Design and development of new pressure sensors for aerodynamic applications

This artwork reports on two different projects that were carried out during the three years of Doctor of the Philosophy course. In the first years a project regarding Capacitive Pressure Sensors Array for Aerodynamic Applications was developed in the Applied Aerodynamic research team of the Second Faculty of Engineering, University of Bologna, Forlì, Italy, and in collaboration with the ARCES laboratories of the same university. Capacitive pressure sensors were designed and fabricated, investigating theoretically and experimentally the sensor’s mechanical and electrical behaviours by means of finite elements method simulations and by means of wind tunnel tests. During the design phase, the sensor figures of merit are considered and evaluated for specific aerodynamic applications. The aim of this work is the production of low cost MEMS-alternative devices suitable for a sensor network to be implemented in air data system. The last two year was dedicated to a project regarding Wireless Pressure Sensor Network for Nautical Applications. Aim of the developed sensor network is to sense the weak pressure field acting on the sail plan of a full batten sail by means of instrumented battens, providing a real time differential pressure map over the entire sail surface. The wireless sensor network and the sensing unit were designed, fabricated and tested in the faculty laboratories. A static non-linear coupled mechanical-electrostatic simulation, has been developed to predict the pressure versus capacitance static characteristic suitable for the transduction process and to tune the geometry of the transducer to reach the required resolution, sensitivity and time response in the appropriate full scale pressure input A time dependent viscoelastic error model has been inferred and developed by means of experimental data in order to model, predict and reduce the inaccuracy bound due to the viscolelastic phenomena affecting the Mylar® polyester film used for the sensor diaphragm. The development of the two above mentioned subjects are strictly related but presently separately in this artwork.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Assimilation of Meteosat Second Generation (MSG) satellite data in a regional numerical weather prediction model using a one-dimensional variational approach

The quality of temperature and humidity retrievals from the infrared SEVIRI sensors on the geostationary Meteosat Second Generation (MSG) satellites is assessed by means of a one dimensional variational algorithm. The study is performed with the aim of improving the spatial and temporal resolution of available observations to feed analysis systems designed for high resolution regional scale numerical weather prediction (NWP) models. The non-hydrostatic forecast model COSMO (COnsortium for Small scale MOdelling) in the ARPA-SIM operational configuration is used to provide background fields. Only clear sky observations over sea are processed. An optimised 1D–VAR set-up comprising of the two water vapour and the three window channels is selected. It maximises the reduction of errors in the model backgrounds while ensuring ease of operational implementation through accurate bias correction procedures and correct radiative transfer simulations. The 1D–VAR retrieval quality is firstly quantified in relative terms employing statistics to estimate the reduction in the background model errors. Additionally the absolute retrieval accuracy is assessed comparing the analysis with independent radiosonde and satellite observations. The inclusion of satellite data brings a substantial reduction in the warm and dry biases present in the forecast model. Moreover it is shown that the retrieval profiles generated by the 1D–VAR are well correlated with the radiosonde measurements. Subsequently the 1D–VAR technique is applied to two three–dimensional case–studies: a false alarm case–study occurred in Friuli–Venezia–Giulia on the 8th of July 2004 and a heavy precipitation case occurred in Emilia–Romagna region between 9th and 12th of April 2005. The impact of satellite data for these two events is evaluated in terms of increments in the integrated water vapour and saturation water vapour over the column, in the 2 meters temperature and specific humidity and in the surface temperature. To improve the 1D–VAR technique a method to calculate flow–dependent model error covariance matrices is also assessed. The approach employs members from an ensemble forecast system generated by perturbing physical parameterisation schemes inside the model. The improved set–up applied to the case of 8th of July 2004 shows a substantial neutral impact.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Genome characterization through a mathematical model of the genetic code: an analysis of the whole chromosome 1 of A. thaliana

The objective of this work is to characterize the genome of the chromosome 1 of A.thaliana, a small flowering plants used as a model organism in studies of biology and genetics, on the basis of a recent mathematical model of the genetic code. I analyze and compare different portions of the genome: genes, exons, coding sequences (CDS), introns, long introns, intergenes, untranslated regions (UTR) and regulatory sequences. In order to accomplish the task, I transformed nucleotide sequences into binary sequences based on the definition of the three different dichotomic classes. The descriptive analysis of binary strings indicate the presence of regularities in each portion of the genome considered. In particular, there are remarkable differences between coding sequences (CDS and exons) and non-coding sequences, suggesting that the frame is important only for coding sequences and that dichotomic classes can be useful to recognize them. Then, I assessed the existence of short-range dependence between binary sequences computed on the basis of the different dichotomic classes. I used three different measures of dependence: the well-known chi-squared test and two indices derived from the concept of entropy i.e. Mutual Information (MI) and Sρ, a normalized version of the “Bhattacharya Hellinger Matusita distance”. The results show that there is a significant short-range dependence structure only for the coding sequences whose existence is a clue of an underlying error detection and correction mechanism. No doubt, further studies are needed in order to assess how the information carried by dichotomic classes could discriminate between coding and noncoding sequence and, therefore, contribute to unveil the role of the mathematical structure in error detection and correction mechanisms. Still, I have shown the potential of the approach presented for understanding the management of genetic information.

