15 resultados para Basophil Degranulation Test -- methods

em AMS Tesi di Dottorato - Alm@DL - Università di Bologna


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The work undertaken in this PhD thesis is aimed at the development and testing of an innovative methodology for the assessment of the vulnerability of coastal areas to marine catastrophic inundation (tsunami). Different approaches are used at different spatial scales and are applied to three different study areas: 1. The entire western coast of Thailand 2. Two selected coastal suburbs of Sydney – Australia 3. The Aeolian Islands, in the South Tyrrhenian Sea – Italy I have discussed each of these cases study in at least one scientific paper: one paper about the Thailand case study (Dall’Osso et al., in review-b), three papers about the Sydney applications (Dall’Osso et al., 2009a; Dall’Osso et al., 2009b; Dall’Osso and Dominey-Howes, in review) and one last paper about the work at the Aeolian Islands (Dall’Osso et al., in review-a). These publications represent the core of the present PhD thesis. The main topics dealt with are outlined and discussed in a general introduction while the overall conclusions are outlined in the last section.

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Which event study methods are best in non-U.S. multi-country samples? Nonparametric tests, especially the rank and generalized sign, are better specified and more powerful than common parametric tests, especially in multi-day windows. The generalized sign test is the best statistic but must be applied to buy-and-hold abnormal returns for correct specification. Market-adjusted and market-model methods with local market indexes, without conversion to a common currency, work well. The results are robust to limiting the samples to situations expected to be problematic for test specification or power. Applying the tests that perform best in simulation to merger announcements produces reasonable results.

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The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.

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Among the experimental methods commonly used to define the behaviour of a full scale system, dynamic tests are the most complete and efficient procedures. A dynamic test is an experimental process, which would define a set of characteristic parameters of the dynamic behaviour of the system, such as natural frequencies of the structure, mode shapes and the corresponding modal damping values associated. An assessment of these modal characteristics can be used both to verify the theoretical assumptions of the project, to monitor the performance of the structural system during its operational use. The thesis is structured in the following chapters: The first introductive chapter recalls some basic notions of dynamics of structure, focusing the discussion on the problem of systems with multiply degrees of freedom (MDOF), which can represent a generic real system under study, when it is excited with harmonic force or in free vibration. The second chapter is entirely centred on to the problem of dynamic identification process of a structure, if it is subjected to an experimental test in forced vibrations. It first describes the construction of FRF through classical FFT of the recorded signal. A different method, also in the frequency domain, is subsequently introduced; it allows accurately to compute the FRF using the geometric characteristics of the ellipse that represents the direct input-output comparison. The two methods are compared and then the attention is focused on some advantages of the proposed methodology. The third chapter focuses on the study of real structures when they are subjected to experimental test, where the force is not known, like in an ambient or impact test. In this analysis we decided to use the CWT, which allows a simultaneous investigation in the time and frequency domain of a generic signal x(t). The CWT is first introduced to process free oscillations, with excellent results both in terms of frequencies, dampings and vibration modes. The application in the case of ambient vibrations defines accurate modal parameters of the system, although on the damping some important observations should be made. The fourth chapter is still on the problem of post processing data acquired after a vibration test, but this time through the application of discrete wavelet transform (DWT). In the first part the results obtained by the DWT are compared with those obtained by the application of CWT. Particular attention is given to the use of DWT as a tool for filtering the recorded signal, in fact in case of ambient vibrations the signals are often affected by the presence of a significant level of noise. The fifth chapter focuses on another important aspect of the identification process: the model updating. In this chapter, starting from the modal parameters obtained from some environmental vibration tests, performed by the University of Porto in 2008 and the University of Sheffild on the Humber Bridge in England, a FE model of the bridge is defined, in order to define what type of model is able to capture more accurately the real dynamic behaviour of the bridge. The sixth chapter outlines the necessary conclusions of the presented research. They concern the application of a method in the frequency domain in order to evaluate the modal parameters of a structure and its advantages, the advantages in applying a procedure based on the use of wavelet transforms in the process of identification in tests with unknown input and finally the problem of 3D modeling of systems with many degrees of freedom and with different types of uncertainty.

