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.

Letteratura postmoderna e comunicazione pubblicitaria, problemi teorici e analisi testuali

Premise: In the literary works of our anthropological and cultural imagination, the various languages and the different discursive practices are not necessarily quoted, expressly alluded to or declared through clear expressive mechanisms; instead, they rather constitute a substratum, a background, now consolidated, which with irony and intertextuality shines through the thematic and formal elements of each text. The various contaminations, hybridizations and promptings that we find in the expressive forms, the rhetorical procedures and the linguistic and thematic choices of post-modern literary texts are shaped as fluid and familiar categories. Exchanges and passages are no longer only allowed but also inevitable; the post-modern imagination is made up of an agglomeration of discourses that are no longer really separable, built up from texts that blend and quote one another, composing, each with its own specificities, the great family of the cultural products of our social scenario. A literary work, therefore, is not only a whole phenomenon, delimited hic et nunc by a beginning and an ending, but is a fragment of that complex, dense and boundless network that is given by the continual interrelations between human forms of communication and symbolization. The research hypothesis: A vision is delineated of comparative literature as a discipline attentive to the social contexts in which texts take shape and move and to the media-type consistency that literary phenomena inevitably take on. Hence literature is seen as an open systematicity that chooses to be contaminated by other languages and other discursive practices of an imagination that is more than ever polymorphic and irregular. Inside this interpretative framework the aim is to focus the analysis on the relationship that postmodern literature establishes with advertising discourse. On one side post-modern literature is inserted in the world of communication, loudly asserting the blending and reciprocal contamination of literary modes with media ones, absorbing their languages and signification practices, translating them now into thematic nuclei, motifs and sub-motifs and now into formal expedients and new narrative choices; on the other side advertising is chosen as a signification practice of the media universe, which since the 1960s has actively contributed to shaping the dynamics of our socio-cultural scenarios, in terms which are just as important as those of other discursive practices. Advertising has always been a form of communication and symbolization that draws on the collective imagination – myths, actors and values – turning them into specific narrative programs for its own texts. Hence the aim is to interpret and analyze this relationship both from a strictly thematic perspective – and therefore trying to understand what literature speaks about when it speaks about advertising, and seeking advertising quotations in post-modern fiction – and from a formal perspective, with a search for parallels and discordances between the rhetorical procedures, the languages and the verifiable stylistic choices in the texts of the two different signification practices. The analysis method chosen, for the purpose of constructive multiplication of the perspectives, aims to approach the analytical processes of semiotics, applying, when possible, the instruments of the latter, in order to highlight the thematic and formal relationships between literature and advertising. The corpus: The corpus of the literary texts is made up of various novels and, although attention is focused on the post-modern period, there will also be ineludible quotations from essential authors that with their works prompted various reflections: H. De Balzac, Zola, Fitzgerald, Joyce, Calvino, etc… However, the analysis focuses the corpus on three authors: Don DeLillo, Martin Amis and Aldo Nove, and in particular the followings novels: “Americana” (1971) and “Underworld” (1999) by Don DeLillo, “Money” (1984) by Martin Amis and “Woobinda and other stories without a happy ending” (1996) and “Superwoobinda” (1998) by Aldo Nove. The corpus selection is restricted to these novels for two fundamental reasons: 1. assuming parameters of spatio-temporal evaluation, the texts are representative of different socio-cultural contexts and collective imaginations (from the masterly glimpses of American life by DeLillo, to the examples of contemporary Italian life by Nove, down to the English imagination of Amis) and of different historical moments (the 1970s of DeLillo’s Americana, the 1980s of Amis, down to the 1990s of Nove, decades often used as criteria of division of postmodernism into phases); 2. adopting a perspective of strictly thematic analysis, as mentioned in the research hypothesis, the variations and the constants in the novels (thematic nuclei, topoi, images and narrative developments) frequently speak of advertising and inside the narrative plot they affirm various expressions and realizations of it: value ones, thematic ones, textual ones, urban ones, etc… In these novels the themes and the processes of signification of advertising discourse pervade time, space and the relationships that the narrator character builds around him. We are looking at “particle-characters” whose endless facets attest the influence and contamination of advertising in a large part of the narrative developments of the plot: on everyday life, on the processes of acquisition and encoding of the reality, on ideological and cultural baggage, on the relationships and interchanges with the other characters, etc… Often the characters are victims of the implacable consequentiality of the advertising mechanism, since the latter gets the upper hand over the usual processes of communication, which are overwhelmed by it, wittingly or unwittingly (for example: disturbing openings in which the protagonist kills his or her parents on the basis of a spot, former advertisers that live life codifying it through the commercial mechanisms of products, sons and daughters of advertisers that as children instead of playing outside for whole nights saw tapes of spots.) Hence the analysis arises from the text and aims to show how much the developments and the narrative plots of the novels encode, elaborate and recount the myths, the values and the narrative programs of advertising discourse, transforming them into novel components in their own right. And also starting from the text a socio-cultural reference context is delineated, a collective imagination that is different, now geographically, now historically, and from comparison between them the aim is to deduce the constants, the similarities and the variations in the relationship between literature and advertising.

