Study of the molecular mechanism(s) underlying the antitumoral and highly pleiotropic functions of the human RNASET2 protein, a phylogenetically conserved extracellular RNase.

The RNASET2 gene encodes for the only human secreted acid ribonuclease of the T2 family. This gene maps in 6q27, a region that is consistently found rearranged in many solid and hematological tumors. Experimental data gathered in our laboratory have demonstrated the role of RNASET2 as a tumor suppressor gene which is endowed with several functions including chemotactic and possibly activating activities toward the monocyte/macrophage population. In a mouse xenograft model, we have recently reported that inoculation of human ovarian cancer-derived cells forced to overexpress RNASET2 led to the development of small, growth-suppressed tumors, characterized by a strong infiltrate of host-derived innate immunity cells, which have been identified as mainly M1 polarized macrophages. By contrast, control cells not expressing RNASET2, developed large, fast-growing tumors with no sign of macrophage infiltration. Further investigations proved that recombinant RNASET2 (produced in either baculovirus or Pichia pastoris expression system) displayed a marked chemotactic activity in vitro, most likely dependent on a subfamily of G-protein Coupled Receptors associated with inhibitory G protein. Taken together, these results led us to suggest that RNASET2-mediated in vivo tumor suppression is a non-cell-autonomous process which result in the recruitment of macrophages into the tumor mass. Based on such evidences, we decided to further investigate the relation between RNASET2 and cells belonging to the monocyte-macrophages lineage. Most of the experiments we planned to carry out involved the use of purified recombinant human RNASET2, hence, the first step of the project was the development of a reliable supply system. Two different sources of production and purification of human RNASET2 protein were already available in our lab (P. pastoris and BEVS), however, a full exploitation of these reagents was precluded by several limitations inherent to both systems. To overcome these limitation, we decided to focus our efforts on improving the P. pastoris expression system, which was more cost-effective and easier to handle in term of available facilities. To solve the problem, we added another 6XHis tag to the construct coding for the human RNASET2 and managed to significantly improve the recovery of the input protein. Considering the downstream applications of the purified human RNASET2, contamination of the preparation by metals (especially Ni, Cd, Zn) and endotoxin are important factor to control in order to properly assess a relationship between a specific treatment (in our case the addition of recombinant human RNASET2) and immune cell response. Applying the proper care in the preparation of the buffers and the handling of the sample we manage to achieve a low endotoxin content and an undetectable level of contamination by metals (Ni, Cd, Zn). As previously mentioned, several studies have reported the consistent tumor suppressive role for several members of the T2 RNase protein family and the recruitment of immune cells seems to be involved in this suppression. In order to improve our knowledge of these functional features of T2 RNases and at the same time to evaluate their evolutionary conservation, we tested the role of human recombinant RNASET2 in the activation and recruitment of immune cells by injecting recombinant human RNASET2 in the body wall of Hirudo verbena a useful model system for our purposes. After rRNASET2 injection, a significant increase in the production of collagen fibrils and a consequent remodeling of the muscle layers was observed. The resulting massive production of connective tissues is then used as a scaffold for immune cells migration and for proper orientation of growth of new vessels. Injection of human recombinant RNASET2 was also shown to induce a massive migration of cells belonging to the macrophages lineage (characterized as HmAIF-1+ and CD68+ cells) within 24 h, coupled to the formation of new blood vessels. The observed inflammatory response was specifically dependent of recombinant RNASET2 injection, since infiltrating macrophages and neo-vessel formation were not observed following injection of either PBS or rRNASET2 protein that was pre-incubated with a neutralizing anti-RNASET2 antibody. Taken together, these data strongly suggest that rRNASET2 injection in leeches induces a marked inflammatory response characterized by macrophage recruitment. The data gathered in our lab in several in vitro and in vivo experimental systems strongly suggest the occurrence of an RNASET2-based intercellular cross-talk, whose molecular mechanisms are nevertheless largely unknown. As a first step in the logical sequence of events culminating in the above mentioned RNASET2 biological activities, we hypothesized the occurrence