Studio della trasduzione del Segnale Nucleare Inositide-Dipendente: identificazione di eEF1A2 come nuovo Fosfosubstrato di PKC βI

Introduction Phospholipase Cb1 (PLC-β1) is a key player in the regulation of nuclear inositol lipid signaling and of a wide range of cellular functions, such as proliferation and differentiation (1,2,3). PLCb1 signaling depends on the cleavage of phosphatidylinositol 4,5-bisphosphate and the formation of the second messengers diacylglycerol and Inositol tris-phosphate which activate canonical protein kinase C (cPKC) isoforms. Here we describe a proteomic approach to find out a potential effector of nuclear PLC-b1 dependent signaling during insulin stimulated myogenic differentiation. Methods Nuclear lysates obtained from insulin induced C2C12 myoblasts were immunoprecipitated with anti-phospho-substrate cPKC antibody. Proteins, stained with Comassie blue, were excised, digested and subsequently analysed in LC-MS/MS. For peptide sequence searching, the mass spectra were processed and analyzed using the Mascot MS/MS ion search program with the NCBI database. Western blotting, GST-pull down and co-immunoprecipitation were performed to study the interaction between eEF1A2 and cPKCs. Site direct mutagenesis was performed to confirm the phosphorylated motif recognized by the antibody. Immunofluorescence analysis, GFP-tagged eEF1A2 vector and subcellular fractionation were performed to study nuclear localization and relative distribution of eEF1A2. Results We have previously shown that PLC-β1 is greatly increased at the nuclear level during insulin-induced myoblasts differentiation and that this nuclear localization is essential for induction of differentiation. Thus, nuclear proteins of insulin stimulated C2C12 myoblasts, were immunoprecipitated with an anti-phospho-substrate cPKC antibody. After Electrophoretic gel separation of proteins immunoprecipitated, several molecules were identified by LC-MS/MS. Among these most relevant and unexpected was eukaryotic elongation factor 1 alpha 2 (eEF1A2). We found that eEF1A2 is phosphorylated by PKCb1 and that these two molecules coimmunolocalized at the nucleolar level. eEF1A2 could be phosphorylated in many sites among which both threonine and serine residues. By site direct mutagenesis we demonstrated that it is the serine residue of the motif recognized by the antibody that is specifically phosphorylated by PKCb1. The silencing of PLCb1 gives rise to a reduction of expression and phosphorylation levels of eEF1A2 indicating this molecule as a target of nuclear PLCb1 regulatory network during myoblasts differentiation.

The dark side of the board: advances in chess Kriegspiel

While imperfect information games are an excellent model of real-world problems and tasks, they are often difficult for computer programs to play at a high level of proficiency, especially if they involve major uncertainty and a very large state space. Kriegspiel, a variant of chess making it similar to a wargame, is a perfect example: while the game was studied for decades from a game-theoretical viewpoint, it was only very recently that the first practical algorithms for playing it began to appear. This thesis presents, documents and tests a multi-sided effort towards making a strong Kriegspiel player, using heuristic searching, retrograde analysis and Monte Carlo tree search algorithms to achieve increasingly higher levels of play. The resulting program is currently the strongest computer player in the world and plays at an above-average human level.

Differenze nella prestazione tecnico-tattica tra diverse categorie di atleti nel tennistavolo di alto livello

