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Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factorsBedini, Andrea <1979> 06 June 2008 (has links)
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.
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Genomic and non genomic effects of elevated concentration of anabolic steroids in human neuronal cellsGuarino, Goffredo <1979> 06 June 2008 (has links)
Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter
the expression and function of neurotransmitter systems and contribute to neuronal cell death.
This effect can explain the behavioural changes, drug dependence and neuro degeneration
observed in steroid abuser.
Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent
downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma
cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide.
Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated
cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that
nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human
MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene
transcription. These results suggest that nandrolone may regulate MOPr expression through
post-transcriptional mechanisms requiring the AR.
Cito-toxicity assays demonstrated a time- and concentration dependent decrease of
cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is
independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was
observed in cells treated with Nandrolone 10-6M for 48h.
Collectively, these data support the existence of two cellular mechanisms that might
explain the neurological syndromes observed in steroids abuser.
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L'infiammazione intestinale nell'animale sperimentale come modello per lo sviluppo di nuovi farmaci: ruolo della via tachichininergica nelle malattie infiammatorie intestinaliUrsino, Maria Grazia <1979> 27 March 2008 (has links)
Background: Several lines of evidence showed that inflammation is associated with changes
in the expression of tachykinins both in human and animal models. Tachykinins, including
substance P (SP), are small peptides expressed in the extrinsic primary afferent nerve fibres
and enteric neurons of the gut: they exert their action through three distinct receptors, termed
NK1, NK2 and NK3. SP modulates intestinal motility and enteric secretion, acting
preferentially through the NK1 receptor. SP neural network and NK1 receptor expression are
increased in patients with inflammatory bowel disease, and similar changes were observed in
experimental models of inflammation. The 2,4 Dinitrobenzene Sulphonic Acid (DNBS) model
of colitis is useful to study innate immunity, non-specific inflammation and wound healing; it
has been suggested that the transmural inflammation seen in this model resembles that found
in Crohns disease and can therefore be used to study what cells and mediators are involved in
this type of inflammation.
Aim: To test the possible protective effect of the NK1 receptor antagonist SSR140333 on:
1) acute model of intestinal inflammation; 2) reactivation of DNBS-induced colitis in rats.
Methods: Acute colitis was induced in male SD rats by intrarectal administration of DNBS
(15 mg/rat in 50% ethanol). Reactivation of colitis was induced by intrarectal injections of
DNBS on day 28 (7.5 mg/rat in 35% ethanol). Animals were sacrificed on day 6 (acute colitis)
and 29 (reactivation of colitis). SSR140333 (10 mg/kg) was administered orally starting from
the day before the induction of colitis for 7 days (acute colitis) or seven days before the
reactivation of colitis. Colonic damage was assessed by means of macroscopic and
microscopic scores, myeloperoxidase activity (MPO) and TNF-α tissue levels. Enzyme
immunoassay was used to measure colonic substance P levels. Statistical analysis was
performed using analysis of variance (one-way or two-way, as appropriate) with the
Bonferronis correction for multiple comparisons.
Results: DNBS administration impaired body weight gain and markedly increased all
inflammatory parameters (p<0.01). Treatment with SSR140333 10 mg/kg significantly
counteracted the impairment in body weight gain, decreased macroscopic and histological
scores and reduced colonic myeloperoxidase activity (p<0.01). Drug treatment counteracted
TNF-α tissue levels and colonic SP concentrations (acute model). Similar results were
obtained administering the NK1 receptor antagonist SSR140333 (3 and 10 mg/kg) for 5 days,
starting the day after the induction of colitis. Intrarectal administration of DNBS four weeks
after the first DNBS administration resulted in reactivation of colitis, with increases in
macroscopic and histological damage scores and increase in MPO activity. Preventive
treatment with SSR140333 10 mg/kg decreased macroscopic damage score, significantly
reduced microscopic damage score but did not affect MPO activity.
Conclusions: Treatment with SSR140333 significantly reduced intestinal damage in acute
model of intestinal inflammation in rats. The NK1 receptor antagonist SSR140333 was also
able to prevent relapse in experimental colitis. These results support the hypothesis of SP
involvement in intestinal inflammation and indicate that NK receptor antagonists may have a
therapeutic potential in inflammatory bowel disease.
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Messa a punto di metodi per lo studio della plasticità neuronale del sistema nervoso entericoAlessandri, Marco <1976> 27 March 2008 (has links)
No description available.
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Nuove strategie farmacologiche per il superamento della farmacoresistenza in chemioterapia antitumoraleSciuscio, Davide <1979> 27 March 2008 (has links)
No description available.
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Effects of cannabidiol and cannabis extracts in models of convulsion and excitotoxicityUtan, Aneli <1974> 27 March 2008 (has links)
No description available.
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Isotiocianati come potenziali farmaci antileucemici: identificazione in vitro ed ex vivo del profilo molecolare e cellulareLenzi, Monia <1977> 27 March 2008 (has links)
Il presente studio ha come obbiettivo lo sviluppo di composti di origine naturale come potenziali farmaci antitumorali, attraverso la definizione dei loro specifici target cellulari e molecolari su diversi modelli cellulari ad alta predittività.
