Ruolo della fosfolipasi C-β1 nel differenziamento miogenico: identificazione di nuovi target nucleari

The expression of phospholipase C-β1 (PLC-β1) and cyclin D3 is highly induced during skeletal myoblast differentiation. We have previously shown that PLC-β1 activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-β1 is a crucial regulator of mouse cyclin D3 gene. Here we report that PLC-β1 catalytic activity plays a role in the increase of cyclin D3 levels and in the induction of differentiation of C2C12 skeletal muscle cells. PLC-β1 mutational analysis revealed the importance of His331 and His378 for the catalytic activity. We show that following insulin administration, cyclin D3 mRNA levels are lower in cells overexpressing the PLC-β1 catalytically inactive form, as compared to wild type cells. We describe a novel signaling pathway elicited by PLC-β1 that modulates Activator Protein-1 (AP-1) activity. Indeed, gel mobility shift assays indicate that there is a c-jun binding site located in cyclin D3 promoter region specifically regulated by PLC-β1 and that c-jun binding activity is significantly increased by insulin stimulation and PLC-β1 overexpression. Moreover, mutation of c-jun/AP-1 binding site decreases the basal cyclin D3 promoter activity and eliminates its induction by insulin and PLC-β1 overexpression. Interestingly, we observed that the ectopic expression of the Inositol Polyphosphate Multikinase (IPMK) in C2C12 myoblasts enhances cyclin D3 gene expression and that the mutation of c-jun site in cyclin D3 promoter determines an impairment of IPMK-dependent promoter induction. These results indicate that PLC-β1 activates a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation through IPMK signaling.

Gli affidamenti c.d. in house nell'ordinamento comunitario e nazionale con particolare riferimento al settore dei trasporti pubblici

Il presente progetto di ricerca analizza quella particolare forma di affidamento diretto dei servizi pubblici denominata in house providing e si articola in tre sezioni. Nella prima sezione viene analizzata la disciplina dei servizi pubblici locali nell’ordinamento italiano mediante un excursus normativo dai primi del 900 ad oggi; la seconda sezione è dedicata alla disciplina dell’affidamento dei servizi pubblici locali di trasporto; la terza sezione, infine, descrive l’in house providing e l’elaborazione pretoria di tale istituto operata dalla giurisprudenza comunitaria. Come noto, la pubblica amministrazione può soddisfare le sue esigenze secondo due diverse modalità: ricorrendo al libero mercato come qualsiasi altro operatore economico oppure auto-producendo i beni e i servizi di cui necessita. Infatti, nonostante il diritto comunitario imponga il rispetto del principio di tutela della concorrenza, lascia impregiudicato il potere di auto-organizzazione in capo alle pubbliche amministrazioni negli Stati membri, le quali potranno scegliere di agire “in economia” o di ricorrere alle prestazioni di operatori terzi. Con la locuzione di derivazione comunitaria in house providing si definisce quel modello organizzativo mediante il quale le pubbliche amministrazioni realizzano le attività di loro competenza attraverso i propri organismi, cioè senza ricorrere al libero mercato per procurarsi i lavori, i servizi e le forniture ad esse occorrenti o per erogare alla collettività prestazioni di pubblico servizio, in deroga ai principi comunitari sulla tutela della concorrenza stabiliti nel Trattato istitutivo della Comunità Europea, che invece imporrebbero lo svolgimento di gare ad evidenza pubblica per l'affidamento di tali servizi. Tuttavia, come chiarito dalla giurisprudenza comunitaria e nazionale, affinché la procedura di gara non sia necessaria, occorre che tra l’amministrazione e il prestatore ci sia sostanziale identità, nonostante le distinte personalità giuridiche, in modo tale da configurare il contratto tra le stesse intercorso come un atto di organizzazione interna.