Nell’ambito della ricerca scientifica nel campo dello sport, la Performance Analysis si sta ritagliando un crescente spazio di interesse. Per Performance Analysis si intende l’analisi della prestazione agonistica sia dal punto di vista biomeccanico che dal punto di vista dell’analisi notazionale. In questa tesi è stata analizzata la prestazione agonistica nel tennistavolo attraverso lo strumento dell’analisi notazionale, partendo dallo studio degli indicatori di prestazione più importanti dal punto di vista tecnico-tattico e dalla loro selezione attraverso uno studio sull’attendibilità nella raccolta dati. L’attenzione è stata posta quindi su un aspetto tecnico originale, il collegamento spostamenti e colpi, ricordando che una buona tecnica di spostamento permette di muoversi rapidamente nella direzione della pallina per effettuare il colpo migliore. Infine, l’obbiettivo principale della tesi è stato quello di confrontare le tre categorie di atleti selezionate: alto livello mondiale maschile (M), alto livello junior europeo (J) ed alto livello mondiale femminile (F). La maggior parte delle azioni cominciano con un servizio corto al centro del tavolo, proseguono con una risposta in push (M) o in flik di rovescio (J). Il colpo che segue è principalmente il top spin di dritto dopo un passo pivot o un top di rovescio senza spostamento. Gli alteti M e J contrattaccano maggiormente con top c. top di dritto e le atlete F prediligono colpi meno spregiudicati, bloccando di rovescio e proseguendo con drive di rovescio. Attraverso lo studio della prestazione di atleti di categorie e generi diversi è possibile migliorare le scelte strategiche prima e durante gli incontri. Le analisi statistiche multivariate (modelli log-lineari) hanno permesso di validare con metodo scientifico sia le procedure già utilizzate in letteratura che quelle innovative messe a punto per la prima volta in occasione di questo studio.

La Previsione dei Consumi Elettrici nel Mercato Energetico Italiano : un nuovo modello empirico con struttura triangolare

L'obiettivo principale della tesi è lo sviluppo di un modello empirico previsivo di breve periodo che sia in grado di offrire previsioni precise ed affidabili dei consumi di energia elettrica su base oraria del mercato italiano. Questo modello riassume le conoscenze acquisite e l'esperienza fatta durante la mia attuale attività lavorativa presso il Romagna Energia S.C.p.A., uno dei maggiori player italiani del mercato energetico. Durante l'ultimo ventennio vi sono stati drastici cambiamenti alla struttura del mercato elettrico in tutto il mondo. Nella maggior parte dei paesi industrializzati il settore dell'energia elettrica ha modificato la sua originale conformazione di monopolio in mercato competitivo liberalizzato, dove i consumatori hanno la libertà di scegliere il proprio fornitore. La modellazione e la previsione della serie storica dei consumi di energia elettrica hanno quindi assunto un ruolo molto importante nel mercato, sia per i policy makers che per gli operatori. Basandosi sulla letteratura già esistente, sfruttando le conoscenze acquisite 'sul campo' ed alcune intuizioni, si è analizzata e sviluppata una struttura modellistica di tipo triangolare, del tutto innovativa in questo ambito di ricerca, suggerita proprio dal meccanismo fisico attraverso il quale l'energia elettrica viene prodotta e consumata nell'arco delle 24 ore. Questo schema triangolare può essere visto come un particolare modello VARMA e possiede una duplice utilità, dal punto di vista interpretativo del fenomeno da una parte, e previsivo dall'altra. Vengono inoltre introdotti nuovi leading indicators legati a fattori meteorologici, con l'intento di migliorare le performance previsive dello stesso. Utilizzando quindi la serie storica dei consumi di energia elettrica italiana, dall'1 Marzo 2010 al 30 Marzo 2012, sono stati stimati i parametri del modello dello schema previsivo proposto e valutati i risultati previsivi per il periodo dall'1 Aprile 2012 al 30 Aprile 2012, confrontandoli con quelli forniti da fonti ufficiali.

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

Mechanisms Contributing to Tyrosin Kinase Inhibitor Resistance in GISTs: Toward a Personalized Therapy

