A mouse model to study inducible oncogene cooperation <i>in vivo</i>

The current model for cancer development envisions cells under going a series of genetic mutations and/or alterations which result in their inability to respond normally to intracellular and extracellular signals that control proliferation, differentiation and death. The number of required genetic alterations varies for different types of cancer and it is likely that further changes occur during its progression to increased malignancy. Thus, cancer is not a static disease but during the development and progression of tumour, multiple changes occur in two kinds of genes: oncogenes and tumour suppressor genes. Oncogene-products can be classified as growth factors, growth factor receptors, Ras oncoproteins, cytoplasmic protein kinases, transcription factors, anti-apoptotic proteins. In particular, the ras oncogene family includes three members: N-ras, K-ras, H-ras. In non-transformed cells, Ras protein, belonging to G-protein family, transduces growth signals from external to the internal environment. In fact, when activated, Ras exchanges GDP with GTP and this allosteric change allows binding of Ras effector molecules and transduction of signalling cascades.R as activity is required for cell cycle progression. In cancer it has been observed that this oncogene is constitutively activated by mutations and induces the cell to enter into cell-cycle also in the absence of growth signals. Among the transcription factors, a gene involved in many tumours is myc. This transcription factor plays a key role in cell proliferation as its target proteins include many positive regulators of the cell-cycle. In tumour cells the protein product of this oncogene is overexpressed. The cooperationb etweenm ultiple oncogenesa nd/orl oss of tumour suppressors from different functional classes is necessary for transformation to proceed. In fact, it was observed that, although overexpression of a single oncogene does not transform wild-type mouse embryonic fibroblasts, combinations of myc and H-rasVAL12, can induce cellular transformation and the cells expressing both oncogenes displayeda markedp roliferative advantage. In thyroid, neoplastic transformation generates several different histotypes of tumours, ranging from poorly aggressive and well-differentiated, to highly malignant and undifferentiated anaplastic cancers. The aim of my thesis was to study the tumorigenesis induced by oncogenes and the oncogene cooperation in vivo during the gradual passage from a poorly aggressive to a much more aggressive tumour. To this end a mouse model expressing the two oncogenes H-rasVAL12 and c-myc (referred as ras and myc) in a tissue-specific as well as in a conditional manner was generated. For this purpose, the coding sequences of the two oncogenes were fused in a bicistronic construct and an IRES (Internal Ribosome Entry Sequence or Site) was inserted between them, to ensure the expression of the second oncogene. The construct was inserted under the control of the promoter of the ubiquitously expressed genes ROSA26 and Eeflal. In order to express these oncogenes in a tissue-specific manner, the transcription of the two oncogenesis preventedb y a STOP sequencef lanked by two LoxP sites. Such a STOP sequence can be removed by Cre recombinase protein. The transgenic mice were crossed with mice expressing Cre in a tissue-specific manner. Two strains of transgenic mice expressing Cre in thyroid cells were used: 1. transgenic mice for TgCre, in which Cre is expressed under the control of Tg promoter after the development of the thyroid; 2. Pax8Cre, in which the Cre sequence is inserted in the Pax8 locus and is expressed during the early stages of the thyroid development. In such a manner the oncogenes were expressed only in thyroid cells, but were still inactive. In particular, ras was fused to the mutated ligand binding domain of the estradiol receptor that is sensitive to tamoxifen and not to endogenous estradiol; while mycwas fused to the mutated ligand binding domain of the progesterone receptor (hPR891) that is sensitive to RU486 and not to endogenous progesterone. With thesef usedo ncogeneist is possible to activate only Ras( with tamoxifen) or only Myc (with RU486) or both (providing both tamoxifen and RU486). Moreover the activity of two oncogenes might be used to immortalize mouse cell lines in culture.</br

Calcium regulates HCC proliferation as well as EGFR recycling/degradation and could be a new therapeutic target in HCC

Calcium is the most abundant element in the human body. Its role is essential in physiological and biochemical processes such as signal transduction from outside to inside the cell between the cells of an organ, as well as the release of neurotransmitters from neurons, muscle contraction, fertilization, bone building, and blood clotting. As a result, intra- and extracellular calcium levels are tightly regulated by the body. The liver is the most specialized organ of the body, as its functions, carried out by hepatocytes, are strongly governed by calcium ions. In this work, we analyze the role of calcium in human hepatoma (HCC) cell lines harboring a wild type form of the Epidermal Growth Factor Receptor (EGFR), particularly its role in proliferation and in EGFR downmodulation. Our results highlight that calcium is involved in the proliferative capability of HCC cells, as its subtraction is responsible for EGFR degradation by proteasome machinery and, as a consequence, for EGFR intracellular signaling downregulation. However, calcium-regulated EGFR signaling is cell line-dependent. In cells responding weakly to the epidermal growth factor (EGF), calcium seems to have an opposite effect on EGFR internalization/degradation mechanisms. These results suggest that besides EGFR, calcium could be a new therapeutic target in HCC

Bollettino Sismico Italiano: Analisys of Early Aftershocks of the 2016 MW 6.0 Amatrice, MW 5.9 Visso and MW 6.5 Norcia earthquakes in Central Italy

