OVOL2 impairs RHO GTPase signaling to restrain mitosis and aggressiveness of Anaplastic Thyroid Cancer

Background: Anaplastic Thyroid Cancer (ATC) is an undifferentiated and aggressive tumor that often originates from well-Differentiated Thyroid Carcinoma (DTC) through a trans-differentiation process. Epithelial-to-Mesenchymal Transition (EMT) is recognized as one of the major players of this process. OVOL2 is a transcription factor (TF) that promotes epithelial differentiation and restrains EMT during embryonic development. OVOL2 loss in some types of cancers is linked to aggressiveness and poor prognosis. Here, we aim to clarify the unexplored role of OVOL2 in ATC. Methods: Gene expression analysis in thyroid cancer patients and cell lines showed that OVOL2 is mainly associated with epithelial features and its expression is deeply impaired in ATC. To assess OVOL2 function, we established an OVOL2-overexpression model in ATC cell lines and evaluated its effects by analyzing gene expression, proliferation, invasion and migration abilities, cell cycle, specific protein localization through immunofluorescence staining. RNA-seq profiling showed that OVOL2 controls a complex network of genes converging on cell cycle and mitosis regulation and Chromatin Immunoprecipitation identified new OVOL2 target genes. Results: Coherently with its reported function, OVOL2 re-expression restrained EMT and aggressiveness in ATC cells. Unexpectedly, we observed that it caused G2/M block, a consequent reduction in cell proliferation and an increase in cell death. This phenotype was associated to generalized abnormalities in the mitotic spindle structure and cytoskeletal organization. By RNA-seq experiments, we showed that many pathways related to cytoskeleton and migration, cell cycle and mitosis are profoundly affected by OVOL2 expression, in particular the RHO-GTPase pathway resulted as the most interesting. We demonstrated that RHO GTPase pathway is the central hub of OVOL2-mediated program in ATC and that OVOL2 transcriptionally inhibits RhoU and RhoJ. Silencing of RhoU recapitulated the OVOL2-driven phenotype pointing to this protein as a crucial target of OVOL2 in ATC. Conclusions: Collectively, these data describe the role of OVOL2 in ATC and uncover a novel function of this TF in inhibiting the RHO GTPase pathway interlacing its effects on EMT, cytoskeleton dynamics and mitosis

FSHD muscular dystrophy Region Gene 1 binds Suv4-20h1 histone methyltransferase and impairs myogenesis

Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD Region Gene 1 (FRG1) since its over-expression in mice, X. laevis and C. elegans leads to muscular dystrophy-like defects, suggesting that FRG1 plays a relevant role in muscle biology. Here we show that, when overexpressed, FRG1 binds and interferes with the activity of the histone methyltransferase Suv4-20h1 both in mammals and Drosophila. Accordingly, FRG1 over-expression or Suv4-20h1 knockdown inhibits myogenesis. Moreover, Suv4-20h KO mice develop muscular dystrophy signs. Finally, we identify the FRG1/Suv4-20h1 target Eid3 as a novel myogenic inhibitor that contributes to the muscle differentiation defects. Our study suggests a novel role of FRG1 as epigenetic regulator of muscle differentiation and indicates that Suv4-20h1 has a gene-specific function in myogenesis

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming

Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes

!CHAOS: A cloud of controls

The paper is aimed to present the !CHAOS open source project aimed to develop a prototype of a national private Cloud Computing infrastructure, devoted to accelerator control systems and large experiments of High Energy Physics (HEP). The !CHAOS project has been financed by MIUR (Italian Ministry of Research and Education) and aims to develop a new concept of control system and data acquisition framework by providing, with a high level of abstraction, all the services needed for controlling and managing a large scientific, or non-scientific, infrastructure. A beta version of the !CHAOS infrastructure will be released at the end of December 2015 and will run on private Cloud infrastructures based on OpenStack

Multiplexed Fiber-Optic Interferometric System With Down-Lead Insensitive Fiber-Optic Probes

A Fiber-Optic Interferometric Sensor with multiplexing capability and noise free behaviour of the leading fiber is presented. The FOIS is based on a differential scheme, where both the reference and the sensing beam are guided by the same polarization maintaining fiber. A microprobe realized with-stacked optics technology permits the measurements in harsh environment. A full comparison of the noise-rejection capability of the realized FOIS versus a conventional ones is made. The experimental results confirm the high-noise rejection features of the developed FOIS

La prosocialità

Capitolo di un manuale di psicologia sociale che analizza le determinanti individuali e situazionali dei comportamenti prosocial

Strategic Planning Process in Public Universities: Critical Issues and "Loosely Coupled" Practice

Beginning in the late 1980s, the university system in Italy underwent an unprecedented process of change that, 40 years later, is still evolving. With the aim of aligning the Italian system to European standards and being more competitive, several legislative provisions have been enacted during the last 30 years that have revolutionized many of the established practices regarding university management. However, it must be highlighted that regulations alone have not always been able to generate substantial changes in the governance practices of universities (Cantele et al. 2012). Often, the impact has merely been a sterile adhesion to legislative obligations. With regard to the topic of this paper, compliance with the legislator's intentions would require considering strategic plans as real managerial mechanisms and practices. This imposes a shift of focus from the document that must be produced to meet quality assurance requirements to the organizational process that must be carried out to make the plan a tool that can effectively support governance in strategic management. Many universities, not just in Italy, have a strategic plan, but this is not sufficient to say that a good management practice has been introduced. Proponents of strategic planning emphasise the importance of process in improving strategic thinking. In particular, process studies assume that the key to understanding the effectiveness (or otherwise) of strategic planning may lie in seeing it as a complex, longitudinal approach to knowledge and action (Mintzberg 2007; Ferlie and Ongaro 2015), where governance engagement is key (Wahyudi, 2009). Unfortunately, in most Italian universities, governance shows mistrust towards these practices, due to a widespread resistance to change in defence of the old bureaucracy, which is, moreover, contested, feeding a vicious circle that undermines the development of an effective strategic planning process. In light of this premise, the objective of the paper is to propose an approach to effectively manage the strategic plan development process in public universities. The paper is articulated in 6 paragraphs. In the first paragraph, the peculiarities of public universities are presented, and in the second and third paragraph, the relevant literature is analysed. In particular, we refer to the literature on strategic planning and control in universities, focusing on the conditions of success/failure of strategic planning found in reality. Having identified the most recurrent anomalies and making use of the theories of organization that consider errors as real learning opportunities (McClamorch et al. 2001; Senge 1990; Schein 1985; Weick and Sutcliffe 2007). In the fourth paragraph, the research project is presented. The research method is detailed and the case of an Italian medium-sized university is described. This case seems particularly interesting because the university was completely lacking a strategic plan and, in proximity to the visit of the National Agency for the Evaluation of Universities and Research (ANVUR) for periodic assessment and accreditation (October 2019), needed to produce a credible and defensible plan in a relatively short time. The evaluation of the strategic plan submitted to ANVUR was very positive, and its implementation at this institution (2020) is currently (2021) under total control by the monitoring team. In the last two paragraphs, the case discussion and some concluding remarks are presented