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Here I will focus on three main topics that best address and include the projects I have been working in during my three year PhD period that I have spent in different research laboratories addressing both computationally and practically important problems all related to modern molecular genomics. The first topic is the use of livestock species (pigs) as a model of obesity, a complex human dysfunction. My efforts here concern the detection and annotation of Single Nucleotide Polymorphisms. I developed a pipeline for mining human and porcine sequences. Starting from a set of human genes related with obesity the platform returns a list of annotated porcine SNPs extracted from a new set of potential obesity-genes. 565 of these SNPs were analyzed on an Illumina chip to test the involvement in obesity on a population composed by more than 500 pigs. Results will be discussed. All the computational analysis and experiments were done in collaboration with the Biocomputing group and Dr.Luca Fontanesi, respectively, under the direction of prof. Rita Casadio at the Bologna University, Italy. The second topic concerns developing a methodology, based on Factor Analysis, to simultaneously mine information from different levels of biological organization. With specific test cases we develop models of the complexity of the mRNA-miRNA molecular interaction in brain tumors measured indirectly by microarray and quantitative PCR. This work was done under the supervision of Prof. Christine Nardini, at the “CAS-MPG Partner Institute for Computational Biology” of Shangai, China (co-founded by the Max Planck Society and the Chinese Academy of Sciences jointly) The third topic concerns the development of a new method to overcome the variety of PCR technologies routinely adopted to characterize unknown flanking DNA regions of a viral integration locus of the human genome after clinical gene therapy. This new method is entirely based on next generation sequencing and it reduces the time required to detect insertion sites, decreasing the complexity of the procedure. This work was done in collaboration with the group of Dr. Manfred Schmidt at the Nationales Centrum für Tumorerkrankungen (Heidelberg, Germany) supervised by Dr. Annette Deichmann and Dr. Ali Nowrouzi. Furthermore I add as an Appendix the description of a R package for gene network reconstruction that I helped to develop for scientific usage (http://www.bioconductor.org/help/bioc-views/release/bioc/html/BUS.html).

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Background Decreased exercise capacity, and reduction in peak oxygen uptake are present in most patients affected by hypertrophic cardiomyopathy (HCM) . In addition an abnormal blood pressure response during a maximal exercise test was seen to be associated with high risk for sudden cardiac death in adult patients affected by HCM. Therefore exercise test (CPET) has become an important part of the evaluation of the HCM patients, but data on its role in patients with HCM in the pediatric age are quite limited. Methods and results Between 2004 and 2010, using CPET and echocardiography, we studied 68 children (mean age 13.9 ± 2 years) with HCM. The exercise test was completed by all the patients without adverse complications. The mean value of achieved VO2 max was 31.4 ± 8.3 mL/Kg/min which corresponded to 77.5 ± 16.9 % of predicted range. 51 patients (75%) reached a subnormal value of VO2max. On univariate analysis the achieved VO2 as percentage of predicted and the peak exercise systolic blood pressure (BP) Z score were inversely associated with max left ventricle (LV) wall thickness, with E/Ea ratio, and directly related with Ea and Sa wave velocities No association was found with the LV outflow tract gradient. During a mean follow up of 2.16 ± 1.7 years 9 patients reached the defined clinical end point of death, transplantation, implanted cardioverter defibrillator (ICD) shock, ICD implantation for secondary prevention or myectomy. Patients with peak VO2 < 52% or with peak systolic BP Z score < -5.8 had lower event free survival at follow up. Conclusions Exercise capacity is decreased in patients with HCM in pediatric age and global ventricular function seems being the most important determinant of exercise capacity in these patients. CPET seems to play an important role in prognostic stratification of children affected by HCM.

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This Ph.D. thesis focuses on the investigation of some chemical and sensorial analytical parameters linked to the quality and purity of different categories of oils obtained by olives: extra virgin olive oils, both those that are sold in the large retail trade (supermarkets and discounts) and those directly collected at some Italian mills, and lower-quality oils (refined, lampante and “repaso”). Concurrently with the adoption of traditional and well-known analytical procedures such as gas chromatography and high-performance liquid chromatography, I carried out a set-up of innovative, fast and environmentally-friend methods. For example, I developed some analytical approaches based on Fourier transform medium infrared spectroscopy (FT-MIR) and time domain reflectometry (TDR), coupled with a robust chemometric elaboration of the results. I investigated some other freshness and quality markers that are not included in official parameters (in Italian and European regulations): the adoption of such a full chemical and sensorial analytical plan allowed me to obtain interesting information about the degree of quality of the EVOOs, mostly within the Italian market. Here the range of quality of EVOOs resulted very wide, in terms of sensory attributes, price classes and chemical parameters. Thanks to the collaboration with other Italian and foreign research groups, I carried out several applicative studies, especially focusing on the shelf-life of oils obtained by olives and on the effects of thermal stresses on the quality of the products. I also studied some innovative technological treatments, such as the clarification by using inert gases, as an alternative to the traditional filtration. Moreover, during a three-and-a-half months research stay at the University of Applied Sciences in Zurich, I also carried out a study related to the application of statistical methods for the elaboration of sensory results, obtained thanks to the official Swiss Panel and to some consumer tests.

