Molecular interactions: metal ions and protein chaperones in the urease system from Helicobacter pylori

Although nickel is a toxic metal for living organisms in its soluble form, its importance in many biological processes recently emerged. In this view, the investigation of the nickel-dependent enzymes urease and [NiFe]-hydrogenase, especially the mechanism of nickel insertion into their active sites, represent two intriguing case studies to understand other analogous systems and therefore to lead to a comprehension of the nickel trafficking inside the cell. Moreover, these two enzymes have been demonstrated to ensure survival and colonization of the human pathogen H. pylori, the only known microorganism able to proliferate in the gastric niche. The right nickel delivering into the urease active site requires the presence of at least four accessory proteins, UreD, UreE, UreF and UreG. Similarly, analogous process is principally mediated by HypA and HypB proteins in the [NiFe]-hydrogenase system. Indeed, HpHypA and HpHypB also have been proposed to act in the activation of the urease enzyme from H. pylori, probably mobilizing nickel ions from HpHypA to the HpUreE-HpUreG complex. A complete comprehension of the interaction mechanism between the accessory proteins and the crosstalk between urease and hydrogenase accessory systems requires the determination of the role of each protein chaperone that strictly depends on their structural and biochemical properties. The availability of HpUreE, HpUreG and HpHypA proteins in a pure form is a pre-requisite to perform all the subsequent protein characterizations, thus their purification was the first aim of this work. Subsequently, the structural and biochemical properties of HpUreE were investigated using multi-angle and quasi-elastic light scattering, as well as NMR and circular dichroism spectroscopy. The thermodynamic parameters of Ni2+ and Zn2+ binding to HpUreE were principally established using isothermal titration calorimetry and the importance of key histidine residues in the process of binding metal ions was studied using site-directed mutagenesis. The molecular details of the HpUreE-HpUreG and HpUreE-HpHypA protein-protein assemblies were also elucidated. The interaction between HpUreE and HpUreG was investigated using ITC and NMR spectroscopy, and the influence of Ni2+ and Zn2+ metal ions on the stabilization of this association was established using native gel electrophoresis, light scattering and thermal denaturation scanning followed by CD spectroscopy. Preliminary HpUreE-HpHypA interaction studies were conducted using ITC. Finally, the possible structural architectures of the two protein-protein assemblies were rationalized using homology modeling and docking computational approaches. All the obtained data were interpreted in order to achieve a more exhaustive picture of the urease activation process, and the correlation with the accessory system of the hydrogenase enzyme, considering the specific role and activity of the involved protein players. A possible function for Zn2+ in the chaperone network involved in Ni2+ trafficking and urease activation is also envisaged.

The Architecture of the Persian Period in the Levant

The earliest scholars were not concerned about preparing extensive investigations linking the Persian-period building remains excavated in the entire Levant together. Moreover, the research interests of scholars caused some impediments to the study of this period viz in the last decades; the Achaemenid period has been neglected by the scholars who -in turn- focused on the earlier and later periods for religious reasons. Too, while some regions have been studied abundantly, but it was not the case in other areas, which makes our knowledge is incomplete. From the explanation side, some scholars try to interpret the architectural remains from an ethnic perspective or unsubstantiated personal fancies, so their arguments were utterly lacking any objectivity. This thesis explores what are the Persian architectural and ornamental impacts on the Levantine architecture and the relations between Persian-period sites in Syria-Palestine region. Too, the architectural remains and their contents benefited us to clarify the settlement patterns in the regions being discussed. The author analyzed the ground plans of the buildings and their architectural features and ornamental motifs by conducting a descriptive, analytical, and interpretative study. He also conducted comparisons with similar buildings outside the Levant, especially in Fars to obtain a more comprehensive and systematic study, and then extracting any direct or indirect Persian influences. This has given us a better understanding of the nature of the social, political, and religious life in the entire Levant and the knowledge gap has been bridged to a satisfying extent. This study has demonstrated a few of the Achaemenid impacts, especially on the northern coastline of the Levant.

