Università degli Studi di Napoli Federico Il Open Archive

    THE COPROLOGICAL DIAGNOSIS OF GASTROINTESTINAL NEMATODE INFECTIONS IN SMALL RUMINANTS

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    Although representing a significant economic and welfare burden to the global ruminant livestock industry, GIN infections in small ruminants are often neglected and implementation in research, diagnosis and surveillance of these parasites is still poor, mainly in the matter of diagnostic methods and their use/interpretation. The accurate diagnosis (and interpretation) of GIN directly supports parasite control strategies and is of relevance for investigations into parasite biology, ecology and epidemiology (Roeber et al., 2013). This aspect is now particularly important given the problems associated with anthelmintic resistance (AR) in GIN populations of small ruminants worldwide (Roeber et al., 2013 a,b). Various methods are employed for the ante mortem diagnosis of GIN infections in small ruminants. These include the observation of clinical signs indicative of disease (although non-pathognomonic), coprological diagnosis (faecal egg count – FEC), biochemical and/or serological, and molecular diagnostic approaches (reviewed in Roeber et al., 2013). However, still now, faecal egg count (FEC) techniques remain the most common laboratory methods for the diagnosis of GIN in small ruminants. Also for FEC, widespread standardization of many laboratory techniques does not exist, and most diagnostic, research and teaching facilities apply their own modifications to published protocols (Kassai, 1999). Although these techniques are regarded to be standard diagnostic procedures, there is a lack of detailed studies of diagnostic performance, including the diagnostic sensitivity, specificity and/or repeatability (Roeber et al., 2013). Furthermore, many aspects concerning factors affecting FEC (e.g. season of sampling, sampling period, consistency of faeces, fecundity of worms, etc., as well as interpretation of FEC) have poorly been investigated so far. These are the reasons that motivated me in choosing “The coprological diagnosis of gastrointestinal nematode infections in small ruminants” as topic of my PhD thesis to help optimize the use and interpretation of FEC in small ruminants