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This work presents exact algorithms for the Resource Allocation and Cyclic Scheduling Problems (RA&CSPs). Cyclic Scheduling Problems arise in a number of application areas, such as in hoist scheduling, mass production, compiler design (implementing scheduling loops on parallel architectures), software pipelining, and in embedded system design. The RA&CS problem concerns time and resource assignment to a set of activities, to be indefinitely repeated, subject to precedence and resource capacity constraints. In this work we present two constraint programming frameworks facing two different types of cyclic problems. In first instance, we consider the disjunctive RA&CSP, where the allocation problem considers unary resources. Instances are described through the Synchronous Data-flow (SDF) Model of Computation. The key problem of finding a maximum-throughput allocation and scheduling of Synchronous Data-Flow graphs onto a multi-core architecture is NP-hard and has been traditionally solved by means of heuristic (incomplete) algorithms. We propose an exact (complete) algorithm for the computation of a maximum-throughput mapping of applications specified as SDFG onto multi-core architectures. Results show that the approach can handle realistic instances in terms of size and complexity. Next, we tackle the Cyclic Resource-Constrained Scheduling Problem (i.e. CRCSP). We propose a Constraint Programming approach based on modular arithmetic: in particular, we introduce a modular precedence constraint and a global cumulative constraint along with their filtering algorithms. Many traditional approaches to cyclic scheduling operate by fixing the period value and then solving a linear problem in a generate-and-test fashion. Conversely, our technique is based on a non-linear model and tackles the problem as a whole: the period value is inferred from the scheduling decisions. The proposed approaches have been tested on a number of non-trivial synthetic instances and on a set of realistic industrial instances achieving good results on practical size problem.

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Obiettivi: Valutare la prevalenza dei diversi genotipi di HPV in pazienti con diagnosi di CIN2/3 nella Regione Emilia-Romagna, la persistenza genotipo-specifica di HPV e l’espressione degli oncogeni virali E6/E7 nel follow-up post-trattamento come fattori di rischio di recidiva/persistenza o progressione di malattia; verificare l’applicabilità di nuovi test diagnostici biomolecolari nello screening del cervicocarcinoma. Metodi: Sono state incluse pazienti con citologia di screening anormale, sottoposte a trattamento escissionale (T0) per diagnosi di CIN2/3 su biopsia mirata. Al T0 e durante il follow-up a 6, 12, 18 e 24 mesi, oltre al Pap test e alla colposcopia, sono state effettuate la ricerca e la genotipizzazione dell'HPV DNA di 28 genotipi. In caso di positività al DNA dei 5 genotipi 16, 18, 31, 33 e/o 45, si è proceduto alla ricerca dell'HPV mRNA di E6/E7. Risultati preliminari: Il 95.8% delle 168 pazienti selezionate è risultato HPV DNA positivo al T0. Nel 60.9% dei casi le infezioni erano singole (prevalentemente da HPV 16 e 31), nel 39.1% erano multiple. L'HPV 16 è stato il genotipo maggiormente rilevato (57%). Il 94.3% (117/124) delle pazienti positive per i 5 genotipi di HPV DNA sono risultate mRNA positive. Abbiamo avuto un drop-out di 38/168 pazienti. A 18 mesi (95% delle pazienti) la persistenza dell'HPV DNA di qualsiasi genotipo era del 46%, quella dell'HPV DNA dei 5 genotipi era del 39%, con espressione di mRNA nel 21%. Abbiamo avuto recidiva di malattia (CIN2+) nel 10.8% (14/130) a 18 mesi. Il pap test era negativo in 4/14 casi, l'HPV DNA test era positivo in tutti i casi, l'mRNA test in 11/12 casi. Conclusioni: L'HR-HPV DNA test è più sensibile della citologia, l'mRNA test è più specifico nell'individuare una recidiva. I dati definitivi saranno disponibili al termine del follow-up programmato.

