Multiple stellar populations in globular clusters with photometry and low resolution spectroscopy

Our view of Globular Clusters has deeply changed in the last decade. Modern spectroscopic and photometric data have conclusively established that globulars are neither coeval nor monometallic, reopening the issue of the formation of such systems. Their formation is now schematized as a two-step process, during which the polluted matter from the more massive stars of a first generation gives birth, in the cluster innermost regions, to a second generation of stars with the characteristic signature of fully CNO-processed matter. To date, star-to-star variations in abundances of the light elements (C, N, O, Na) have been observed in stars of all evolutionary phases in all properly studied Galactic globular clusters. Multiple or broad evolutionary sequences have also been observed in nearly all the clusters that have been observed with good signal-to-noise in the appropriate photometric bands. The body of evidence suggests that spreads in light-element abundances can be fairly well traced by photometric indices including near ultraviolet passbands, as CNO abundance variations affect mainly wavelengths shorter than ~400 nm owing to the rise of some NH and CN molecular absorption bands. Here, we exploit this property of near ultraviolet photometry to trace internal chemical variations and combined it with low resolution spectroscopy aimed to derive carbon and nitrogen abundances in order to maximize the information on the multiple populations. This approach has been proven to be very effective in (i) detecting multiple population, (ii) characterizing their global properties (i.e., relative fraction of stars, location in the color-magnitude diagram, spatial distribution, and trends with cluster parameters) and (iii) precisely tagging their chemical properties (i.e., extension of the C-N anticorrelation, bimodalities in the N content).

Biochemistry in healthy and neoplastic human tissues: metabolic alteration revealed by HR-MAS nuclear magnetic resonance spectroscopy

