Ruolo anti-apoptotico della tirosin-chinasi Lyn nella leucemia linfatica cronica a cellule B

B-chronic lymphocytic leukemia (B-CLL) is the commonest leukemia in the Western world and is characterized by the clonal expansion of small CD5+ B lymphocytes in the pheripheral blood, bone marrow and lymphoid organs, due to defective apoptosis. One of the crucial factors for the survival of B-CLL cells is the anomalous activity of a few enzymes such as the lipide kinase PI3K, the Ser/Thr kinase PKC and the tyrosine-kinases Syk and Lyn. My research focus was Src family kinase Lyn, which is overexpressed, constitutively active and delocalized in the cytosol within an aberrant multiprotein complex, in association with the molecular chaperone Hsp90, which in turn accounts for the high level of tyrosine-phosphorylation, ultimately contributing to the cancer phenotype of B-CLL cells. The aim of my PhD work was to identify Lyn’s cytosolic substrates potentially involved in the apoptosis resistance mechanisms observed in leukemia cells. Using biochemical and biomolecular methods as well as a bioinformatics approach, two potential substrates of Lyn were identified, namely the cysteine protease caspase-8 and the protein phosphatase-2A (PP2A). Firstly, we established that in B-CLL, the inactive zymogen of caspase-8, procaspase-8, is phosphorylated at Tyr380 by the cytosolic pool of Lyn, which brings about its inhibition and its structural rearrangements in a dymeric cytosolic form. The phosphorylation of Tyr380 of caspase-8 is abolished by the use of both the Lyn inhibitor dasatinib and the Hsp90 inhibitor geldanamycin, the latter of which also leads to the disassembly of the aberrant cytosolic complex and the drop in the Lyn’s cytosolic activity, resulting in the caspase-dependent apoptosis. Secondly, we verified that Lyn negatively affects the activity of one of the key factors in the modulation of cell survival signals, PP2A. Phosphorylation of the catalytic subunit of PP2A at Tyr307 causes inhibition of the phosphatase activity not only per se but also strengthening the interaction of PP2A with its cellular inhibitor SET, which is also overexpressed in B-CLL cells. As a result, PP2A is stably preserved in an inhibited form, which contributes to the persistence of prosurvival signals, in particular those mediated by the serine/threonine kinase Akt. Moreover, to identify new compounds able to activate PP2A in order to contrast survival signals in B-CLL, we tested fingolimod (FTY720), which is already known as a PP2A activator, currently in use in the treatment of multiple sclerosis and recently emerging as a pro-apoptotic factor in different types of cancer cells. Notably, the mechanism of action consists in the ability of fingolimod to bind to SET and hence remove this cellular inhibitor of PP2A from its catalytic subunit. Since this drug is used as an immunosuppressive agent, by virtue of its provoking the degradation of the sphingosine-1-phosphate receptor, and shows cardiovascular side effects, it might prove unsuitable in a more complex therapeutic strategy. Therefore, fingolimod structural analogues which could not interfere with the sphingosine receptors, but could still destabilize the PP2A/SET complex were designed and synthesized. One of these compounds, MP07-66, showed an effectiveness similar to fingolimod, as to both the ability to disrupting the PP2A/SET complex and inhibit the protein kinase Akt, this latter being vital in preserving the survival signals. This compound also was able to induce apoptosis in leukemia cells, which effect was further enhanced by the combination with dasatinib by a synergistic mechanism, further confirming the role of phosphorylation in the stability of PP2A/SET. In conclusion, this work identifies PP2A and Caspase 8 as substrates of the cytosolic aberrant activity of Lyn, confirming the key role of this tyrosine kinase in supporting multiple anti-apoptotic signals in the B-CLL. The molecular mechanisms of apoptosis resistance here identified may be potential targets in the development of new therapies for clinical use

