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Although the number of inactive students is rather high in Italian University (35%), the figures are distinctively lower for medical students (14% of inactive students and 3-5% of renouncing). A recent survey among medical students at La Sapienza University of Rome has singled out the following as the main causes of graduation delay: difficulties in studying (43%); delayed matriculation (due to controversies in the application competition) (16%); psychological upset (13%); family (6%) or health (3%) problems; and economical difficulties (3%). The causes of graduation delay have been debated by the National Conference of the Undergraduate Curricula Presidents in a Forum held in Novara. Four types of causes and solutions have been discussed: i) teaching causes and active tutoring; ii) psychological upset and counselling; iii) social, cultural and economic hardships and counter window interventions; and iv) disproportion between didactic load and learning capabilities, and educational interventions. In conclusion, the need for monitoring student upset and for reducing disciplinary Learning objects (subject to quick obsolescence) in favour of methodological ones (that favour a lifelong learning) has been assessed.

This Forum is a part of the tetralogy of educational events devoted to “Training of teachers, supervisors and students to leadership and teamwork”. In a tetralogy the so called “Pedagogical pills” are mini-lectures, the workshops are based on experiential learning, forums are the privileged environment in which the Conference co-constructs its shared knowledge, since they are small group activities focused on sharing and discussion of real life experience. In this forum we identified some critical situations in which teamwork asks for a special attention, specially when the team is inter-professional. The following articles report on the most significant and common elements emerged from the discussion. The considered environments were the team of teachers of an integrated course, the relationship between teachers and administrative- technical personnel dn the inter-professional collaboration, probably the most challenging context in the near future.

Adiponectin (Acrp30) is an adipocyte-secreted hormone with pleiotropic metabolic effects, whose reduced levels were related to development and progression of several malignancies. We looked at the presence of Acrp30 receptors in human glioblastomas (GBM), hypothesizing a role for Acrp30 also in this untreatable cancer. Here we demonstrate that human GBM express Acrp30 receptors (AdipoR1 and AdipoR2), which are often co-expressed in GBM samples (70% of the analyzed tumors). To investigate the effects of Acrp30 on GBM growth, we used human GBM cell lines U87-MG and U251, expressing both AdipoR1 and AdipoR2 receptors. In these cells, Acrp30 treatment inhibits DNA synthesis and cell proliferation rate, inducing arrest in G1 phase of the cell cycle. These effects were correlated to a sustained activation of ERK1/2 and Akt kinases, upon Acrp30 treatment. Our results suggest that Acrp30 may represent a novel endogenous negative regulator of GBM cell proliferation, to be evaluated for the possible development of novel pharmacological approaches. © 2014 Wiley Periodicals, Inc.

NTP1 Background: The dynamic intra- and inter-chromosomal links between specific loci contribute to the creation of cell type-specific gene expression profiles and to gene regulation during differentiation processes. Looping is implicated in bringing together far upstream or downstream regions with the gene promoter and body sites, and in establishing contacts between the 5' and 3' ends of genes, since 3' end-processing factors interact with components of the transcriptional machinery. The tumor suppressor PRDM2/RIZ gene plays a role in controlling cellular processes, such as cell cycle progression and regulation of development. The retinoblastoma proteinineracting zinc-finger gene (RIZ) is estrogen responsive and has two alternative promoters, the more downstream of which, promoter 2, is nearby to an EREsequence and is involved in estrogen receptor transcriptional activation. Methods: With the innovative DNA-Picked Chromatin (DPC) assay after timecourse of 17-ßestradiol (E2) induction of MCF-7 breast cancer cells, we highlight preferential interaction between hormone-responsive RIZ promoter and the polyadenylation sites. Gene expression analysis of induced cell RNA was performed with qRT-PCR assay. Results: Within 60’ of E2 treatment of cells, we have observed increased exon segments, exons 9a and 10 (alternative polyA site), linked to isolated promoter 2 and concomitant decrease of exon10 to RIZ promoter 1. The exon 9a shows a low association to RIZ promoter 1 without E2. qRT-PCR also demonstrated increased exon 9a-containing transcripts. Conclusions: The E2 remodels the chromatin architecture of PRDM2/RIZ gene locus to create a loop for the mRNA transcription with polyA-exon 9a, leading to the production of oncogenic variants.