The Amatrice-Visso-Norcia seismic sequence is the most important of the last 30 years in Italy. The seismic sequence started on 24 August, 2016 and still is ongoing in central Apennines. At the end of February 2017 more than 57,000 events were located, 80,000 events up to the end of September 2017 (Fig. 1). The mainshocks of the sequence occurred on 24 August 2016 (Mw 6.0 and Mw 5.4), 26 October 2016 (Mw 5.4 and Mw 5.9), 30 October 2016 (Mw 6.5), 18 January 2017 (four earthquakes Mw≥ 5.0). In this seismic sequence, all the waveforms recorded by temporary stations deployed by the SISMIKO emergency group (stations T12**; Moretti et al., 2016) where available in real- time at the surveillance room of INGV. Because of the high level of seismicity and the dense seismic network installed in the region, more than 150 events per day were located at the end of February 2017; still 60 events per day were located up to the end of August 2017.The Amatrice-Visso-Norcia is the most important seismic sequence since 2015, the time when the analysis procedures of the BSI group (Bollettino Sismico Italiano) were revised (Nardi et al., 2015). BSI is now available every four months on the web: bulletins contain revised earthquakes (location and magnitude) with ML≥ 1.5, quasi-real time revision of ML≥ 3.5 earthquakes and phase arrivals from waveforms recorded on seismic stations available from the European Integrated Data Archive (EIDA), (Mazza et al., 2012). These last procedures allow the integration of signals from temporary seismic stations (Moretti et al., 2014) installed by the emergency group SISMIKO (Moretti and Sismiko working group, 2016), even when they are not in real time transmission, if they are rapidly archived in EIDA, together with real time signals from the seismic stations of the permanent INGV network. The analysis strategy of the BSI group for the Amatrice -Visso - Norcia seismic sequence (AVN.s.s in the following) was to select the earthquakes located in the box with min/max latitude: 42.2/43.2 - and min/max longitude: 12.4/14.1 to prepare a special volume of BSI on the seismic sequence.PublishedTrieste, Italy1SR. TERREMOTI - Servizi e ricerca per la Societ

Bollettino Sismico Italiano gennaio – aprile 2019

Nel periodo che va dal 1 gennaio al 30 aprile 2019, gli analisti del BSI hanno revisionato tutti gli eventi di magnitudo M≥1.5, mentre i parametri dei terremoti di magnitudo inferiore a tale soglia di revisione sono stati calcolati in tempo reale, nella sala di sorveglianza sismica di Roma.Istituto Nazionale di Geofisica e Vulcanologia - Dipartimento di Protezione CivilePublished4IT. Banche dat

Bollettino Sismico Italiano settembre – dicembre 2018

Gli analisti del BSI hanno revisionato tutti gli eventi di magnitudo M≥1.5, localizzati dal 1 settembre al 31 dicembre 2018. I parametri dei terremoti di magnitudo inferiore a tale soglia di revisione, sono quelli calcolati in tempo reale, nella sala di sorveglianza sismica di Roma.Istituto Nazionale di Geofisica e Vulcanologia - Dipartimento di Protezione CivilePublished4IT. Banche dat

Bollettino Sismico Italiano gennaio – aprile 2018

Nel primo quadrimestre 2018 si sono verificati in Italia cinque eventi di magnitudo superiore o uguale a 4.0, di cui nessuno di magnitudo superiore a 5.0. Due di essi, avvenuti il 4 (MW 4.0) e il 10 aprile (MW 4.6), hanno interessato la zona della sequenza dell’Italia centrale, in provincia di Macerata. Un terremoto di magnitudo MW 4.3 è avvenuto in provincia di Campobasso, il 25 aprile, ad una profondità di 29 km. Infine due terremoti profondi, avvenuti il 12 febbraio (ML 4.4, con profondità di 379 km) e il 7 marzo (ML 4.0, con profondità di 294 km), hanno interessato il Tirreno Meridionale, al largo della costa calabra.Istituto Nazionale di Geofisica e Vulcanologia - Dipartimento di Protezione CivilePublished4IT. Banche dat

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19

Bollettino Sismico Italiano maggio – agosto 2017

I parametri dei terremoti registrati dalla Rete Sismica Nazionale Italiana, localizzati nelle sale di monitoraggio di Roma, Napoli e Catania, sono immediatamente disponibili sul web, alla pagina http://terremoti.ingv.it/, e nell’Italian Seismological Instrumental and parametric Data-base (ISIDe working group (2016) version 1.0, DOI: 10.13127/ISIDe). Gli analisti del Bollettino Sismico Italiano (BSI) ricontrollano i parametri dei terremoti localizzati, inserendo pesi e polarità degli arrivi delle onde sismiche e integrando, inoltre, i dati letti in sala con tutti quelli disponibili nel sistema di acquisizione. Dal 1985 i dati del bollettino sono consultabili nel data-base ISIDe.Istituto Nazionale di Geofisica - Dipartimento di Protezione CivilePublished4IT. Banche dat