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.

Thermochronologic and geodynamic evolution of the Pontides: Sakarya terrane (Karakaya Complex) and Istanbul terrane, Turkey.

An integrated array of analytical methods -including clay mineralogy, vitrinite reflectance, Raman spectroscopy on carbonaceous material, and apatite fission-track analysis- was employed to constrain the thermal and thermochronological evolution of selected portions of the Pontides of northern Turkey. (1) A multimethod investigation was applied for the first time to characterise the thermal history of the Karakaya Complex, a Permo-Triassic subduction-accretion complex cropping out throughout the Sakarya Zone. The results indicate two different thermal regimes: the Lower Karakaya Complex (Nilüfer Unit) -mostly made of metabasite and marble- suffered peak temperatures of 300-500°C (greenschist facies); the Upper Karakaya Complex (Hodul and the Orhanlar Units) –mostly made of greywacke and arkose- yielded heterogeneous peak temperatures (125-376°C), possibly the result of different degree of involvement of the units in the complex dynamic processes of the accretionary wedge. Contrary to common belief, the results of this study indicate that the entire Karakaya Complex suffered metamorphic conditions. Moreover, a good degree of correlation among the results of these methods demonstrate that Raman spectroscopy on carbonaceous material can be applied successfully to temperature ranges of 200-330°C, thus extending the application of this method from higher grade metamorphic contexts to lower grade metamorphic conditions. (2) Apatite fission-track analysis was applied to the Sakarya and the İstanbul Zones in order to constrain the exhumation history and timing of amalgamation of these two exotic terranes. AFT ages from the İstanbul and Sakarya terranes recorded three distinct episodes of exhumation related to the complex tectonic evolution of the Pontides. (i) Paleocene - early Eocene ages (62.3-50.3 Ma) reflect the closure of the İzmir-Ankara ocean and the ensuing collision between the Sakarya terrane and the Anatolide-Tauride Block. (ii) Late Eocene - earliest Oligocene (43.5-32.3 Ma) ages reflect renewed tectonic activity along the İzmir-Ankara. (iii) Late Oligocene- Early Miocene ages reflect the onset and development of the northern Aegean extension. The consistency of AFT ages, both north and south of the tectonic contact between the İstanbul and Sakarya terranes, suggest that such terranes were amalgamated in pre-Cenozoic times. (3) Fission-track analysis was also applied to rock samples from the Marmara region, in an attempt to constrain the inception and development of the North Anatolian Fault system in the region. The results agree with those from the central Pontides. The youngest AFT ages (Late Oligocene - early Miocene) were recorded in the western portion of the Marmara Sea region and reflect the onset and development of northern Aegean extension. Fission-track data from the eastern Marmara Sea region indicate rapid Early Eocene exhumation induced by the development of the İzmir-Ankara orogenic wedge. Thermochronological data along the trace of the Ganos Fault –a segment of the North Anatolian Fault system- indicate the presence of a tectonic discontinuity active by Late Oligocene time, i.e. well before the arrival of the North Anatolian Fault system in the area. The integration of thermochronologic data with preexisting structural data point to the existence of a system of major E-W-trending structural discontinuities active at least from the Late Oligocene. In the Early Pliocene, inception of the present-day North Anatolian Fault system in the Marmara region occurred by reactivation of these older tectonic structures. 


Multi-level models and infrastructures for simulating biological system development

The hierarchical organisation of biological systems plays a crucial role in the pattern formation of gene expression resulting from the morphogenetic processes, where autonomous internal dynamics of cells, as well as cell-to-cell interactions through membranes, are responsible for the emergent peculiar structures of the individual phenotype. Being able to reproduce the systems dynamics at different levels of such a hierarchy might be very useful for studying such a complex phenomenon of self-organisation. The idea is to model the phenomenon in terms of a large and dynamic network of compartments, where the interplay between inter-compartment and intra-compartment events determines the emergent behaviour resulting in the formation of spatial patterns. According to these premises the thesis proposes a review of the different approaches already developed in modelling developmental biology problems, as well as the main models and infrastructures available in literature for modelling biological systems, analysing their capabilities in tackling multi-compartment / multi-level models. The thesis then introduces a practical framework, MS-BioNET, for modelling and simulating these scenarios exploiting the potential of multi-level dynamics. This is based on (i) a computational model featuring networks of compartments and an enhanced model of chemical reaction addressing molecule transfer, (ii) a logic-oriented language to flexibly specify complex simulation scenarios, and (iii) a simulation engine based on the many-species/many-channels optimised version of Gillespie’s direct method. The thesis finally proposes the adoption of the agent-based model as an approach capable of capture multi-level dynamics. To overcome the problem of parameter tuning in the model, the simulators are supplied with a module for parameter optimisation. The task is defined as an optimisation problem over the parameter space in which the objective function to be minimised is the distance between the output of the simulator and a target one. The problem is tackled with a metaheuristic algorithm. As an example of application of the MS-BioNET framework and of the agent-based model, a model of the first stages of Drosophila Melanogaster development is realised. The model goal is to generate the early spatial pattern of gap gene expression. The correctness of the models is shown comparing the simulation results with real data of gene expression with spatial and temporal resolution, acquired in free on-line sources.