ST2. PRDM Gene Products in Testicular Germ Cell Tumors E. di Zazzo1, C. Porcile1, C. De Rosa2, A. Marino1, S. Bartollino1, C. Abbondanza2, B. Moncharmont1 1Università degli Studi del Molise, Campobasso, Italy; 2Seconda Università degli studi di Napoli, Naples, Italy Background: Testicular germ cell tumors (TGCT) originate from primordial germ cells blocked at different stages during maturation, reflecting different histological tumor subtypes. A common genetic alteration in TGCT is a deletion of chromosome 1 short arm, where the PRDM2 gene, a member of positive regulatory domain gene family, is located. Moreover recent studies demonstrated that members of PRDM gene family have an essential role in the early stages of testicular development. The aim of this study is to evaluate PRDM gene family members for a possible tumorsuppressor function in TGCT. Methods: PRDM gene expression was assessed by mRNA RT-PCR. Cells were treated with 100 nM 17β-Estradiol (E2), 100 nM DHT or 10 uM RA in serum free medium for 24h. RNA interference was performed using BLOCK-iT™ Pol II miR RNAi system. Proliferation assay was performed with propidium iodide staining and FACS analysis. Results: In GC1 mouse spermatogonial cells treatment with proliferation agents 5α-dihydrotestosterone (DHT) and E2 reduced PRDM2/RIZ1 expression levels whereas PRDM2 total forms showed no variation; the same treatment significantly increased PRDM4 and PRDM10 expression levels. Silencing PRDM2 gene expression by RNA interference increased PRDM10 expression levels and reduced the proliferation rate of spermatogonia. Conclusions: In spermatogonia as in MCF-7 cell line, E2 and DHT regulate PRDM2 gene expression suggesting that PRDM2 gene products could mediate the effect of these agents on cell cycle progression. PRDM4 and PRDM10 are also responsive to steroid hormones and PRDM10 probably cooperates with PRDM2, as demonstrated by the increase of its expression levels after PRDM2 gene silencing.

Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm.

Immunocytochemical demonstration of estrogen receptors in 115 human breast cancer specimens was performed using mouse monoclonal antibodies against estrogen receptor and avidin-biotin as the displaying system. The antibody indicated a highly heterogeneous endowment of neoplastic cells with estrogen receptor at both nuclear and cytoplasmic levels. The percentage of labeled cells within each tumor specimen was recorded to compare this immunocytochemical assay with the biochemical assay of estrogen receptors by the dextran-coated charcoal method. A significant correlation was observed between these two assays. The present results show that estrogen receptors can be confidently demonstrated at the single cell level, thus providing additional information to quantitative biochemical assays. Their prognostic and therapeutic predictive powers may be usefully integrated, particularly in view of the heterogeneous distribution of receptors among cancer cells.

Objective Glioblastoma (GBM) is the most aggressive and common form of primary brain cancer. Survival is poor and improved treatment options are urgently needed. Dual specificity phosphatase-6 (DUSP6) is actively involved in oncogenesis showing unexpected tumor-promoting properties in human glioblastoma, contributing to the development and expression of the full malignant and invasive phenotype. The purpose of this study was to assess if DUSP6 activates epithelial-to-mesenchymal transition (EMT) in glioblastoma and its connection with the invasive capacity. Results We found high levels of transcripts mRNA by qPCR analysis in a panel of primary GBM compared to adult or fetal normal tissues. At translational levels, these data correlate with high protein expression and long half-life values by cycloheximide-chase assay in immunoblot experiments. Next, we demonstrate that DUSP6 gene is involved in epithelial-to-mesenchymal transition (EMT) in GBM by immunoblot characterization of the mesenchymal and epithelial markers. Vimentin, N-Cadherin, E-Cadherin and fibronectin were measured with and without DUSP6 over-expression, and in response to several stimuli such as chemotherapy treatment. In particular, the high levels of vimentin were blunted at increasing doses of cisplatin in condition of DUSP6 over-expression while N-Cadherin contextually increased. Finally, DUSP6 per se increased invasion capacity of GBM. Overall, our data unveil the DUSP6 involvement in invasive mesenchymal-like properties in GBM.

