Analysis of the biological functions of Kidins220: from cells to organisms.

In this work, I have characterised the biological functions of Kidins220 (Kinase D interacting substrate of 220 kDa) in both an in vitro and in vivo context. Kidins220 is a conserved membrane protein mainly expressed in neuronal cells, which has been implicated in the process of neuronal differentiation in response to neurotrophic stimuli. In the first part of this study, I present an analysis of the molecular mechanism regulating the intracellular trafficking of Kidins220 in a rat pheochromocytoma cell line (PC 12), which upon treatment with nerve growth factor (NGF) differentiates into neuronal-like cells resembling sympathetic neurons. Kidins220 transport to neurite tips is driven by the microtubule-dependent motor complex kinesin-1. Perturbation of Kidins220 trafficking in this cellular system reduces the activation of the signalling cascades initiated by NGF and impairs neuronal differentiation. I was interested in understanding the mechanisms that might modulate the association of Kidins220 to kinesin-1. Kidins220 recruits kinesin-1 via a novel binding motif, which does not share any similarities with the known kinesin-1- interacting signatures. I found that Abelson tyrosine kinase (Abl) phosphorylates Kidins220 at the level of this kinesin interacting motif, thus inhibiting the binding to kinesin light chain. These preliminary results suggest that phosphorylation might act as a molecular switch, to mediate the release of Kidins220 from the kinesin-1 motor complex. In the second part of this work, I analysed the effects of Kidins220 depletion in living organisms. I tackled this problem by targeting Kidins220 by RNA interference in Drosophila melanogaster. In an independent approach, I have generated a construct, based on the Cre/LoxP recombination system, for the conditional knockout of the Kidins220 gene in mice. Kidins220 null animals die at late stages of embryonic development, and display severe cardiovascular and neurological defects. Their phenotype is described in detail using a variety of immunohistochemical and cell biological approaches. This work therefore presents new evidence supporting the notion that Kidins220 plays an important role in regulating not only neuronal function and differentiation, but also other key developmental processes such as cardiac development

Mild Inactivation of RE-1 Silencing Transcription Factor (REST) Reduces Susceptibility to Kainic Acid-Induced Seizures

RE-1 Silencing Transcription factor (REST) controls several steps in neural development by modulating the expression of a wide range of neural genes. Alterations in REST expression have been associated with the onset of epilepsy; however, whether such alterations are deleterious or represent a protective homeostatic response remains elusive. To study the impact of REST modulation on seizure propensity, we developed a tool for its negative modulation in vivo. The tool is composed of the paired-amphipathic helix 1 (PAH1) domain, a competitive inhibitor of REST activation by mSin3, fused to the light-oxygen-voltage sensing 2 (LOV2) domain of Avena sativa phototropin 1, a molecular switch to alternatively hide or expose the PAH1 inhibitor. We employed the C450A and I539E light-independent AsLOV2 variants to mimic the closed (inactive) and open (active) states of LOV2-PAH1, respectively. Recombinant AAV1/2 viral particles (rAAVs) allowed LOV2-PAH1 expression in HEK293T cells and primary neurons, and efficiently transduced hippocampal neurons in vivo. mRNA expression analysis revealed an increased expression of several neuronal genes in the hippocampi of mice expressing the open probe. AAV-transduced mice received a single dose of kainic acid (KA), a treatment known to induce a transient increase of REST levels in the hippocampus. Remarkably, mice expressing the active variant displayed a reduced number of KA-induced seizures, which were less severe compared to mice carrying the inactive probe. These data support the validity of our tool to modulate REST activity in vivo and the potential impact of REST modulation on epileptogenesis

Engineering REST-Specific Synthetic PUF Proteins to Control Neuronal Gene Expression: A Combined Experimental and Computational Study

