Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

La psicologia della Gestalt. Ricerche teoriche e sperimentali sulla percezione sonora e musicale (1890-1939)

The aim of the present work is a historical survey on Gestalt trends in psychological research between late 19th and the first half of 20th century with privileged reference to sound and musical perception by means of a reconsideration of experimental and theoretical literature. Ernst Mach and Christian von Ehrenfels gave rise to the debate about Gestaltqualität which notably grew thanks to the ‘Graz School’ (Alexius Meinong, Stephan Witasek, Anton Faist, Vittorio Benussi), where the object theory and the production theory of perception were worked out. Stumpf’s research on Tonpsychologie and Franz Brentano’s tradition of ‘act psychology’ were directly involved in this debate, opposing to Wilhelm Wundt’s conception of the discipline; this clearly came to light in Stumpf’s controversy with Carl Lorenz and Wundt on Tondistanzen. Stumpf’s concept of Verschmelzung and his views about consonance and concordance led him to some disputes with Theodor Lipps and Felix Krueger, lasting more than two decades. Carl Stumpf was responsible for education of a new generation of scholars during his teaching at the Berlin University: his pupils Wolfgang Köhler, Kurt Koffka and Max Wertheimer established the so-called ‘Berlin School’ and promoted the official Gestalt theory since the 1910s. After 1922 until 1938 they gave life and led together with other distinguished scientists the «Psychologische Forschung», a scientific journal in which ‘Gestalt laws’ and many other acoustical studies on different themes (such as sound localization, successive comparison, phonetic phenomena) were exposed. During the 1920s Erich Moritz von Hornbostel gave important contributions towards the definition of an organic Tonsystem in which sound phenomena could find adequate arrangement. Last section of the work contains descriptions of Albert Wellek’s studies, Kurt Huber’s vowel researches and aspects of melody perception, apparent movement and phi-phenomenon in acoustical field. The work contains also some considerations on the relationships among tone psychology, musical psychology, Gestalt psychology, musical aesthetics and musical theory. Finally, the way Gestalt psychology changed earlier interpretations is exemplified by the decisive renewal of perception theory, the abandon of Konstanzannahme, some repercussions on theory of meaning as organization and on feelings in musical experience.

Il "caso" Maroncelli. Organizzazione del consenso e vincoli massonici nell'Italia radicale e nella New York della Tammany Hall, 1840-1890