An architecture for network acceleration as a service in the cloud continuum

The pervasive availability of connected devices in any industrial and societal sector is pushing for an evolution of the well-established cloud computing model. The emerging paradigm of the cloud continuum embraces this decentralization trend and envisions virtualized computing resources physically located between traditional datacenters and data sources. By totally or partially executing closer to the network edge, applications can have quicker reactions to events, thus enabling advanced forms of automation and intelligence. However, these applications also induce new data-intensive workloads with low-latency constraints that require the adoption of specialized resources, such as high-performance communication options (e.g., RDMA, DPDK, XDP, etc.). Unfortunately, cloud providers still struggle to integrate these options into their infrastructures. That risks undermining the principle of generality that underlies the cloud computing scale economy by forcing developers to tailor their code to low-level APIs, non-standard programming models, and static execution environments. This thesis proposes a novel system architecture to empower cloud platforms across the whole cloud continuum with Network Acceleration as a Service (NAaaS). To provide commodity yet efficient access to acceleration, this architecture defines a layer of agnostic high-performance I/O APIs, exposed to applications and clearly separated from the heterogeneous protocols, interfaces, and hardware devices that implement it. A novel system component embodies this decoupling by offering a set of agnostic OS features to applications: memory management for zero-copy transfers, asynchronous I/O processing, and efficient packet scheduling. This thesis also explores the design space of the possible implementations of this architecture by proposing two reference middleware systems and by adopting them to support interactive use cases in the cloud continuum: a serverless platform and an Industry 4.0 scenario. A detailed discussion and a thorough performance evaluation demonstrate that the proposed architecture is suitable to enable the easy-to-use, flexible integration of modern network acceleration into next-generation cloud platforms.

Sviluppo di modelli motore e veicolo per l'analisi di strategie di controllo in applicazioni Software e Hardware In the Loop

This work describes the development of a simulation tool which allows the simulation of the Internal Combustion Engine (ICE), the transmission and the vehicle dynamics. It is a control oriented simulation tool, designed in order to perform both off-line (Software In the Loop) and on-line (Hardware In the Loop) simulation. In the first case the simulation tool can be used in order to optimize Engine Control Unit strategies (as far as regard, for example, the fuel consumption or the performance of the engine), while in the second case it can be used in order to test the control system. In recent years the use of HIL simulations has proved to be very useful in developing and testing of control systems. Hardware In the Loop simulation is a technology where the actual vehicles, engines or other components are replaced by a real time simulation, based on a mathematical model and running in a real time processor. The processor reads ECU (Engine Control Unit) output signals which would normally feed the actuators and, by using mathematical models, provides the signals which would be produced by the actual sensors. The simulation tool, fully designed within Simulink, includes the possibility to simulate the only engine, the transmission and vehicle dynamics and the engine along with the vehicle and transmission dynamics, allowing in this case to evaluate the performance and the operating conditions of the Internal Combustion Engine, once it is installed on a given vehicle. Furthermore the simulation tool includes different level of complexity, since it is possible to use, for example, either a zero-dimensional or a one-dimensional model of the intake system (in this case only for off-line application, because of the higher computational effort). Given these preliminary remarks, an important goal of this work is the development of a simulation environment that can be easily adapted to different engine types (single- or multi-cylinder, four-stroke or two-stroke, diesel or gasoline) and transmission architecture without reprogramming. Also, the same simulation tool can be rapidly configured both for off-line and real-time application. The Matlab-Simulink environment has been adopted to achieve such objectives, since its graphical programming interface allows building flexible and reconfigurable models, and real-time simulation is possible with standard, off-the-shelf software and hardware platforms (such as dSPACE systems).