Gli isotiocianati, contenuti nei vegetali appartenenti alla famiglia delle Crucifereae, sono dotati di una comprovata capacità di inibire la formazione di tumori in modelli animali preventivamente trattati con cancerogeni. Questa attività è riconducibile principalmente alla modulazione degli enzimi coinvolti nell’attivazione/detossificazione di xenobiotici e ad effetti citostatici e citossici, osservati su numerose linee cellulari.
Un isotiocianato particolarmente promettente è il sulforafane (SFN). La ricerca condotta durante il periodo di dottorato si è, quindi, focalizzata sull’isotiocianato SFN e in particolare sulla sua capacità di modulare specifici eventi cellulari e molecolari coinvolti nel processo di leucemogenesi.
Inizialmente è stato indagato il potenziale citostatico e citotossico del SFN su una linea cellulare T linfoblastoide (cellule Jurkat), con particolare attenzione agli effetti sulla proliferazione cellulare, all’induzione di apoptosi/necrosi e all’analisi di alcuni dei meccanismi molecolari coinvolti negli effetti citostatici e citotossici dell’isotiocianato ( livelli proteici di p53, bax e bcl-2).
Successivamente, poiché requisiti fondamentali di un antitumorale sono selettività d’azione e scarsa tossicità, è stato indagato il potenziale citostatico e citotossico dell’isotiocianato SFN sulla controparte non trasformata delle cellule leucemiche T linfoblastoidi, analizzando gli stessi eventi studiati su cellule tumorali e alcuni dei meccanismi molecolari coinvolti (livelli proteici di ciclina D2, ciclina D3, chinasi ciclina dipendente (CDK) 4 e CDK6 ).
Il SFN si è dimostrato in grado di indurre apoptosi sulle cellule Jurkat e di inibirne la proliferazione, mediante un blocco in fase G2/M del ciclo cellulare e un incremento dei livelli di p53 e bax. Il SFN è in grado di indurre effetti citostatici e citotossici anche su linfociti T non trasformati. Tuttavia, le dosi necessarie per esibire tali effetti sono ben più elevate di quelle attive su cellule leucemiche.
Una tappa importante nello sviluppo di un farmaco antitumorale è, la definizione, dove possibile, dei suoi effetti in un modello ex vivo, altamente predittivo di quella che sarà la risposta farmacologica in vivo. Sono stati quindi valutati gli effetti del SFN su colture primarie di blasti provenienti da pazienti affetti da diversi tipi di leucemia , sia mieloide che linfoblastica.
Il SFN non sembra possedere alcuna attività su campioni da pazienti affetti da LLC, mentre un importante attività proapoptotica si registra nei campioni da pazienti affetti da LMA, dove l’effetto del SFN è sorprendentemente marcato anche su campioni da pazienti multiresistenti.
L’attività dell’isotiocianato sui campioni da pazienti affetti da LLA è decisamente più marcata sul campione da paziente affetto da LLA a cellule B, mentre sul campione di Leucemia Acuta Bifenotipica l’effetto proapoptotico del SFN si registra dopo tempi di trattamento brevi piuttosto che dopo tempi di trattamento più lunghi.
In conclusione, i risultati ottenuti evidenziano che il SFN possiede un’interessante attività antileucemica in vitro e, dato di particolare rilevanza, anche ex vivo.
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Development of screening assays to test novel integrin antagonists in allergic inflammationSpartà, Antonino Maria <1977> 05 May 2009 (has links)
Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and
to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action.
Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils.
Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may
play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.
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Coinvolgimento del recettore NOP nei meccanismi molecolari attivati da esposizione cronica a cannabinoidi, oppiacei ed alcolCannarsa, Rosalia <1975> 24 March 2009 (has links)
No description available.
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Nuovo modello animale per lo studio della working memory: validazione farmacologica e applicazioniLocchi, Federica <1980> 24 March 2009 (has links)
The present study was performed to validate a spatial working memory task using pharmacological manipulations. The water escape T-maze, which combines the advantages of the Morris water maze and the T-maze while minimizes the disadvantages, was used. Scopolamine, a drug that affects cognitive function in spatial working memory tasks, significantly decreased the rat performance in the present delayed alternation task. Since glutamate neurotransmission plays an important role in the maintaining of working memory, we evaluated the effect of ionotropic and metabotropic glutamatergic receptors antagonists, administered alone or in combination, on rat behaviour. As the acquisition and performance of memory tasks has been linked to the expression of the immediately early gene cFos, a marker of neuronal activation, we also investigated the neurochemical correlates of the water escape T-maze after pharmacological treatment with glutamatergic antagonists, in various brain areas. Moreover, we focused our attention on the involvement of perirhinal cortex glutamatergic neurotransmission in the acquisition and/or
consolidation of this particular task. The perirhinal cortex has strong and reciprocal connections with both specific cortical sensory areas and some memory-related structures, including the hippocampal formation and amygdala. For its peculiar position, perirhinal cortex has been recently regarded as a key region in working memory processes, in particular in providing temporary maintenance of information. The effect of perirhinal cortex lesions with ibotenic acid on the acquisition and consolidation of the water escape T-maze task was evaluated. In conclusion, our data suggest that the water escape T-maze could be considered a valid, simple and quite fast method to assess spatial working memory, sensible to pharmacological
manipulations. Following execution of the task, we observed cFos expression in several brain regions. Furthermore, in accordance to literature, our results suggest that glutamatergic neurotransmission plays an important role in the acquisition and consolidation of working memory processes.
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