Rainfall spatial predictions: a two-part model and its assessment

Spatial prediction of hourly rainfall via radar calibration is addressed. The change of support problem (COSP), arising when the spatial supports of different data sources do not coincide, is faced in a non-Gaussian setting; in fact, hourly rainfall in Emilia-Romagna region, in Italy, is characterized by abundance of zero values and right-skeweness of the distribution of positive amounts. Rain gauge direct measurements on sparsely distributed locations and hourly cumulated radar grids are provided by the ARPA-SIMC Emilia-Romagna. We propose a three-stage Bayesian hierarchical model for radar calibration, exploiting rain gauges as reference measure. Rain probability and amounts are modeled via linear relationships with radar in the log scale; spatial correlated Gaussian effects capture the residual information. We employ a probit link for rainfall probability and Gamma distribution for rainfall positive amounts; the two steps are joined via a two-part semicontinuous model. Three model specifications differently addressing COSP are presented; in particular, a stochastic weighting of all radar pixels, driven by a latent Gaussian process defined on the grid, is employed. Estimation is performed via MCMC procedures implemented in C, linked to R software. Communication and evaluation of probabilistic, point and interval predictions is investigated. A non-randomized PIT histogram is proposed for correctly assessing calibration and coverage of two-part semicontinuous models. Predictions obtained with the different model specifications are evaluated via graphical tools (Reliability Plot, Sharpness Histogram, PIT Histogram, Brier Score Plot and Quantile Decomposition Plot), proper scoring rules (Brier Score, Continuous Rank Probability Score) and consistent scoring functions (Root Mean Square Error and Mean Absolute Error addressing the predictive mean and median, respectively). Calibration is reached and the inclusion of neighbouring information slightly improves predictions. All specifications outperform a benchmark model with incorrelated effects, confirming the relevance of spatial correlation for modeling rainfall probability and accumulation.

Statistical analysis of the error associated with the simplification of the stratigraphy in geotechnical models

The uncertainties in the determination of the stratigraphic profile of natural soils is one of the main problems in geotechnics, in particular for landslide characterization and modeling. The study deals with a new approach in geotechnical modeling which relays on a stochastic generation of different soil layers distributions, following a boolean logic – the method has been thus called BoSG (Boolean Stochastic Generation). In this way, it is possible to randomize the presence of a specific material interdigitated in a uniform matrix. In the building of a geotechnical model it is generally common to discard some stratigraphic data in order to simplify the model itself, assuming that the significance of the results of the modeling procedure would not be affected. With the proposed technique it is possible to quantify the error associated with this simplification. Moreover, it could be used to determine the most significant zones where eventual further investigations and surveys would be more effective to build the geotechnical model of the slope. The commercial software FLAC was used for the 2D and 3D geotechnical model. The distribution of the materials was randomized through a specifically coded MatLab program that automatically generates text files, each of them representing a specific soil configuration. Besides, a routine was designed to automate the computation of FLAC with the different data files in order to maximize the sample number. The methodology is applied with reference to a simplified slope in 2D, a simplified slope in 3D and an actual landslide, namely the Mortisa mudslide (Cortina d’Ampezzo, BL, Italy). However, it could be extended to numerous different cases, especially for hydrogeological analysis and landslide stability assessment, in different geological and geomorphological contexts.

Assessing the fit of unidimensional IRT models for binary data under model misspecification

Model misspecification affects the classical test statistics used to assess the fit of the Item Response Theory (IRT) models. Robust tests have been derived under model misspecification, as the Generalized Lagrange Multiplier and Hausman tests, but their use has not been largely explored in the IRT framework. In the first part of the thesis, we introduce the Generalized Lagrange Multiplier test to detect differential item response functioning in IRT models for binary data under model misspecification. By means of a simulation study and a real data analysis, we compare its performance with the classical Lagrange Multiplier test, computed using the Hessian and the cross-product matrix, and the Generalized Jackknife Score test. The power of these tests is computed empirically and asymptotically. The misspecifications considered are local dependence among items and non-normal distribution of the latent variable. The results highlight that, under mild model misspecification, all tests have good performance while, under strong model misspecification, the performance of the tests deteriorates. None of the tests considered show an overall superior performance than the others. In the second part of the thesis, we extend the Generalized Hausman test to detect non-normality of the latent variable distribution. To build the test, we consider a seminonparametric-IRT model, that assumes a more flexible latent variable distribution. By means of a simulation study and two real applications, we compare the performance of the Generalized Hausman test with the M2 limited information goodness-of-fit test and the Likelihood-Ratio test. Additionally, the information criteria are computed. The Generalized Hausman test has a better performance than the Likelihood-Ratio test in terms of Type I error rates and the M2 test in terms of power. The performance of the Generalized Hausman test and the information criteria deteriorates when the sample size is small and with a few items.