HIV-1 ed il reticolo endoplasmatico: aspetti del ciclo replicativo e riduzione dell'infettivita'

Durante il suo ciclo replicativo, HIV-1 sfrutta buona parte del macchinario biosintetico cellulare per la produzione di RNA e proteine che verranno poi assemblati a formare la progenie virale. La maggior parte delle proteine codificate nel genoma di HIV-1 vengono sintetizzate dai ribosomi liberi, fatta eccezione per Vpu ed il precursore Env/gp160, dotate di un segnale di secrezione per l’immissione nell’ER (Endoplasmic Reticulum). La glicoproteina virale gp160, prima di giungere alla membrana plasmatica, transita attraverso l’apparato di secrezione cellulare dove subisce ampie modificazioni post-traduzionali. In aggiunta è sottoposto ad un taglio proteolitico ad opera di furin-proteasi nell’apparato del Golgi, dando origine a due frammenti, gp120 e gp41, che rimanendo uniti non covalentemente costituiscono il contro-recettore virale. Esso viene esposto sull’envelope del virione maturo ed è in grado di legare CD4 e CCR5/CXCR4, rispettivamente recettore e corecettori di HIV-1, permettendo l’ingresso del capside nella cellula e quindi l’infezione. Impedendo la corretta maturazione del precursore Env, ad esempio promuovendo la sua degradazione durante il transito attraverso il reticolo endoplasmatico, ci si attende di ottenere una progenie virale non infettiva. Tali virus infatti non esporrebbero sul loro envelope i recettori essenziali per il legame e l’ingresso nelle cellule bersaglio. È stato dunque messo a punto un sistema volto alla degradazione selettiva di proteine in transito nell’ER durante la loro sintesi che si basa sull’utilizzo di proteine chimeriche indicate come “degradine”, caratterizzate da due porzioni funzionali: un dominio di legame al target, che può essere un scFv (single chain variable fragment) derivato da un anticorpo monoclonale diretto contro il target oppure un ligando/recettore in grado di interagire con la proteina di interesse; un dominio in grado di indurre la degradazione della molecola bersaglio, una volta che questa si è legata alla degradina. Quest’ultima porzione è costituita da una forma tronca della proteina cellulare SEL1L, che fisiologicamente svolge un ruolo fondamentale nella promozione dell’ERAD (ER-Associated Degradation) di proteine non correttamente ripiegate transitanti nel reticolo endoplasmatico. In questo studio per ottenere l’effetto desiderato si sono impiegate entrambe le tipologie di domini di riconoscimento del target: scFv diretti contro gp120 e diverse porzioni di CD4, di cui è nota l’interazione con Env a livello dell’ER. Attraverso esperimenti di trasfezione transiente di colture cellulari si è potuto verificare che le degradine specifiche per gp160 sono in grado di ridurre significativamente i livelli intracellulari di proteina, inducendo specificamente ed efficacemente la sua degradazione. Si è andata di conseguenza a valutare l’infettività di vettori lentivirali prodotti in linee cellulari di packaging esprimenti le degradine. I saggi condotti hanno dimostrato che la degradazione della proteina virale gp160 in fase di produzione del vettore porta effettivamente alla formazione di virioni non infettivi. Questo fatto è dovuto alla mancata incorporazione di contro-recettori sull’envelope delle particelle virali in formazione. Nell’ambito dello studio della degradazione di proteine localizzate nel reticolo endoplasmatico, nel corso del progetto è stato inoltre analizzato il comportamento della proteina Vpu di HIV-1. Questa proteina virale è infatti in grado di indurre la degradazione di due proteine cellulari, CD4 e BST-2/Tetherin, sfruttando probabilmente parte del medesimo macchinario cellulare necessario al funzionamento delle degradine. Tale attività è fondamentale per il completamento del ciclo replicativo di HIV-1 e per la produzione di progenie infettiva. In particolare Tetherin è stata recentemente identificata come fattore cellulare ad attività antivirale. Per poter dunque fornire ulteriori dettagli su questi processi degradativi si è valutata la retrotraslocazione dall’ER al citoplasma delle due proteine in diverse condizioni ed in presenza di Vpu. Tali dati sono stati ottenuti tramite una tecnologia che permette di biotinilare specificamente le molecole che hanno raggiunto il citosol a seguito della dislocazione. È così possibile discriminare con accuratezza la frazione di proteina che ha avuto effettivamente accesso a tale compartimento cellulare. In questo modo si è riusciti ad approfondire questi meccanismi di degradazione, confermando il ruolo fondamentale del proteasoma nell’eliminazione di CD4 ed indicando il suo coinvolgimento nella degradazione di Tetherin, come indicato da lavori presenti in letteratura. Si sono inoltre potute raccogliere interessanti informazioni riguardo allo stato di folding delle due molecole durante il processo di dislocazione. In particolare, lo studio dello stato di ossidazione dei ponti disolfuro in esse contenuti suggerisce che le due proteine siano parzialmente ripiegate durante l’attraversamento della membrana dell’ER Nel corso di questo studio si sono dunque chiariti alcuni aspetti del ruolo del reticolo endoplasmatico nella replicazione di HIV-1. Si è inoltre proposto come potenziale approccio terapeutico un metodo per ridurre l’infettività virale sfruttando un sistema di degradazione agente in tale compartimento cellulare

