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CDKL5 and neuronal morphological defects: novel perspectives of microtubule related drugs
No full textMutations in the CDKL5 gene have been found in individuals with a rare neurodevelopmental disorder. CDKL5 is a serine/threonine kinase whose functions are still not fully understood. An aberrant neuronal morphology linked to Cdkl5 has been observed both in Cdkl5--‐silenced primary neurons as well as in Cdkl5--‐null brains strongly suggesting that these defects may underlie the cognitive impairment characterizing both patients and mice devoid of the kinase. The molecular basis of these defects is still far from understood but altered cytoskeletal dynamics are likely to be involved. The identification of IQGAP1 as novel CDKL5 interactor may provide a key to understand such neuronal defects. IQGAP1 promotes microtubule dynamics through its association with Rac1 and CLIP--‐170, whose activity on MTs might play a significant role in the regulation of cytoskeletal dynamics in cycling cells and neurons. Considering how the loss of CDKL5 negatively impacts on cellular and neuronal morphology, we envisaged that this kinase could regulate cytoskeletal dynamics acting directly or indirectly on CLIP--‐170. Indeed, CLIP--‐170 is known to regulate several aspects of neuronal morphology such as axon outgrowth, dendritic arborization and growth cone organization. By bridging the actin cytoskeleton to MTs, CLIP--‐170 coordinates proper cytoskeletal dynamics.
In the current study, we analysed the role of CDKL5 in the regulation of CLIP--‐170 activity. Through a FRET analysis on COS7 cells we demonstrated that the loss of CDKL5 causes CLIP--‐170 to be mainly in its closed inactive conformation, thus reducing its interaction with MTs. Furthermore, using Cdkl5--‐ KO primary hippocampal neurons we demonstrated that the loss of CDKL5 is detrimental for the correct progression of several steps of neuronal maturation. Moreover, we demonstrated that such defects can be restored upon treatment with pregnenolone (a neurosteroid which stabilizes the extended conformation of CLIP--‐170, increasing its affinity for MTs and promoting their polymerization) and Pregnenolone--‐Methyl--‐Ether, a synthetic non--‐metabolizable P5 derivative. We speculate that the positive effects of the two compounds are likely to be due to an activation of CLIP--‐ 170 functionality and a concomitant increase in MT dynamics
Development of saporin-based therapeutic options for the treatment of solid tumors.
No full textUp to now, cancer represents one of the most challenging disease to treat, mostly due to its ability to adapt and negatively respond to current available therapies. Over the past 30 years, Ribosome Inactivating Proteins (RIPs) have attracted great interest in the scientific community for their therapeutic potential. Indeed, such toxins have been extensively used as potent and versatile therapeutic weapons due to important advantages compared to chemotherapeutics: for instance, they act independently form cell cycle, killing both quiescent and dividing cells, limiting the development of cancer resistance. However, the successful application of toxins-based therapeutics to solid tumors remains to be demonstrated. In this study, we explored the potential use of the plant-RIP saporin (SAP) for the treatment of solid tumors. In particular, we propose two different strategies, in which SAP is selectively and safely conveyed to malignant cells either in the form of recombinant protein through genetically fused targeting moieties, or throughout cell-derived extracellular vesicles as vehicles. Overall our data indicate that SAP-based recombinant proteins are promising antitumoral therapeutic options. Applied as single or combined treatment, as well as used together with traditional therapeutics, they appropriately address both intra and inter- tumor heterogeneity. In addition, the use of exosomes as SAP nanocarriers is a promising strategy to improve safety and drug delivery to tumor cells
Metal nanoparticle permeation through the plasma membrane: Xenopus laevis oocytes as novel tools for membrane permeability evaluation and physico-chemical characterization of particle properties.
No full textIn this Ph.D. thesis several kinds of metal-based NPs were tested in Xenopus laevis oocytes, in order to understand the metal NP related toxicity. The mechanism of the metals entrance in the cell and the passive permeation hypothesis were investigated. The results can be categorized as follows:
• The metallic NPs tested (Co, Fe, Ni) dissolve more than their oxide counterparts; however, they are unstable in suspension and their aggregation rate is fast. All these particles are not able to induce modifications in membrane biophysical parameters and in intracellular metal concentration, thereby excluding a direct membrane crossing mechanism for their internalization.
