Development and Characterisation of PC12 Cell Lines Allowing Inducible Expression of Prion Proteins Carrying Pathogenic Mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP)p gene that are presumed to favor conversion of PrP into a neurotoxic isoform (PrpSc) Several cellular models of inherited prion diseases have been Q developed in which mutant PrP costitutively expressed acquires PrPSc-like properties but is not cytotoxic. However, the use of constitutive models does not exclude the potential toxicity of mutant PrP, as it can be speculated that only J clones resistant to PrP toxicity are selected after clonal selection. To rule out n this possibility a tetracycline-inducible (Tet-on) model was developed in this thesis, in which PrP expression is switched on after clonal selection. cDNAs encoding mouse wild-type PrP, as well as mouse PrP homologues of the human D178N (D177N/M128 and D177NA/128) and nine-octapeptide (PG14) mutations under the control of the tetracycline-responsive element, were transfected in PC12 Tet-on cells. The Tet-on system was chosen based on the lack of pleiotropic or toxic effects and because it allows a tightly regulated expression of the protein of interest. To ensure unwanted background expression of PrP in the absence of inducer, cells were co-transfected with J pTet-tTS, encoding the tet-controlled transcriptional silencer. The D177N and PG14 mutants expressed in this system displayed biochemical properties reminiscent of PrP^^, including detergent insolubility and low protease resistance. Low levels of mutant PrPs were detected on the cell surface by confocal immunofluorescence analysis compared to wild-type PrP. These mutant displayed reticular intracellular distribution, suggesting impaired delivery to the cell surface and retention in the endoplasmic reticulum (ER). Evaluation of cellular viability revealed a decrement in cell survival after 96 h of mutant PrP expression, associated with an increased number of apoptotic cells. This effect was not fully consistent and was not exacerbated by Q neuronal differentiation with NGF or by treating cells with FI2O2, tunicamycin, or by removing serum. The results presented in this thesis also demonstrate that expression of PrP does not protect PC 12 Tet-on cells from such stresses. To explore the possibility that expression of mutant PrPs triggered toxic -1 response pathways in the ER, the mRNA level of ER stress markers was measured. The results shown in this thesis indicate that, although the altered intracellular distribution of mutant PrPs suggested retention of the protein in the endoplasmic reticulum, ER stress responses were not detected

Ripensare le professioni politecniche: dalla pratica alla formazione? / Rethinking polytechnic professions: from practice to education?

L’articolo propone una riflessione collettiva sul senso e sui modi della formazione e della pratica professionale a partire dalla constatazione di come le crisi, le sfide e le tecnologie di oggi superino i perimetri che caratterizzano le specializzazioni di ieri. Inevitabilmente è l’individuazione degli strumenti, delle strategie e delle risorse da impegnare per raggiungere un obiettivo tanto ambizioso quanto urgente. In una contingenza in cui le istituzioni pubbliche e accademiche riconoscono esplicitamente il valore dell’interdisciplinarità, il presente contributo esplora lo scarto che esiste tra il modo in cui l’interdisciplinarità viene insegnata e quello in cui viene attuata in esperienze professionali. L’articolo affronta questa questione analizzando attraverso le parole dei protagonisti i meccanismi attraverso cui l’innovazione si produce in esperienze professionali virtuose sviluppate fuori, per poi interrogarsi su come queste possano nutrire le sperimentazioni in corso dentro le università. A collective reflection on the meaning and modalities of education and professional practice today originates from the observation that today’s crises, challenges and technologies exceed the perimeters that characterize yesterday’s specializations. Inevitably, opaque is the identification of the tools, strategies, and resources to be committed to achieve an objective that is as ambitious as it is urgent. In a contingency in which public and academic institutions explicitly recognise the value of interdisciplinarity, this contribution explores the gap that exists between the way interdisciplinarity is taught and the way it is implemented in professional experiences. To address this issue, the article starts from analysing through the words of the protagonists the mechanisms through which innovation is produced in virtuous professional experiences developed outside, and then questions how these can nourish the experiments taking place inside universities

Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases

Progettare la creazione di valore. Verso una trasformazione dei quartieri periferici

L’articolo propone un’integrazione di metodi e fonti relativi, da un lato, all’ambito della progettazione architettonica e, dall’altro, alle discipline estima-tive. Analizzando alcuni trend emergenti nella domanda in ambito residenziale, posti in evidenza dalla pandemia di Covid-19 ma riconducibili a fenomeni carat-terizzati da traiettorie molto più lunghe, la ricerca ambisce a evidenziare alcune criticità e lacune che caratterizzano le attuali pratiche progettuali e valutative e fare così nuova luce sul potenziale valore di spazi tipicamente sottoutilizzati – e sottostimati – ma altamente trasformabili. Il testo introduce così una riflessione di carattere critico-metodologico, che offre una nuova prospettiva per l’analisi di quartieri caratterizzati da un alto livello di “periferizzazione”

A Smo/Gli multitarget hedgehog pathway inhibitor impairs tumor growth

Pharmacological Hedgehog (Hh) pathway inhibition has emerged as a valuable anticancer strategy. A number of small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector glioma-associated oncogene 1 (Gli1) has been designed and developed. In a recent study, we exploited the high versatility of the natural isoflavone scaffold for targeting the Hh signaling pathway at multiple levels showing that the simultaneous targeting of Smo and Gli1 provided synergistic Hh pathway inhibition stronger than single administration. This approach seems to effectively overcome the drug resistance, particularly at the level of Smo. Here, we combined the pharmacophores targeting Smo and Gli1 into a single and individual isoflavone, compound 22, which inhibits the Hh pathway at both upstream and downstream level. We demonstrate that this multitarget agent suppresses medulloblastoma growth in vitro and in vivo through antagonism of Smo and Gli1, which is a novel mechanism of action in Hh inhibition

New Transparent Laser-Drilled Fluorine-doped Tin Oxide covered Quartz Electrodes for Photo-Electrochemical Water Splitting

A new-designed transparent, conductive and porous electrode was developed for application in a compact laboratory-scale proton exchange membrane (PEM) photo-electrolyzer. The electrode is made of a thin transparent quartz sheet covered with fluorine-doped tin oxide (FTO), in which an array of holes is laser-drilled to allow water and gas permeation. The electrical, morphological, optical and electrochemical characterization of the drilled electrodes is presented in comparison with a non-drilled one. The drilled electrode exhibits, in the visible region, a good transmittance (average value of 62%), a noticeable reflectance due to the light scattering effect of the hole-drilled internal region, and a higher effective surface area than the non-drilled electrode. The proof-of-concept of the applicability of the drilled electrode was achieved by using it as a support for a traditional photocatalyst (i.e. commercial TiO2 nanoparticles). The latter, coupled with a polymeric electrolyte membrane (i.e.Nafion 117) and a Pt counter electrode, forms a transparent membrane electrode assembly (MEA), with a good conductivity, wettability and porosity. Electrochemical impedance spectroscopy (EIS) was used as a very powerful tool to gain information on the real active surface of the new drilled electrode and the main electrochemical parameters driving the charge transfer reactions on it. This new electrode architecture is demonstrated to be an ideal support for testing new anodic and cathodic photoactive materials working in tandem configuration for solar fuels production by water photo-electrolysis

Tiny events: tales of urban domesticity from Lingotto, a former working-class neighborhood in Turin

Article published by 12 authors, coordinated by Filippo De Pieri and Gaia Caramellino, as "The Housing History Collective