Complexity of N-Myc transcriptional function in childhood neuroblastoma

Myc is a transcription factor that can activate transcription of several hundreds genes by direct binding to their promoters at specific DNA sequences (E-box). However, recent studies have also shown that it can exert its biological role by repressing transcription. Such studies collectively support a model in which c-Myc-mediated repression occurs through interactions with transcription factors bound to promoter DNA regions but not through direct recognition of typical E-box sequences. Here, we investigated whether N-Myc can also repress gene transcription, and how this is mechanistically achieved. We used human neuroblastoma cells as a model system in that N-MYC amplification/over-expression represents a key prognostic marker of this tumour. By means of transcription profile analyses we could identify at least 5 genes (TRKA, p75NTR, ABCC3, TG2, p21) that are specifically repressed by N-Myc. Through a dual-step-ChIP assay and genetic dissection of gene promoters, we found that N-Myc is physically associated with gene promoters in vivo, in proximity of the transcription start site. N-Myc association with promoters requires interaction with other proteins, such as Sp1 and Miz1 transcription factors. Furthermore, we found that N-Myc may repress gene expression by interfering directly with Sp1 and/or with Miz1 activity (i.e. TRKA, p75NTR, ABCC3, p21) or by recruiting Histone Deacetylase 1 (Hdac1) (i.e. TG2). In vitro analyses show that distinct N-Myc domains can interact with Sp1, Miz1 and Hdac1, supporting the idea that Myc may participate in distinct repression complexes by interacting specifically with diverse proteins. Finally, results show that N-Myc, through repressed genes, affects important cellular functions, such as apoptosis, growth, differentiation and motility. Overall, our results support a model in which N-Myc, like c-Myc, can repress gene transcription by direct interaction with Sp1 and/or Miz1, and provide further lines of evidence on the importance of transcriptional repression by Myc factors in tumour biology.

Recupero della funzione renale in pazienti con acute kidney injury (AKI) sottoposti a terapia sostitutiva renale

L’insufficienza renale acuta(AKI) grave che richiede terapia sostitutiva, è una complicanza frequente nelle unità di terapia intensiva(UTI) e rappresenta un fattore di rischio indipendente di mortalità. Scopo dello studio é stato valutare prospetticamente, in pazienti “critici” sottoposti a terapie sostitutive renali continue(CRRT) per IRA post cardiochirurgia, la prevalenza ed il significato prognostico del recupero della funzione renale(RFR). Pazienti e Metodi:Pazienti(pz) con AKI dopo intervento di cardiochirurgia elettivo o in emergenza con disfunzione di due o più organi trattati con CRRT. Risultati:Dal 1996 al 2011, 266 pz (M 195,F 71, età 65.5±11.3aa) sono stati trattati con CRRT. Tipo di intervento: CABG(27.6%), dissecazione aortica(33%), sostituzione valvolare(21.1%), CABG+sostituzione valvolare(12.6%), altro(5.7%). Parametri all’inizio del trattamento: BUN 86.1±39.4, creatininemia(Cr) 3.96±1.86mg/dL, PAM 72.4±13.6mmHg, APACHE II score 30.7±6.1, SOFAscore 13.7±3. RIFLE: Risk (11%), Injury (31.4%), Failure (57.6%). AKI oligurica (72.2%), ventilazione meccanica (93.2%), inotropi (84.5%). La sopravvivenza a 30 gg ed alla dimissione è stata del 54.2% e del 37.1%. La sopravvivenza per stratificazione APACHE II: <24=85.1 e 66%, 25-29=63.5 e 48.1%, 30-34=51.8 e 31.8%, >34=31.6 e 17.7%. RFR ha consentito l’interruzione della CRRT nel 87.8% (86/98) dei survivors (Cr 1.4±0.6mg/dL) e nel 14.5% (24/166) dei nonsurvivors (Cr 2.2±0.9mg/dL) con un recupero totale del 41.4%. RFR è stato osservato nel 59.5% (44/74) dei pz non oligurici e nel 34.4% dei pz oligurici (66/192). La distribuzione dei pz sulla base dei tempi di RFR è stata:<8=38.2%, 8-14=20.9%, 15-21=11.8%, 22-28=10.9%, >28=18.2%. All’analisi multivariata, l’oliguria, l’età e il CV-SOFA a 7gg dall’inizio della CRRT si sono dimostrati fattori prognostici sfavorevoli su RFR(>21gg). RFR si associa ad una sopravvivenza elevata(78.2%). Conclusioni:RFR significativamente piu frequente nei pz non oligurici si associa ad una sopravvivenza alla dimissione piu elevata. La distribuzione dei pz in rapporto ad APACHE II e SOFAscore dimostra che la sopravvivenza e RFR sono strettamente legati alla gravità della patologia.