This thesis is focused on the metabolomic study of human cancer tissues by ex vivo High Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. This new technique allows for the acquisition of spectra directly on intact tissues (biopsy or surgery), and it has become very important for integrated metabonomics studies. The objective is to identify metabolites that can be used as markers for the discrimination of the different types of cancer, for the grading, and for the assessment of the evolution of the tumour. Furthermore, an attempt to recognize metabolites, that although involved in the metabolism of tumoral tissues in low concentration, can be important modulators of neoplastic proliferation, was performed. In addition, NMR data was integrated with statistical techniques in order to obtain semi-quantitative information about the metabolite markers. In the case of gliomas, the NMR study was correlated with gene expression of neoplastic tissues. Chapter 1 begins with a general description of a new “omics” study, the metabolomics. The study of metabolism can contribute significantly to biomedical research and, ultimately, to clinical medical practice. This rapidly developing discipline involves the study of the metabolome: the total repertoire of small molecules present in cells, tissues, organs, and biological fluids. Metabolomic approaches are becoming increasingly popular in disease diagnosis and will play an important role on improving our understanding of cancer mechanism. Chapter 2 addresses in more detail the basis of NMR Spectroscopy, presenting the new HR-MAS NMR tool, that is gaining importance in the examination of tumour tissues, and in the assessment of tumour grade. Some advanced chemometric methods were used in an attempt to enhance the interpretation and quantitative information of the HR-MAS NMR data are and presented in chapter 3. Chemometric methods seem to have a high potential in the study of human diseases, as it permits the extraction of new and relevant information from spectroscopic data, allowing a better interpretation of the results. Chapter 4 reports results obtained from HR-MAS NMR analyses performed on different brain tumours: medulloblastoma, meningioms and gliomas. The medulloblastoma study is a case report of primitive neuroectodermal tumor (PNET) localised in the cerebellar region by Magnetic Resonance Imaging (MRI) in a 3-year-old child. In vivo single voxel 1H MRS shows high specificity in detecting the main metabolic alterations in the primitive cerebellar lesion; which consist of very high amounts of the choline-containing compounds and of very low levels of creatine derivatives and N-acetylaspartate. Ex vivo HR-MAS NMR, performed at 9.4 Tesla on the neoplastic specimen collected during surgery, allows the unambiguous identification of several metabolites giving a more in-depth evaluation of the metabolic pattern of the lesion. The ex vivo HR-MAS NMR spectra show higher detail than that obtained in vivo. In addition, the spectroscopic data appear to correlate with some morphological features of the medulloblastoma. The present study shows that ex vivo HR-MAS 1H NMR is able to strongly improve the clinical possibility of in vivo MRS and can be used in conjunction with in vivo spectroscopy for clinical purposes. Three histological subtypes of meningiomas (meningothelial, fibrous and oncocytic) were analysed both by in vivo and ex vivo MRS experiments. The ex vivo HR-MAS investigations are very helpful for the assignment of the in vivo resonances of human meningiomas and for the validation of the quantification procedure of in vivo MR spectra. By using one- and two dimensional experiments, several metabolites in different histological subtypes of meningiomas, were identified. The spectroscopic data confirmed the presence of the typical metabolites of these benign neoplasms and, at the same time, that meningomas with different morphological characteristics have different metabolic profiles, particularly regarding macromolecules and lipids. The profile of total choline metabolites (tCho) and the expression of the Kennedy pathway genes in biopsies of human gliomas were also investigated using HR-MAS NMR, and microfluidic genomic cards. 1H HR-MAS spectra, allowed the resolution and relative quantification by LCModel of the resonances from choline (Cho), phosphorylcholine (PC) and glycerolphorylcholine (GPC), the three main components of the combined tCho peak observed in gliomas by in vivo 1H MRS spectroscopy. All glioma biopsies depicted an increase in tCho as calculated from the addition of Cho, PC and GPC HR-MAS resonances. However, the increase was constantly derived from augmented GPC in low grade NMR gliomas or increased PC content in the high grade gliomas, respectively. This circumstance allowed the unambiguous discrimination of high and low grade gliomas by 1H HR-MAS, which could not be achieved by calculating the tCho/Cr ratio commonly used by in vivo 1H MR spectroscopy. The expression of the genes involved in choline metabolism was investigated in the same biopsies. The present findings offer a convenient procedure to classify accurately glioma grade using 1H HR-MAS, providing in addition the genetic background for the alterations of choline metabolism observed in high and low gliomas grade. Chapter 5 reports the study on human gastrointestinal tract (stomach and colon) neoplasms. The human healthy gastric mucosa, and the characteristics of the biochemical profile of human gastric adenocarcinoma in comparison with that of healthy gastric mucosa were analyzed using ex vivo HR-MAS NMR. Healthy human mucosa is mainly characterized by the presence of small metabolites (more than 50 identified) and macromolecules. The adenocarcinoma spectra were dominated by the presence of signals due to triglycerides, that are usually very low in healthy gastric mucosa. The use of spin-echo experiments enable us to detect some metabolites in the unhealthy tissues and to determine their variation with respect to the healthy ones. Then, the ex vivo HR-MAS NMR analysis was applied to human gastric tissue, to obtain information on the molecular steps involved in the gastric carcinogenesis. A microscopic investigation was also carried out in order to identify and locate the lipids in the cellular and extra-cellular environments. Correlation of the morphological changes detected by transmission (TEM) and scanning (SEM) electron microscopy, with the metabolic profile of gastric mucosa in healthy, gastric atrophy autoimmune diseases (AAG), Helicobacter pylori-related gastritis and adenocarcinoma subjects, were obtained. These ultrastructural studies of AAG and gastric adenocarcinoma revealed lipid intra- and extra-cellularly accumulation associated with a severe prenecrotic hypoxia and mitochondrial degeneration. A deep insight into the metabolic profile of human healthy and neoplastic colon tissues was gained using ex vivo HR-MAS NMR spectroscopy in combination with multivariate methods: Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). The NMR spectra of healthy tissues highlight different metabolic profiles with respect to those of neoplastic and microscopically normal colon specimens (these last obtained at least 15 cm far from the adenocarcinoma). Furthermore, metabolic variations are detected not only for neoplastic tissues with different histological diagnosis, but also for those classified identical by histological analysis. These findings suggest that the same subclass of colon carcinoma is characterized, at a certain degree, by metabolic heterogeneity. The statistical multivariate approach applied to the NMR data is crucial in order to find metabolic markers of the neoplastic state of colon tissues, and to correctly classify the samples. Significant different levels of choline containing compounds, taurine and myoinositol, were observed. Chapter 6 deals with the metabolic profile of normal and tumoral renal human tissues obtained by ex vivo HR-MAS NMR. The spectra of human normal cortex and medulla show the presence of differently distributed osmolytes as markers of physiological renal condition. The marked decrease or disappearance of these metabolites and the high lipid content (triglycerides and cholesteryl esters) is typical of clear cell renal carcinoma (RCC), while papillary RCC is characterized by the absence of lipids and very high amounts of taurine. This research is a contribution to the biochemical classification of renal neoplastic pathologies, especially for RCCs, which can be evaluated by in vivo MRS for clinical purposes. Moreover, these data help to gain a better knowledge of the molecular processes envolved in the onset of renal carcinogenesis.