The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [Su(var)3-9, enhancer-of-zeste and trithorax] domain of histone methyltransferases (HMTs). These genes are involved in epigenetic regulation of gene expression through their intrinsic HMTase activity or via interactions with other chromatin modifying enzymes. In this way they control a broad spectrum of biological processes, including proliferation and differentiation control, cell cycle progression, and maintenance of immune cell homeostasis. In cancer, tumor-specific dysfunctions of PRDM genes alter their expression by genetic and/or epigenetic modifications. A common characteristic of most PRDM genes is to encode for two main molecular variants with or without the PR domain. They are generated by either alternative splicing or alternative use of different promoters and play opposite roles, particularly in cancer where their imbalance can be often observed. In this scenario, PRDM proteins are involved in cancer onset, invasion, and metastasis and their altered expression is related to poor prognosis and clinical outcome. These functions strongly suggest their potential use in cancer management as diagnostic or prognostic tools and as new targets of therapeutic intervention.

The famous sentence “Après moi le déluge” (“After me the flood”), attributed to Louis XV, king of France, makes reference to the perception that one’s own successor is not going to be suitable to the task. The situation seems to apply to plenty of the Italian Presidents of the Undergraduate Curriculum in Medicine, who continually succeed one another, with the new President too often bound to restart from the beginning. The most obvious solution to this problem is to institutionalize the handover process. However, the presence of collegiate bodies, helping the President to manage the Curriculum, is another important tool to ensure continuity between the succeeding Presidents. Technical Committee for Planning teaching and education (TCP) was created in 2000 by the Permanent Conference of Presidents of the Undergraduate Curricula in Medicine and every Italian Undergraduate Curriculum should have such a structure. However, the functions attributed to TCP are changing because of the new tasks the President is nowadays facing, from faculty development, to the quality assurance, to the needs to second the paradigm shift from a teacher-based teaching to a student-centred learning. As a consequence, TCP requirements are changing and it is time to give birth to a 2.0 TCP to help Presidents to face nowadays challenges.

The article describes the ideas discussed during the labs organized by the Educational Team at the 131 CPPCLMMC Conference. The educational issues discussed during the three ateliers were: flipped classroom, video use in medical education and medical humanities. These three areas can be seen as prototypical examples of students centred teaching strategies. As result of the groups activities, the positive role of active and cooperative learning, the relevance of the faculty for identify the educational goals and related didactic strategies, the role of medical humanities in fostering a patient-centred doctor, was pointed out.

Prostate cancer is one of the most commonly diagnosed cancers in men, and androgen deprivation therapy still represents the primary treatment for prostate cancer patients. This approach, however, frequently fails and patients develop castration-resistant prostate cancer, which is almost untreatable. Cancer cells are characterized by a hierarchical organization, and stem/progenitor cells are endowed with tumor-initiating activity. Accumulating evidence indicates that prostate cancer stem cells lack the androgen receptor and are, indeed, resistant to androgen deprivation therapy. In contrast, these cells express classical (α and/or ß) and novel (GPR30) estrogen receptors, which may represent new putative targets in prostate cancer treatment. In the present review, we discuss the still-debated mechanisms, both genomic and non-genomic, by which androgen and estradiol receptors (classical and novel) mediate the hormonal control of prostate cell stemness, transformation, and the continued growth of prostate cancer. Recent preclinical and clinical findings obtained using new androgen receptor antagonists, anti-estrogens, or compounds such as enhancers of androgen receptor degradation and peptides inhibiting non-genomic androgen functions are also presented. These new drugs will likely lead to significant advances in prostate cancer therapy.