of a putative receptor for RNASET2. The common idea behind all our approaches was to covalently link RNASET2 (either the endogenous protein or the human recombinant protein) to any interactor and, after purification of the complex, to perform a MS analysis to characterize the interactants. As a first approach, we carried out chemical modification of purified recombinant RNASET2 with different compounds (SDA, LC-SDA, Sulfo SDA, Sulfo LC-SDA) in order to add a diazirine ring to all primary ammines of the target protein. The resulting modified RNASET2 protein could then, in principle, be crosslinked to all electron donors available following UV light activation. The modified protein was then incubated with RNASET2-silenced OVCAR3 cells and analyzed by western blot following UV exposure. Despite several attempts with this approach, we could not observe any shift in the molecular weight of RNASET2. Considering that most of the result obtained show evidence of a tropism of RNASET2 for cells belonging to the monocyte/macrophage lineage, with our last approach we tested a promyelocytic cell line. We exposed the U937 cells preincubated with rRNASET2 to a chemical crosslinkers (BS3) able to covalently link two or more proteins in close proximity. By this approach we managed to see a shift in the molecular weight of the RNASET2, but unfortunately in the next step we couldn't purify any detectable complex. Considering that TAMs are usually forced by the tumor cells to acquire an M2-polarized pro-tumorigenic phenotype, a key finding of our research group is that tumor xenografts that are suppressed in their growth following RNASET2 overexpression display a strong infiltration of M1-polarized macrophages which are known to show anti-tumorigenic properties. Therefore, the last part of this PhD program was dedicated to investigate the molecular/cellular bases of the interaction between RNASET2 and immune cells, in particular to describe a possible involvement of this protein in driving macrophages polarization. The main idea was to develop a system to reliably produce M0, M1 and M2 macrophages that could then be used to test the effect of RNASET2 looking at their transcriptional profile. To this end, we chose a well-known cellular model (THP-1 cell line) and, in order to remove any bias from future analysis, we selected a pool of cells silenced for the expression of RNASET2. THP-1 is a promyelocytic cell line therefore in order to effectively produce polarized cells we needed to differentiate it into macrophages. Using the guidelines found in the literature we managed, by treating THP-1 cells with PMA 5 ng/ml for 48 h, to produce M0 macrophages. Subsequently, we developed a protocol to effectively drive naive macrophages to either M1 or M2 phenotype. We followed the suggested guidelines and assessed the efficacy of our system by testing a small panel of known marker of polarization (TNF, CXCL10, CCL19 for M1 polarization and CCL22, MRC1, MSR1 for M2 polarization). Considering the final aim of the developed assay, we tested whether our macrophages, especially M1 and M2, still retained the plasticity to change their profile according to external stimuli. M0 cells were exposed to both M1 and M2 stimuli; the resulting profile was neither M1 nor M2, suggesting that the cells prepared with our system still retained the characteristic/desired plasticity to properly respond to external signals. The data gathered so far support the idea that the system developed might be useful to study the involvement of RNASET2 in macrophages polarization. To summarize, during these three years of my PhD program I was involved in many different parts of a project aimed at deepen our understanding of the mechanism underlying RNASET2 tumor suppression. Although I wasn't able to identify any putative receptor for RNASET2 experimental evidence suggests the existence of a molecular mechanism involved in the \u201csensing\u201d of the RNASET2 protein. Further studies are clearly needed in order to clarify this issue and the recent finding of RNASET2-containing exosomes is a promising avenue of investigation. The data collected in Hirudo strengthen our hypothesis of a close relationship between Immune cells (in particular cells belonging to monocyte/macrophages lineage) and RNASET2 but, more importantly these results open new research opportunities to study the involvement of T2 ribonucleases in inflammation. From a strictly technical point of view, I was able to improve the system of production and purification of the recombinant human protein, making it possible the use of RNASET2 in conditions previously inaccessible (interaction with immune cells/injection in organism). To conclude I contributed to laid the bases for future studies aiming at dissecting the involvement of RNASET2 in macrophages polarization and I did it by developing a simple assay that might test the "polarizing potential" of RNASET2 (but potentially of any compound)