Structure of the Southern Tyrrhenian subduction system: insights from seismological analysis of anisotropy and attenuation

The Southern Tyrrhenian subduction system shows a complex interaction among asthenospheric flow, subducting slab and overriding plate. To shed light on the deformations and mechanical properties of the slab and surrounding mantle, I investigated seismic anisotropy and attenuation properties through the subduction region. I used both teleseisms and slab earthquakes, analyzing shear-wave splitting on SKS and S phases, respectively. The fast polarization directions φ, and the delay time, δt, were retrieved using the method of Silver and Chan [1991. SKS and S φ reveal a complex anisotropy pattern across the subduction zone. SKS-rays sample primarily the sub-slab region showing rotation of fast directions following the curved shape of the slab and very strong anisotropy. S-rays sample mainly the slab, showing variable φ and a smaller δt. SKS and S splitting reveals a well developed toroidal flow at SW edge of the slab, while at its NE edge the pattern is not very clear. This suggests that the anisotropy is controlled by the slab rollback, responsible for about 100 km slab parallel φ in the sub-slab mantle. The slab is weakly anisotropic, suggesting the asthenosphere as main source of anisotropy. To investigate the physical properties of the slab and surrounding regions, I analyzed the seismic P and S wave attenuation. By inverting high-quality S-waves t* from slab earthquakes, 3D attenuation models down to 300 km were obtained. Attenuation results image the slab as low-attenuation body, but with heterogeneous QS and QP structure showing spot of high attenuation , between 100-200 km depth, which could be due dehydration associated to the slab metamorphism. A low QS anomaly is present in the mantle wedge beneath the Aeolian volcanic arc and could indicate mantle melting and slab dehydration.

General effective medium model for the complex permittivity extraction with an open-ended coaxial probe in presence of a multilayer material under test

The work presented in this thesis is focused on the open-ended coaxial-probe frequency-domain reflectometry technique for complex permittivity measurement at microwave frequencies of dispersive dielectric multilayer materials. An effective dielectric model is introduced and validated to extend the applicability of this technique to multilayer materials in on-line system context. In addition, the thesis presents: 1) a numerical study regarding the imperfectness of the contact at the probe-material interface, 2) a review of the available models and techniques, 3) a new classification of the extraction schemes with guidelines on how they can be used to improve the overall performance of the probe according to the problem requirements.

Resonances in the two centers Coulomb system

In this work we investigate the existence of resonances for two-centers Coulomb systems with arbitrary charges in two and three dimensions, defining them in terms of generalized complex eigenvalues of a non-selfadjoint deformation of the two-center Schrödinger operator. After giving a description of the bifurcation of the classical system for positive energies, we construct the resolvent kernel of the operators and we prove that they can be extended analytically to the second Riemann sheet. The resonances are then defined and studied with numerical methods and perturbation theory.

Patent Strategies and R&D in Complex Product Industries

The study aims at providing a framework conceptualizing patenting activities under the condition of intellectual property rights fragmentation. Such a framework has to deal with the interrelated problems of technological complexity in the modern patent landscape. In that respect, ex-post licensing agreements have been incorporated into the analysis. More precisely, by consolidating the right to use patents required for commercialization of a product, private market solutions, such as cross-licensing agreements and patent pools help firms to overcome problems triggered by the intellectual property rights fragmentation. Thereby, private bargaining between parties as such cannot be isolated from the legal framework. A result of this analysis is that policies ignoring market solutions and only focusing on static gains can mitigate the dynamic efficiency gains as induced by the patent system. The evidence found in this thesis supports the opinion that legal reforms that aim to decrease the degree of patent protection or to lift it all together can hamper the functioning of the current system.