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The research field of the Thesis is the evaluation of motor variability and the analysis of motor stability for the assessment of fall risk. Since many falls occur during walking, a better understanding of motor stability could lead to the definition of a reliable fall risk index aiming at measuring and assessing the risk of fall in the elderly, in the attempt to prevent traumatic events. Several motor variability and stability measures are proposed in the literature, but still a proper methodological characterization is lacking. Moreover, the relationship between many of these measures and fall history or fall risk is still unknown, or not completely clear. The aim of this thesis is hence to: i) analyze the influence of experimental implementation parameters on variability/stability measures and understand how variations in these parameters affect the outputs; ii) assess the relationship between variability/stability measures and long- short-term fall history. Several implementation issues have been addressed. Following the need for a methodological standardization of gait variability/stability measures, highlighted in particular for orbital stability analysis through a systematic review, general indications about implementation of orbital stability analysis have been showed, together with an analysis of the number of strides and the test-retest reliability of several variability/stability numbers. Indications about the influence of directional changes on measures have been provided. The association between measures and long/short-term fall history has also been assessed. Of all the analyzed variability/stability measures, Multiscale entropy and Recurrence quantification analysis demonstrated particularly good results in terms of reliability, applicability and association with fall history. Therefore, these measures should be taken in consideration for the definition of a fall risk index.

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A first phase of the research activity has been related to the study of the state of art of the infrastructures for cycling, bicycle use and methods for evaluation. In this part, the candidate has studied the "bicycle system" in countries with high bicycle use and in particular in the Netherlands. Has been carried out an evaluation of the questionnaires of the survey conducted within the European project BICY on mobility in general in 13 cities of the participating countries. The questionnaire was designed, tested and implemented, and was later validated by a test in Bologna. The results were corrected with information on demographic situation and compared with official data. The cycling infrastructure analysis was conducted on the basis of information from the OpenStreetMap database. The activity consisted in programming algorithms in Python that allow to extract data from the database infrastructure for a region, to sort and filter cycling infrastructure calculating some attributes, such as the length of the arcs paths. The results obtained were compared with official data where available. The structure of the thesis is as follows: 1. Introduction: description of the state of cycling in several advanced countries, description of methods of analysis and their importance to implement appropriate policies for cycling. Supply and demand of bicycle infrastructures. 2. Survey on mobility: it gives details of the investigation developed and the method of evaluation. The results obtained are presented and compared with official data. 3. Analysis cycling infrastructure based on information from the database of OpenStreetMap: describes the methods and algorithms developed during the PhD. The results obtained by the algorithms are compared with official data. 4. Discussion: The above results are discussed and compared. In particular the cycle demand is compared with the length of cycle networks within a city. 5. Conclusions

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Foodborne diseases impact human health and economies worldwide in terms of health care and productivity loss. Prevention is necessary and methods to detect, isolate and quantify foodborne pathogens play a fundamental role, changing continuously to face microorganisms and food production evolution. Official methods are mainly based on microorganisms growth in different media and their isolation on selective agars followed by confirmation of presumptive colonies through biochemical and serological test. A complete identification requires form 7 to 10 days. Over the last decades, new molecular techniques based on antibodies and nucleic acids allow a more accurate typing and a faster detection and quantification. The present thesis aims to apply molecular techniques to improve official methods performances regarding two pathogens: Shiga-like Toxin-producing Escherichia coli (STEC) and Listeria monocytogenes. In 2011, a new strain of STEC belonging to the serogroup O104 provoked a large outbreak. Therefore, the development of a method to detect and isolate STEC O104 is demanded. The first objective of this work is the detection, isolation and identification of STEC O104 in sprouts artificially contaminated. Multiplex PCR assays and antibodies anti-O104 incorporated in reagents for immunomagnetic separation and latex agglutination were employed. Contamination levels of less than 1 CFU/g were detected. Multiplex PCR assays permitted a rapid screening of enriched food samples and identification of isolated colonies. Immunomagnetic separation and latex agglutination allowed a high sensitivity and rapid identification of O104 antigen, respectively. The development of a rapid method to detect and quantify Listeria monocytogenes, a high-risk pathogen, is the second objective. Detection of 1 CFU/ml and quantification of 10–1,000 CFU/ml in raw milk were achieved by a sample pretreatment step and quantitative PCR in about 3h. L. monocytogenes growth in raw milk was also evaluated.

