COINVOLGIMENTO DEL DOMINIO BRCT DI PARP1 NELLA VIA APOPTOTICA p53 DIPENDENTE INDOTTA DA RAGGI UV

Poly(ADP-ribose) polymerase-1 (PARP-1) is one of the most abundant proteins within mammalian cells. PARP-1 is an abundant nuclear enzyme involved in DNA repair and transcriptional regulation and is now recognized as a key regulator of cell survival and cell death as well as a master component of a number of transcription factors involved in tumor development and inflammation. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. Despite many interesting studies that implicate PARP-1 in transcription, chromatin remodelling, apoptosis, DNA repair and several neurological disorders, its precise role is still unclear. The aim of my studies is represented by the involvement of the PARP-1 domain, BRCT in the induction of p53-dependent apoptotic pathway. The p53 protein is one of the most important tumor suppressor proteins. Normally, the p53 protein is in a latent state. However, in case of DNA damage, nucleotide depletion or hypoxia ,when its activity is required, p53 becomes rapidly activated and initiates transcription of pro-apoptotic and cell cycle arrest-inducing target genes. By means of the aid of T7 phage display have been the expression of a library of peptides able to bind Phosphopeptides; through biopanning and after several cycle of selection 18 clones were isolated of which 9 phosphopeptides bound p53, but not able to bind non phosphorilated p53. The clones obtained were then amplified and further selected with an electrophoretic run; sequenced and the subsequent alignment of the sequences with those of protein database SwissProt: 2 clones (S4p5311 and S4p5316) results correspond to PARP-1 protein sequences, BRCT domain. Therefore we developed a model of cell death, characterized by cells U2OS (Human Bone Osteosarcoma) treated with UV and after 24 hours of exposure we obtained a mortality of 50-60%. We then analyzed the morphological and immunochemistry markers that confirmed, our hypothesis, Death by UV irradiation happen by apoptosis; final confirmation is given to us by the study of cell vitality by colorimetric assay. Moreover we have studied how changes in the expression of p53 protein following treatment with UV, revealing an increase in time-dependent beginning 3 hours after treatment and with a maximum to 24 hours. Furthermore by Affinity Purification, performed with functionalized beads with GST-BRCT group were incubated with cell lysates from cultures at different times of treatment. BRCT-p53 interaction occurs with a very different kinetics from the expression of p53. We supposed that the key event which regulates the interaction between p53 and BRCT is a phosphorylation and this interaction is able to reduce cell death after exposure to UV. To test whether the BRCT-p53 interaction could also occur within cells, we transfected some culture cell by using Lipofectamine, with two separate vectors. One, able to express a GFP and the other BRCT-GFP fusion protein. Then using immunoprecipitation technique we were able to verify the interaction occurred within the cell, between BRCT and p53, and we discovered a protective effect against apoptosis induced by UV radiation. We didn’t see the same effect on the cells transfected only with the GFP vector, and again on a different line cells called H1299 which is p3 negative In conclusion, we have validated the model of cell death developed by us for apoptosis experiment and we have characterized the interaction between BRCT and p53 in several biological condition. For the future we would like to discover if the phosphorylation represent the main even that regulates the interaction between p53 and BRCT, and how this mechanism performs its protective effect

Biomedical Image Classification via Dynamically Early Stopped Artificial Neural Network

It is well known that biomedical imaging analysis plays a crucial role in the healthcare sector and produces a huge quantity of data. These data can be exploited to study diseases and their evolution in a deeper way or to predict their onsets. In particular, image classification represents one of the main problems in the biomedical imaging context. Due to the data complexity, biomedical image classification can be carried out by trainable mathematical models, such as artificial neural networks. When employing a neural network, one of the main challenges is to determine the optimal duration of the training phase to achieve the best performance. This paper introduces a new adaptive early stopping technique to set the optimal training time based on dynamic selection strategies to fix the learning rate and the mini-batch size of the stochastic gradient method exploited as the optimizer. The numerical experiments, carried out on different artificial neural networks for image classification, show that the developed adaptive early stopping procedure leads to the same literature performance while finalizing the training in fewer epochs. The numerical examples have been performed on the CIFAR100 dataset and on two distinct MedMNIST2D datasets which are the large-scale lightweight benchmark for biomedical image classification

Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention - Insights into the STRATEGY study

ObjectivesThe purpose of this study was to evaluate the value of platelet reactivity (PR) in predicting the response to treatment and outcome in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention assisted by glycoprotein (GP) IIb/IIIa inhibition.BackgroundThere is limited prognostic information on the role of spontaneous or drug-modulated PR in STEMI patients.MethodsThe PR was measured with Platelet Function Analyzer (PFA)-100 and light transmission aggregometry (LTA) using adenosine diphosphate as agonist in 70 consecutive STEMI patients at entry (PR-T0), 10 min after GP IIb/IIIa bolus (PR-T1), and discharge (PR-T2) and in 30 stable angina (SA) patients (PR-SA). Complete platelet inhibition (CPI) was based on closure time >300 s by PFA-100 and percentage inhibition of platelet aggregation >95% by LTA. Clinical, electrocardiographic, and angiographic responses to treatment during 1-year follow-up were collected.ResultsAccording to both techniques, PR-T0 was higher than: 1) PR-T2 and PR-SA; 2) in those without CPI at T1; and 3) in patients with final Thrombolysis In Myocardial Infarction (TIMI) flow grade <3. The PR-T0 assessed with PFA-100 correlated with: 1) corrected TIMI frame count (r = −0.6, p < 0.001); 2) ST-segment resolution (r = 45, p < 0.001); and 3) creatine kinase-MB (r = −0.47, p < 0.001). At 1 year, patients with high PR-T0 showed an adjusted 5- to 11-fold increase in the risk of death, reinfarction, and target vessel revascularization (hazard ratio [HR] 11, 95% confidence interval [CI] 1.5 to 78 [p = 0.02] in PFA-100; HR 5.2, 95% CI 1.1 to 23 [p = 0.03] in LTA).ConclusionsThe PR at entry affects response to GP IIb/IIIa inhibition, mechanical treatment, and long-term outcome in STEMI patients undergoing primary intervention

