Modulation of p53 activities by the prolyl-isomerase Pin1 and the bromodomain protein Brd7

2006/2007ABSTRACT: MODULATION OF p53 ACTIVITIES BY THE PROLYL-ISOMERASE PIN1 AND THE BROMODOMAIN PROTEIN BRD7 The tumour suppressor p53 belongs to a family of transcription factors that play key roles in maintaining genomic stability and cellular homeostasis. The orchestration of the appropriate cellular responses depends on the fine regulation of p53’s functions through post-translational modifications and interaction with other proteins. In many years of intense study a considerable knowledge on p53 activity has been achieved, yet a greater insight is needed on the specificity of its response. In the first part of this thesis a novel mechanism in the regulation of p53-mediated apoptotic response has been disclosed. It has been demonstrated that upon severe stress signalling p53 dissociates from iASPP, an anti-apoptotic co-factor that inhibits p53 apoptotic functions, and that key roles in this process are played by the prolyl isomerase Pin1. Moreover, it emerged that phosphorylation at p53 Ser46 is required for Pin1-mediated dissociation of the p53-iASPP complex thus providing a mechanistic explanation for the relevance of this site in p53 mediated apoptosis. Notably, the role of Pin1 in assisting the dissociation of p53 from iASPP appears to be independent from Pin1-induced acetylation of p53 and dissociation from Mdm2, further confirming that Pin1 may modulate p53 activity at different levels. A different approach to gain insight on the mechanisms governing p53 regulation is the analysis of p53 protein interaction profiles. The bromodomain containing protein Brd7 was identified as a common interactor of the p53 family proteins in a yeast two hybrid screening conducted in our lab. The presence of the bromodomain and evidences from literature made Brd7 a promising candidate for modulating the p53 pathway at the transcriptional level. This protein and its functional interaction with p53 have been therefore characterized in the second part of this thesis. Upon depletion of Brd7 expression in cells it has been demonstrated that Brd7 is required for efficient cell-cycle arrest in U2OS cells upon challenging with genotoxic stimuli. This effect appeared to be due to a reduction in p21 expression that occurred upon Brd7 depletion and under stress condition. The down-regulation of p21 as a consequence of Brd7 silencing occurred at the transcriptional level and proved to be p53-depedent. Taken together the data reported in the second part of this thesis suggest a role for Brd7 as a positive regulator of p53 transcriptional activity during cell-cycle arrest response and that this function might be exerted by regulating p53-mediated transcription on a chromatin context. Further analysis is needed to dissct the role of this functional interaction. Yet, preliminary investigation suggest that Brd7 might be an important modulator of p53 response and that it can be an important means for p53 to crosstalk with other signalling pathway. Together, the data presented in this thesis contribute to achieve greater knowledge on the mechanism that govern p53 response. As the p53 pathway is compromised to some degree in almost all human cancers, this would be also of great relevance in designing new targeted strategies for cancer treatment.Riassunto: Modulazione delle attività di p53 da parte della prolyl isomerasi Pin1 e della proteina contenente dominio Bromo Brd7 L’oncosoppressore p53 appartiene a una famiglia di fattori di trascrzione che svolge un ruolo fondamentale nel mantenimento della stabilità genomica e dell’omeostasi cellulare. L’attuazione di un’appropriata risposta cellulare dipende molto dalla regolazione fine delle funzioni di p53. Ciò avviene attraverso modificazioni post-traduzionali e interazioni con altre proteine cellulari. In molti anni di intenso studio si è raggiunta una notevole conoscenza sull’ attività di p53 ma ancora non è stato definito cosa regoli la specificità della risposta. Nella prima parte di questa tesi è stato portato alla luce un nuovo meccanismo nella regolazione della risposta apoptotica mediata da p53. E’ stato dimostrato che, in seguito a stress intensi, p53 si dissocia da iASPP, un co-fattore anti-apoptotico che inibisce le funzioni apoptotiche di p53 e che la prolyl-isomerasi in1 gioca un ruolo fondamentale in questo processo. In aggiunta è emerso che la fosforilazione di p53 al residuo Ser46 è necessaria al distacco di p53 da iASPP mediato da Pin1, fornendo una spiegazione meccanicistica alla nota rilevanza di questo siro per l’apoptosi mediata da p53. Interessantemente, il ruolo di Pin1 nell’assistere la dissociazione di p53 da iASPP sembra essere indipendente dalla sua capacità di favorirne l’acetilazione e il distacco da Mdm2, così confermando che Pin1 può modulare l’attività di p53 a diversi livelli. Un differente approccio per avere delucidazioni sui meccanismi che governano la regolazione di p53 è l’analisi del profilo di interazione proteica. La proteina Brd7 (contenente dominio Bromo) è stata identificata come comune interattore dei membri della famiglia di p53 in uno screening di doppio ibrido in lievito, condotto nel nostro laboratorio. La presenza del dominio Bromo e alcune evidenze di letteratura hanno reso Brd7 un candidato promettente come modulatore trascrizionale della via di segnalazione di p53. Brd7 e la sua interazione funzionale con p53 sono stati caratterizzati nella seconda parte di questa tesi. Dopo aver bloccato l’espressione di Brd7 in cellule è stato dimostrato che Brd7 è necessario per un efficiente arresto del ciclo cellulare in seguito a danni genotossici. Questo effetto sembra essere dovuto ad una riduzione nell’espressione di p21 che avviene I seguito alla deplezione di Brd7 e in condizioni di stress.Tale riduzione avviene a livello trascrizionale ed è p53-dipendente. Insieme I dati riportati nella seconda parte della tesi indicano un ruolo per Brd7 come regolatore positivo dell’attività trascrizionale di p53 durante l’arresto del ciclo cellulare e ciò può avvenire attraverso una regolazione a livello della cromatina. Insieme, I dati presentati in questa tesi contribuiscono ad una maggiore conoscenza sui meccanismi che governano la risposta di p53. Dato che la via di segnalazione di p53 è compromessa a qaulche livello in quasi tutti i tumori, questo potrebbe essere rilevante per disegnare nuove terapie mirate per la cura del cancro.197