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In the past two decades the work of a growing portion of researchers in robotics focused on a particular group of machines, belonging to the family of parallel manipulators: the cable robots. Although these robots share several theoretical elements with the better known parallel robots, they still present completely (or partly) unsolved issues. In particular, the study of their kinematic, already a difficult subject for conventional parallel manipulators, is further complicated by the non-linear nature of cables, which can exert only efforts of pure traction. The work presented in this thesis therefore focuses on the study of the kinematics of these robots and on the development of numerical techniques able to address some of the problems related to it. Most of the work is focused on the development of an interval-analysis based procedure for the solution of the direct geometric problem of a generic cable manipulator. This technique, as well as allowing for a rapid solution of the problem, also guarantees the results obtained against rounding and elimination errors and can take into account any uncertainties in the model of the problem. The developed code has been tested with the help of a small manipulator whose realization is described in this dissertation together with the auxiliary work done during its design and simulation phases.

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Heavy Liquid Metal Cooled Reactors are among the concepts, fostered by the GIF, as potentially able to comply with stringent safety, economical, sustainability, proliferation resistance and physical protection requirements. The increasing interest around these innovative systems has highlighted the lack of tools specifically dedicated to their core design stage. The present PhD thesis summarizes the three years effort of, partially, closing the mentioned gap, by rationally defining the role of codes in core design and by creating a development methodology for core design-oriented codes (DOCs) and its subsequent application to the most needed design areas. The covered fields are, in particular, the fuel assembly thermal-hydraulics and the fuel pin thermo-mechanics. Regarding the former, following the established methodology, the sub-channel code ANTEO+ has been conceived. Initially restricted to the forced convection regime and subsequently extended to the mixed one, ANTEO+, via a thorough validation campaign, has been demonstrated a reliable tool for design applications. Concerning the fuel pin thermo-mechanics, the will to include safety-related considerations at the outset of the pin dimensioning process, has given birth to the safety-informed DOC TEMIDE. The proposed DOC development methodology has also been applied to TEMIDE; given the complex interdependence patterns among the numerous phenomena involved in an irradiated fuel pin, to optimize the code final structure, a sensitivity analysis has been performed, in the anticipated application domain. The development methodology has also been tested in the verification and validation phases; the latter, due to the low availability of experiments truly representative of TEMIDE's application domain, has only been a preliminary attempt to test TEMIDE's capabilities in fulfilling the DOC requirements upon which it has been built. In general, the capability of the proposed development methodology for DOCs in delivering tools helping the core designer in preliminary setting the system configuration has been proven.

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Negli ultimi anni, i limiti sempre più stringenti sulle emissioni inquinanti dei gas di scarico, hanno portato ad un notevole aumento della complessità dei motori a combustione interna. Questa complicazione determina un aumento esponenziale del numero di test da effettuare nella sala prova. I metodi tipici di gestione dei test non possono più essere utilizzati, ma è essenziale creare un sistema che ottimizzi le prove. Per ridurre drasticamente il tempo di esecuzione, è necessario implementare un'architettura in grado di facilitare lo scambio di dati tra i sistemi presenti nella sala prova, e, in aggiunta, definire le strategie di automazione dei test. L'approccio a taluni metodi si presenta ancora complicato in molti gruppi di sviluppo di strategie di controllo motore, anche se, una volta sviluppati, portano e a grandi benefici durante la fase di test. Il lavoro illustra i metodi implementati per la gestione di queste strategie. Prima si descrive l'approccio utilizzato nella calibrazione di anticipo di accensione per mantenere livelli accettabili di detonazione durante il processo di calibrazione. Successivamente è mostrato il sistema di automazione dei test che consente il pieno controllo del punto di funzionamento del motore, la gestione dell'acquisizione e la verifica della stabilità delle condizioni ottenute. L'ultima parte mostra sistemi di prototipazione rapida per la gestione di componenti innovatici del motore.