Multidimensional analysis of complex networks

Complex Networks analysis turn out to be a very promising field of research, testified by many research projects and works that span different fields. Those analysis have been usually focused on characterize a single aspect of the system and a study that considers many informative axes along with a network evolve is lacking. We propose a new multidimensional analysis that is able to inspect networks in the two most important dimensions, space and time. To achieve this goal, we studied them singularly and investigated how the variation of the constituting parameters drives changes to the network as a whole. By focusing on space dimension, we characterized spatial alteration in terms of abstraction levels. We proposed a novel algorithm that, by applying a fuzziness function, can reconstruct networks under different level of details. We verified that statistical indicators depend strongly on the granularity with which a system is described and on the class of networks. We keep fixed the space axes and we isolated the dynamics behind networks evolution process. We detected new instincts that trigger social networks utilization and spread the adoption of novel communities. We formalized this enhanced social network evolution by adopting special nodes (called sirens) that, thanks to their ability to attract new links, were able to construct efficient connection patterns. We simulated the dynamics of the system by considering three well-known growth models. Applying this framework to real and synthetic networks, we showed that the sirens, even when used for a limited time span, effectively shrink the time needed to get a network in mature state. In order to provide a concrete context of our findings, we formalized the cost of setting up such enhancement and provided the best combinations of system's parameters, such as number of sirens, time span of utilization and attractiveness.

Evaluation of Human Exposure to Magnetic Fields Generated by Electric Power Systems in Complex Configurations

The international growing concern for the human exposure to magnetic fields generated by electric power lines has unavoidably led to imposing legal limits. Respecting these limits, implies being able to calculate easily and accurately the generated magnetic field also in complex configurations. Twisting of phase conductors is such a case. The consolidated exact and approximated theory regarding a single-circuit twisted three-phase power cable line has been reported along with the proposal of an innovative simplified formula obtained by means of an heuristic procedure. This formula, although being dramatically simpler, is proven to be a good approximation of the analytical formula and at the same time much more accurate than the approximated formula found in literature. The double-circuit twisted three-phase power cable line case has been studied following different approaches of increasing complexity and accuracy. In this framework, the effectiveness of the above-mentioned innovative formula is also examined. The experimental verification of the correctness of the twisted double-circuit theoretical analysis has permitted its extension to multiple-circuit twisted three-phase power cable lines. In addition, appropriate 2D and, in particularly, 3D numerical codes for simulating real existing overhead power lines for the calculation of the magnetic field in their vicinity have been created. Finally, an innovative ‘smart’ measurement and evaluation system of the magnetic field is being proposed, described and validated, which deals with the experimentally-based evaluation of the total magnetic field B generated by multiple sources in complex three-dimensional arrangements, carried out on the basis of the measurement of the three Cartesian field components and their correlation with the field currents via multilinear regression techniques. The ultimate goal is verifying that magnetic induction intensity is within the prescribed limits.

Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.

Operating System Contribution to Composable Timing Behaviour in High-Integrity Real-Time Systems

The development of High-Integrity Real-Time Systems has a high footprint in terms of human, material and schedule costs. Factoring functional, reusable logic in the application favors incremental development and contains costs. Yet, achieving incrementality in the timing behavior is a much harder problem. Complex features at all levels of the execution stack, aimed to boost average-case performance, exhibit timing behavior highly dependent on execution history, which wrecks time composability and incrementaility with it. Our goal here is to restitute time composability to the execution stack, working bottom up across it. We first characterize time composability without making assumptions on the system architecture or the software deployment to it. Later, we focus on the role played by the real-time operating system in our pursuit. Initially we consider single-core processors and, becoming less permissive on the admissible hardware features, we devise solutions that restore a convincing degree of time composability. To show what can be done for real, we developed TiCOS, an ARINC-compliant kernel, and re-designed ORK+, a kernel for Ada Ravenscar runtimes. In that work, we added support for limited-preemption to ORK+, an absolute premiere in the landscape of real-word kernels. Our implementation allows resource sharing to co-exist with limited-preemptive scheduling, which extends state of the art. We then turn our attention to multicore architectures, first considering partitioned systems, for which we achieve results close to those obtained for single-core processors. Subsequently, we shy away from the over-provision of those systems and consider less restrictive uses of homogeneous multiprocessors, where the scheduling algorithm is key to high schedulable utilization. To that end we single out RUN, a promising baseline, and extend it to SPRINT, which supports sporadic task sets, hence matches real-world industrial needs better. To corroborate our results we present findings from real-world case studies from avionic industry.