The Presidents of the Italian Undergraduate Curricula in Medicine have faced the problem of remediation in a workshop held in Udine on the 22nd September 2017. Presidents have been subdivided into small groups, have been given detailed data about a hypothetic undergraduate curriculum and have been asked to devise remediation strategies adequate to the case study proposed. Data included general information about the School of Medicine, its structure and scientific excellences, and amount and composition of teaching staff. Information has been given also on the region where the School is placed, its population and social-economical parameters, and on the number and origin of medical students. Entity of students’ graduation delay has been detailed. Undergraduate curriculum has been illustrated giving detailed information about the examinations: their number, weight in credits, deployment through the curriculum, and number of students passing them in due time, with minimum, maximum and average score. Rules for students entry blocks for the following year and information about the consistency of tutoringcounselling services and of teachers continuous education strategies have been finally given. The Presidents of Undergraduate Curricula in Medicine have reached the conclusions that they should obtain the necessary detailed information about entity and causes of their students’ graduation delay. Remediation strategies should include active and peer-to-peer tutoring and forms of flexibility in assessment design and curriculum re-modelling.

In order to understand the oncogenic properties of retinoblastoma-interacting zinc-finger (RIZ) gene products, we produced an MCF-7-derived cell line expressing a fusion protein containing the zinc-finger (aa 359–497) domain of RIZ protein (MCF-7/znf). The Zn-finger domain contains three of the eight putative Zn-finger motifs and is located in proximity of the E1A-like domain containing the Rb protein-binding motif. The MCF-7/znf cells showed a higher growth rate than the parental or the control cell lines, both in hormone-deprived conditions or upon estrogen stimulation. Furthermore, they were less sensitive to the growth inhibitory effect of anti-estrogens and showed a higher level of expression of cyclin D1 and A. The expressed Zn-finger domain recombinant product was localized in the nucleus and in the nucleoli and its expression modified the pattern of actin staining in the cytoplasm. In conclusion the presented results indicated that the Zn-finger domain could be endowed with the putative oncogenic activity of RIZ2 gene product.

Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor a and activate ERK1/2 upon E-2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-alpha on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of beta-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival.

Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co-activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced in vivo , in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control.

PRDM (PRDI-BF1 and RIZ homology domain containing) protein family members are characterized by the presence of a PR domain and a variable number of Zn-finger repeats. Experimental evidence has shown that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals like steroid hormones and control the expression of growth factors. PRDM proteins therefore have a pivotal role in the transduction of signals that control cell proliferation and differentiation and consequently neoplastic transformation. In this review, we describe pathways in which PRDM proteins are involved and the molecular mechanism of their transcriptional regulation.

Struttura e metabolismo delle lipoproteine plasmatiche, Enzimi e trasportatori chiave del metabolismo delle lipoproteine, Recettori delle lipoproteine, Metabolismo delle lipoproteine contenenti apoB, Biogenesi delle HDL e trasporto inverso del colesterolo, Principali malattie del metabolismo lipidico.

""The retinoblastoma protein-interacting zinc-finger (RIZ) gene, also known as PRDM2, encodes two protein products, RIZ1 and RIZ2, differing for the presence of a 202 aa domain, called PR domain, at the N-terminus of the RIZ1 molecule. While the histone H3 K9 methyltransferase activity of RIZ1 is associated with the negative control of cell proliferation, no information is currently available on either expression regulation of the RIZ2 form or on its biological activity. RIZ proteins act as ER co-activators and promote optimal estrogen response in female reproductive tissues. In estrogen-responsive cells, 17-beta estradiol modulates RIZ gene expression producing a shift in the balanced expression of the two forms. Here, we demonstrate that an estrogen-responsive element (ERE) within the RIZ promoter 2 is regulated in a ligand-specific manner by ERa, through both the AF1 and AF2 domains. The pattern of ERa binding, histone H4 acetylation, and histone H3 cyclical methylation of lysine 9 was comparable to other estrogen-regulated promoters. Association of topoisomerase II beta with the RIZ promoter 2 confirmed the transcriptional activation induced by estrogen. We hypothesize that RIZ2, acting as a negative regulator of RIZ1 function, mediates the proliferative effect of estrogen through regulation of survival and differentiation gene expression. J. Cell. Physiol. 227: 964975, 2012. (C) 2011 Wiley Periodicals, Inc.""