• The metal oxide NPs which retain a population below 200 nm (Fe3O4, Co3O4) in suspension in experimental medium induced significant modifications in membrane biophysical parameters and in intracellular metal concentration. The effects are recorded only when the population of NPs smaller than 200 nm is present, namely only within 5 min from the particles addition to the medium; after this time range, effects on membrane become milder until they disappear, stating the complete fast recovery of the membrane.
• The metal oxide NPs that never go below 200 nm (NiO, Fe2O3) in suspension in the experimental medium do not cross the membrane and do not modify neither intracellular metal concentration nor membrane biophysical parameters.
• The surface coating modifies particles behavior in suspension in the experimental medium. The non-covalent coating by BSA stabilized the 200 nm population for Co3O4 and Fe3O4 NPs but abolished the modifications in intracellular metal concentration and in membrane biophysical parameters. The covalent coating by APTES of Fe2O3 NPs partially stabilized particles around 100 nm but failed to induce membrane crossing. The role of particle surface is thus crucial to achieve direct membrane crossing.
Many of the studies conducted nowadays on direct membrane crossing rely on models, both in silico as well as in vitro, that are limited in replicating the biological complexity of the cells. Using voltage clamp and fluorescent probe on Xenopus Laevis oocytes may lay the foundation for an innovative screening platform, allowing to study the internalization and the effects on membrane. Xenopus laevis oocytes are easy to collect, maintain and prepare. They are flexible and adaptable to different culture conditions, allowing to study interactions in controlled environment.
Moreover, Xenopus laevis oocytes can also be a useful tool to study and optimize targeted internalization. Their historical use as heterologous expression system is well known, selectively express target proteins to study specific internalization pathways of new modified nanoparticles will be one of the new goals for the therapeutic use of nanomaterial
Integrated multidisciplinary approaches to investigate geochemical anomalies in waters.
No full textWater is a fundamental need for human and environmental benefits, and its inorganic quality is a mandatory standard. Potentially Toxic Elements (PTEs), due to their high toxicity need to be evaluated. Consequently, in case of possibly alarming concentration, the causes (natural or anthropogenic) need to be clarified, to evaluate possible remediation techniques.
Integrated multidisciplinary approaches are needed to clearly understand causes of anomalous PTEs concentrations in waters, coupling chemical, hydrogeological and statistical tools, especially in a dynamic comportment as water.
In this dissertation integrated approaches will be applied to understand sources of anomalous concentrations of PTEs in waters. Firstly, PTEs sources in an alpine catchment are evaluated through the application multivariate statistics and the analysis of water and sediment to evaluate the natural load of PTEs.
Also, hydrogeochemical anomalies caused by a seismic sequence in Central Italy are explained through the application of a temporal trend analysis of hydrogeochemical and seismic variables. In this way, a conceptual model of PTEs release after the shocks is proposed.
The collection of these case studies highlights the need of a complete, integrated, and multidisciplinary approach to deal with geochemical data. These approaches still require a good knowledge of lithology and a big data set of chemical variables, but could fit as a first step to create leaching and transport models
Assessment of portable and miniaturized sensors for the monitoring of human exposure to air pollutants
Full text linkIn the last years, several in-field campaigns have been conducted using portable and miniaturized monitors to evaluate the personal exposure to different pollutants. In general, this kind of monitors are characterized by worse metrological performance if compared to the traditional standard methods. Despite this disadvantage, portable and miniaturized monitors could be easily used across different applications, because their advantageous features, such as the capability to provide real-time measurement, the high spatial and temporal resolution of acquired data, the ability to adapt to different experimental designs and, especially, the ability to follow the subject in any activity. Finally, portable and miniaturized instruments can provide data acquired in the respiratory zone of the subject, following therefore the practices for a correct exposure assessment. Obviously, the best compromise between the analytical gold standard (in terms of precision, accuracy and instrumental sensitivity) and the gold standard in regard to the exposure assessment should be chosen.