Development of strategies for vascular damage repair in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive and rare disease with so far unclear pathogenesis, limited treatment options and poor prognosis. Unbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is an important hallmark of PAH. In this research Sodium butyrate (BU) has been evaluated in vitro and in vivo models of PAH. This histone deacetylase inhibitor (HDACi) counteracted platelet-derived growth factor (PDGF)-induced ki67 expression in PASMCs, and arrested cell cycle mainly at G0/G1 phases. Furthermore, BU reduced the transcription of PDGFRbeta, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing and pulmonary artery ring assays indicated that BU inhibited PDGF-induced PASMC migration. BU strongly inhibited PDGF-induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculinA. In vivo, BU showed efficacy in monocrotaline-induced PAH in rats. Indeed, the HDACi reduced both thickness of distal pulmonary arteries and right ventricular hypertrophy. Besides these studies, Serial Analysis of Gene Expression (SAGE) has be used to obtain complete transcriptional profiles of peripheral blood mononuclear cells (PBMCs) isolated from PAH and Healthy subjects. SAGE allows quantitative analysis of thousands transcripts, relying on the principle that a short oligonucleotide (tag) can uniquely identify mRNA transcripts. Tag frequency reflects transcript abundance. We enrolled patients naïve for a specific PAH therapy (4 IPAH non-responder, 3 IPAH responder, 6 HeritablePAH), and 8 healthy subjects. Comparative analysis revealed that significant differential expression was only restricted to a hundred of down- or up-regulated genes. Interestingly, these genes can be clustered into functional networks, sharing a number of crucial features in cellular homeostasis and signaling. SAGE can provide affordable analysis of genes amenable for molecular dissection of PAH using PBMCs as a sentinel, surrogate tissue. Altogether, these findings may disclose novel perspectives in the use of HDACi in PAH and potential biomarkers.

A multiparental cross population for mapping QTL for relevant agronomic traits in durum wheat (Triticum durum Desf.)

Multiparental cross designs for mapping quantitative trait loci (QTL) in crops are efficient alternatives to conventional biparental experimental populations because they exploit a broader genetic basis and higher mapping resolution. We describe the development and deployment of a multiparental recombinant inbred line (RIL) population in durum wheat (Triticum durum Desf.) obtained by crossing four elite cultivars characterized by different traits of agronomic value. A linkage map spanning 2,663 cM and including 7,594 single nucleotide polymorphisms (SNPs) was produced by genotyping 338 RILs with a wheat-dedicated 90k SNP chip. A cluster file was developed for correct allele calling in the framework of the tetraploid durum wheat genome. Based on phenotypic data collected over four field experiments, a multi-trait quantitative trait loci (QTL) analysis was carried out for 18 traits of agronomic relevance (including yield, yield-components, morpho-physiological and seed quality traits). Across environments, a total of 63 QTL were identified and characterized in terms of the four founder haplotypes. We mapped two QTL for grain yield across environments and 23 QTL for grain yield components. A novel major QTL for number of grain per spikelet/ear was mapped on chr 2A and shown to control up to 39% of phenotypic variance in this cross. Functionally different QTL alleles, in terms of direction and size of genetic effect, were distributed among the four parents. Based on the occurrence of QTL-clusters, we characterized the breeding values (in terms of effects on yield) of most of QTL for heading and maturity as well as yield component and quality QTL. This multiparental RIL population provides the wheat community with a highly informative QTL mapping resource enabling the dissection of the genetic architecture of multiple agronomic relevant traits in durum wheat.