Heat transfer in drop-laden turbulence

Heat transfer by large deformable drops in a turbulent flow is a complex and rich in physics system, in which drops deformation, breakage and coalescence influence the transport of heat. We study this problem coupling direct numerical simulations (DNS) of turbulence, with a phase-field method for the interface description. Simulations are run at fixed shear Reynolds and Weber numbers. To evaluate the influence of microscopic flow properties, like momentum/thermal diffusivity, on macroscopic flow properties, like mean temperature or heat transfer rates, we consider four different values of the Prandtl number, which is the momentum to thermal diffusivity ratio: Pr=1, Pr=2, Pr=4 and Pr=8. The drops volume fraction is Phi=5.4% for all cases. Drops are initially warmer than the turbulent carrier fluid, and release heat at different rates, depending on the value of Pr, but also on their size and on their own dynamics (topology, breakage, drop-drop interaction). Computing the time behavior of the drops and carrier fluid average temperatures, we clearly show that an increase of Pr slows down the heat transfer process. We explain our results by a simplified phenomenological model: we show that the time behavior of the drops average temperature is self similar, and a universal behavior can be found upon rescaling by t/Pr^2/3

Assessment of the IEQ in Two High Schools by Means of Monitoring, Surveys and Dynamic Simulation

Abstract This work presents an assessment of schools' indoor environmental quality, based on investigations carried out in three Italian classrooms in Treviso, in the North-East of Italy. A first monitoring campaign was performed during the mid season (May-June), a second one during the heating period (January-February). At a first stage, the study was focused on two different approaches, an objective and a subjective one, in order to compare the objective responses with the occupants' subjective sensations. The first method consisted of physical observations and field measurements of thermal environmental parameters, used to calculate Fanger's comfort indices and to apply a comfort adaptive model. The subjective approach was managed by giving students and teachers a survey about their personal judgment concerning the level of comfort perceived. Finally, a simulation model has been built-up and calibrated using the indoor values of air temperature and air humidity trends collected by data loggers. A generic optimization program has been used to calibrate the thermal model. The responses from measurements, surveys and simulations were integrated, analyzed and compared, obtaining a good agreement between the three approaches in assessing the classroom thermal comfort category

Cues to opening mechanisms from in silico electric field excitation of cx26 hemichannel and in vitro mutagenesis studies in HeLa transfectans

Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family

Effectiveness of load-level isolation system for pallet racking systems

Recent Italian earthquakes have shown the high seismic vulnerability of pallet racking systems. In the down-aisle direction and in the absence of bracing systems, these structures are very flexible moment-resisting frames. Instead, in the cross-aisle direction they consist of slender trusses, stiffened by various bracing systems; the latter, although necessary for lateral stability, attract significant seismic accelerations, which can cause the stored goods to fall, posing a threat to human safety. To reduce this risk while increasing the rack structural performance, some mitigation systems were proposed, based on specific base-isolation or dissipation devices. In this paper, an innovative passive control system is investigated, i.e., the Load-Level Isolation System (LLIS), which consists of applying isolators to the load level to control the movement of pallets in the cross-aisle direction. The LLIS is based on the Tuned Mass Damper (TMD) strategy and exploits the high payload of these structures. Among the major uncertainties of this system are the amount of isolated mass and its position within the structure. Therefore, for a case study rack, the LLIS parameters (isolation stiffness and damping ratio) are optimized for various arrangements of this system, considering cases with one or two isolated levels. The applied optimization procedure is an extension of the classical TMD approaches. The effectiveness of the optimized LLISs is then investigated through bidirectional Time-History analyses on 3D Finite Element rack models. The results show that using the LLIS, even on a single load-level, can greatly reduce the upright stresses and the cross-aisle displacements and accelerations, and that the optimal position of the LLIS is in the upper part of the rack

Digital twin for civil engineering systems: an exploratory review for distributed sensing updating