Regulation of gene transcription is an essential mechanism for differentiation and adaptation of organisms. A key actor in this regulation process is the repressor element 1 (RE1)-silencing transcription factor (REST), a transcriptional repressor that controls more than 2000 putative target genes, most of which are neuron-specific. With the purpose of modulating REST expression, we exploited synthetic, ad hoc designed, RNA binding proteins (RBPs) able to specifically target and dock to REST mRNA. Among the various families of RBPs, we focused on the Pumilio and FBF (PUF) proteins, present in all eukaryotic organisms and controlling a variety of cellular functions. Here, a combined experimental and computational approach was used to design and test 8- and 16-repeat PUF proteins specific for REST mRNA. We explored the conformational properties and atomic features of the PUF-RNA recognition code by Molecular Dynamics simulations. Biochemical assays revealed that the 8- and 16-repeat PUF-based variants specifically bind the endogenous REST mRNA without affecting its translational regulation. The data also indicate a key role of stacking residues in determining the binding specificity. The newly characterized REST-specific PUF-based constructs act as excellent RNA-binding modules and represent a versatile and functional platform to specifically target REST mRNA and modulate its endogenous expression

Control of Au nanoantenna emission enhancement of magnetic dipolar emitters by means of VO2 phase change layers

Active, ultra-fast external control of the emission properties at the nanoscale is of great interest for chip-scale, tunable and efficient nanophotonics. Here we investigated the emission control of dipolar emitters coupled to a nanostructure made of an Au nanoantenna, and a thin vanadium dioxide (VO2) layer that changes from semiconductor to metallic state. If the emitters are sandwiched between the nanoantenna and the VO2 layer, the enhancement and/or suppression of the nanostructure’s magnetic dipole resonance enabled by the phase change behavior of the VO2 layer can provide a high contrast ratio of the emission efficiency. We show that a single nanoantenna can provide high magnetic field in the emission layer when VO2 is metallic, leading to high emission of the magnetic dipoles; this emission is then lowered when VO2 switches back to semiconductor. We finally optimized the contrast ratio by considering different orientation, distribution and nature of the dipoles, as well as the influence of a periodic Au nanoantenna pattern. As an example of a possible application, the design is optimized for the active control of an Er3+ doped SiO2 emission layer. The combination of the emission efficiency increase due to the plasmonic nanoantenna resonances and the ultra-fast contrast control due to the phase-changing medium can have important applications in tunable efficient light sources and their nanoscale integration

Tuning ZnO nanorods photoluminescence through atmospheric plasma treatments

Room temperature atmospheric plasma treatments are widely used to activate and control chemical functionalities at surfaces. Here, we investigated the effect of atmospheric pressure plasma jet (APPJ) treatments in reducing atmosphere (Ar/1 parts per thousand H-2 mixture) on the photoluminescence (PL) properties of single crystal ZnO nanorods (NRs) grown through hydrothermal synthesis on fluorine-doped tin oxide glass substrates. The results were compared with a standard annealing process in air at 300 degrees C. Steady-state photoluminescence showed strong suppression of the defect emission in ZnO NRs for both plasma and thermal treatments. On the other side, the APPJ process induced an increase in PL quantum efficiency (QE), while the annealing does not show any improvement. The QE in the plasma treated samples was mainly determined by the near band-edge emission, which increased 5-6 fold compared to the as-prepared samples. This behavior suggests that the quenching of the defect emission is related to the substitution of hydrogen probably in zinc vacancies (V-Zn), while the enhancement of UV emission is due to doping originated by interstitial hydrogen (H-i), which diffuses out during annealing. Our results demonstrate that atmospheric pressure plasma can induce a similar hydrogen doping as ordinarily used vacuum processes and highlight that the APPJ treatments are not limited to the surfaces but can lead to subsurface modifications. APPJ processes at room temperature and under ambient air conditions are stable, convenient, and efficient methods, compared to thermal treatments to improve the optical and surface properties of ZnO NRs, and remarkably increase the efficiency of UV emission. (c) 2019 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Fabrication of biocompatible free-standing nanopatterned films for primary neuronal cultures

Devising and constructing biocompatible devices for nervous system regeneration is an extremely challenging task. Besides tackling the issue of biocompatibility, biomaterials for neuroscience applications should mimic the complex environment of the extracellular matrix, which in vivo provides neurons with a series of cues and signals to guide cells towards their appropriate targets. In this work, a novel nanopatterned biocompatible poly-ε-caprolactone (PCL) film is realized to assist the attachment and growth of primary hippocampal neurons. Costly and time-consuming processes can be avoided using plasma-surface nanotexturing obtained by a mixed gas SF6/Ar at -5 °C. The intrinsic composition and line topography of nanopatterned PCL ensure healthy development of the neuronal network, as shown by confocal microscopy, by analysing the expression of a range of neuronal markers typical of mature cultures, as well as by scanning electron microscopy. In addition, we show that surface nanopatterning improves differentiation of neurons compared to flat PCL films, while no neural growth was observed on either flat or nanopatterned substrates in the absence of a poly-d-lysine coating. Thus, we successfully optimized a nanofabrication protocol to obtain nanostructured PCL layers endowed with several mechanical and structural characteristics that make them a promising, versatile tool for future tissue engineering studies aimed at neural tissue regeneration

