria syndrome,
cardiovascular pathologies, and muscular dystrophy. A new category of pathologies has
been defined, the laminopathies, which are caused by mutations in the gene encoding for A-type
lamins. As the majority of medical issues, lamins, and all their related aspects can be considered as
a quite complex problem. Indeed, there are many facets to explore, and this definitely requires a
multidisciplinary approach. One of the most intriguing aspects concerning lamins is their remarkable
contribute to cells mechanics. Over the years, this has led to the speculation of the so-called
“structural hypothesis”, which attempts to elucidate the etiology and some features of the laminopathies.
Among the various techniques tried to figure out the role of lamins in the cells mechanics,
the AFM has been already successfully applied, proving its versatility. Therefore, the present work
aims both to highlight the qualities of AFM and to review the most relevant knowledge about lamins,
in order to promote the study of the latter, taking advantage from the forme
Rezension zu: Ilaria Bignamini/Clare Hornsby, Digging and Dealing in Eighteenth-Century Rome. With additional research by Irma Della Giovampaola and Jonathan Yarker (New Haven and London 2010)
No full textData of cost-optimal solutions and retrofit design methods for school renovation in a warm climate
No full textAbstract"Efficient Solutions and Cost-Optimal Analysis for Existing School Buildings" (Paolo Maria Congedo, Delia D’Agostino, Cristina Baglivo, Giuliano Tornese, Ilaria Zacà) [1] is the paper that refers to this article. It reports the data related to the establishment of several variants of energy efficient retrofit measures selected for two existing school buildings located in the Mediterranean area. In compliance with the cost-optimal analysis described in the Energy Performance of Buildings Directive and its guidelines (EU, Directive, EU 244,) [2,3], these data are useful for the integration of renewable energy sources and high performance technical systems for school renovation. The data of cost-efficient high performance solutions are provided in tables that are explained within the following sections.The data focus on the describe school refurbishment sector to which European policies and investments are directed. A methodological approach already used in previous studies about new buildings is followed (Baglivo Cristina, Congedo Paolo Maria, D׳Agostino Delia, Zacà Ilaria, 2015; IlariaZacà, Delia D’Agostino, Paolo Maria Congedo, Cristina Baglivo; Baglivo Cristina, Congedo Paolo Maria, D’Agostino Delia, Zacà Ilaria, 2015; Ilaria Zacà, Delia D’Agostino, Paolo Maria Congedo, Cristina Baglivo, 2015; Paolo Maria Congedo, Cristina Baglivo, IlariaZacà, Delia D’Agostino,2015) [4–8]. The files give the cost-optimal solutions for a kindergarten (REF1) and a nursery (REF2) school located in Sanarica and Squinzano (province of Lecce Southern Italy). The two reference buildings differ for construction period, materials and systems.The eleven tables provided contain data about the localization of the buildings, geometrical features and thermal properties of the envelope, as well as the energy efficiency measures related to walls, windows, heating, cooling, dhw and renewables. Output values of energy consumption, gas emission and costs are given for a financial and a macro-economic analysis.This data article provides 288 and 96 combinations for REF1 and REF2, respectively. The output values are obtained using the software ProCasaClima 2015v.2.0
Author Correction: Gluten consumption and inflammation affect the development of celiac disease in at-risk children
No full textThe original version of this Article contained an error in the spelling of the authors Renata Auricchio, Ilaria Calabrese, Martina Galatola, Donatella Cielo, Fortunata Carbone, Marianna Mancuso, Giuseppe Matarese, Riccardo Troncone, Salvatore Auricchio & Luigi Greco which were incorrectly given as Auricchio Renata, Calabrese Ilaria, Galatola Martina, Cielo Donatella, Carbone Fortunata, Mancuso Marianna, Matarese Giuseppe, Troncone Riccardo, Auricchio Salvatore & Greco Luigi. The original article has been corrected
Environmental policies and social impacts. Suggestions from Europeans cities
No full textAre we really sure that green and sustainable are always good for everyone? Ilaria Beretta tries to go beyond the rhetoric of sustainability to understand more deeply the interconnection between economy, society and environment in urban contexts, in the belief that only empirical research and the analysis of data can lead to a correct reading, which is as objective as possible, of the complex reality in which we live. The author, through a comparison between European and U.S. American cities, focuses in particular on policies related to climate change and land use, showing how these, if not properly monitored in their social effects, risk contributing to the tightening of social inequalities, through processes such as ecological and green gentrification. The volume is structured in three parts. In the first section, she illustrates the main European Union’s sustainability and environmental urban policies, focusing on climate change and land use strategies. The second chapter provides a conceptual framework through the environmental justice paradigm, as it applies in Europe and in U.S. Finally, the third chapter offers an empirical analysis of ecological and green gentrification processes, comparing American and European realities, and highlighting how context-specific differences can have profound effects on the manifestation (or not) of these phenomena
Mechanical Properties of 3T3 Fibroblasts due to Fixation Assessed Using Atomic Force Microscopy
No full textIn this paper we analyzed how the Mechanical Properties of 3T3 Fibroblasts change due to Fixation procedure. This was assessed using Atomic Force Microscop
Metabolic and proliferative cells activity on different substrates
No full textScaffolds for tissue engineering can be either natural or synthetic materials. The latter allow control of chemical, physical and mechanical properties and also provide support and shape, however they are not of biological origin, and therefore could not promote cell adhesion. This problem does not occur in natural materials however, they have the drawback of having non-suitable mechanical properties and tend to deteriorate too fast. In this work we study the influence of different substrate on the metabolic as well as proliferative cells activity. In particular 4 substrates have been considered: (i) medical grade StageFlexer (silicone elastomer), (ii) Polydimethylsiloxane (PDMS), (iii) PDMS with a layer of carbon nanotubes (CNT’s) and (iv) PDMS with a layer of ceramic whiskers. The results of both tests (metabolic and proliferative capacity) showed that the PDMS without any surface treatment, is the worst of the tested substrates. The reason is to be found in the fact that the PDMS is highly hydrophobic and therefore cells have low adhesion to the substrate. This represents a major limitation of PDMS and its functionalization is necessary to improve cell adhesion. Cells placed on PDMS samples with CNT’s show higher metabolic activity and proliferative capacity, compared to the PDMS and PDMS treated with fibronectin. However, the best outcomes have occurred with the PDMS substrate coated with ceramic whiskers
Investigating Cell-Substrate and Cell–Cell Interactions by Means of Single-Cell-Probe Force Spectroscopy
No full textCell adhesion forces are typically a mixture of specific and nonspecific cell substrate and cell–cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the
time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins
(S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion
forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell–cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 lg/mL (FN20) and 100 lg/mL (FN100), which impact the number of substrate receptors
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