Triaxial tests on frozen ground: formulation and modelling

Artificial Ground Freezing (AGF) is a controllable process that can be used by engineers to stabilise temporarily the ground, provide structural support and/or exclude groundwater from an excavation until construction of the final lining provides permanent stability and water tightness. In this work, the process of ground freezing is studied using a constitutive model that encompasses frozen and unfrozen behaviour within a unified effective-stress-based framework and employs a combination of ice pressure, liquid water pressure and total stress as state variables. The parameters of the constitutive model are calibrated against experimental data obtained from samples retrieved during construction of Napoli underground, in which AGF was extensively used to excavate in granular soils and weak fractured rock below the ground water table

Adaptive Threshold Selection for Set Membership State Estimation with Quantized Measurements

State estimation for discrete-time linear systems with quantized measurements is addressed. By exploiting the set-theoretic nature of the information provided by the quantizer, the problem is cast in the set membership estimation setting. Motivated by the possibility of suitably tuning the quantizer thresholds in sensor networks, the optimal design of adaptive quantizers is formulated in terms of the minimization of the radius of information associated to the state estimation problem. The optimal solution is derived for first-order systems and the result is exploited to design adaptive quantizers for generic systems, minimizing the size of the feasible output signal set. Then, the minimum number of sensor thresholds for which the adaptive quantizers guarantee asymptotic boundedness of the state estimation uncertainty is established. Threshold adaptation mechanisms based on several types of outer approximations of the feasible state set are also proposed. The effectiveness of the designed adaptive quantizers is demonstrated on numerical tests involving a specific case study and randomly generated systems, highlighting the trade off between the resulting estimation uncertainty and the computational burden required by recursive set approximations

Artificial ground freezing of a volcanic ash: laboratory tests and modelling

The use of artificial ground freezing (AGF) to form earth support systems has had applications worldwide. These cover a variety of construction problems, including the formation of frozen earth walls to support deep excavations, structural underpinning for foundation improvement and temporary control of ground water in construction processes. On one hand, the main advantage of AGF as a temporary support system in comparison to other support methods, such as those based on injections of chemical or cement grout into the soil, is the low impact on the surrounding environment as the refrigerating medium required to obtain AGF is circulated in pipes and exhausted in the atmosphere or re-circulated without contamination of the ground water. On the other hand, the available methods may vary significantly in their sustainability and complexity in terms of times and costs required for their installation and maintenance. The ability to predict the effects induced by AGF on granular materials is therefore crucial to assessing construction time and cost and to optimising the method. In this work, the thermo-hydro-mechanical processes induced by artificial freezing of a soil body are studied using a constitutive model that encompasses frozen and unfrozen behaviour within a unified effective-stress-based framework. It makes use of a combination of ice pressure, liquid water pressure and total stress as state variables. The model is validated and calibrated using the results of a series of laboratory tests on natural samples of a volcanic ash (Pozzolana) retrieved during construction of Napoli underground, where the technique of AGF was used extensively to stabilise temporarily the ground and control the ground water