Environmental weathering of natural and artificial stones used in historical architecture: influence of microstructure and new restoration methods

For some study cases (the Cathedral of Modena, Italy, XII-XIV century; the Ducal Palace in Mantua, Italy, XVI century; the church of San Francesco in Fano, Italy, XIV-XIX century), considered as representative of the use of natural and artificial stones in historical architecture, the complex interaction between environ-mental aggressiveness, materials’ microstructural characteristics and degradation was investigated. From the results of such analyses, it was found that materials microstructure plays a fundamental role in the actual extent to which weathering mechanisms affect natural and artificial stones. Consequently, the need of taking into account the important role of material microstructure, when evaluating the environmental aggressiveness to natural and artificial stones, was highlighted. Therefore, a possible quantification of the role of microstructure on the resistance to environmental attack was investigated. By exposing stone samples, with significantly different microstructural features, to slightly acidic aqueous solutions, simulating clean and acid rain, a good correlation between weight losses and the product of carbonate content and specific surface area (defined as the “vulnerable specific surface area”) was found. Alongside the evaluation of stone vulnerability, the development of a new consolidant for weathered carbonate stones was undertaken. The use of hydroxya-patite, formed by reacting the calcite of the stone with an aqueous solution of di-ammonium hydrogen phosphate, was found to be a promising consolidating tech-nique for carbonates stones. Indeed, significant increases in the mechanical prop-erties can be achieved after the treatment, which has the advantage of simply con-sisting in a non-hazardous aqueous solution, able to penetrate deeply into the stone (> 2 cm) and bring significant strengthening after just 2 days of reaction. Furthermore, the stone sorptivity is not eliminated after treatment, so that water and water vapor exchanges between the stone and the environment are not com-pletely blocked.

Molecular and functional characterization of the chemotactic genes in the PCBs-degrader Pseudomonas pseudoalcaligenes KF707

Although bacteria represent the simplest form of life on Earth, they have a great impact on all living beings. For example the degrader bacterium Pseudomonas pseudoalcaligenes KF707 is used in bioremediation procedures for the recovery of polluted sites. Indeed, KF707 strain is know for its ability to degrade biphenyl and polychlorinated biphenyls - to which is chemotactically attracted - and to tolerate the oxydative stress due to toxic metal oxyanions such as tellurite and selenite. Moreover, in bioremediation processes, target compounds can be easily accessible to KF707 through biofilm formation. All these considerations suggest that KF707 is such a unique microorganism and this Thesis work has been focused on determining the molecular nature of some of the peculiar physiological traits of this strain. The genome project provided a large set of informations: putative genes involved in the degradation of aromatic and toxic compounds and associated to stress response were identified. Notably, multiple chemotactic operons and cheA genes were also found. Deleted mutants in the cheA genes were constructed and their role in motility, chemotaxis and biofilm formation were assessed and compared to those previously attributed to a cheA1 gene in a KF707 mutant constructed by a mini-Tn5 transposon insertion and which was impaired in motility and biofilm development. The results of this present Thesis work, taken together, were interpreted to suggest that in Pseudomonas pseudoalcaligenes KF707 strain, multiple factors are involved in these networks and they might play different roles depending on the environmental conditions. The ability of KF707 strain to produce signal molecules possibly involved in cell-to-cell communication, was also investigated: lack of a lux-like QS system - which is conversely widely present in Gram negative bacteria – keeps open the question about the actual molecular nature of KF707 quorum sensing mechanism.

Use of shared memory in the context of embedded multi-core processors: exploration of the technology and its limits

Modern embedded systems embrace many-core shared-memory designs. Due to constrained power and area budgets, most of them feature software-managed scratchpad memories instead of data caches to increase the data locality. It is therefore programmers’ responsibility to explicitly manage the memory transfers, and this make programming these platform cumbersome. Moreover, complex modern applications must be adequately parallelized before they can the parallel potential of the platform into actual performance. To support this, programming languages were proposed, which work at a high level of abstraction, and rely on a runtime whose cost hinders performance, especially in embedded systems, where resources and power budget are constrained. This dissertation explores the applicability of the shared-memory paradigm on modern many-core systems, focusing on the ease-of-programming. It focuses on OpenMP, the de-facto standard for shared memory programming. In a first part, the cost of algorithms for synchronization and data partitioning are analyzed, and they are adapted to modern embedded many-cores. Then, the original design of an OpenMP runtime library is presented, which supports complex forms of parallelism such as multi-level and irregular parallelism. In the second part of the thesis, the focus is on heterogeneous systems, where hardware accelerators are coupled to (many-)cores to implement key functional kernels with orders-of-magnitude of speedup and energy efficiency compared to the “pure software” version. However, three main issues rise, namely i) platform design complexity, ii) architectural scalability and iii) programmability. To tackle them, a template for a generic hardware processing unit (HWPU) is proposed, which share the memory banks with cores, and the template for a scalable architecture is shown, which integrates them through the shared-memory system. Then, a full software stack and toolchain are developed to support platform design and to let programmers exploiting the accelerators of the platform. The OpenMP frontend is extended to interact with it.

