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Analisi paleosismica e definizione del terremoto caratteristico nella Pianura Padana, con particolare riferimento al settore pedemontano lombardo.
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Cell delivery of Met docking site peptides inhibit angiogenesis and vascular tumor growth.
No full textAngiogenesis is a very complex biological process that drives the formation of new blood vessels starting from pre-existing one. In adulthood, it occurs both in physiological and pathological processes.
Pathological angiogenesis is involved in several diseases, including cancer. In the last decade, the concept of anti-angiogenic therapy has been developed as a strategy to treat cancer inducing the inhibition of new vessels that provide sustenance to the tumor. Recently, Met receptor and its ligand HGF (Hepatocyte Growth Factor) has been proposed as target for the anti-angiogenic therapy.
HGF and its receptor Met are responsible for a wide variety of cellular responses, both physiologically during embryo development and tissue homeostasis, and pathologically, particularly during tumor growth and dissemination. In cancer, Met can act as an oncogene on tumor cells, as well as a pro-angiogenic factor activating endothelial cells and inducing
new vessel formation. Molecules interfering with Met activity could be valuable agents for anti-cancer treatment simultaneously inhibiting oncogenesis and angiogenesis.
The aim of this study has been the identification of peptides able to interfere with HGF-induced angiogenesis.
We synthesized three different peptides containing the Met docking site fused to the internalization sequences of Antennapedia homeodomain or of Tat transactivation domain which were used to deliver peptides into living cells.
In vitro we treated human endothelial cells with peptides and we quantified proliferation. We then evaluated the ability of peptides to interfere with HGF-induced migration, invasion and morphogenesis of endothelial cells. By western blotting analysis we investigated whether these peptides affect Met activation and downstream signalling. In vivo we performed matrigel sponge assay and Kaposi’s sarcoma xenograft to test the peptides efficacy on angiogenesis and tumor growth.
We observed that in endothelial cells internalized peptides inhibited ligand-dependent cell proliferation, motility, invasiveness and morphogenesis in vitro, to an even greater extent and with much less toxicity than the Met inhibitor PHA-665752, which correlated with interference of HGF-dependent downstream signalling, as shown by reduction in ERK1/2 activation (Cantelmo et al.). In vivo, the peptides inhibited HGF-induced angiogenesis in the matrigel sponge assay and impaired xenograft tumor growth and vascularization in Kaposi’s sarcoma (Cantelmo et al.).
These data show that interference with the Met receptor intracellular sequence impairs HGF-induced angiogenesis, suggesting the use of antidocking site compounds as a therapeutic strategy to counteract angiogenesis in cancer as well as in other diseases
Phylogeography of Italian barbels (Cyprinidae, Barbus) inferred by mitochondrial and nuclear markers.
No full textSpecies of the genus Barbus, being primary freshwater fishes intolerant of salt water, are of great value for biogeographic studies since their dispersal strictly depends on geological
evolution of the landmasses (i.e. catchments watershed, mountain chains and fluctuations of sea level). In Italian peninsula four specie are formally recognized: B. caninus, B. balcanicus, B. plebejus and B. tyberinus. Their genetic relationships were assessed using both mitochondrial and nuclear markers. The study was carried out as first developing new nuclear primers for the S7 ribosomal protein and the Growth hormone genes (Gh); then performing a SNPs characterization of these loci on 18 populations (264 specimens in total). Results from nuclear sequences were then compared with those from partial sequences of the Cytochrome b mitochondrial gene (733 bp). Recovered phylogenies were congruent with the current morphology-based systematic and taxonomy. Results highlighted the close relationships between species belonging to the fluvio-lacustrine ecological group: B. plebejus and B. tyberinus and the high genetic distance between species belonging to the riverine group: B. caninus and B. balcanicus. Moreover findings were congruent with hypotheses of partial permeability of principal biogeographic barriers (Alpine and the Apennine chains) to freshwater fish fauna.
Successively the influence of different ecological preferences on gene flow was tested for B. caninus and B. tyberinus on 6 and 7 populations respectively. Results pointed out that the riverine B. caninus has higher structured populations than B. tyberinus, probably due to the different dispersion ability and the different habitat colonized. Moreover, for the first time, molecular evidences were shown about hybridization events occurring between B. caninus and B. plebejus, B. tyberinus and B. barbus
Functional characterization of the tumor antagonizing gene RNASET2.
