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Materiaie genetico, alleli, mutazione e variabilità Mutazione; tipi e classificazione Mutazioni spontanee, mutazioni indotte e agenti mutageni Ripazione del DNA meccanismi di riparo del DNA Conseguenze delle mutazioni mutaz/on/ puntiformi nelle regioni codificanti mutaz/oni nelle regioni non codificanti del gene Ricombinazione e trasposizione come eventi mutazionali crossing over ineguale,elementi mobil, espansione delle ripetizioni di tnnudeotidi Mutazioni cromosomiche (variazioni della struttura dei cromosomi) Tecniche per l'identificazione di mutazioni cromosomiche Mutazioni genomiche (variazioni del numero dei cromosomi) Variazioni del numero dei cromosomi nella specie umana Disomia uniparentale Mutazioni ed ingegneria genetica

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro and/or in vivo tissues with morphological and functional features similar to the biological tissue of the human body [1]. In the attempt to construct suitable tissue models, a critical step is the setting of 3D scaffolds that mimic the supportive structures of a natural extracellular matrix microenvironment into which cells are normally embedded. In this context, the generation of 3D cultures of brain cells is of particular interest. For instance, the poly L‐lactic acid (PLLA) polymer is wildly used because of its biocompatible and biodegradable potential; the PLLA scaffold topography simulates the natural extracellular matrix (ECM) and can make it a good candidate for neural tissue engineering [2]. To achieve this goal, in this study, PLLA scaffold with characteristics of bioactivity was prepared via thermally-induced phase separation (TIPS) [3], and utilized as substrate for primary rat astrocytes 3D growth. To assess the cells spatial distribution and morphology within the scaffolds, the structures were characterized by scanning electron microscopy. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Different scaffold morphologies and coatings such as collagen I and IV, and fibronectin were tested in order to evaluate their influence on astrocyte growth, morphology and EV production. To evaluate these effects on astrocyte morphology on the PLLA scaffolds, TEM preparation was also performed. Cells were present in all regions of the scaffold, they were observed to adhere, grow and penetrating into the interior region of the scaffold, acquiring their typical morphology. In addition, they also secrete EVs as in vivo [4]. Their ability to produce EVs was demonstrated by both TEM and SEM analyses, which revealed intracellular MVBs and EVs compatible in size with exosomes. The results revealed that the porous sheath of PLLA allowed cell migration inside the scaffold and that the one coated with collagen IV, served as very good matrices for astrocytes, suggesting that the chosen conditions could be a good starting point for the preparation of 3D brain cell co culture systems useful for clinical applications. References [1] R. Langer, JP. Vacanti. Science. 14 260 (1993): 920-926 [2] M. Soleimani, S. Nadri, I. Shabani. Int. J. Dev. Biol. 54 (2010) 1295-1300 [3] GA. Mannella, G. Conoscenti, F. Carfì Pavia, V. La Carrubba, V. Brucato. Mater Lett 160 (2015) 31-33 [4] G. Schiera, CM. Di Liegro, I. Di Liegro. Biomed Res Int (2015): 152926

Several studies have clearly demonstrated that within a heterogeneous tumor mass the inter-clonal cooperation between metastatic and non-metastatic cells can facilitate the tumor progression. Recent accumulating evidence has highlighted that tumor-derived exosomes (TDEs) play a relevant role as mediator of the inter-clonal collaborative cooperation affecting the properties of both tumor and non-tumor component. In this context, our goal was to understand if exosomes derived from highly metastatic cells may influence the behaviour of less aggressive tumor cells and the properties of endothelium. We found that metastatic SW620 cells transfer through exosomes (SW620Exos) their round/amoeboid phenotype to elongated non-metastatic SW480 cells also inducing the increase of the motile and invasive activities. Moreover, SW620Exos caused endothelial hyperpermeability by altering the junctional complexes in HUVECs. The SWATH-based quantitative proteomic analysis highlighted that SW620Exos were significant enriched in several proteins related to the RhoA/ROCK sig¬naling, known to induce the amoeboid motility as well as the destabilization of endothelial junctional complexes. According to this data, we found that the treatment with ExoSW620 elicited in both SW480 cells and HUVECs the increase of RhoA activity, while the induced morphological and functional effects were reverted by co-treatment with a specific ROCK inhibitor. RacGap1 and thrombin were identified as putative key mediators of the effects induced by SW620Exos in target cells. Taken together our data indicates that within a heterogeneous tumor mass exosomes released by metastatic cells affect the features of both tumor and non-tumor cell components, thus contributing to accelerate the metastatic cascade.

Adenocarcinomas and their associated lymphovascular emboli metastasize as tight aggregates of tumor cells. Using a xenograft model of human inflammatory breast cancer (Mary-X), which exhibited florid lymphovascular emboli in mice and high density tumor aggregates (spheroids) in vitro, we previously demonstrated that both emboli and spheroids are mediated by an intact overexpressed E-cadherin axis which regulates homotypic tumor cell adhesion. We now report that Mary-X spheroids secrete 5-10 fold higher levels of microvesicles (MVs) than most other carcinoma cell lines. Despite the high density of tumor cells and the strong juxtaposition of tumor cells to each other in the spheroids, intercellular spaces exist which contain numerous MVs. These intraspheroidal (IS) MVs entrapped between the E-cadherin adherens junctions can be isolated by dissociating the spheroids in Ca+2-free media with EDTA. These IS MVs compared to the extraspheroidal (ES) MVs found in the conditioned media (CM) comprise a significant (approximately 25%) subpopulation of MVs which persisted for several days in culture (day 3: 0.48 v 2.0; day 7: 1.1 v 3.9; day 14: 0.64 v 3.2 (# x 10E10)). The IS MVs differed in size compared to the ES MVs. By nanoparticle tracking analysis (Nanosight), IS MV mean volume was 79 nm compared with the ES MV mean volume of 152 nm. These size differences were also confirmed by transmission electron microscopy (TEM), though both were smaller (50 nm v 110 nm). The proteome profile of the IS MVs can be readily compared with that of the ES MVs found in CM. Cellular target differences between the IS MVs and the ES MVs can also be studied. It would be attractive to hypothesize that IS MVs preferentially function in autocrine signaling whereas ES MVs signal in a paracrine manner.