The impact of phosphoinositide metabolic enzymes in glioblastoma: a tale of phosphoinositide-specific phospholipase C PLCβ1 and 5-phosphatase SKIP

Background: Glioblastoma multiforme (GBM) is one of the deadliest and most aggressive form of primary brain tumor. Unfortunately, current GBM treatment therapies are not effective in treating GBM patients. They usually experience very poor prognosis with a median survival of approximately 12 months. Only 3-5% survive up to 3 years or more. A large-scale gene profile study revealed that several genes involved in essential cellular processes are altered in GBM, thus, explaining why existing therapies are not effective. The survival of GBM patients depends on understanding the molecular and key signaling events associated with these altered physiological processes in GBM. Phosphoinositides (PI) form just a tiny fraction of the total lipid content in humans, however they are implicated in almost all essential biological processes, such as acting as second messengers in spatio-temporal regulation of cell signaling, cytoskeletal reorganization, cell adhesion, migration, apoptosis, vesicular trafficking, differentiation, cell cycle and post-translational modifications. Interestingly, these essential processes are altered in GBM. More importantly, incoming reports have associated PI metabolism, which is mediated by several PI phosphatases such as SKIP, lipases such as PLCβ1, and other kinases, to regulate GBM associated cellular processes. Even as PLCβ1 and SKIP are involved in regulating aberrant cellular processes in several other cancers, very few studies, of which majority are in-silico-based, have focused on the impact of PLCβ1 and SKIP in GBM. Hence, it is important to employ clinical, in vitro, and in vivo GBM models to define the actual impact of PLCβ1 and SKIP in GBM. AIM: Since studies of PLCβ1 and SKIP in GBM are limited, this study aimed at determining the pathological impact of PI metabolic enzymes, PLCB1 and SKIP, in GBM patient samples, GBM cell line models, and xenograft models for SKIP. Results: For the first time, this study confirmed through qPCR that PLCβ1 gene expression is lower in human GBM patient samples. Moreover, PLCβ1 gene expression inversely correlates with pathological grades of glioma; it decreases as glioma grades increases or worsens. Silencing PLCβ1 in U87MG GBM cells produces a dual impact in GBM by participating in both pro-tumoral and anti-tumoral roles. PLCβ1 knockdown cells were observed to have more migratory abilities, increased cell to extracellular matrix (ECM) adhesion, transition from epithelial phenotype to mesenchymal phenotype through the upregulation of EMT transcription factors Twist1 and Slug, and mesenchymal marker, vimentin. On the other hand, p-Akt and p-mTOR protein expression were downregulated in PLCβ1 knockdown cells. Thus, the oncogenic pathway PI3K/Akt/mTOR pathway is inhibited during PLCβ1 knockdown. Consistently, cell viability in PLCβ1 knockdown cells were significantly decreased compared to controls. As for SKIP, this study demonstrated that about 48% of SKIP colocalizes with nuclear PtdIns(4,5)P2 to nuclear speckles and that SKIP knockdown alters nuclear PtdIns(4,5)P2 in a cell-type dependent manner. In addition, SKIP silencing increased tumor volume and weight in xenografts than controls by reducing apoptosis and increasing viability. All in all, these data confirm that PLCβ1 and SKIP are involved in GBM pathology and a complete understanding of their roles in GBM may be beneficial.

The conceptualisation of music in ancient Greek thought (V century B.C. – II century B.C.)

The focus of this dissertation is the analysis of the music-related philosophical passages from the 5th century B.C. to the 2nd century B.C. It aims to provide a multifaceted view towards music as a cultural phenomenon, which is based primarily on the philological and culturological explorations instead of the technical-musicological approach. The texts from our selected period attest that mousikē had an extremely broad conceptualisation which led to the attribution of the different, sometimes completely opposite value: from an insignificant performative practice to an activity which corresponds to the divine laws and directly affects the human soul. The discussed testimonia provide evidence of defining music both as an exclusively acoustic phenomenon and as a philosophically significant concept that oversteps the sonic definition. Our sources clearly demonstrate that mousikē was a polysemous term: it was understood as an interdisciplinary form of art (as the arts of the Muses), though it was also used to indicate the exclusively instrumental music or a philosophical concept, which does not necessarily define sound as its essential quality. The aim of this dissertation is to clarify the arguments behind each of these positions, to analyse whether such different modes of conceptualisation are compatible among themselves, and to see how they fit together into explaining what was understood as music in Antiquity. In this thesis we explore the conceptual framework of mousikē and analyse what enabled the musical thought to be worthy of the attention of the greatest philosophical minds. We will demonstrate that it was not the sound or the artistic practices that were central in the philosophical thought on music, but instead the embedded structural qualities that have correspondence to the universal proportions of the cosmic world and which are perceptible to the listeners through the medium of sound.