No full textSeveral types of cancer including solid tumors and blood malignancies are associated with alterations in different regions of chromosome 6. In particular, the 6q region has been frequently found to be deleted in a wide range of solid tumors, including ovarian carcinoma. Therefore, this chromosomal region has been extensively investigated in order to identifying putative tumor suppressor genes. In specific, a minimal region of deletion for ovarian cancer has been defined in the peritelomeric 6q27 region, and our group has recently isolated from this region the RNASET2 gene, coding for a secreted ribonuclease belonging to the Rh/T2/S acid ribonuclease family.
The RNASET2 gene was found to be down-regulated at the transcriptional level in several primary ovarian tumors and ovarian cancer cell lines and its potential for tumor growth inhibition has been subsequently tested in vivo in a highly tumorigenic and metastatic Hey3Met2 ovarian cancer cell line.
To this aim, athymic mice were injected with Hey3Met2 cell clones stably transfected with expression vectors encoding wild-type or a catalytically inactive mutant RNASET2 protein. Strikingly, wild-type RNASET2-expressing cells formed smaller tumors in mice and gave rise to a lower number of metastasis when compared to control cells transfected with the vector only. Moreover, mutant RNASET2-expressing cells also displayed a similar or even more increased inhibition of tumorigenicity when compared to wild-type RNASET2, suggesting that catalytic activity is not involved in control of tumor suppression and metastasis.
With the purpose of further defining the biological role of RNASET2, we have undertaken a functional characterization of this gene, by comparing RNASET2-overespressing cells behavior both in vitro and in vivo. In sharp contrast with previous in vivo observations, several in vitro tests such as proliferation, clonogenicity, growth in soft agar and apoptosis assays failed to reveal any change in cancer-related phenotypes in the same RNASET2-overexpressing cell line, thus suggesting a non-cell autonomous activity for RNASET2 as a tumor suppressor.
This “asymmetric” in vivo/in vitro property has been recently ascribed to a novel class of tumor suppressor genes, called tumor antagonizing genes (TAGs), whose oncosuppressive activity is carried out only in vivo by induction of microenvironmental remodeling.
To confirm this hypothesis we turned to a xenograft assay and, as expected, Hey3Met2 cells expressing wild-type or mutant RNASET2 were not able to support tumor growth when compared to control mice.
Strikingly, a detailed histological analysis of tumor sections revealed a different architectural pattern between control mice and RNASET2-expressing tumors. The former showed a solid and diffuse growth with strong component of human tumor cells, whereas in the latter a more organized structure and a 2 massive infiltration of host stromal cells mainly derived from monocyte/macrophage lineage has been found.
In order to establish whether these cells could be functionally responsible for RNASET2-mediated tumor suppression, a further in vivo xenograft assay with Rag-2-/- γc-/- mice was performed, since these mice lack both lymphocytes and NK cells, thus retaining innate immunological response only through macrophages. As previously observed in nude mice, RNASET2-expressing cells were again clearly suppressed in tumorigenicity, further suggesting a functional role for macrophages recruitment in tumor suppression mediated by RNASET2.
To definitively confirm the involvement of this cell lineage, in an additional in vivo experiment Rag-2-/- γc-/- mice were pretreated with the macrophages-depleting agent clodronate before inoculation of Hey3Met2 cells. Untreated mice showed once again a clear effect of tumor suppression exerted by RNASET2 whereas in clodronate-treated mice a very similar tumor development was noticed in both control and RNASET2-expressing cells. Finally, immunohistochemical analysis showed that recruited immune cells mainly belong to M1 subtype of anti-tumorigenic macrophages, suggesting a possible mechanism of tumor suppression exerted by this secreted ribonuclease.
In order to extend RNASET2 functional characterization, beside the experimental model described above a complementary research approach based on gene silencing was carried out, aimed at studying how the lack of RNASET2 could affect tumor development and providing a powerful tool for further investigations.