Patient safety subcultures among nursing home staff in Italy: a cross-sectional study

Nursing home (NH) residents are vulnerable subjects and highly susceptible to adverse events. Knowledge of patient safety culture (PSC) is essential for an organization to ensure patient safety. However, research on PSC in NHs, and its variability among staff, is still scarce. This study aimed to explore whether and how PSC differed among NH staff (Managers, Nurses, Direct Care Staff, Support Staff, Administrative Staff and Other Providers) in the Autonomous Province of Trento, Italy. This study employed a cross-sectional design and collected data from 1145 NH providers using the Nursing Home Survey on Patient Safety Culture (NHSPSC). Data were analyzed using linear mixed models, with each of the 12 NHSPSC domains as a response variable. The majority of the respondents (61.6%) were Direct Care Staff members. ‘Feedback and Communication about Incidents’ and ‘Overall Perceptions of Resident Safety’ were the domains with the highest proportions of positive answers (PPAs). For most staff categories, ‘Staffing’ was the domain with the lowest PPA. Support Staff showed significantly lower scores in the majority of domains (8/12). Shorter job tenure, fewer weekly working hours, working mostly during the day and working in highly specialized areas were associated with higher scores in several domains. Interventions to improve PSC must consider the differences between professional groups. Further research is needed to explore the relationship between job-related features and perceptions of patient safety among NH workers

Patient safety in the eyes of aspiring healthcare professionals: a systematic review of their attitudes

A culture of safety is important for the delivery of safe, high-quality care, as well as for healthcare providers' wellbeing. This systematic review aimed to describe and synthesize the literature on patient safety attitudes of the next generation of healthcare workers (health professional students, new graduates, newly registered health professionals, resident trainees) and assess potential differences in this population related to years of study, specialties, and gender. We screened four electronic databases up to 20 February 2020 and additional sources, including weekly e-mailed search alerts up to 18 October 2020. Two independent reviewers conducted the search, study selection, quality rating, data extraction, and formal narrative synthesis, involving a third reviewer in case of dissent. We retrieved 6606 records, assessed 188 full-texts, and included 31 studies. Across articles, healthcare students and young professionals showed overwhelmingly positive patient safety attitudes in some areas (e.g., teamwork climate, error inevitability) but more negative perceptions in other domains (e.g., safety climate, disclosure responsibility). Women tend to report more positive attitudes. To improve safety culture in medical settings, health professions educators and institutions should ensure education and training on patient safety

Pathophysiology of motor dysfunction in Parkinson\u2019s Disease as the rationale for drug treatment and rehabilitation