We isolated and characterized nanovesicles from edible Citrus limon with size and composition similar to mammalian-derived exosomes. Furthermore we show an in vitro and in vivo anti-proliferative and pro-apoptotic effect of these vesicles. This study opens to the possibility of using this natural plant-derived nanovesicles as antineoplastic agents.

The tunic is a peculiar integumentary tissue covering the epidermis of ascidians and other tunicates. It is an extracellular matrix whose outermost surface is revealed to be a continuos layer called tunic cuticle, composed of electron dense fibrous materials. Mechanisms occurring during the growth of the animal after metamorphosis, must assure the maintenance of the thin tunic cuticle density and integrity as it represents the primary body surface barrier preventing the mechanical and microbial assaults. Investigations by optical and electron microscopy on tunic samples from young Ciona intestinalis, allowed us to study morphological relationship between the tunic cuticle architecture and granulocytic cellular groups that are located next to it and the involvement of them on its formation.

Ascidian swimming larvae are entirely surrounded by a hyaline extracellular coat, called as tunic or test, on which numerous test cells adhere. The functional role played by test cells in larvae of various ascidian species consists in depositing submicroscopic structures known as ornaments and/or substances of proteoglycan nature in the larval test surface. The deposition of ornaments would render the larval test hydrophilic and thus allow the larvae to swim being immersed in sea water. Ultrastructural investigations reported in literature on larvae of Cionidae and Ascididae families have not evidenced the presence of ornaments in the swimming larval test. For these Ascididae families it has been hypothesized that test cells secrete an amorphous substance that would let them to adhere to larval tunic. In order to clarify the functional role played by test cells of swimming larvae of the Ascididae family, ultrastructural and cytochemical investigations have been carried out on test cells of Ascidia malaca swimming larvae. The ultrastructural observations have evidenced that these cells are metabolically active and show an amoeboidic behaviour as they mainly adhere to the surface of the test. Their cytoplasm is characterized by the presence of a Golgi and large granules that gradually empty their contents and release the same on the test surface. The cytochemical investigastions carried out at ultrastructural level have evidenced that the substances secreted by test cells and deposited on the larval test consisting of glycosaminoglycans. According to the data reported in literature the results of the present investigations confirm that the deposition of glycosaminoglycans enables the adhesion of test cells on the surface of larval tunic and would render the Ascidia malaca larva hydrophilic and able to swim being immersed in sea water.

Following an inflammatory stimulus such as the injection of erythrocytes into the tunic of Ciona intestinalis, hemocytes respond to events occurring in the tunic within a short time, and infiltrate the inflamed tissue being massively involved in the acute inflammatory response and capsule formation. The proliferative response of circulating hemocytes and pharynx assume particular interest as it is significantly enhanced during these defence reactions. Microscopy clearly may contribute to extend our understanding of the phenomenon showing interesting mitotic figures and hematogenic nodules with proliferative characteristics.

libro di biologia e genetica

libro di testo

Screening of subtelomeric rearrangements in autism spectrum disorder. Identification of a partial trisomy of 13q in a patient.

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro tissues with morphological and functional features similar to the biological tissue of the human body. Polymeric materials can be used in contact with biological systems in replacing destroyed tissue by transplantation [1]. Several biopolymers, including poly L (lactic acid) (PLLA), have been used in biomedical applications to set scaffolds with ductile proprieties and biodegradation kinetics [2]. In particular, the PLLA scaffold topography mimics the natural extracellular matrix and makes it a good candidate for neural tissue engineering. We report about of 3D system the PLLA porous scaffolds prepared via thermally-induced phase separation (TIPS) [3], and utilized as substrate for primary rat astrocytes 3D growth. Interestingly astrocytes adapt well to these porous matrices, not only remaining on the surface, but also penetrating inside the scaffolds. They colonize the matrix acquiring a typical star-like morphology; they form cell contacts and, in addition produce EVs as in vivo [4]. These results suggest that the chosen conditions could be a good starting point for 3D brain culture systems. PLLA scaffolds could be further enriched to host two or three different brain cell types, in order to set an in vitro model of blood brain barrier. The future use of co-culture systems may be involved in drug delivery studies, and in the formulation of new therapeutic strategies for the treatment of neurological diseases. [1]Langer R, Vacanti JP. Tissue engineering. Science. 1993; 260: 920 [2]Nejati E, et al. Appl. Sci. Manuf. 2008; 39: 1589–1596 [3]Scaffaro R, et al. J. Mech. Behav. Biomed. Mater. 2016; 54:8-20 [4]Schiera G, et al. Biomed Res Int 2015: 152926, 2015