Structural and biochemical studies of Sporosarcina pasteurii UreE: a nickel-chaperone involved in the urease activation process

Urease is a nickel-dependent enzyme that catalyzes hydrolysis of urea in the last step of organic nitrogen mineralization. Its active site contains a dinuclear center for Ni(II) ions that must be inserted into the apo-enzyme through the action of four accessory proteins (UreD, UreE, UreF, UreG) leading to activation of urease. UreE, acting as a metallo-chaperone, delivers Ni(II) to the preformed complex of apo-urease-UreDFG and has the capability to enhance the GTPase activity of UreG. This study, focused on characterization of UreE from Sporosarcina pasteurii (SpUreE), represents a piece of information on the structure/mobility-function relationships that control nickel binding by SpUreE and its interaction with SpUreG. A calorimetric analysis revealed the occurrence of a binding event between these proteins with positive cooperativity and a stoichiometry consistent with the formation of the (UreE)2-(UreG)2 hetero-oligomer complex. Chemical Shift Perturbations induced by the protein-protein interaction were analyzed using high-resolution NMR spectroscopy, which allowed to characterize the molecular details of the protein surface of SpUreE involved in the complex formation with SpUreG. Moreover, backbone dynamic properties of SpUreE, determined using 15N relaxation analysis, revealed a general mobility in the nanoseconds time-scale, with the fastest motions observed at the C-termini. The latter analysis made it possible for the first time to characterize of the C-terminal portions, known to contain key residues for metal ion binding, that were not observed in the crystal structure of UreE because of disorder. The residues belonging to this portion of SpUreE feature large CSPs upon addition of SpUreG, showing that their chemical environment is directly affected by protein-protein interaction. Metal ion selectivity and affinity of SpUreE for cognate Ni(II) and non cognate Zn(II) metal ions were determined, and the ability of the protein to select Ni(II) over Zn(II), in consistency with the proposed role in Ni(II) cations transport, was established.

Upgrading Bio-Platform Molecules in the Gas-Phase: From Levulinic Acid to Bio-Chemicals

Levulinic acid (LA) is a polyfunctional molecule obtained from biomass. Because of its structure, the United States Department of energy classified LA as one of the top 12 building block chemicals. Typically, it is valorized through chemical reduction to γ-valerolactone (GVL). It is usually done with H2 in batch systems with high H2 pressures and noble metal catalysts, making it expensive and less applicable. Therefore, alternative approaches such as catalytic transfer hydrogenation (CTH) through the Meerwein–Ponndorf–Verley (MPV) reaction over heterogeneous catalysts have been studied. This uses organic molecules (alcohols) which act as a hydride transfer agent (H-donor), to reduce molecules containing carbonyl groups. Given the stability of the intermediate, reports have shown the batch liquid-phase CTH of levulinate esters with secondary alcohols, and remarkable results (GVL yield) have been obtained over ZrO2, given the need of a Lewis acid (LASites) and base pair for CTH. However, there were no reports of the continuous gas-phase CTH of levulinate esters. Therefore, high surface area ZrO2 was tested for gas-phase CTH of methyl levulinate (ML) using ethanol, methanol and isopropanol as H-donors. Under optimized conditions with ethanol (250 ℃), the reaction is selective towards GVL (yield 70%). However, heavy carbonaceous materials over the catalyst surface progressively blocked LASites changing the chemoselectivity. The in situ regeneration of the catalyst permitted a partial recovery of the LASites and an almost total recovery of the initial catalytic behavior, proving the deactivation reversible. Tests with methanol were not promising (ML conversion 35%, GVL yield 4%). As expected, using isopropanol provided complete conversion and a GVL yield of 80%. The reaction was also tested using bioethanol derived from agricultural waste. In addition, a preliminary study was performed for the hydrogenolysis of polyols to produce bioethanol, were Pd-Fe catalyst promoted the ethanol selective (37%) hydrogenolysis of glycerol.