    ENGINEERING METAL BINDING SITES IN DE NOVO DESIGNED FOUR-HELIX BUNDLES

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    A single polypeptide chain may access to an astronomical amount of conformers[1]. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, which can afford the complete set of chemical reactions needed to support life[2]. These structural templates are so stable to allow several mutations without disruption of the global folding even reaching the ability to bind several exogenous cofactors. In this perspective metal cofactors play a crucial role in regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals adopting only few ligands and slightly different geometries[3]. Thus, understanding how the fine-tuning of the scaffolds hosting them imparts the wide spectrum of reactivity is of crucial interest both in the fields of structural biology and bioinorganic chemistry. Several scaffolds and metal binding motifs are object of intense work in the literature[4]. In this PhD work, we focused on the four-helix bundle as scaffold for metal binding sites in the context of protein de novo design, to obtain basic biochemical components for biosensing or catalysis. In order to accomplish our objectives we chose to expand the designable space of a well-characterized de novo designed family of metalloproteins, the DFs (Due-Ferro). DF1, the progenitor of the family, bears a dicarboxylate bridged dinuclear metal center in its hydrophobic core constituted by the four-helix bundle unit[5]. We investigated the opportunity to find new chemical properties[6,7] by loosening on the one side and tightening on the other the C2 symmetric environment of the metal cofactor, giving raise to two new class of de novo designed proteins: DF-Click and QF. Previous attempts to design asymmetrical DFs gave successful results[8,9], even resulting in catalytic activity towards different oxidation reaction[8] upon redesign of the metal binding site[7]. We implemented an alternative synthetic approach to generate new DF analogues by means of copper catalyzed azide-alkyne cycloaddition (CuAAC), also known as “Click Chemistry”[10]. Our design strategy led to a helix-loop-helix heterodimer, obtained through side chain chemical ligation, affording the DF-Click series. The design, synthesis and spectroscopic characterization of an analogue of this series named DF-Click1, which was inspired to the hydroxylase component of the bacterial multicomponent monooxygenases (BMMs)[11], was completed during the PhD research activity by means of solution NMR, circular dichroism, UV-Visible absorption spectroscopy. Further, as we were interested in the metal properties induced by an asymmetrical site, we also evaluated the opportunity to design a model with a higher degree of symmetry. We aimed to the synthesis of a D2 symmetric homotetramer able to bind a tetranuclear metal cofactor. We named this novel de novo designed protein class QF (Quattro Ferro). It consists of four helix strands each one bearing the ExxH binding motif as in helix 2 of DF1. It was inspired to the cubane like Mn-O cluster found in Photosystem II, where four manganese ions provide the oxidative equivalents necessary for the four electrons oxidation of water. During the PhD work, the design process, synthesis and spectroscopic analysis of four analogues were accomplished: W45Y27 and W45Y28, which derive from the ab initio design of a protein crystal with predetermined topology; QF6 and QF33, whose design derives from the serendipitous finding of an already deposited crystal structure of a GCN4 mutant with a similar crystal topology, but without the desired polarity. A combination of NMR and X-Ray analysis confirms the correctness of the design. Spectroscopic analysis intended to ascertain the metal binding stoichiometry and geometry is under course. References (1) Dill, K. A.; Chan, H. S. Nat. Struct. Mol. Biol. 1997, 4, 10. (2) Sillitoe, I.; Cuff, A. L.; Dessailly, B. H.; Dawson, N. L.; Furnham, N.; Lee, D.; Lees, J. G.; Lewis, T. E.; Studer, R. A.; Rentzsch, R.; Yeats, C.; Thornton, J. M.; Orengo, C. A. Nucleic Acids Res. 2012, 41, D490. (3) Bertini, I. Biological Inorganic Chemistry: Structure and Reactivity; University Science Books, 2007. (4) Lu, Y.; Berry, S. M.; Pfister, T. D. Chem. Rev. 2001, 101, 3047. (5) Maglio, O.; Nastri, F.; Martin de Rosales, R. T.; Faiella, M.; Pavone, V.; DeGrado, W. F.; Lombardi, A. Comptes Rendus Chim. 2007, 10, 703. (6) Faiella, M.; Andreozzi, C.; de Rosales, R. T. M.; Pavone, V.; Maglio, O.; Nastri, F.; DeGrado, W. F.; Lombardi, A. Nat Chem Biol 2009, 5, 882. (7) Reig, A. J.; Pires, M. M.; Snyder, R. A.; Wu, Y.; Jo, H.; Kulp, D. W.; Butch, S. E.; Calhoun, J. R.; Szyperski, T.; Solomon, E. I.; Degrado, W. F. Nat. Chem. 2012, 4, 900. (8) Kaplan, J.; DeGrado, W. F. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11566. (9) Calhoun, J. R.; Kono, H.; Lahr, S.; Wang, W.; DeGrado, W. F.; Saven, J. G. J. Mol. Biol. 2003, 334, 1101. (10) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2004. (11) Tinberg, C. E.; Lippard, S. J. Acc. Chem. Res. 2011, 44, 28