Cardinal motor features of Parkinson\u2019s disease (PD) include bradykinesia, rest tremor, and rigidity, which appear in the early stages of the disease and largely depend on dopaminergic nigrostriatal denervation. Intermediate and advanced PD stages are characterized by motor fluctuations and dyskinesia, which depend on complex mechanisms secondary to severe nigrostriatal loss and to the problems related to oral levodopa absorption, and motor and nonmotor symptoms and signs that are secondary to marked dopaminergic loss and multisystem neurodegeneration with damage to nondopaminergic pathways. Nondopaminergic dysfunction results in motor problems, including posture, balance and gait disturbances, and fatigue, and nonmotor problems, encompassing depression, apathy, cognitive impairment, sleep disturbances, pain, and autonomic dysfunction. There are a number of symptomatic drugs for PD motor signs, but the pharmacological resources for nonmotor signs and symptoms are limited, and rehabilitation may contribute to their treatment. The present review will focus on classical notions and recent insights into the neuropathology, neuropharmacology, and neurophysiology of motor dysfunction of PD. These pieces of information represent the basis for the pharmacological, neurosurgical, and rehabilitative approaches to PD

skin reaction in antiviral therapy for chronic hepatitis c a role for polyethylene glycol interferon

In the past decade, different modalities of antiviral therapy have been adopted aimed at eradicating hepatitis C virus infection. Initially, interferon was used in monotherapy, then interferon combined with ribavirin and amantadine. Recently, interferon has been conjugated with polyethylene glycol to allow optimization of its pharmacokinetic properties and to improve its antiviral activity. This study focused on the characteristics of the skin reactions that we observed in 27 patients with naive hepatitis C who received polyethylene glycol interferon-ribavirin-amantadine or polyethylene glycol interferon-ribavirin and in 10 previous non-responders to interferon monotherapy who were retreated with triple therapy. In 9 patients (7 on triple therapy) dermatitis-like lesions were observed, and in 5 the severity of the lesions necessitated withdrawal from therapy

MicroRNA profiling of paediatric AML with FLT-ITD or MLL-rearrangements: Expression signatures and in vitro modulation of miR-221-3p and miR-222-3p with BRD4/HATs inhibitors.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain‑containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of CDKN1B, a known target of miR-221-3p and miR-222-3p, increase in apoptosis and downregulation of miR-221-3p and miR-222-3p expression in CD34+ AML primary cells. Altogether, these findings suggested that several miRs expression signatures at diagnosis may be used for risk stratification and as relapse prediction biomarkers in paediatric AML outlining that epigenetic drugs, could represent a novel therapeutic strategy for high-risk paediatric patients with AML. For these epigenetic drugs, additional research for enhancing activity, bioavailability and safety is needed

ZnO-mediated regioselective C-arylsulfonylation of indoles: A facile solvent-free synthesis of 2- and 3-sulfonylindoles and preliminary evaluation of their activity against drug-resistant mutant HIV-1 reverse transcriptases (RTs)

A ZnO-mediated one-pot solvent-free protocol for the regioselective C-arylsulfonylation of indoles is described and some novel derivatives were tested on wild type and non-nucleoside inhibitor resistant K103N and Y181C HIV-1 reverse transcriptases (RTs)

Improving the culture of safety among healthcare workers: Integration of different instruments to gain major insights and drive effective changes

The Safety Attitude Questionnaire (SAQ) and Manchester Patient Safety Framework (MaPSaF) are known as effective tools to assess patient safety culture and climate and develop targeted strategies. However, they are seldom applied in an integrated way. The aim of this study was to conduct an implementation project through a novel use of both instruments to gain unique insights. The Italian version of MaPSaF and SAQ were administered to 1,759 healthcare workers from three Italian hospitals (response rate: MaPSaF 70.5%, SAQ 61.6%). MaPSaF evaluation proved an overall bureaucratic level of patient safety culture. SAQ scores showed a predominance of neutral scores (75.99%). The dimension perception of management gained the lowest mean score (53.32), while Stress recognition obtained the highest (75.17). Safety climate perception differed significantly among groups: working in a small hospital, in a medical department, and being a physician were associated with the most positive results. The majority (67.1%) of responders to both MaPSaF and SAQ considered the two instruments as providing with different and complementary information. Overall, results showed that an integrated approach in the evaluation of an organisation's safety culture may result useful for an in-depth analysis of the criticalities and the adoption of appropriate improvement strategies