Optimal energy management and control of an industrial microgrid with plug-in electric vehicles

An industrial microgrid (IMG) consists in a microgrid involving manufacturer plants which are usually equipped with distributed generation facilities, industrial electric vehicles, energy storage systems, etc. In this paper, the problem of IMG efficient operation in presence of plug-in electric vehicles is addressed. To this purpose, schedule of the different device operations of IMGs has to be optimally computed, minimizing the operation cost while guaranteeing electrical network stability and production constraints. Such a problem is formulated in a receding horizon framework involving dynamic optimal power flow equations. Uncertainty affecting plug-in electric vehicles is handled by means of a chance constraint approach. The obtained nonconvex problem is then approximately solved by exploiting suitable convex relaxation techniques. Numerical simulations have been performed showing computational feasibility and robustness of the proposed approach against increased penetration of electric vehicles

Why teach “Bioethics and Human Rights” to healthcare professions undergraduates?

This article highlights the importance of teaching “bioethics and human rights” to undergraduate students seeking health care degrees and illustrates how this topic fits well within these programs of studies. Historical, cultural, anthropological and practical reasons support teaching these topics as enrichment of medical training. The years after the Second World War showed how bioethics, human rights and medicine are closely intertwined. Moreover the relationship between human rights and bioethics has grown ever closer increasingly involving medicine and health care professionals. The authors observe that medical students have to face a cultural pluralism in bioethics and biolaw and we give students the opportunity to develop their critical thinking and logical argumentation abilities as well as their interest in academic research. Furthermore, the authors – who draw up briefly the experience of the Institute of Bioethics at the Faculty of Medicine and Surgery of the UCSC (Rome) - assert the necessity to help medical students to be respectful of patients in every clinical setting. It is therefore of utmost importance to train students to focus on the ethical dimension of care and to make good ethical decisions even in dilemmatic cases. To achieve this outcome, healthcare professionals should possess an integral vision of their work (technical and humanistic competence) and sharp skills to reflect in depth, avoiding superficiality and negligence. From this perspective, the teaching of “bioethics and human rights” could be very useful

Design and realization of the CUFF - clenching upper-limb force feedback wearable device for distributed mechano-tactile stimulation of normal and tangential skin forces

Rendering forces to the user is one of the main goals of haptic technology. While most force-feedback interfaces are robotic manipulators, attached to a fixed frame and designed to exert forces on the users while being moved, more recent haptic research introduced two novel important ideas. On one side, cutaneous stimulation aims at rendering haptic stimuli at the level of the skin, with a distributed, rather than, concentrated approach. On the other side, wearable haptics focuses on highly portable and mobile devices, which can be carried and worn by the user as the haptic equivalent of an mp3 player. This paper presents a light and simple wearable device (CUFF) for the distributed mechano-tactile stimulation of the user's arm skin with pressure and stretch cues, related to normal and tangential forces, respectively. The working principle and the mechanical and control implementation of the CUFF device are presented. Then, after a basic functional validation, a first application of the device is shown, where it is used to render the grasping force of a robotic hand (the Pisa/IIT SoftHand). Preliminary results show that the device is capable to deliver in a reliable manner grasping force information, thus eliciting a good softness discrimination in users and enhancing the overall grasping experience

Post-Collisional Tectonomagmatic Evolution, Crustal Reworking and Ore Genesis along a Section of the Southern Variscan Belt: The Variscan Mineral System of Sardinia (Italy)