    A MEASUREMENT ARCHITECTURE BASED ON COMPRESSIVE SAMPLING FOR THE MONITORING OF ELECTRICAL POWER TRANSMISSION GRIDS

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    In recent years significant change in the power grid for the distribution of electrical energy have been observed. Thanks to liberalization of the electrical market and the incentives offered to the production of energy from renewable sources, the number of medium and small producers has increased. Moreover, with the development of communication technology, the transmission grid has been increasingly equipped with automated devices capable of monitoring and transmitting some information about the grid and, in some cases, to control the actuation devices connected to the grid itself. In this scenario, the term smart grid was born, used to describe energy networks that can automatically monitor energy flows and adjust to changes in energy supply and demand accordingly. When coupled with smart metering systems, smart grids reach consumers and suppliers by providing information on real-time consumption. As an example, consumers can adapt their energy usage to different energy prices throughout the day with personal smart meters, saving money on their energy bills by consuming more energy in lower price periods. As it can be expected, the wider the grid, the larger the number of smart meters required for its monitoring. However, deploying thousands of such devices turns rapidly to be too expensive. Consequently, the realization of the distributed measurement system could not be economically sustainable. To overcome the considered limitations, the research activities have been focused on the possibility to move towards a different approach for monitoring grid deployed on wide geographical areas, exploiting the advantages of an innovative acquisition paradigm: the Compressed Sampling (CS) . CS is a signal processing technique for efficiently acquiring and reconstructing a signal from far fewer samples than those required by the Shannon-Nyquist sampling theorem. In particular, the proposed architecture consists of low cost nodes mandated only to sample and digitize a limited number of input signal samples and transmit them to a central measurement unit, thus saving the costs related to large memory supports and expensive digital processing units. Once the samples are received, the central unit recovers the signal spectrum thanks to CS-based algorithm and carries out the desired measurements. Thanks to the CS-based approach, it is possible to design and realize a measurement node, characterized by reduced memory depth and only one ADC, suitable for poly-phase system with neutral wire that allows meeting the requirements of a distributed measurement system. Numerical and experimental test verified highlight the capability of CS-based acquisition approach of correctly measuring the root-mean-square amplitude of voltage waveforms and assuring simultaneous multi-channel acquisitions. Finally, the compliance of the measurement node based on CS approach with the current Power Quality standards is assessed and discussed

    Ubiquitin-dependent control of Kinase Suppressor of Ras 1 (KSR1) signaling by the RING ligase praja2.

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    Kinase Suppressor of Ras 1 (KSR1) is an evolutionally conserved protein kinase that plays a fundamental role in mitogenic pathway. In response to Ras activation, KSR1 assembles a tripartite kinase complex that optimally transfers signals generated at cell membrane to downstream ERK signaling. The role of KSR1 in Ras signaling has been largely explored. However, the impact of attenuating signals on KSR1 was still elusive. Here, I contributed to identify a novel mechanism of ERK attenuation based on the ubiquitin-dependent control of KSR1. Stimulation of membrane receptors by growth factor induced a rapid poly-ubiquitination of KSR1, which paralleled the decay of ERK signaling. We identified praja2 as the principal E3 ligase that ubiquitinates KSR1. Interfering with praja2 expression or activity impeded KSR1 ubiquitination and sustained ERK signaling. Thus, the dynamic interplay between the ubiquitin system and scaffold components of the Ras pathway contributes to shape the profile of ERK signaling, profoundly impacting on fundamental aspects of cell behavior

    SPERM DNA INTEGRITY IN BUFFALO, BULL AND STALLION

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    The interest in sperm DNA integrity evaluation and its relationship to subfertility and infertility loaded to development of several sperm DNA assays. The aim of this study was to compare several sperm DNA assays in buffaloes, bulls and stallions, and to identify the relationships between those DNA assays and traditional sperm features. In Italian Mediterranean Buffalo (IMB) bulls traditional sperm features (motility, viability, acrosome integrity and morphology), sperm DNA integrity (neutral Comet assay, Sperm Bos Halomax-SBH, and Sperm Chromatin Structure Assay-SCSA), fertility in vivo and their relationships, were evaluated. The neutral Comet assay was correlated to sperm motility, viability, coiled tails and presence of epithelial cells. The SCSA measures were correlated to sperm viability, bent midpieces and distal droplets. SBH was correlated to Non-Viable Acrosome Damaged-NVAD (r= 0.60; P0.05). Pregnancy rate at 30 d, 45 d and calving rate were 57%, 55% and 45%, respectively. Receiver Operating Characteristic (ROC) Curve evidenced significant values for sperm motility, distal droplets, Non-Viable Acrosome Damaged, Standard Deviation-αt (SD-αt) and neutral comet measures of Olive Tail moment and tail moment, %DNA in head (and tail) and tail area (P0.05). This study identified a decrease in sperm DNA quality using both the neutral Comet assay and the SCSA, which was not identified using traditional measures of sperm quality