Cell Population Growth Kinetics in the Presence of Stochastic Heterogeneity of Cell Phenotype

Recent studies at individual cell resolution have revealed phenotypic heterogeneity in nominally clonal tumor cell populations. The heterogeneity affects cell growth behaviors, which can result in departure from the idealized uniform exponential growth of the cell population. Here we measured the stochastic time courses of growth of an ensemble of populations of HL60 leukemia cells in cultures, starting with distinct initial cell numbers to capture a departure from the {uniform exponential growth model for the initial growth (``take-off'')}. Despite being derived from the same cell clone, we observed significant variations in the early growth patterns of individual cultures with statistically significant differences in growth dynamics, which could be explained by the presence of inter-converting subpopulations with different growth rates, and which could last for many generations. Based on the hypothesis of existence of multiple subpopulations, we developed a branching process model that was consistent with the experimental observations

Measurement of the top quark forward-backward production asymmetry and the anomalous chromoelectric and chromomagnetic moments in pp collisions at √s = 13 TeV

Abstract The parton-level top quark (t) forward-backward asymmetry and the anomalous chromoelectric (d̂ t) and chromomagnetic (μ̂ t) moments have been measured using LHC pp collisions at a center-of-mass energy of 13 TeV, collected in the CMS detector in a data sample corresponding to an integrated luminosity of 35.9 fb−1. The linearized variable AFB(1) is used to approximate the asymmetry. Candidate t t ¯ events decaying to a muon or electron and jets in final states with low and high Lorentz boosts are selected and reconstructed using a fit of the kinematic distributions of the decay products to those expected for t t ¯ final states. The values found for the parameters are AFB(1)=0.048−0.087+0.095(stat)−0.029+0.020(syst),μ̂t=−0.024−0.009+0.013(stat)−0.011+0.016(syst), and a limit is placed on the magnitude of | d̂ t| < 0.03 at 95% confidence level. [Figure not available: see fulltext.

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Measurement of prompt open-charm production cross sections in proton-proton collisions at root s=13 TeV

The production cross sections for prompt open-charm mesons in proton-proton collisions at a center-of-mass energy of 13TeV are reported. The measurement is performed using a data sample collected by the CMS experiment corresponding to an integrated luminosity of 29 nb(-1). The differential production cross sections of the D*(+/-), D-+/-, and D-0 ((D) over bar (0)) mesons are presented in ranges of transverse momentum and pseudorapidity 4 < p(T) < 100 GeV and vertical bar eta vertical bar < 2.1, respectively. The results are compared to several theoretical calculations and to previous measurements.Peer reviewe

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV

Search for Physics beyond the Standard Model in Events with Overlapping Photons and Jets

Results are reported from a search for new particles that decay into a photon and two gluons, in events with jets. Novel jet substructure techniques are developed that allow photons to be identified in an environment densely populated with hadrons. The analyzed proton-proton collision data were collected by the CMS experiment at the LHC, in 2016 at root s = 13 TeV, and correspond to an integrated luminosity of 35.9 fb(-1). The spectra of total transverse hadronic energy of candidate events are examined for deviations from the standard model predictions. No statistically significant excess is observed over the expected background. The first cross section limits on new physics processes resulting in such events are set. The results are interpreted as upper limits on the rate of gluino pair production, utilizing a simplified stealth supersymmetry model. The excluded gluino masses extend up to 1.7 TeV, for a neutralino mass of 200 GeV and exceed previous mass constraints set by analyses targeting events with isolated photons.Peer reviewe