SURGERY IN MALIGNANT GERM CELL TUMORS OF CHILDHOOD. RESULTS OF THE SECOND ITALIAN COOPERATIVE STUDY TCG 98

Analysis of treatment and results of the patientsenrolled in the Italian TCG-98 Study, still open and comparison of data with those of the previous Studt TCG-9

Interfacing Graphene-Based Materials With Neural Cells

The scientific community has witnessed an exponential increase in the applications of graphene and graphene-based materials in a wide range of fields, from engineering to electronics to biotechnologies and biomedical applications. For what concerns neuroscience, the interest raised by these materials is two-fold. On one side, nanosheets made of graphene or graphene derivatives (graphene oxide, or its reduced form) can be used as carriers for drug delivery. Here, an important aspect is to evaluate their toxicity, which strongly depends on flake composition, chemical functionalization and dimensions. On the other side, graphene can be exploited as a substrate for tissue engineering. In this case, conductivity is probably the most relevant amongst the various properties of the different graphene materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation, which holds a great potential in regenerative medicine. In this review, we try to give a comprehensive view of the accomplishments and new challenges of the field, as well as which in our view are the most exciting directions to take in the immediate future. These include the need to engineer multifunctional nanoparticles (NPs) able to cross the blood-brain-barrier to reach neural cells, and to achieve on-demand delivery of specific drugs. We describe the state-of-the-art in the use of graphene materials to engineer three-dimensional scaffolds to drive neuronal growth and regeneration in vivo, and the possibility of using graphene as a component of hybrid composites/multi-layer organic electronics devices. Last but not least, we address the need of an accurate theoretical modeling of the interface between graphene and biological material, by modeling the interaction of graphene with proteins and cell membranes at the nanoscale, and describing the physical mechanism(s) of charge transfer by which the various graphene materials can influence the excitability and physiology of neural cells

Polarized Raman mapping and phase-transition by CW excitation for fast purely optical characterization of VO2 thin films

Vanadium dioxide has attracted much interest due to the drastic change of the electrical and optical properties it exhibits during the transition from the semiconductor state to the metallic state, which takes place at a critical temperature of about 68 °C. Much study has been especially devoted to developing advanced fabrication methodologies to improve the performance of VO2 thin films for phase-change applications in optical devices. Films structural and morphological characterisation is normally performed with expensive and time consuming equipment, as x-ray diffractometers, electron microscopes and atomic force microscopes. Here we propose a purely optical approach which combines Polarized Raman Mapping and Phase-Transition by Continuous Wave Optical Excitation (PTCWE) to acquire through two simple measurements structural, morphological and thermal behaviour information on polycrystalline VO2 thin films. The combination of the two techniques allows to reconstruct a complete picture of the properties of the films in a fast and effective manner, and also to unveil an interesting stepped appearance of the hysteresis cycles probably induced by the progressive stabilization of rutile metallic domains embedded in the semiconducting monoclinic matrix

The November 2017 Mw 5.5 Pohang earthquake: a possible case of induced seismicity in South Korea

The Mw 5.5 earthquake that struck South Korea in November 2017 was one of the largest and most damaging events in this country over the last century. Its proximity to an Enhanced Geothermal Systems site, where high pressure hydraulic injection had been performed during the previous two years, raises the possibility that this earthquake was anthropogenic. We have combined seismological and geodetic analyses to characterize the mainshock and its largest aftershocks, constrain the geometry of this seismic sequence and shed light on its casual factors. According to our analysis it seems plausible that the occurrence of this earthquake was influenced by these industrial activities. Finally we found that the earthquake transferred static stress to larger nearby faults, potentially increasing the seismic hazard in the area