Circulating biomarkers in familial cerebral cavernous malformation

Background Cerebral Cavernous Malformation (CCM) is a rare cerebrovascular disease, characterized by the presence of multiple vascular malformations that may result in intracerebral hemorrhages (ICHs), seizure(s), or focal neurological deficits (FND). Familial CCM (fCCM) is due to loss of function mutations in one of the three independent genes KRIT1 (CCM1), Malcavernin (CCM2), or Programmed Cell death 10 (PDCD10/CCM3). The aim of this study was to identify plasma protein biomarkers of fCCM to assess the severity of the disease and predict its progression.Methods Here, we have investigated plasma samples derived from n = 71 symptomatic fCCM patients (40 female/31 male) and n =17 healthy donors (HD) (9 female/8 male) of the Phase 1/2 Treat_CCM trial, using multiplexed protein profiling approaches.Findings Biomarkers as sCD14 (p = 0.00409), LBP (p = 0.02911), CXCL4 (p = 0.038), ICAM-1 (p = 0.02013), ANG2 (p = 0.026), CCL5 (p = 0.00403), THBS1 (p = 0.0043), CRP (p = 0.0092), and HDL (p = 0.027), were significantly different in fCCM compared to HDs. Of note, sENG (p = 0.011), THBS1 (p = 0.011) and CXCL4 (p = 0.011), were correlated to CCM genotype. sROBO4 (p = 0.014), TM (p = 0.026) and CRP (p = 0.040) were able to predict incident adverse clinical events, such as ICH, FND or seizure. GDF-15, FLT3L, CXCL9, FGF-21 and CDCP1, were identified as predictors of the formation of new MRI-detectable lesions over 2-year follow-up. Furthermore, the functional relevance of ang2, thbs1, robo4 and cdcp1 markers was validated by zebrafish pre-clinical model of fCCM.Interpretation Overall, our study identifies a set of biochemical parameters to predict CCM progression, suggesting biological interpretations and potential therapeutic approaches to CCM disease

Reprogramming of miRNA networks in cancer and leukemia.

We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network

Factor XIIIA-V34L and Factor XIIIB-H95R Gene Variants: Effects on Survival in Myocardial Infarction Patients

It has been demonstrated recently that coagulation factor XIII (FXIII) plays an extraordinary role in myocardial healing after infarction, improving survival in a mouse model. Common FXIII gene variants (i.e. FXIIIA-V34L and FXIIIB-H95R) significantly influence the molecular activity. To evaluate whether there is a relationship between the two FXIII gene variants and survival in patients after myocardial infarction (MI), V34L and H95R were PCR-genotyped in a cohort of 560 MI cases and follow-up was monitored. Cases with ST-segment elevation MI (STEMI) were 416 (74.3%) and 374 of these were treated with primary percutaneous coronary intervention (PCI) (89.9%). The remaining 144 patients showed non-ST-segment elevation MI (NSTEMI) at enrollment. The combined endpoint was the occurrence of death, re-infarction, and heart failure. Kaplan-Meier analysis at one year yielded an overall rate for adverse events of 24.5% with a lower incidence in the L34-carriers (28.8% vs 17.1%; log-rank, P = 0.00025), similar to that of the 416 STEMI (23.8%) being (28.0% and 16.9%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Primary PCI-group had a slight lower incidence (22.9%) of adverse events (26.8% and 17.1%; VV34- and L34-carriers respectively; log-rank, P = 0.009). During hospitalization, 506 patients received PCI (374 primary PCI and 132 elective PCI). Significance was conserved also in the overall PCI-group (28.6% and 17.8%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Similar findings were observed at 30 days follow-up. Cases carrying both FXIII variants had improved survival rate (log-rank, P = 0.019). On the other hand, minor bleeding complications were found increased in L34-carriers (P = 0.0001) whereas major bleeding complications were not. Finally, more direct evidence on the role of FXIII molecule on survival might come from the fact that despite significant FXIII antigen reductions observed in cases after MI, regardless the FXIII genotype considered, L34-carriers kept almost normal FXIII activity (VV34- vs L34-carriers; P < 0.001). We conclude that FXIII L34-allele improves survival after MI in all the groups analyzed, possibly through its higher activity associated with assumable positive effects on myocardial healing and recovered functions. Genetically determined higher FXIII activity might influence post-MI outcome. This paves the way for using FXIII molecules to improve myocardial healing, recovery of functions, and survival after infarction