We live in an environment of ever-growing demand for transport networks, which also have ageing infrastructure. However, it is not feasible to replace all the infrastructural assets that have surpassed their service lives. The commonly established alternative is increasing their durability by means of Structural Health Monitoring (SHM)-based maintenance and serviceability. Amongst the multitude of approaches to SHM, the Digital Twin model is gaining increasing attention. This model is a digital reconstruction (the Digital Twin) of a real-life asset (the Physical Twin) that, in contrast to other digital models, is frequently and automatically updated using data sampled by a sensor network deployed on the latter. This tool can provide infrastructure managers with functionalities to monitor and optimize their asset stock and to make informed and data-based decisions, in the context of day-to-day operative conditions and after extreme events. These data not only include sensor data, but also include regularly revalidated structural reliability indices formulated on the grounds of the frequently updated Digital Twin model. The technology can be even pushed as far as performing structural behavioral predictions and automatically compensating for them. The present exploratory review covers the key Digital Twin aspects—its usefulness, modus operandi, application, etc.—and proves the suitability of Distributed Sensing as its network sensor component.This research was funded by Fondazione CARITRO Cassa di Risparmio di Trento e Rovereto, grant number 2021.0224.Peer ReviewedPostprint (published version

mGlu1 Receptors Monopolize the Synaptic Control of Cerebellar Purkinje Cells by Epigenetically Down-Regulating mGlu5 Receptors

In cerebellar Purkinje cells (PCs) type-1 metabotropic glutamate (mGlu1) receptors play a key role in motor learning and drive the refinement of synaptic innervation during postnatal development. The cognate mGlu5 receptor is absent in mature PCs and shows low expression levels in the adult cerebellar cortex. Here we found that mGlu5 receptors were heavily expressed by PCs in the early postnatal life, when mGlu1α receptors were barely detectable. The developmental decline of mGlu5 receptors coincided with the appearance of mGlu1α receptors in PCs, and both processes were associated with specular changes in CpG methylation in the corresponding gene promoters. It was the mGlu1 receptor that drove the elimination of mGlu5 receptors from PCs, as shown by data obtained with conditional mGlu1α receptor knockout mice and with targeted pharmacological treatments during critical developmental time windows. The suppressing activity of mGlu1 receptors on mGlu5 receptor was maintained in mature PCs, suggesting that expression of mGlu1α and mGlu5 receptors is mutually exclusive in PCs. These findings add complexity to the the finely tuned mechanisms that regulate PC biology during development and in the adult life and lay the groundwork for an in-depth analysis of the role played by mGlu5 receptors in PC maturation

Targeted activation of the SHP-1/PP2A signaling axis elicits apoptosis of chronic lymphocytic leukemia cells

Lyn, a member of the Src family of kinases, is a key factor in the dys-regulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn's action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia

Lyn sustains oncogenic signaling in chronic lymphocytic leukemia by strengthening SET-mediated inhibition of PP2A.

Aberrant protein kinase activities, and the consequent dramatic increase of Ser/Thr and -Tyr phosphorylation, promote the deregulation of the survival pathways in chronic lymphocytic leukemia (CLL), which is crucial to the pathogenesis and progression of the disease. In this study, we show that the tumor suppressor Protein Phosphatase 2A (PP2A), one of the major Ser/Thr phosphatase, is in an inhibited form due to the synergistic contribution of two events, the interaction with its physiological inhibitor SET and the phosphorylation of Y307 of the catalytic subunit of PP2A. The latter event is mediated by Lyn, a Src family kinase previously found to be overexpressed, delocalized and constitutively active in CLL cells. This Lyn/PP2A axis accounts for the persistent high level of phosphorylation of the phosphatase's targets and represents a key connection linking phosphotyrosine- and phosphoserine/threonine-mediated oncogenic signals. The data herein presented show that the disruption of the SET/PP2A complex by a novel FTY720-analogue (MP07-66) devoid of immunosuppressive effects leads to the reactivation of PP2A, which in turn triggers apoptosis of CLL cells. When used in combination with SFK inhibitors, the action of MP07-66 is synergistically amplified, providing a new option in the therapeutic strategy for CLL patients