Le variazioni anatomiche: La vera immagine dell'anatomia dell'uomo

La studio dell’Anatomia umana presenta una varietà di sfaccettature, che sono alla base della reale comprensione del corpo umano; ovvero la vera anatomia non è quella rappresentata nei testi ma quella che appare durante la dissezione o nelle più sofisticate analisi di immagine. Lo scopo di questa tesi è stato quello di rivisitare alcune situazioni vascolari che possono andare incontro a variazioni e cercare di comprendere, anche con l’aiuto della bibliografia, se tali variazioni possono essere causa o epifenomeni di patologie a carico delle arterie affette dalle variazioni stesse o di territori da esse dipendenti per l’afflusso sanguigno. E’ stata condotta una analisi su preparati cadaverici in particolare in tre distretti: a) addome e tripode celiaco/mesenterica superiore; b) circolo cerebrale; d) orco aortico.

Evaluation of 3D cell culture systems for host-pathogen interaction studies

Traditional cell culture models have limitations in extrapolating functional mechanisms that underlie strategies of microbial virulence. Indeed during the infection the pathogens adapt to different tissue-specific environmental factors. The development of in vitro models resembling human tissue physiology might allow the replacement of inaccurate or aberrant animal models. Three-dimensional (3D) cell culture systems are more reliable and more predictive models that can be used for the meaningful dissection of host–pathogen interactions. The lung and gut mucosae often represent the first site of exposure to pathogens and provide a physical barrier against their entry. Within this context, the tracheobronchial and small intestine tract were modelled by tissue engineering approach. The main work was focused on the development and the extensive characterization of a human organotypic airway model, based on a mechanically supported co-culture of normal primary cells. The regained morphological features, the retrieved environmental factors and the presence of specific epithelial subsets resembled the native tissue organization. In addition, the respiratory model enabled the modular insertion of interesting cell types, such as innate immune cells or multipotent stromal cells, showing a functional ability to release pertinent cytokines differentially. Furthermore this model responded imitating known events occurring during the infection by Non-typeable H. influenzae. Epithelial organoid models, mimicking the small intestine tract, were used for a different explorative analysis of tissue-toxicity. Further experiments led to detection of a cell population targeted by C. difficile Toxin A and suggested a role in the impairment of the epithelial homeostasis by the bacterial virulence machinery. The described cell-centered strategy can afford critical insights in the evaluation of the host defence and pathogenic mechanisms. The application of these two models may provide an informing step that more coherently defines relevant molecular interactions happening during the infection.

Studio sul ruolo dei fattori genetici coinvolti nella Valvola Aortica Bicuspide e/o nell'Aneurisma dell'Aorta Toracica

La Valvola Aortica Bicuspide (BAV) rappresenta la più comune anomalia cardiaca congenita, con un’incidenza dello 0,5%-2% nella popolazione generale. Si caratterizza per la presenza di due cuspidi valvolari anziché tre e comprende diverse forme. La BAV è frequentemente associata agli aneurismi dell’aorta toracica (TAA). La dilatazione dell’aorta espone al rischio di sviluppare le complicanze aortiche acute. Materiali e metodi Sono stati reclutati 20 probandi consecutivi sottoposti a chirurgia della valvola aortica e dell'aorta ascendente presso l'Unità di Chirurgia Cardiaca di Policlinico S.Orsola-Malpighi di TAA associata a BAV. Sono stati esclusi individui con una condizione sindromica predisponente l’aneurisma aortico. Ciascun familiare maggiorenne di primo grado è stato arruolato nello studio. L’analisi di mutazioni dell’intero gene ACTA2 è stata eseguita con la tecnica del “bidirectional direct sequencing”. Nelle forme familiari, l’intera porzione codificante del genoma è stata eseguita usando l’exome sequencing. Risultati Dopo il sequenziamento di tutti i 20 esoni e giunzioni di splicing di ACTA2 nei 20 probandi, non è stata individuata alcuna mutazione. Settantasette familiari di primo grado sono stati arruolati. Sono state identificate cinque forme familiari. In una famiglia è stata trovata una mutazione del gene MYH11 non ritenuta patogenetica. Conclusioni La mancanza di mutazioni, sia nelle forme sporadiche sia in quelle familiari, ci suggerisce che questo gene non è coinvolto nello sviluppo della BAV e TAA e, l’associazione che è stata riportata deve essere considerata occasionale. L’architettura genetica della BAV verosimilmente dovrebbe consistere in svariate differenti varianti genetiche che interagiscono in maniera additiva nel determinare un aumento del rischio.