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract. This work considers the pharmacological response in GIST patients treated with imatinib by two different angles: the genetic and somatic point of view. We analyzed polymorphisms influence on treatment outcome, keeping in consideration SNPs in genes involved in drug transport and folate pathway. Naturally, all these intriguing results cannot be considered as the only main mechanism in imatinib response. GIST mainly depends by oncogenic gain of function mutations in tyrosin kinase receptor genes, KIT or PDGFRA, and the mutational status of these two genes or acquisition of secondary mutation is considered the main player in GIST development and progression. To this purpose we analyzed the secondary mutations to better understand how these are involved in imatinib resistance. In our analysis we considered both imatinib and the second line treatment, sunitinib, in a subset of progressive patients. KIT/PDGFRA mutation analysis is an important tool for physicians, as specific mutations may guide therapeutic choices. Currently, the only adaptations in treatment strategy include imatinib starting dose of 800 mg/daily in KIT exon-9-mutated GISTs. In the attempt to individualize treatment, genetic polymorphisms represent a novelty in the definition of biomarkers of imatinib response in addition to the use of tumor genotype. Accumulating data indicate a contributing role of pharmacokinetics in imatinib efficacy, as well as initial response, time to progression and acquired resistance. At the same time it is becoming evident that genetic host factors may contribute to the observed pharmacokinetic inter-patient variability. Genetic polymorphisms in transporters and metabolism may affect the activity or stability of the encoded enzymes. Thus, integrating pharmacogenetic data of imatinib transporters and metabolizing genes, whose interplay has yet to be fully unraveled, has the potential to provide further insight into imatinib response/resistance mechanisms.

Interactions between the gut microbiota, short-chain fatty acids and the immune system in pediatric patients undergoing hematopoietic stem cell transplantation

The gut microbiota (GM) is essential for human health and contributes to several diseases; indeed it can be considered an extension of the self and, together with the genetic makeup, determines the physiology of an organism. In this thesis has been studied the peripheral immune system reconstitution in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) in the early phase; in parallel, have been also explored the gut microbiota variations as one of the of primary factors in governing the fate of the immunological recovery, predisposing or protecting from complications such as the onset of acute graft-versus-host disease (GvHD). Has been demonstrated, to our knowledge for the first time, that aHSCT in pediatric patients is associated to a profound modification of the GM ecosystem with a disruption of its mutualistic asset. aGvHD and non-aGvHD subjects showed differences in the process of GM recovery, in members abundance of the phylum Bacteroidetes, and in propionate fecal concentration; the latter are higher in the pre-HSCT composition of non-GvHD subjects than GvHD ones. Short-chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients and distribute systemically from the gut to blood. For this reason, has been studied their effect in vitro on human DCs, the key regulators of our immune system and the main player of aGvHD onset. Has been observed that propionate and, particularly, butyrate show a strong and direct immunomodulatory activity on DCs reducing inflammatory markers such as chemokines and interleukins. This study, with the needed caution, suggests that the pre-existing GM structure can be protective against aGvHD onset, exerting its protective role through SCFAs. They, indeed, may regulate cell traffic within secondary lymphoid tissues, influence T cell development during antigen recognition, and, thus, directly shape the immune system.

Relevance of the pig stomach for the detection of dietary factors and gut maturation and control

In this thesis two approaches were applied to achieve a double general objective. The first chapter was dedicated to the study of the distribution of the expression of genes of several bitter and fat receptor in several gastrointestinal tracts. A set of 7 genes for bitter taste and for 3 genes for fat taste was amplified with real-time PCR from mRNA extracted from 5 gastrointestinal segments of weaned pigs. The presence of gene expression for several chemosensing receptors for bitter and fat taste in different compartments of the stomach confirms that this organ should be considered a player for the early detection of bolus composition. In the second chapter we investigated in young pigs the distribution of butyrate-sensing olfactory receptor (OR51E1) receptor along the GIT, its relation with some endocrine markers, its variation with age, and after interventions affecting the gut environment and intestinal microbiota in piglets and in different tissues. Our results indicate that OR51E1 is strictly related to the normal GIT enteroendocrine activity. In the third chapter we investigated the differential gene expression between oxyntic and pyloric mucosa in seven starter pigs. The obtained data indicate that there is significant differential gene exression between oxintic of the young pig and pyloric mucosa and further functional studies are needed to confirm their physiological importance. In the last chapter, thymol, that has been proposed as an oral alternative to antibiotics in the feed of pigs and broilers, was introduced directly into the stomach of 8 weaned pigs and sampled for gastric oxyntic and pyloric mucosa. The analysis of the whole transcript expression shoes that the stimulation of gastric proliferative activity and the control of digestive activity by thymol can influence positively gastric maturation and function in the weaned pigs.