SECONDARY STRUCTURES OF DNA AND PNA: FROM ORDERED SUPRAMOLECULAR GQUADRUPLEXES TO PNA/DNA HETERODUPLEXES

Oligonucleotides and oligonucleotide analogues are extremely versatile molecules from a biotechnological point of view, since they could be used as scaffolds for the development of new drugs in therapeutics, but also for the development of new materials in nanotechnology. The present thesis is included in this context and it is aimed at the discovery of all potential applications of these molecules. In particular, my work is mainly focused on: ✓ The synthesis and characterization of a novel G-rich ODN sequence for the development of long G-wires; ✓ The synthesis and characterization of PNA sequences to be used in a novel approach for the treatment of Cystic Fibrosis; ✓ The synthesis and characterization of a short sequence of PNA conjugated to a novel bifunctional linker aimed at the obtainment of PNA-based quadruplex

Peptide Nucleic Acids as miRNA Target Protectors for the Treatment of Cystic Fibrosis

Cystic Fibrosis (CF) is one of the most common life shortening conditions in Caucasians. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene which result in reduced or altered CFTR functionality. Several microRNAs (miRNAs) downregulate the expression of CFTR, thus causing or exacerbating the symptoms of CF. In this context, the design of anti-miRNA agents represents a valid functional tool, but its translation to the clinic might lead to unpredictable side effects because of the interference with the expression of other genes regulated by the same miRNAs. Herein, for the first time, is proposed the use of peptide nucleic acids (PNAs) to protect specific sequences in the 3'UTR (untranslated region) of the CFTR messenger RNA (mRNA) by action of miRNAs. Two PNAs (7 and 13 bases long) carrying the tetrapeptide Gly-SerP-SerP-Gly at their C-end, fully complementary to the 3'UTR sequence recognized by miR-509-3p, have been synthesized and the structural features of target PNA/RNA heteroduplexes have been investigated by spectroscopic and molecular dynamics studies. The co-transfection of the pLuc-CFTR-3´UTR vector with different combinations of PNAs, miR-509-3p, and controls in A549 cells demonstrated the ability of the longer PNA to rescue the luciferase activity by up to 70% of the control, thus supporting the use of suitable PNAs to counteract the reduction in the CFTR expression

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Il consumo sostenibile nella visione dei Gruppi di Acquisto Solidale di Roma

In questo articolo si presentano i risultati preliminari di una ricerca sulle filiere alimentari alternative e in particolare sui Gruppi di Acquisto Solidale (GAS) nella città di Roma. I GAS sono un movimento di consumatori critici che guidano le loro scelte di consumo in base a principi di solidarietà e rispetto dell'ambiente. In questa ricerca sono considerati come nicchie di innovazione capaci di suggerire soluzioni efficaci ai problemi di insostenibilità dei sistemi agro-industriali dominanti. La ricerca, basata su analisi di dati, documenti e interviste in profondità, si propone di ricostruire la storia, illustrare le caratteristiche socio-demografiche degli aderenti ai GAS e soprattutto indagare sulla loro visione del consumo sostenibile utilizzando come strumento di analisi le cinque dimensioni che secondo Seyfang (2009) lo caratterizzano: comprare locale, ridurre l’impatto ambientale, costruire solidarietà e comunità, agire in modo collettivo, creare nuove infrastrutture. La ricerca è finanziata dai fondi PRIN 2008

Different methylation and MicroRNA expression pattern in male and female familial breast cancer

Epigenetic regulation, has been very scarcely explored in familial breast cancer (BC). In the present study RASSF1A and RAR beta promoter methylation and miR17, miR21, miR 124, and let-7a expression were investigated to highlight possible differences of epigenetic regulation between male and female familial BC, also in comparison with sporadic BC. These epigenetic alterations were studied in 56 familial BC patients (27 males and 29 females) and in 16 female sporadic cases. RASSF1A resulted more frequently methylated in men than women (76% vs. 28%, respectively, P=0.0001), while miR17 and let-7a expression frequency was higher in women than in men (miR17: 66% in women vs. 41% in men, P<0.05; let-7a: 45% in women vs. 15% in men, P=0.015). RASSF1A methylation affected 27.6% of familial BC while 83% of familial cases showed high expression of the gene (P=0.025); on the contrary, only 17% of familial BC presented RAR beta methylation and 55% of familial cases overexpressed this gene (P=0.005). Moreover, miR17, miR21, and let-7a resulted significantly overexpressed in familial compared to sporadic BC. RASSF1A overexpression (86% vs. 65%, P=0.13) and RAR beta overexpression (57% vs. 32%, P=0.11) were higher in BRCA1/2 carriers even if not statistical significance was reached. BRCA mutation carriers also demonstrated significant overexpression of: miR17 (93% vs. 35%, P=0.0001), let-7a (64% vs. 16%, P=0.002), and of miR21 (100% vs. 65%, P=0.008). In conclusion, the present data suggest the involvement of RASSF1A in familial male BC, while miR17 and let-7a seem to be implied in familial female BC. J. Cell. Physiol. 228: 12641269, 2013. (c) 2012 Wiley Periodicals, Inc