Leonardo Ricci in the United States (1952-1972). A twenty-year American transfer as a turning experience in teaching and design.

The research solved the historiographic lacuna about Leonardo Ricci’s work in the United States focusing on the span 1952-1972 as a fundamental period for the architect's research, which moved from the project for the community space to macrostructures. The considered period is comprised between Ricci’s first travel to the United States and the date of his resignation from the University of Florida, one year before his resignation from the deanship of the faculty of architecture of Florence (1973). The research retraced philologically the stages of Ricci’s activity in the U.S.A. unveiling the premises and results of his American transfer, and to what extent it marked a turning period for his work as educator and designer and for the wider historiographic contest of the Sixties. The American transfer helped him grounding his belief in avoiding a priori morphological results in favor of what he called the “form-act” design method. Ricci’s research in the U.S.A. is described in his books Anonymous (XX century) and City of the Earth (unpublished). In them and in Ricci’s projects one common thread is traceable: the application of the “form-act” as the best tool to conceive urban design, a discipline established in the United States during Ricci’s first stay at M.I.T., in which he encountered the balance point between architecture and urban planning, between the architect’s sign and his being anonymous, between the collective and the individual dimension. With the notions of “anonymous architecture” and “form-act”, Urban Design and “open work” are the key words to understand Ricci’s work in the United States and in Italy. Urban design’s main goal to design the city as a collective work of art was the solution of that dychothomous research that enlivened Ricci’s work and one possible answer to that tension useful for him to seek the truth of architecture.

Development, mechanical characterization and qualification in liquid lead of stainless steels for structural applications in the future fission and fusion nuclear reactors

The research activity carried out in the Brasimone Research Center of ENEA concerns the development and mechanical characterization of steels conceived as structural materials for future fission reactors (Heavy Liquid Metal IV Generation reactors: MYRRHA and ALFRED) and for the future fusion reactor DEMO. Within this framework, two parallel lines of research have been carried out: (i) characterization in liquid lead of steels and weldings for the components of the IV Generation fission reactors (GIV) by means of creep and SSRT (Slow Strain Rate Tensile) tests; (ii) development and screening on mechanical properties of RAFM (Reduced Activation Ferritic Martensitic) steels to be employed as structural materials of the future DEMO fusion reactor. The doctoral work represents therefore a comprehensive report of the research carried out on nuclear materials both from the point of view of the qualification of existing (commercial) materials for their application in the typical environmental conditions of 4th generation fission reactors operating with lead as coolant, and from the point of view of the metallurgical study (with annexed microstructural and mechanical characterization of the selected compositions / Thermo Mechanical Treatment (TMT) options) of new compositional variants to be proposed for the “Breeding Blanket” of the future DEMO Fusion Reactor.

Evaluation of deambulation in a protected environment and in the public green in limb amputated subjects treated through a new osteointegration method

The limb amputation is one of the oldest surgical procedures performed and it still represents an event that drastically changes the life of an individual. Despite the technological progress, the difficulties related to the realization and daily use of the socket remain very common. Among the different technologies adopted in the prosthetic field, this project focused on the osseointegration technique. This technique consists in implanting a stem within the medullary canal of the amputated skeletal segment that extends outside the amputation stump with a prosthesis, later connected to the metal extension. The objective of this PhD project is to treat and to evaluate selected patients with osseointegrated prosthetic implants for the treatment of lower limb amputations. Patients are recruited at the Rizzoli Orthopaedic Institute and at the Prosthesis - INAIL center of Vigorso (Budrio) during outpatient visits, while the surgical procedure is performed by the same expert surgeon in the II Orthopaedic and Traumatology Clinic of the Rizzoli Orthopaedic Institute. The project is still ongoing, to date three patients had completed both procedures, but due to various personal problems, just one of them is included in the analysis. This patient increased his percentage of prosthesis use and the level of mobility with an overall improvement of quality of live after the procedure. The osseointegration technique represents a promising alternative method of treatment for amputees who are not satisfied with their socket prosthesis. In the coming years it will continue the collection of clinical, radiographic and kinematic data of subjects undergoing this procedure in order to perform a long-term monitoring of both clinical outcomes and quality of life.