Biomechanics of sprint running: a methodological contribution

Sports biomechanics describes human movement from a performance enhancement and an injury reduction perspective. In this respect, the purpose of sports scientists is to support coaches and physicians with reliable information about athletes’ technique. The lack of methods allowing for in-field athlete evaluation as well as for accurate joint force estimates represents, to date, the main limitation to this purpose. The investigations illustrated in the present thesis aimed at providing a contribution towards the development of the above mentioned methods. Two complementary approaches were adopted: a Low Resolution Approach – related to performance assessment – where the use of wearable inertial measurement units is exploited during different phases of sprint running, and a High Resolution Approach – related to joint kinetics estimate for injury prevention – where subject-specific, non-rigid constraints for knee joint kinematic modelling used in multi-body optimization techniques are defined. Results obtained using the Low Resolution Approach indicated that, due to their portability and inexpensiveness, inertial measurement systems are a valid alternative to laboratory-based instrumentation for in-field performance evaluation of sprint running. Using acceleration and angular velocity data, the following quantities were estimated: trunk inclination and angular velocity, instantaneous horizontal velocity and displacement of a point approximating the centre of mass, and stride and support phase durations. As concerns the High Resolution Approach, results indicated that the length of the anterior cruciate and lateral collateral ligaments decreased, while that of the deep bundle of the medial collateral ligament increased significantly during flexion. Variations of the posterior cruciate and the superficial bundle of the medial collateral ligament lengths were concealed by the experimental indeterminacy. A mathematical model was provided that allowed the estimate of subject-specific ligament lengths as a function of knee flexion and that can be integrated in a multi-body optimization procedure.

The gaseous halo of the Milky Way

In the last decade, sensitive observations have revealed that disc galaxies are surrounded by multiphase gaseous halos produced by the circulation of gas from the discs to the environment and vice-versa. This Thesis is a study of the gaseous halo of the Milky Way carried out via the modelling of the HI emission and the available absorption-line data. We fitted simple kinematical models to the HI LAB Survey and found that the Galaxy has a massive (~3x10^8 Mo) HI halo extending a few kiloparsecs above the plane. This layer rotates more slowly than the disc and shows a global inflow motion, a kinematics similar to that observed in the HI halos of nearby galaxies. We built a dynamical model of the galactic fountain to reproduce the properties of this layer. In this model, fountain clouds are ejected from the disc by SN feedback and - as suggested by hydrodynamical simulations - triggers the cooling of coronal gas, which is entrained by the cloud wakes and accretes onto the disc when the clouds fall back. For a proper choice of the parameters, the model reproduces well the HI data and predicts an accretion of coronal gas onto the disc at a rate of 2 Mo/yr. We extended this model to the warm-hot component of the halo, showing that most of the ion absorption features observed towards background sources are consistent with being produced in the turbulent wakes that lag behind the fountain clouds. Specifically, the column densities, positions, and velocities of the absorbers are well reproduced by our model. Finally, we studied the gas content of galaxies extracted from a cosmological N-body+SPH simulation, and found that an HI halo with the forementioned properties is not observed, probably due ti the relatively low resolution of the simulations.

Development of new tools and devices for CMB and foreground data analysis and future experiments