Therefore, in brief, principal aims of this thesis are (i) to evaluate the on-field performances of portable and miniaturized monitors for gaseous pollutants and airborne PM and (ii) to use these monitors in exposure assessment studies and (iii) to understand if data acquired via portable and miniaturized monitors could be useful in other fields of application, such as epidemiological studies or toxicological studies, in which the evaluation of the inhaled dose of pollutants could play a key role
Existence, non existence and uniqueness results for higher order elliptic systems
Full text linkIn this dissertation, we deal with Hamiltonian Lane-Emden type systems where in place of the Laplace operator we take into account the polyharmonic operator. Recallthatthepolyharmonicoperatordoesnotalwayssatisfyamaximumprinciple. Indeed, if we take Dirichlet boundary conditions, the maximum principle is known to hold only on a ball or small deformations of a ball, whereas it fails on ellipses with sufficiently big ratio of half axes. On the one hand, if the operators in the two equations have the same order, the problem is variational. In this case, we prove some existence and non-existence results under Dirichlet boundary conditions on a sufficiently smooth bounded domain. We also consider more general nonlinearities than power-like. The existence result is achieved by means of a Linking Theorem on suitable fractional Sobolev spaces. On the other hand, if the operators have different orders the problem turns out to be non-variational: here we prove some existence results on a ball by means of a different approach, precisely we exploit degree theory and a blow-up analysis, combined with a suitably adapted moving planes technique and Liouville-type theorems. Moreover, we prove uniqueness of solutions to equations and systems up to order eight on a ball endowed with Dirichlet boundary conditions. The proof extends the classical argument by Gidas-Ni-Nirenberg
Discriminative feature learning for multimodal classification
Full text linkThe purpose of this thesis is to tackle two related topics: multimodal classification and objective functions to improve the discriminative power of features.
First, I worked on image and text classification tasks and performed many experiments to show the effectiveness of different approaches available in literature.
Then, I introduced a novel methodology which can classify multimodal documents using singlemodal classifiers merging textual and visual information into images and a novel loss function to improve separability between samples of a dataset.
Results show that exploiting multimodal data increases performances on classification tasks rather than using traditional single-modality methods.
Moreover the introduced GIT loss function is able to enhance the discriminative power of features, lowering intra-class distance and raising inter-class distance between samples of a multiclass dataset
Numerical methods for all-speed flows in fluid-dynamics and non-linear elasticity
Full text linkIn this thesis we are concerned with the numerical simulation of compressible materials flows, including gases, liquids and elastic solids. These materials are described by a monolithic Eulerian model of conservation laws, closed by an hyperelastic state law that includes the different behaviours of the considered materials. A novel implicit relaxation scheme to solve compressible flows at all speeds is proposed, with Mach numbers ranging from very small to the order of unity. The scheme is general and has the same formulation for all the considered materials, since a direct dependence on the state law is avoided via the relaxation. It is based on a fully implicit time discretization, easily implemented thanks to the linearity of the transport operator in the relaxation system. The spatial discretization is obtained by a combination of upwind and centered schemes in order to recover the correct numerical viscosity in different Mach regimes. The scheme is validated with one and two dimensional simulations of fluid flows and of deformations of compressible solids. We exploit the domain discretization through Cartesian grids, allowing for massively parallel computations (HPC) that drastically reduce the computational times on 2D test cases. Moreover, the scheme is adapted to the resolution on adaptive grids based on quadtrees, implementing adaptive mesh refinement techinques. The last part of the thesis is devoted to the numerical simulation of heterogeneous multi-material flows. A novel sharp interface method is proposed, with the derivation of implicit equilibrium conditions. The aim of the implicit framework is the solution of weakly compressible and low Mach flows, thus the proposed multi-material conditions are coupled with the implicit relaxation scheme that is solved in the bulk of the flow.