Our working hypothesis considers RNASET2 inactivation as a critical step in ovarian cancer progression, therefore cancer cell lines endowed with a low in vivo tumorigenic potential and expressing high endogenous RNASET2 levels might represent an ideal model system to challenge this hypothesis. In such a context, RNAi-mediated gene knockdown could provide additional information on this topic.
RNAi-mediated silencing of the RNASET2 gene has therefore been attempted in the human ovarian carcinoma cell line OVCAR3, which displays the above mentioned features.
Complete knockdown of RNASET2 in OVCAR3 cell line was successfully achieved and preliminary in vitro tests are in keeping with our previous data, showing no significant differences in proliferation and clonogenic potential in vitro in RNASET2-silenced when compared to control clones, and further supporting a “non-cell autonomous” behavior of RNASET2.
Gene silencing of RNASET2 also allowed us to studying both the intracellular routing and likely the mechanism(s) by which extracellular RNASET2 affects in vivo cell behavior, demonstrating that providing an external source of RNASET2 to silenced OVCAR3 cells resulted in the binding of the ribonuclease to the cell surface and its subsequently internalization
Exploring accretion theory with a new subclass of high mass x-ray binaries: interpretation of integral observations of the supergiant fast x-ray transients.
No full textIn the last eight years the INTEGRAL satellite has allowed to discover a new class of transient X-ray binaries associated with OB supergiants called Supergiant Fast X-ray Transients (SFXTs). They display a high dynamic range, spanning 3-5 orders of magnitude from a quiescent luminosity of 1032 erg s-1 up to a peak luminosity of 1036 - 1037 erg s-1, and they spend most of the time in an intermediate aring level of emission at around 1033 - 1034 erg s-1. The enigmatic properties emerging from this class of sources raise interesting problems to the standard theories for the accretion of matter onto compact objects, and to the properties of OB supergiants in SFXTs. The aim of this thesis is to gain more information about the peculiar transient behaviour of SFXTs and to explore the accretion mechanisms involved in these enigmatic X-ray sources. I present a new clumpy wind model for OB supergiants, with both spherical and non-spherical geometry, that I have developed to investigate the e_ects of accretion from a clumpy wind on the X-ray variability of SFXTs. I assume that the clumps are con_ned by ram pressure of the ambient gas, and I assume that a fraction of the stellar wind is in form of clumps with power law mass and radius distributions. Then, I compute the expected X-ray lightcurves in the framework of the Bondi-Hoyle accretion theory, modi_ed to take into account the presence of clumps, and I apply this model to reproduce the X-ray lightcurves of _ve representative HMXBs: two persistent supergiant systems (Vela X-1 and 4U 1700-377), and the SFXTs IGR J16479-4514, IGR J11215-5952 and IGR J18483-0311. The model can reproduce the observed lightcurves well, but requiring in all cases an overall mass loss from the supergiants about a factor 3-10 smaller than the values inferred from UV lines studies that assume homogeneous wind, but in agreement with recent studies that assume clumpy winds.
Then, I report the systematic analysis of all INTEGRAL observations from 2003 to 2009 of 14 SFXTs (con_rmed and candidates), implying a net exposure time of about 30 Ms. This analysis led to discover several new outbursts from SFXTs. I discuss the e_ects of X-ray photoionization on the accretion in close binary systems, and I show that, because of X-ray photoionization, there is a high probability of formation of transient accretion discs from the capture of angular momentum in IGR J16479-4514. This result suggests that the formation of transient accretion discs could be partly responsible for the aring activity in SFXTs with narrow orbits or in SFXTs with higher orbital periods and high eccentricities, when the neutron star is close to periastron. I also propose an alternative way to explain the origin of ares with peculiar shapes observed with INTEGRAL applying the intermittent accretion model of Lamb et al. (1977). Then, I report the study of the candidate SFXT IGR J16418-4532, for which I obtain a re_ned estimate of the orbital period from Swift/BAT data, and a re_ned estimate of the spin period of the neutron star from INTEGRAL data. I con_rm the presence of a region of the orbital lightcurve with a low ux, probably due to an eclipse, or due to the onset of the centrifugal inhibition of accretion. The uncertainties of the results from infrared observations do not allow an assessment of the spectral class of the counterpart of IGR J16418-4532, which could be a main sequence, giant, or O8.5 supergiant. From considerations involving the expected X-ray luminosities, the duration of the likely eclipse, and the onset of the centrifugal inhibition of accretion, I _nd that in all these cases it is possible to exclude the presence of a O8.5 V star, and the presence of a supergiant is favoured
Ecology and conservation of red-backed shrike (Lanius collurio) in semi-natural habitats of north-western Italy.