Pharmacological targeting of P-selectin to halt the progression of myelofibrosis in the Gata1low mouse model

Primary myelofibrosis is a clonal hematopoietic disorder characterized by marked degrees of systemic inflammation. The release of pro-inflammatory factors by clonal hematopoietic cell populations cause the remodeling of a specialized microenvironment, defined niche, in which the hematopoietic stem cells reside. The main source of pro-inflammatory cytokines is represented by malignant megakaryocytes. The bone marrow and spleen from myelofibrosis patients, as well as those from the Gata1low mouse model of the disease, contain increased number of abnormal megakaryocytes. These cells express on their surface high levels of the adhesion receptor P-selectin that, by triggering a pathological megakaryocyte-neutrophil emperipolesis, lead to increased bioavailability of TGF-β1 in the microenvironment and disease progression. Gata1low mice develop with age a phenotype similar to that of patients with myelofibrosis. We previously demonstrated that deletion of the P-selectin gene in Gata1low mice prevented the development of the myelofibrotic phenotype in these mice. In the current study, we tested the hypothesis that pharmacological inhibition of P-selectin may rescue the fibrotic phenotype of Gata1low mice. To test this hypothesis, we have investigated the phenotype expressed by old Gata1low mice treated with the anti-mouse monoclonal antibody against P-selectin RB40.34, alone or in combination with the JAK2 inhibitor Ruxolitinib. The results showed that the combined therapy normalized the phenotype of Gata1low mice with limited toxicity by reducing fibrosis, TGF-β1 and CXCL1 content in the BM and spleen and by restoring hematopoiesis in the bone marrow and the normal architecture of the spleen. In conclusion, pharmacological inhibition of P-selectin was effective in targeting malignant megakaryocytes and the microenvironmental abnormalities that affect the hematopoietic stem cell compartment in this model. These results suggest that P-selectin and JAK1/2 inhibitors in combination may represent a valid therapeutic option for patients with myelofibrosis.

Analysis of the long-term subsidence in the urban area of Bologna

Land subsidence in urban areas represents a widespread geological hazard and a pressing challenge for modern society. This research focuses on the subsidence process affecting the city of Bologna (Italy). Since the 1960s, Bologna has experienced ground deformation due to aquifers overexploitation that peaked during the 1970s with rates of 10 cm/year. Despite a general reduction in these rates over the subsequent decades, thanks to groundwater regulations policies, recent data underscore a substantial subsidence resurgence. To reconstruct the subsurface stratigraphic architecture of Bologna’s urban area and generate a 3D geological model, a multidisciplinary approach centred on a stratigraphic analysis relying on the lithofacies criterion was adopted. The convergence of the analyses within this framework resulted in partitioning the study area into three geological domains exhibiting unique morphological features and depositional stacking patterns. Subsequently, since long-term data are crucial for a comprehensive understanding of ongoing subsidence, a methodology was developed to generate cumulative ground displacement time series and maps by integrating ground-based and remotely-sensed measurements. While the reconstructed long-term subsidence field consistently aligns with the primary geological variations summarised in the 3D model, the generated cumulative displacement curves systematically match pluriannual trends observed in groundwater level and pumping monitoring data. Lastly, to evaluate the expression of the observed relationships from a geotechnical perspective, a series of one-dimensional subsidence calculations were conducted considering the mechanical properties of the investigated deposits and piezometric data. These analyses provided valuable insight into the overall mechanical behaviour of the existing soils, as well as the post-pumping groundwater level and pore pressure distributions, consistent with field data. The methodological approach employed enables a comprehensive analysis of land subsidence in urban areas, allowing the exploration of individual factors governing the deformation process and their interactions, even within complex stratigraphic and hydrogeological environments such as Bologna’s urban area.