Il viaggio celeste di Paolo (2Cor 12,1-10). Particolarità e funzionalità di una forma di contatto con il soprannaturale

L’obiettivo principale di questa ricerca è quello di affrontare le questioni critiche poste all’esegesi da uno dei passi paolini più discussi. Grazie all’impiego delle categorie connesse all’esperienza religiosa, si vuole argomentare la possibilità di leggere il viaggio celeste in termini di modello culturale attraverso il quale interpretare un’esperienza di contatto con il soprannaturale. L’approccio individuato può consentire di rivalutare la funzione di tale narrazione non solo nel contesto di 2 Corinzi, bensì anche in relazione ai paralleli letterari di matrice tanto giudaica e protocristiana quanto greco-romana. Conseguenza di simile analisi ad ampio raggio è il riconoscimento dell’importanza attribuita da Paolo ad esperienze di questo tipo, come testimoniato da alcuni brani centrali delle sue lettere.

L'inno avestico agli Aməsha Spəṇtas (Yasht 2). Testo critico e traduzione con commentario storico religioso

L'inno dedicato ai sette Amesha Spəṇta è parte della produzione avestica recenziore, e si compone in gran parte di porzioni testuali riprese da altri testi avestici a loro volta di formazione tardiva. Lo Yašt si divide in tre parti principali: le stanze 0-10; 11-14; e infine la stanza 15 che comprende la formula di chiusura tipica degli inni avestici. La prima sezione (2.0-10) è composta dalla formula di apertura, incompleta rispetto a quelle dei restanti inni, seguita dai primi sette capitoli di entrambi i Sīh-rōzag compresi i Gāh. Le stanze centrali (11-14) si caratterizzano per l'assenza di passi gemelli, un elevato numero di hapax e di arcaismi formali e inoltre, una grande variabilità nella tradizione manoscritta. Si tratta di una formula magica per esorcizzare/allontanare demoni e stregoni, che doveva essere recitata per sette volte. Tale formula probabilmente rappresentava in origine un testo autonomo che veniva recitato assieme ad altri testi avestici. La versione a noi pervenuta comprende la recitazione di parte di entrambi i Sīh-rōzag, ma è molto probabile che tale arrangement sia soltanto una sequenza recitativa che doveva coesistere assieme ad altre. Attualmente la formula magica viene recitata principalmente assieme allo Yasna Haptaŋhāiti, senza le restanti stanze dell'inno nella sua versione geldneriana. Il testo sembra nascere come formula magica la quale venne recitata assieme a diversi testi avestici come per esempio parti dello Sīh-rōzag. In un periodo impossibile da stabilire con certezza la versione viene fissata nella forma a noi pervenuta nella maggior parte dei manoscritti e per la sua affinità formale probabilmente interpretato come inno e perciò incluso nell'innario avestico.

Modelling the infrastructure of the “Śivadharma Database”

A Digital Scholarly Edition is a conceptually and structurally sophisticated entity. Throughout the centuries, diverse methodologies have been employed to reconstruct a text transmitted through one or multiple sources, resulting in various edition types. With the advent of digital technology in philology, these practices have undergone a significant transformation, compelling scholars to reconsider their approach in light of the web. In the digital age, philologists are expected to possess (too) advanced technical skills to prepare interactive and enriched editions, even though, in most cases, only mechanical or documentary editions are published online. The Śivadharma Database is a web Content Management System (CMS) designed to facilitate the preparation, publication, and updating of Digital Scholarly Editions. By providing scholars with a user-friendly CRUD web application to reconstruct and annotate a text, they can prepare their textus with additional components such as apparatus, notes, translations, citations, and parallels. It is possible by leveraging an annotation system based on HTML and graph data structure. This choice is made because the text entity is multidimensional and multifaceted, even if its sequential presentation constrains it. In particular, editions of South Asian texts of the Śivadharma corpus, the case study of this research, contain a series of phenomena that are difficult to manage formally, such as overlapping hierarchies. Hence, it becomes necessary to establish the data structure best suited to represent this complexity. In Śivadharma Database, the textus is an HTML file readily displayable. Textual fragments, annotated via an interface without requiring philologists to write code and saved in the backend, form the atomic unit of multiple relationships organised in a graph database. This approach enables the formal representation of complex and overlapping textual phenomena, allowing for good annotation expressiveness with minimal effort to learn the relevant technologies during the editing workflow.