No full textSpecies conservation largely depends on knowledge of habitat needs of target species. On the other hand, breeding habitat plays an essential role in the life-history of birds. Many bird species are currently threatened by unfavourable environmental changes or by habitat destruction or degradation prompted by human activities, in particular farmland birds. This dramatic and widespread decline has been related to agricultural intensification, especially through loss of ecological heterogeneity. Also land abandonment, which mainly affects marginal areas and is often indirectly caused by intensification, negatively affects several bird species.
The red-backed shrike Lanius collurio (Aves: Laniidae) is a typical passerine species of Euroasiatic low-intensity farmland landscapes and is one of the species most sensitive to agricultural changes.
This species underwent large declines over most of Europe during the last century, disappearing almost completely from large areas, and being consequently listed in the Annex I of the Birds Directive (79/409/EEC). The main reason for the decline is probably linked to the increased intensification and mechanization of traditional agriculture, which led to unfavourable changes of breeding habitats. However, conditions in the African wintering grounds or migratory stop-over areas may also have affected its population status. Recently, some studies have tried to address habitat determinants of species’ occurrence and breeding performance in many areas, but studies in southern Europe are still scarce and most works deal with central or northern Europe.
Lombardy, northern Italy, includes portions of both the Alpine and Apennine chains, which at intermediate elevations host key populations of red-backed shrike. Lowland areas in the region have undergone heavy agricultural intensification (generally heavier than in the rest of Italy), resulting in wide extensions of specialized cultivations, often with a single crop occupying tens or hundreds of hectares (especially Zea mais), without marginal features (as hedges, shrubs or tree patches).
Therefore, conservation of farmland birds in hilly and mountain areas, where changes of agricultural practices have been less dramatic, is a priority for bird conservation in Italy, and especially in Lombardy. At the same time, land abandonment is dramatically reducing open habitats in hilly and mountain portions of the region, thus leading to further unsuitable modification to the breeding habitats of shrikes and other farmland or ecotonal species.
Red-backed shrike is one of the most threatened species in Lombardy and is listed among conservation priority species by the regional government. Although the species seems to have dramatically declined, for example in Varese and Bergamo provinces, few quantitative data are available to compare the current and previous estimates of population size and distribution.
We developed a multi-level conservation strategy for Lanius collurio in Lombardy, starting from a regional-wide assessment of habitat preferences, whose results were translated into conservation priorities over different spatial scales and in a regional Action Plan for the conservation of the species.
After defining habitat associations, we i) described conservation priorities (at landscape scale) over the whole region, by identifying the most important populations of the species, main areas of potential occurrence and main threats and conservation needs in terms of large-scale habitat change, ii) derived territory-level guidelines for management of different habitats occupied by the species.
Management guidelines include a description of the habitat mosaic virtually representing the most suitable option for the species in different environmental contexts (permanent grasslands, pastures, shrublands, heathlands, vineyards) inhabited by L. collurio in Lombardy and best practices favourable for the species. On average, the definition of proportion of habitat variables in an ideal territory of a breeding pair of red – backed shrike in Lombardy includes: 60% of grazed or mowed grasslands; 20% of bushes; 10% of untilled grasslands; 70 meters of hedges; 60 meters of fences.
Furthermore, for each suitable breeding habitat for the species in Lombardy we determined the ideal mosaic of vegetation in one hectare of surface that can hosts a breeding pair of red-backed shrike (e.g. for permanent grasslands: 73% mowed of grazed grassland; 12% untilled grassland; 10% shrubs; 5% hedges).
Finally, an ‘Action Plan for the conservation of Red-Backed Shrike in Lombardy’ was produced, based on a long-term strategy of conservation of the species in Lombardy region. Its adoption by the regional government of Lombardy is currently under way