    Cooperation and migration for future communication networks

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    5G networks will have to address challenges, related to capacity, data rate, latency, connectivity, costs and Quality of Experience (QoE), that LTE-A Long Term Evolution-Advanced) is not able to properly deal with. For this reason, research community is working to identify the set of key technologies that will enable the future mobile network. Three of these technologies fall in the areas of cooperative approaches for wireless networks, Software Defined Networking (SDN), and Network Function Virtualization (NFV). In this thesis work, some original results concerning the aforementioned technologies will be presented, by reserving a greater emphasis to the first one. Specifically, at first, the performance analysis of Multiple-Input Multiple-Output (MIMO) cooperative multiple-relay systems corrupted by Gaussian noise, in the case of naive (non-optimized) approach for the processing at the source and relay nodes, will be presented. Then, the optimal processing design will be faced based on the minimization of the arithmetic mean, over all the substreams, of the symbol error probabilities. At this point, the design of a new equalizer for a Single-Input Single-Output (SISO) Orthogonal-Frequency Division Multiplexing (OFDM) system affected by impulsive noise will be presented and the impact of the joint employment of cooperation and space-time block coding will be shown. At the end, with reference to the migration network primitive, a simplified mathematical model, that unveils the dependence of the total migration time and downtime of the virtual machine memory transfer on the main WAN parameters (such as capacity, buffering, and propagation delay) will be developed

    POST-EARTHQUAKE ASSESSMENT OF DAMAGED NON-DUCTILE BUILDINGS: DETAILED EVALUATION FOR RATIONAL REPARABILITY DECISIONS

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    Non-ductile reinforced concrete frame structures represent a large portion of the existing building stock all over the world; the lacking of important reinforcement details on such constructions render them vulnerable and a significant source of hazard to life in future earthquakes as well as a of economic losses during moderate to severe seismic ground motions. Ideally, effective risk mitigation may be obtained with structural retrofit or by building demolition and replacement. However, economic and social constraints impair abrupt application of those solutions, requiring the study of alternative policies to encourage owners of more vulnerable structures to undertake risk assessment and mitigation. One important aspect in debate on risk mitigation is the treatment of buildings damaged by earthquakes and their possible reparability; different solutions may be adopted in order to make cities safer and more resilient to earthquakes. Building reparability strongly depends on the expected future performance of damaged building and the required repair costs. After earthquakes, the exhaustive assessment of the costs requires detailed on site surveys to establish the damage level, amount of needed interventions to restore the building in all its structural and nonstructural components and the computation of related costs. On the other hand, analytical prediction of damage level due to earthquakes could help significantly to forecast expected costs; also, applying performance-based earthquake engineering methods can contribute significantly to this scope and further allow the sound evaluation of safety variation due to damage, in addition to costs. The main objective of this research is to explore and test different methods and tools for the assessment of buildings reparability taking into account both the expected safety variation and costs. In particular, two main level of analyses for the assessment of damaged buildings are explored, namely detailed analysis based on non-linear time-histories, that is finalized to accurate estimation of expected safety variation for mainshocks corresponding to increasing return period and related repair costs, and pushover based ones, that allows simplified, practice oriented, assessment of variation of the residual capacity and performance loss due to assigned earthquakes

    Cancer-associated fibroblasts (CAFs) release exosomal microRNAs that dictate an aggressive phenotype in breast cancer cells