Dans cette thèse on s’intéresse à la simulation numérique d’écoulements des matériaux compressibles, voir fluides et solides élastiques. Les matériaux considérés sont décrits avec un modèle monolithique eulérian, fermé avec une loi d’état hyperélastique qui considère les différents comportéments des matériaux. On propose un nouveau schéma de relaxation qui résout les écoulements compressibles dans des différents régimes, avec des nombres de Mach très petits jusqu’à l’ordre 1. Le schéma a une formulation générale qui est la même pour tous le matériaux considérés, parce que il ne dépend pas directement de la loi d’état. Il se base sur une discrétization complétement implicite, facile à implémenter grâce à la linearité de l’opérateur de transport du système de relaxation. La discrétization en éspace est donnée par la combinaison de flux upwind et centrés, pour retrouver la correcte viscosité numérique dans les différents régimes. L’utilisation de mailles cartésiennes pour les cas 2D s’adapte bien à une parallélisation massive, qui permet de réduire drastiquement le temps de calcul. De plus, le schéma a été adapté pour la résolution sur des mailles quadtree, pour implémenter l’adaptivité de la maille avec des critères entropiques. La dernière partie de la thèse concerne la simulation numérique d’écoulements multi-matériaux. On a proposé une nouvelle méthode d’interface “sharp”, en dérivant les conditions d’équilibre en implicite. L’objectif est la résolution d’interfaces physiques dans des régimes faiblement compressibles et avec un nombre de Mach faible, donc les conditions multi-matériaux sont couplées au schéma implicite de relaxation
Correlazione tra markers infiammatori e trattamento con antipsicotici atipici in pazienti affetti da disturbo bipolare
Full text linkThe main aim of this study was to give evidences that the drugs used in the treatment of BD patients can have anti-inflammatory properties. We have longitudinally investigated in 50 BD patients the clinical outcome during six week of treatment and the effects of the drug on inflammatory markers such as CRP and ERS. In addition, in 15 patients, on the basis of the recent data of literature showing a typical anti-inflammatory pattern of quetiapine, we have additionally investigated their ability to affects plasma levels of inflammatory cytokines. A cohort of 50 BD patients were consecutively enrolled at the psychiatric unit of the Cantonal Clinic (Ticino, CH). All patients were visited three times: at each visit, all the subjects were evaluated both for clinical conditions by using clinical psychopathological scales and for biochemical parameters with complete blood formula and PRC and ERS levels. In the subgroup of 15 patients enrolled from October 2017 to May 2018 (same inclusion and exclusion criteria), we have additionally evaluated plasma levels of IL-6 and IFN-γ, and IL-10. We found a significant reduction only for CRP. In the group of 15 patients, the levels of IL-6 were dramatically reduced at all visit time and reached the statistical values for the Δ T2-T0. A direct correlation was found between clinical response and CRP plasma levels after 6 weeks of treatment. The main finding of the present study is that, antipsychotic drugs, in addition to the ability to improve clinical outcome in BP patients, are also able to profoundly affect inflammatory markers
Il bene comune della sicurezza collettiva tra politica criminale e diritto penale.
Full text linkContemporary society is pervaded by a growing request of security, but such need often derives from risk situations that are not real, but only perceived by the community; an important role is played by the media that increas the sense of collective insecurity, constantly broadcasting news on crimes. In light of the above, the government is working to protect the citizen personal safety from the newly perceived causes of risk, seeking new sources of legitimacy. The most used instrument is the Criminal Law: the introduction of new incriminating cases and tightening sanctioning treatments are often the means by which the legal system proposes to resolve emergency situations, to protect the safety of citizens and, therefore, to gain the consent of the members of the community. However, the use of such instrument led some authors to question the re-educational scope of the punishment, determining problems regarding the of the equality and non-discrimination fundamental principles. The research aims to stimulate an analysis regarding the nature of the collective sense of security, in order to formulate a juxtaposition of the common concept. This study can lead to significant applicative consequences, such as the change in the perspective and in the enhancement of the collective security management, with specific focus on the means of restorative justice. The prospected solution is the active participation of the individual in the construction of its social reintegration project, which must also involve the victims and the community. This kind of intervention unequivocally contributes to strengthen the collective sense of security, both from an objective and a subjective point of view