Since the early Paleozoic, numerous metallogenic events produced in the Sardinian massif a singular concentration of mineral deposits of various kinds. Among them, the Variscan metallogenic peak represents a late Paleozoic phase of diffuse ore formation linked to the tectonomagmatic evolution of the Variscan chain. Twomain classes of oresmay primarily be attributed to this peak:(1)mesothermal orogenic-type As-Au ±W± Sb ores, only found in E Sardinia, and (2) intrusion-related Sn-W-Mo-F and base metals-bearing ores found in the whole Sardinian Batholith, but mainly occurring in central–south Sardinia. Both deposit classes formed diachronously during the Variscan post-compressional extension. The orogenic-type ores are related to regional-scale flows ofmineralizing fluids, and the intrusion-related ores occur around fertile intrusions of different granite suites. Metallogenic reconstructions suggest almost entirely crustal processes ofmineralizationwithout a significant contribution fromthemantle. We summarized these processes with a holistic approach and conceptualized the Sardinian Variscan Mineral System (SVMS), a crustal-scale physical system of ore mineralization in the Sardinian basement. The SVMS required suitable metal sources in the crust and diffuse crustal reworking triggered by heat that allowed (a) the redistribution of the original metal budget of the crust in magmas by partial melting and (b) the production of metal-bearing fluids by metamorphic dehydration. Heat transfer in the Sardinian Variscan crust involved shear heating in lithospheric shear zones and the role of mantle uplift as a thermal engine in an extensional tectonic setting. Lithospheric shear zones acted as effective pathways in focusing fluid flow through a large-scale plumbing system into regional-scale structural traps for ores. Pre-Variscan metal sources of metallogenic relevance may have been (1) the magmatic arc and magmatic arc-derived materials of Ordovician age, extensively documented in E Sardinia crust, and (2) an inferred Precambrian crystalline basement lying under the Phanerozoic crustal section, whose presence has been assumed from geophysical data and from petrological and geochemical characteristics of granite suites. At shallower crustal levels, important contributions of metals may have come from pre-Variscan ore sources, such as the Pb-ZnMVT Cambrian ores of SWSardinia or the REE-bearing Upper Ordovician paleoplacers of E Sardinia

Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons.

RanBP2/Nup358 is a member of the large nucleoporin family constituting the nuclear pore complex (NPC). Depending on the cell type and the physiological state, Nup358 interacts with specific partner proteins and influences distinct mechanisms independent of its role in nucleocytoplasmic transport. Here, we provide evidence that Nup358 associates selectively with the axon initial segment (AIS) of mature neurons and mediated by the AIS scaffold ankyrin-G. The N-terminus of Nup358 is found to be sufficient for its localization at the AIS. Further, we show that Nup358 is expressed as two isoforms, one full-length and another shorter form of Nup358. These isoforms differ in their subcellular distribution in neurons and expression level during neuronal development. Overall, the present study highlights an unprecedented localization of Nup358 within the AIS and suggests its involvement in neuronal function

Health technology assessment of pathogen reduction technologies applied to plasma for clinical use

Although existing clinical evidence shows that the transfusion of blood components is becoming increasingly safe, the risk of transmission of known and unknown pathogens, new pathogens or re-emerging pathogens still persists. Pathogen reduction technologies may offer a new approach to increase blood safety. The study is the output of collaboration between the Italian National Blood Centre and the Post-Graduate School of Health Economics and Management, Catholic University of the Sacred Heart, Rome, Italy. A large, multidisciplinary team was created and divided into six groups, each of which addressed one or more HTA domains.Plasma treated with amotosalen + UV light, riboflavin + UV light, methylene blue or a solvent/detergent process was compared to fresh-frozen plasma with regards to current use, technical features, effectiveness, safety, economic and organisational impact, and ethical, social and legal implications. The available evidence is not sufficient to state which of the techniques compared is superior in terms of efficacy, safety and cost-effectiveness. Evidence on efficacy is only available for the solvent/detergent method, which proved to be non-inferior to untreated fresh-frozen plasma in the treatment of a wide range of congenital and acquired bleeding disorders. With regards to safety, the solvent/detergent technique apparently has the most favourable risk-benefit profile. Further research is needed to provide a comprehensive overview of the cost-effectiveness profile of the different pathogen-reduction techniques. The wide heterogeneity of results and the lack of comparative evidence are reasons why more comparative studies need to be performed