Questa tesi parte da un evento “minore” della storia del XIX secolo per tendere, poi, ad alcuni obiettivi particolari. L’evento è costituito da uno strano funerale postumo, quello di Piero Maroncelli, carbonaro finito alla Spielberg, graziato, poi emigrato in Francia e in America, il cui corpo viene sepolto a New York nell’estate del 1846. Quarant’anni dopo, nel 1886, i resti di Maroncelli vengono esumati e – attraverso una “trafila” particolare, di cui è protagonista la Massoneria, da una sponda all’altra dell’Atlantico – solennemente trasferiti nella città natale di Forlì, dove vengono collocati nel pantheon del cimitero monumentale, inaugurato per l’occasione. Gli eventi celebrativi che si consumano a New York e a Forlì sono molto diversi fra loro, anche se avvengono quasi in contemporanea e sotto regie politiche ispirate dal medesimo radicalismo anticlericale. E’ chiaro che Maroncelli è un simbolo e un pretesto: simbolo di un’italianità transnazionale, composta di un “corpo” in transito, fuori dello spazio nazionale, ma appartenente alla patria (quella grande e quella piccola); pretesto per acquisire e rafforzare legami politici, mercè mobilitazioni di massa in un caso fondate sul festival, sulla festa en plein air, nell’altro sui rituali del cordoglio per i “martiri” dell’indipendenza. L’opportunità di comparare questi due contesti – l’Italia, colta nella sua periferia radicale e la New York della Tammany Hall -, non sulla base di ipotesi astratte, ma nella concretezza di un “caso di studio” reale e simultaneo, consente di riflettere sulla pervasività dell’ideologia democratica nella sua accezione ottocentesca, standardizzata dalla Massoneria, e, d’altro canto, sui riti del consenso, colti nelle rispettive tipicità locali. Un gioco di similitudini e di dissomiglianze, quindi. Circa gli obiettivi particolari – al di là della ricostruzione del “caso” Maroncelli -, ho cercato di sondare alcuni temi, proponendone una ricostruzione in primo luogo storiografica. Da un lato, il tema dell’esilio e del trapianto delle esperienze di vita e di relazione al di fuori del proprio contesto d’origine. Maroncelli utilizza, come strumento di identitario e di accreditamento, il fourierismo; la generazione appena successiva alla sua, grazie alla Giovine Italia, possiede già un codice interno, autoctono, cui fare riferimento; alla metà degli anni Cinquanta, ad esso si affiancherà, con successo crescente, la lettura “diplomatica” di ascendenza sabauda. Dall’altro, ho riflettuto sull’aspetto legato ai funerali politici, al cordoglio pubblico, al trasferimento postumo dei corpi. La letteratura disponibile, al riguardo, è assai ricca, e tale da consentire una precisa classificazione del “caso” Maroncelli all’interno di una tipologia della morte laica, ben delineata nell’Italia dell’Ottocento e del primo Novecento. E poi, ancora, ho preso in esame le dinamiche “festive” e di massa, approfondendo quelle legate al mondo dell’emigrazione italiana a Mew York, così distante nel 1886 dall’élite colta di quarant’anni prima, eppure così centrale per il controllo politico della città. Dinamiche alle quali fa da contrappunto, sul versante forlivese, la visione del mondo radicale e massonico locale, dominato dalla figura di Aurelio Saffi e dal suo tentativo di plasmare un’immagine morale e patriottica della città da lasciare ai posteri. Quasi un’ossessione, per il vecchio triumviro della Repubblica romana (morirà nel 1890), che interviene sulla toponomastica, sull’edilizia cemeteriale, sui pantheon civico, sui “ricordi” patriottici. Ho utilizzato fonti secondarie e di prima mano. Anche sulle prime, quantitativamente assai significative, mi sono misurata con un lavoro di composizione e di lettura comparata prima mai tentato. La giustapposizione di chiavi di lettura apparentemente distanti, ma giustificate dalla natura proteiforme e complicata del nostro “caso”, apre, a mio giudizio, interessanti prospettive di ricerca. Circa le fonti di prima mano, ho attinto ai fondi disponibili su Maroncelli presso la Biblioteca comunale di Forlì, alle raccolte del Grande Oriente d’Italia a Roma, a periodici italoamericani assai rari, sparsi in diverse biblioteche italiane, da Milano a Roma. Mi rendo conto che la quantità dei materiali reperiti, sovente molto eterogenei, avrebbero imposto una lettura delle fonti più accurata di quella che, in questa fase della ricerca, sono riuscita a condurre. E’ vero, però, che le suggestioni già recuperabili ad un’analisi mirata al contenuto principale – le feste, la propaganda, il cordoglio – consentono la tessitura di una narrazione non forzata, nella quale il ricordo della Repubblica romana viaggia da una sponda all’altra dell’Atlantica, insieme ai resti di Maroncelli; nella quale il rituale massonico funge da facilitatore e da “mediatore culturale”; nella quale, infine, il linguaggio patriottico e la koinè democratica trans-nazionale riescono incredibilmente a produrre o a incarnare identità. Per quanto tempo? La risposta, nel caso forlivese, è relativamente facile; in quello della “colonia” italiana di New York, presto alterata nella sua connotazione demografica dalla grande emigrazione transoceanica, le cose appaiono più complesse. E, tuttavia, nel 1911, cinquantesimo dell’Unità, qualcuno, nella grande metropoli americana, si sarebbe ricordato di Maroncelli, sia pure in un contesto e con finalità del tutto diverse rispetto al 1886: segno che qualcosa, sotto traccia, era sopravvissuto.