Design and Characterization of New Luminescent Sensors and Labels

The aim of this Ph.D. project has been the design and characterization of new and more efficient luminescent tools, in particular sensors and labels, for analytical chemistry, medical diagnostics and imaging. Actually both the increasing temporal and spatial resolutions that are demanded by those branches, coupled to a sensitivity that is required to reach the single molecule resolution, can be provided by the wide range of techniques based on luminescence spectroscopy. As far as the development of new chemical sensors is concerned, as chemists we were interested in the preparation of new, efficient, sensing materials. In this context, we kept developing new molecular chemosensors, by exploiting the supramolecular approach, for different classes of analytes. In particular we studied a family of luminescent tetrapodal-hosts based on aminopyridinium units with pyrenyl groups for the detection of anions. These systems exhibited noticeable changes in the photophysical properties, depending on the nature of the anion; in particular, addition of chloride resulted in a conformational change, giving an initial increase in excimeric emission. A good selectivity for dicarboxylic acid was also found. In the search for higher sensitivities, we moved our attention also to systems able to perform amplification effects. In this context we described the metal ion binding properties of three photoactive poly-(arylene ethynylene) co-polymers with different complexing units and we highlighted, for one of them, a ten-fold amplification of the response in case of addition of Zn2+, Cu2+ and Hg2+ ions. In addition, we were able to demonstrate the formation of complexes with Yb3+ an Er3+ and an efficient sensitization of their typical metal centered NIR emission upon excitation of the polymer structure, this feature being of particular interest for their possible applications in optical imaging and in optical amplification for telecommunication purposes. An amplification effect was also observed during this research in silica nanoparticles derivatized with a suitable zinc probe. In this case we were able to prove, for the first time, that nanoparticles can work as “off-on” chemosensors with signal amplification. Fluorescent silica nanoparticles can be thus seen as innovative multicomponent systems in which the organization of photophysically active units gives rise to fruitful collective effects. These precious effects can be exploited for biological imaging, medical diagnostic and therapeutics, as evidenced also by some results reported in this thesis. In particular, the observed amplification effect has been obtained thanks to a suitable organization of molecular probe units onto the surface of the nanoparticles. In the effort of reaching a deeper inside in the mechanisms which lead to the final amplification effects, we also attempted to find a correlation between the synthetic route and the final organization of the active molecules in the silica network, and thus with those mutual interactions between one another which result in the emerging, collective behavior, responsible for the desired signal amplification. In this context, we firstly investigated the process of formation of silica nanoparticles doped with pyrene derivative and we showed that the dyes are not uniformly dispersed inside the silica matrix; thus, core-shell structures can be formed spontaneously in a one step synthesis. Moreover, as far as the design of new labels is concerned, we reported a new synthetic approach to obtain a class of robust, biocompatible silica core-shell nanoparticles able to show a long-term stability. Taking advantage of this new approach we also showed the synthesis and photophysical properties of core-shell NIR absorbing and emitting materials that proved to be very valuable for in-vivo imaging. In general, the dye doped silica nanoparticles prepared in the framework of this project can conjugate unique properties, such as a very high brightness, due to the possibility to include many fluorophores per nanoparticle, high stability, because of the shielding effect of the silica matrix, and, to date, no toxicity, with a simple and low-cost preparation. All these features make these nanostructures suitable to reach the low detection limits that are nowadays required for effective clinical and environmental applications, fulfilling in this way the initial expectations of this research project.

Analysis and modeling techniques for ultrasonic tissue characterization

This thesis introduces new processing techniques for computer-aided interpretation of ultrasound images with the purpose of supporting medical diagnostic. In terms of practical application, the goal of this work is the improvement of current prostate biopsy protocols by providing physicians with a visual map overlaid over ultrasound images marking regions potentially affected by disease. As far as analysis techniques are concerned, the main contributions of this work to the state-of-the-art is the introduction of deconvolution as a pre-processing step in the standard ultrasonic tissue characterization procedure to improve the diagnostic significance of ultrasonic features. This thesis also includes some innovations in ultrasound modeling, in particular the employment of a continuous-time autoregressive moving-average (CARMA) model for ultrasound signals, a new maximum-likelihood CARMA estimator based on exponential splines and the definition of CARMA parameters as new ultrasonic features able to capture scatterers concentration. Finally, concerning the clinical usefulness of the developed techniques, the main contribution of this research is showing, through a study based on medical ground truth, that a reduction in the number of sampled cores in standard prostate biopsy is possible, preserving the same diagnostic power of the current clinical protocol.

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.