The discovery of the Cosmic Microwave Background (CMB) radiation in 1965 is one of the fundamental milestones supporting the Big Bang theory. The CMB is one of the most important source of information in cosmology. The excellent accuracy of the recent CMB data of WMAP and Planck satellites confirmed the validity of the standard cosmological model and set a new challenge for the data analysis processes and their interpretation. In this thesis we deal with several aspects and useful tools of the data analysis. We focus on their optimization in order to have a complete exploitation of the Planck data and contribute to the final published results. The issues investigated are: the change of coordinates of CMB maps using the HEALPix package, the problem of the aliasing effect in the generation of low resolution maps, the comparison of the Angular Power Spectrum (APS) extraction performances of the optimal QML method, implemented in the code called BolPol, and the pseudo-Cl method, implemented in Cromaster. The QML method has been then applied to the Planck data at large angular scales to extract the CMB APS. The same method has been applied also to analyze the TT parity and the Low Variance anomalies in the Planck maps, showing a consistent deviation from the standard cosmological model, the possible origins for this results have been discussed. The Cromaster code instead has been applied to the 408 MHz and 1.42 GHz surveys focusing on the analysis of the APS of selected regions of the synchrotron emission. The new generation of CMB experiments will be dedicated to polarization measurements, for which are necessary high accuracy devices for separating the polarizations. Here a new technology, called Photonic Crystals, is exploited to develop a new polarization splitter device and its performances are compared to the devices used nowadays.

Farming and processing stategies for improving poultry meat quality

The purpose of the PhD research was the identification of new strategies of farming and processing, with the aim to improve the nutritional and technological characteristics of poultry meat. Part of the PhD research was focused on evaluation of alternative farming systems, with the aim to increase animal welfare and to improve the meat quality and sensorial characteristics in broiler chickens. It was also assessed the use of innovative ingredients for marination of poultry meat (sodium bicarbonate and natural antioxidants) The research was developed by studying the following aspects: - Meat quality characteristics, oxidative stability and sensorial traits of chicken meat obtained from two different farming systems: free range vs conventional; - Meat quality traits of frozen chicken breast pre-salted using increasing concentrations of sodium chloride; - Use of sodium bicarbonate in comparison with sodium trypolyphosphate for marination of broiler breast meat and phase; - Marination with thyme and orange essential oils mixture to improve chicken meat quality traits, susceptibility to lipid oxidation and sensory traits. The following meat quality traits analyseswere performed: Colour, pH, water holding capacity by conventional (gravimetric methods, pressure application, centrifugation and cooking) and innovative methods (low-field NMR and DSC analysis) ability to absorb marinade soloutions, texture (shear force using different probes and texture profile analysis), proximate analysis (moisture, proteins, lipids, ash content, collagen, fatty acid), susceptibility to lipid oxidation (determinations of reactive substances with thiobarbituric acid and peroxide value), sensorial analysis (triangle test and consumer test).

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

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

Structure determination of proteins and peptides in solution: simulation, chirality and NMR studies

The study of protein fold is a central problem in life science, leading in the last years to several attempts for improving our knowledge of the protein structures. In this thesis this challenging problem is tackled by means of molecular dynamics, chirality and NMR studies. In the last decades, many algorithms were designed for the protein secondary structure assignment, which reveals the local protein shape adopted by segments of amino acids. In this regard, the use of local chirality for the protein secondary structure assignment was demonstreted, trying to correlate as well the propensity of a given amino acid for a particular secondary structure. The protein fold can be studied also by Nuclear Magnetic Resonance (NMR) investigations, finding the average structure adopted from a protein. In this context, the effect of Residual Dipolar Couplings (RDCs) in the structure refinement was shown, revealing a strong improvement of structure resolution. A wide extent of this thesis is devoted to the study of avian prion protein. Prion protein is the main responsible of a vast class of neurodegenerative diseases, known as Bovine Spongiform Encephalopathy (BSE), present in mammals, but not in avian species and it is caused from the conversion of cellular prion protein to the pathogenic misfolded isoform, accumulating in the brain in form of amiloyd plaques. In particular, the N-terminal region, namely the initial part of the protein, is quite different between mammal and avian species but both of them contain multimeric sequences called Repeats, octameric in mammals and hexameric in avians. However, such repeat regions show differences in the contained amino acids, in particular only avian hexarepeats contain tyrosine residues. The chirality analysis of avian prion protein configurations obtained from molecular dynamics reveals a high stiffness of the avian protein, which tends to preserve its regular secondary structure. This is due to the presence of prolines, histidines and especially tyrosines, which form a hydrogen bond network in the hexarepeat region, only possible in the avian protein, and thus probably hampering the aggregation.