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    Despite many novel therapeutic approaches, breast cancer remains one of the leading cause of cancer mortality among women. Recent findings indicate that cancer-associated fibroblasts (CAFs), the major components of the tumor microenvironment, play a crucial role in breast cancer progression, but how they promote tumorigenesis is poorly understood. Increasing evidence indicates that exosomes, membrane vesicles sized 30-100 nm in diameter, play an important role in cell-cell communication. Exosomes and their cargo, including microRNAs (miRs), may vehicolate between cells and affect the biological behavior of recipient cells. Therefore, one alternative mechanism of the promotion of breast cancer progression by CAFs may be through cancer-associated fibroblast-secreted exosomes, which would deliver oncogenic miRs to breast cancer cells. Firstly, to investigate the potential role of miRs in stroma-tumor communication, we compared miR expression profile in exosomes from 2 cancer-associated fibroblasts and 2 normal fibroblasts. We found that in CAF exosomes the levels of miR-21, miR-378e, and miR-143 were increased as compared to normal fibroblast exosomes, and we validated the array data by real-time PCR. By immunofluorescence experiments, we demonstrated that PKH26-labeled-exosomes could be transferred from fibroblasts to a breast cancer epithelial cell line, T47D. Furthermore, to elucidate whether the identified miRs were shuttled into T47D cells via exosomes, we transfected CAFs with cy3-labeled-miRs (cy3-miR-21, cy3-miR-143, cy3-miR-378e), and, then, we isolated the released exosomes. Interestingly, when these exosomes were added to T47D cells, the cy3-miRs were detected in the cytoplasm of T47D cells, and they co-localized with the signals of an exosomal marker, CD63. Then, we demonstrated that TGF-β, apart from its direct role in the activation of normal fibroblasts to CAFs, increased the levels of these miRs in normal fibroblast exosomes. Finally, for the first time, we provided evidence of the role of CAF exosomes and their miR contents in the induction of stemness phenotype in T47D cells. In fact, T47D cells exposed to CAF exosomes or transfected with the identified miRs exhibited a significantly increased capacity to form mammospheres, and increased stem cell and epithelial-mesenchymal transition markers, SOX2, Nanog, Oct3/4, Snail and Zeb. We conclude that CAFs regulate the stemness phenotype of breast cancer cells through exosome-mediated delivery of oncogenic miRs. Our data provide insight into the mechanisms underlying the stemness maintenance in breast cancer

    LE OPERAZIONI STRAORDINARIE IN PENDENZA DI PROCEDURA CONCORSUALE

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    La salvaguardia del complesso aziendale e la continuazione dell’attività è stata resa più facile grazie all’utilizzo, nelle procedure di composizione della crisi d’impresa, di particolari istituti giuridici con i quali si può operare sulla titolarità e sulla struttura organizzativa dell’impresa, le cd. operazioni straordinarie d’impresa . Fino alla riforma del diritto societario tali strumenti sono stati scarsamente utilizzati sia in ambito “concorsuale” che le tra imprese in bonis. Il d. lgs. n. 6/2003, non solo disciplina, ma liberalizza e incentiva il ricorso a tali istituti, anche nel campo della composizione della crisi d’impresa

    Agile All-digital Clock Generators with Spread-Spectrum Capabilities in 28nm technology

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    The spread spectrum clocking (SSC) is an established, effective and efficient technique to reduce the electromagnetic interference (EMI) produced by digital chips. This method allows to reduce the level of EMI of digital circuit by intentionally sweeping the frequency of the clock signal (frequency modulated) within a certain frequency range in order to evenly spread the energy of each clock harmonic over a given bandwidth, reducing in this way the peak power level of radiated EMI. In this thesis, a novel All-digital Spread Spectrum Clock Generator (SSCG) prototype is presented in all its aspects: design, simulation and post-fabrication measurements. The circuit is realized by using a design flow completely based on standard cells simplifying design and porting in new technologies, and is able to perform both discontinuous frequency modulation or complex modulation profiles. The circuit uses four digitally controlled delay lines and two digital delay interpolator driven by a digital modulator to synthesize the output waveform A chip has been implemented in a 28nm CMOS technology. The chip is able to generate signals up to 3.3GHz. The measured peak level reduction of the clock spectrum, at 1GHz output frequency, is 27dB with a 10% modulation depth. The power dissipation is 24mW. In the second part of this thesis the first developed SSCG has been redesigned in order to allow the generation of an output clock signal with a frequency higher than the frequency of the input clock signal. To this purpose a new delay line block has been designed in order to implement the clock frequency multiplication. Furthermore, the injection locking technique is implemented by using a novel DCO-based architecture in order to improve the jitter performance of the circuit. The circuit is designed by using only standard cells and is able to generate an output clock frequency larger of 2GHz
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