Automatic risk evaluation in elderly patients based on Autonomic Nervous System assessment

Dysfunction of Autonomic Nervous System (ANS) is a typical feature of chronic heart failure and other cardiovascular disease. As a simple non-invasive technology, heart rate variability (HRV) analysis provides reliable information on autonomic modulation of heart rate. The aim of this thesis was to research and develop automatic methods based on ANS assessment for evaluation of risk in cardiac patients. Several features selection and machine learning algorithms have been combined to achieve the goals. Automatic assessment of disease severity in Congestive Heart Failure (CHF) patients: a completely automatic method, based on long-term HRV was proposed in order to automatically assess the severity of CHF, achieving a sensitivity rate of 93% and a specificity rate of 64% in discriminating severe versus mild patients. Automatic identification of hypertensive patients at high risk of vascular events: a completely automatic system was proposed in order to identify hypertensive patients at higher risk to develop vascular events in the 12 months following the electrocardiographic recordings, achieving a sensitivity rate of 71% and a specificity rate of 86% in identifying high-risk subjects among hypertensive patients. Automatic identification of hypertensive patients with history of fall: it was explored whether an automatic identification of fallers among hypertensive patients based on HRV was feasible. The results obtained in this thesis could have implications both in clinical practice and in clinical research. The system has been designed and developed in order to be clinically feasible. Moreover, since 5-minute ECG recording is inexpensive, easy to assess, and non-invasive, future research will focus on the clinical applicability of the system as a screening tool in non-specialized ambulatories, in order to identify high-risk patients to be shortlisted for more complex investigations.

Engineering in Transition. Approaches, strategies and technologies for implementing system innovation towards sustainability

With the aim to provide people with sustainable options, engineers are ethically required to hold the safety, health and welfare of the public paramount and to satisfy society's need for sustainable development. The global crisis and related sustainability challenges are calling for a fundamental change in culture, structures and practices. Sustainability Transitions (ST) have been recognized as promising frameworks for radical system innovation towards sustainability. In order to enhance the effectiveness of transformative processes, both the adoption of a transdisciplinary approach and the experimentation of practices are crucial. The evolution of approaches towards ST provides a series of inspiring cases which allow to identify advances in making sustainability transitions happen. In this framework, the thesis has emphasized the role of Transition Engineering (TE). TE adopts a transdisciplinary approach for engineering to face the sustainability challenges and address the risks of un-sustainability. With this purpose, a definition of Transition Technologies is provided as a valid instruments to contribute to ST. In the empirical section, several transition initiatives have been analysed especially at the urban level. As a consequence, the model of living-lab of sustainability has crucially emerged. Living-labs are environments in which innovative technologies and services are co-created with users active participation. In this framework, university can play a key role as learning organization. The core of the thesis has concerned the experimental application of transition approach within the School of Engineering and Architecture of University of Bologna at Terracini Campus. The final vision is to realize a living-lab of sustainability. Particularly, a Transition Team has been established and several transition experiments have been conducted. The final result is not only the improvement of sustainability and resilience of the Terracini Campus, but the demonstration that university can generate solutions and strategies that tackle the complex, dynamic factors fuelling the global crisis.

Fault detection, diagnosis and active fault tolerant control for a satellite attitude control system

Modern control systems are becoming more and more complex and control algorithms more and more sophisticated. Consequently, Fault Detection and Diagnosis (FDD) and Fault Tolerant Control (FTC) have gained central importance over the past decades, due to the increasing requirements of availability, cost efficiency, reliability and operating safety. This thesis deals with the FDD and FTC problems in a spacecraft Attitude Determination and Control System (ADCS). Firstly, the detailed nonlinear models of the spacecraft attitude dynamics and kinematics are described, along with the dynamic models of the actuators and main external disturbance sources. The considered ADCS is composed of an array of four redundant reaction wheels. A set of sensors provides satellite angular velocity, attitude and flywheel spin rate information. Then, general overviews of the Fault Detection and Isolation (FDI), Fault Estimation (FE) and Fault Tolerant Control (FTC) problems are presented, and the design and implementation of a novel diagnosis system is described. The system consists of a FDI module composed of properly organized model-based residual filters, exploiting the available input and output information for the detection and localization of an occurred fault. A proper fault mapping procedure and the nonlinear geometric approach are exploited to design residual filters explicitly decoupled from the external aerodynamic disturbance and sensitive to specific sets of faults. The subsequent use of suitable adaptive FE algorithms, based on the exploitation of radial basis function neural networks, allows to obtain accurate fault estimations. Finally, this estimation is actively exploited in a FTC scheme to achieve a suitable fault accommodation and guarantee the desired control performances. A standard sliding mode controller is implemented for attitude stabilization and control. Several simulation results are given to highlight the performances of the overall designed system in case of different types of faults affecting the ADCS actuators and sensors.