"Growing evidence supports the concept that dynamic intra-and inter-chromosomal links between specific loci contribute to the creation of cell type-specific gene expression profiles. Therefore, analysis of the establishment of peculiar functional correlations between sites, also distant on linear DNA, that govern the transcriptional process appears to be of fundamental relevance. We propose here an experimental approach showing that 17 beta-estradiol-induced transcription associates to formation of loops between the promoter and termination regions of hormone-responsive genes. This strategy reveals as a tool to be also suitably used, in conjunction with automated techniques, for an extensive analysis of sites shared by multiple genes for induced expression."

The nuclear protein methyl-transferase Retinoblastoma-interacting zinc-finger protein 1 (RIZ1) is considered to be a downstream effector of estrogen action in target tissues. Silencing of RIZ1 expression is common in many tumors. We analyzed RIZ1 expression in normal and malignant prostate tissue and evaluated whether estradiol (E2) or dihydrotestosterone (DHT) treatment modulated RIZ1 in cultured prostate epithelial cells (PEC). Moreover, we studied the possible involvement of RIZ1 in estrogen action on the EPN prostate cell line, constitutively expressing both estrogen receptor (ER)-alpha and beta. RIZ1 protein, found in the nucleus of normal PECs by immunohistochemistry, was progressively lost in cancer tissues as the Gleason score increased and was only detected in the cytoplasmic compartment. RIZ1 transcript levels, as assayed by semi-quantitative RT-PCR in primary PEC cultures, were significantly reduced in cancer cells (P < 0.05). In EPN DHT treatment significantly increased RIZ1 transcript and protein levels (P < 0.05); E2 induced a reduction of S phase without significant changes of RIZ1 expression. In E2-treated EPN cell extracts RIZ co-immunoprecipitated with ERbeta and ERalpha. Our data demonstrate that RIZ1 is expressed in normal PECs and down-regulated in cancer cells, with a switch of its sub-cellular localization from the nucleus to the cytoplasm upon cancer grade progression. RIZ1 expression levels in the PECs were modulated by DHT or E2 treatment in vitro. Furthermore, the E2 effects on ER-expressing prostate cells involve RIZ1, which confirms a possible role for ER-mediated pathways in a non-classic E(2)-target tissue.

Estrogen receptor (ER) was purified from calf uterus by immunoaffinity chromatography in the absence of the ligand. The purified ER consists of a mixture of monomer and homodimer forms of 67-kDa hormone-binding subunit (no 90-kDa heat shock protein is present). The purified ER was incubated with a 32P-labeled 61-basepair oligonucleotide containing the sequence of the estrogen response element (ERE) of the Xenopus laevis A2 vitellogenin gene. DNA mobility shift assays showed formation of specific complexes of the ERE containing oligonucleotide with ER, formation which did not require and was not affected by estradiol or antiestrogenic molecules. Both the monomer and the dimer were equally able to interact with the ERE-containing oligonucleotide. Sucrose gradient experiments showed that only the ER monomer is able to interact with an oligonucleotide in which a single mutation destroyed the dyad symmetry of ERE. Multiple symmetric mutations which did not alter the dyad symmetry of ERE nevertheless totally destroyed the ability of the oligonucleotide to form complexes with either the monomeric or dimeric form of ER. These results suggest that ER is able to bind to ERE independently of the presence of estradiol or other proteins and, therefore, that estradiol does not act by modulating the ability of ER to bind to ERE on DNA.