Low-dimensional carbon allotropes: an electron microscopy investigation

The research reported in this manuscript concerns the structural characterization of graphene membranes and single-walled carbon nanotubes (SWCNTs). The experimental investigation was performed using a wide range of transmission electron microscopy (TEM) techniques, from conventional imaging and diffraction, to advanced interferometric methods, like electron holography and Geometric Phase Analysis (GPA), using a low-voltage optical set-up, to reduce radiation damage in the samples. Electron holography was used to successfully measure the mean electrostatic potential of an isolated SWCNT and that of a mono-atomically thin graphene crystal. The high accuracy achieved in the phase determination, made it possible to measure, for the first time, the valence-charge redistribution induced by the lattice curvature in an individual SWCNT. A novel methodology for the 3D reconstruction of the waviness of a 2D crystal membrane has been developed. Unlike other available TEM reconstruction techniques, like tomography, this new one requires processing of just a single HREM micrograph. The modulations of the inter-planar distances in the HREM image are measured using Geometric Phase Analysis, and used to recover the waviness of the crystal. The method was applied to the case of a folded FGC, and a height variation of 0.8 nm of the surface was successfully determined with nanometric lateral resolution. The adhesion of SWCNTs to the surface of graphene was studied, mixing shortened SWCNTs of different chiralities and FGC membranes. The spontaneous atomic match of the two lattices was directly imaged using HREM, and we found that graphene membranes act as tangential nano-sieves, preferentially grafting achiral tubes to their surface.

NMR, Metabonomics and Molecular Profiles: Applications to the Quality Assessment of Foodstuffs

Nuclear Magnetic Resonance (NMR) is a branch of spectroscopy that is based on the fact that many atomic nuclei may be oriented by a strong magnetic field and will absorb radiofrequency radiation at characteristic frequencies. The parameters that can be measured on the resulting spectral lines (line positions, intensities, line widths, multiplicities and transients in time-dependent experi-ments) can be interpreted in terms of molecular structure, conformation, molecular motion and other rate processes. In this way, high resolution (HR) NMR allows performing qualitative and quantitative analysis of samples in solution, in order to determine the structure of molecules in solution and not only. In the past, high-field NMR spectroscopy has mainly concerned with the elucidation of chemical structure in solution, but today is emerging as a powerful exploratory tool for probing biochemical and physical processes. It represents a versatile tool for the analysis of foods. In literature many NMR studies have been reported on different type of food such as wine, olive oil, coffee, fruit juices, milk, meat, egg, starch granules, flour, etc using different NMR techniques. Traditionally, univariate analytical methods have been used to ex-plore spectroscopic data. This method is useful to measure or to se-lect a single descriptive variable from the whole spectrum and , at the end, only this variable is analyzed. This univariate methods ap-proach, applied to HR-NMR data, lead to different problems due especially to the complexity of an NMR spectrum. In fact, the lat-ter is composed of different signals belonging to different mole-cules, but it is also true that the same molecules can be represented by different signals, generally strongly correlated. The univariate methods, in this case, takes in account only one or a few variables, causing a loss of information. Thus, when dealing with complex samples like foodstuff, univariate analysis of spectra data results not enough powerful. Spectra need to be considered in their wholeness and, for analysing them, it must be taken in consideration the whole data matrix: chemometric methods are designed to treat such multivariate data. Multivariate data analysis is used for a number of distinct, differ-ent purposes and the aims can be divided into three main groups: • data description (explorative data structure modelling of any ge-neric n-dimensional data matrix, PCA for example); • regression and prediction (PLS); • classification and prediction of class belongings for new samples (LDA and PLS-DA and ECVA). The aim of this PhD thesis was to verify the possibility of identify-ing and classifying plants or foodstuffs, in different classes, based on the concerted variation in metabolite levels, detected by NMR spectra and using the multivariate data analysis as a tool to inter-pret NMR information. It is important to underline that the results obtained are useful to point out the metabolic consequences of a specific modification on foodstuffs, avoiding the use of a targeted analysis for the different metabolites. The data analysis is performed by applying chemomet-ric multivariate techniques to the NMR dataset of spectra acquired. The research work presented in this thesis is the result of a three years PhD study. This thesis reports the main results obtained from these two main activities: A1) Evaluation of a data pre-processing system in order to mini-mize unwanted sources of variations, due to different instrumental set up, manual spectra processing and to sample preparations arte-facts; A2) Application of multivariate chemiometric models in data analy-sis.