Study of the thermal effects induced by magnetic resonance on endocardial leads: numerical models and experimental validation

Magnetic resonance imaging (MRI) is today precluded to patients bearing active implantable medical devices AIMDs). The great advantages related to this diagnostic modality, together with the increasing number of people benefiting from implantable devices, in particular pacemakers(PM)and carioverter/defibrillators (ICD), is prompting the scientific community the study the possibility to extend MRI also to implanted patients. The MRI induced specific absorption rate (SAR) and the consequent heating of biological tissues is one of the major concerns that makes patients bearing metallic structures contraindicated for MRI scans. To date, both in-vivo and in-vitro studies have demonstrated the potentially dangerous temperature increase caused by the radiofrequency (RF) field generated during MRI procedures in the tissues surrounding thin metallic implants. On the other side, the technical evolution of MRI scanners and of AIMDs together with published data on the lack of adverse events have reopened the interest in this field and suggest that, under given conditions, MRI can be safely performed also in implanted patients. With a better understanding of the hazards of performing MRI scans on implanted patients as well as the development of MRI safe devices, we may soon enter an era where the ability of this imaging modality may be more widely used to assist in the appropriate diagnosis of patients with devices. In this study both experimental measures and numerical analysis were performed. Aim of the study is to systematically investigate the effects of the MRI RF filed on implantable devices and to identify the elements that play a major role in the induced heating. Furthermore, we aimed at developing a realistic numerical model able to simulate the interactions between an RF coil for MRI and biological tissues implanted with a PM, and to predict the induced SAR as a function of the particular path of the PM lead. The methods developed and validated during the PhD program led to the design of an experimental framework for the accurate measure of PM lead heating induced by MRI systems. In addition, numerical models based on Finite-Differences Time-Domain (FDTD) simulations were validated to obtain a general tool for investigating the large number of parameters and factors involved in this complex phenomenon. The results obtained demonstrated that the MRI induced heating on metallic implants is a real risk that represents a contraindication in extending MRI scans also to patient bearing a PM, an ICD, or other thin metallic objects. On the other side, both experimental data and numerical results show that, under particular conditions, MRI procedures might be consider reasonably safe also for an implanted patient. The complexity and the large number of variables involved, make difficult to define a unique set of such conditions: when the benefits of a MRI investigation cannot be obtained using other imaging techniques, the possibility to perform the scan should not be immediately excluded, but some considerations are always needed.

Aging of nuclear power plant cables: in search of non-destructive diagnostic quantities

The safety systems of nuclear power plants rely on low-voltage power, instrumentation and control cables. Inside the containment area, cables operate in harsh environments, characterized by relatively high temperature and gamma-irradiation. As these cables are related to fundamental safety systems, they must be able to withstand unexpected accident conditions and, therefore, their condition assessment is of utmost importance as plants age and lifetime extensions are required. Nowadays, the integrity and functionality of these cables are monitored mainly through destructive test which requires specific laboratory. The investigation of electrical aging markers which can provide information about the state of the cable by non-destructive testing methods would improve significantly the present diagnostic techniques. This work has been made within the framework of the ADVANCE (Aging Diagnostic and Prognostics of Low-Voltage I\&C Cables) project, a FP7 European program. This Ph.D. thesis aims at studying the impact of aging on cable electrical parameters, in order to understand the evolution of the electrical properties associated with cable degradation. The identification of suitable aging markers requires the comparison of the electrical property variation with the physical/chemical degradation mechanisms of polymers for different insulating materials and compositions. The feasibility of non-destructive electrical condition monitoring techniques as potential substitutes for destructive methods will be finally discussed studying the correlation between electrical and mechanical properties. In this work, the electrical properties of cable insulators are monitored and characterized mainly by dielectric spectroscopy, polarization/depolarization current analysis and space charge distribution. Among these techniques, dielectric spectroscopy showed the most promising results; by means of dielectric spectroscopy it is possible to identify the frequency range where the properties are more sensitive to aging. In particular, the imaginary part of permittivity at high frequency, which is related to oxidation, has been identified as the most suitable aging marker based on electrical quantities.

Valorisation of organic waste: new developments from proton nuclear magnetic resonance characterization

The last half-century has seen a continuing population and consumption growth, increasing the competition for land, water and energy. The solution can be found in the new sustainability theories, such as the industrial symbiosis and the zero waste objective. Reducing, reusing and recycling are challenges that the whole world have to consider. This is especially important for organic waste, whose reusing gives interesting results in terms of energy release. Before reusing, organic waste needs a deeper characterization. The non-destructive and non-invasive features of both Nuclear Magnetic Resonance (NMR) relaxometry and imaging (MRI) make them optimal candidates to reach such characterization. In this research, NMR techniques demonstrated to be innovative technologies, but an important work on the hardware and software of the NMR LAGIRN laboratory was initially done, creating new experimental procedures to analyse organic waste samples. The first results came from soil-organic matter interactions. Remediated soils properties were described in function of the organic carbon content, proving the importance of limiting the addition of further organic matter to not inhibit soil processes as nutrients transport. Moreover NMR relaxation times and the signal amplitude of a compost sample, over time, showed that the organic matter degradation of compost is a complex process that involves a number of degradation kinetics, as a function of the mix of waste. Local degradation processes were studied with enhanced quantitative relaxation technique that combines NMR and MRI. The development of this research has finally led to the study of waste before it becomes waste. Since a lot of food is lost when it is still edible, new NMR experiments studied the efficiency of conservation and valorisation processes: apple dehydration, meat preservation and bio-oils production. All these results proved the readiness of NMR for quality controls on a huge kind of organic residues and waste.