Sviluppo di un tomografo multi-energy per lo studio pre-clinico di nuove metodiche diagnostiche finalizzate al riconoscimento precoce della patologia tumorale

A new multi-energy CT for small animals is being developed at the Physics Department of the University of Bologna, Italy. The system makes use of a set of quasi-monochromatic X-ray beams, with energy tunable in a range from 26 KeV to 72 KeV. These beams are produced by Bragg diffraction on a Highly Oriented Pyrolytic Graphite crystal. With quasi-monochromatic sources it is possible to perform multi-energy investigation in a more effective way, as compared with conventional X-ray tubes. Multi-energy techniques allow extracting physical information from the materials, such as effective atomic number, mass-thickness, density, that can be used to distinguish and quantitatively characterize the irradiated tissues. The aim of the system is the investigation and the development of new pre-clinic methods for the early detection of the tumors in small animals. An innovative technique, the Triple-Energy Radiography with Contrast Medium (TER), has been successfully implemented on our system. TER consist in combining a set of three quasi-monochromatic images of an object, in order to obtain a corresponding set of three single-tissue images, which are the mass-thickness map of three reference materials. TER can be applied to the quantitative mass-thickness-map reconstruction of a contrast medium, because it is able to remove completely the signal due to other tissues (i.e. the structural background noise). The technique is very sensitive to the contrast medium and is insensitive to the superposition of different materials. The method is a good candidate to the early detection of the tumor angiogenesis in mice. In this work we describe the tomographic system, with a particular focus on the quasi-monochromatic source. Moreover the TER method is presented with some preliminary results about small animal imaging.

Bioanalytical applications of multicolour bioluminescence imaging: new tools for drug discovery and development

The subject of this thesis is multicolour bioluminescence analysis and how it can provide new tools for drug discovery and development.The mechanism of color tuning in bioluminescent reactions is not fully understood yet but it is object of intense research and several hypothesis have been generated. In the past decade key residues of the active site of the enzyme or in the surface surrounding the active site have been identified as responsible of different color emission. Anyway since bioluminescence reaction is strictly dependent from the interaction between the enzyme and its substrate D-luciferin, modification of the substrate can lead to a different emission spectrum too. In the recent years firefly luciferase and other luciferases underwent mutagenesis in order to obtain mutants with different emission characteristics. Thanks to these new discoveries in the bioluminescence field multicolour luciferases can be nowadays employed in bioanalysis for assay developments and imaging purposes. The use of multicolor bioluminescent enzymes expanded the potential of a range of application in vitro and in vivo. Multiple analysis and more information can be obtained from the same analytical session saving cost and time. This thesis focuses on several application of multicolour bioluminescence for high-throughput screening and in vivo imaging. Multicolor luciferases can be employed as new tools for drug discovery and developments and some examples are provided in the different chapters. New red codon optimized luciferase have been demonstrated to be improved tools for bioluminescence imaging in small animal and the possibility to combine red and green luciferases for BLI has been achieved even if some aspects of the methodology remain challenging and need further improvement. In vivo Bioluminescence imaging has known a rapid progress since its first application no more than 15 years ago. It is becoming an indispensable tool in pharmacological research. At the same time the development of more sensitive and implemented microscopes and low-light imager for a better visualization and quantification of multicolor signals would boost the research and the discoveries in life sciences in general and in drug discovery and development in particular.

Stem Cells as a therapy for myocardial infarction in animal models

Advances in stem cell biology have challenged the notion that infarcted myocardium is irreparable. The pluripotent ability of stem cells to differentiate into specialized cell lines began to garner intense interest within cardiology when it was shown in animal models that intramyocardial injection of bone marrow stem cells (MSCs), or the mobilization of bone marrow stem cells with spontaneous homing to myocardium, could improve cardiac function and survival after induced myocardial infarction (MI) [1, 2]. Furthermore, the existence of stem cells in myocardium has been identified in animal heart [3, 4], and intense research is under way in an attempt to clarify their potential clinical application for patients with myocardial infarction. To date, in order to identify the best one, different kinds of stem cells have been studied; these have been derived from embryo or adult tissues (i.e. bone marrow, heart, peripheral blood etc.). Currently, three different biologic therapies for cardiovascular diseases are under investigation: cell therapy, gene therapy and the more recent “tissue-engineering” therapy . During my Ph.D. course, first I focalised my study on the isolation and characterization of Cardiac Stem Cells (CSCs) in wild-type and transgenic mice and for this purpose I attended, for more than one year, the Cardiovascular Research Institute of the New York Medical College, in Valhalla (NY, USA) under the direction of Doctor Piero Anversa. During this period I learnt different Immunohistochemical and Biomolecular techniques, useful for investigating the regenerative potential of stem cells. Then, during the next two years, I studied the new approach of cardiac regenerative medicine based on “tissue-engineering” in order to investigate a new strategy to regenerate the infracted myocardium. Tissue-engineering is a promising approach that makes possible the creation of new functional tissue to replace lost or failing tissue. This new discipline combines isolated functioning cells and biodegradable 3-dimensional (3D) polymeric scaffolds. The scaffold temporarily provides the biomechanical support for the cells until they produce their own extracellular matrix. Because tissue-engineering constructs contain living cells, they may have the potential for growth and cellular self-repair and remodeling. In the present study, I examined whether the tissue-engineering strategy within hyaluron-based scaffolds would result in the formation of alternative cardiac tissue that could replace the scar and improve cardiac function after MI in syngeneic heterotopic rat hearts. Rat hearts were explanted, subjected to left coronary descending artery occlusion, and then grafted into the abdomen (aorta-aorta anastomosis) of receiving syngeneic rat. After 2 weeks, a pouch of 3 mm2 was made in the thickness of the ventricular wall at the level of the post-infarction scar. The hyaluronic scaffold, previously engineered for 3 weeks with rat MSCs, was introduced into the pouch and the myocardial edges sutured with few stitches. Two weeks later we evaluated the cardiac function by M-Mode echocardiography and the myocardial morphology by microscope analysis. We chose bone marrow-derived mensenchymal stem cells (MSCs) because they have shown great signaling and regenerative properties when delivered to heart tissue following a myocardial infarction (MI). However, while the object of cell transplantation is to improve ventricular function, cardiac cell transplantation has had limited success because of poor graft viability and low cell retention, that’s why we decided to combine MSCs with a biopolimeric scaffold. At the end of the experiments we observed that the hyaluronan fibres had not been substantially degraded 2 weeks after heart-transplantation. Most MSCs had migrated to the surrounding infarcted area where they were especially found close to small-sized vessels. Scar tissue was moderated in the engrafted region and the thickness of the corresponding ventricular wall was comparable to that of the non-infarcted remote area. Also, the left ventricular shortening fraction, evaluated by M-Mode echocardiography, was found a little bit increased when compared to that measured just before construct transplantation. Therefore, this study suggests that post-infarction myocardial remodelling can be favourably affected by the grafting of MSCs delivered through a hyaluron-based scaffold

Elastic Propagation in random media: applications to the imaging of volcano structures

High-frequency seismograms contain features that reflect the random inhomogeneities of the earth. In this work I use an imaging method to locate the high contrast small- scale heterogeneity respect to the background earth medium. This method was first introduced by Nishigami (1991) and than applied to different volcanic and tectonically active areas (Nishigami, 1997, Nishigami, 2000, Nishigami, 2006). The scattering imaging method is applied to two volcanic areas: Campi Flegrei and Mt. Vesuvius. Volcanic and seismological active areas are often characterized by complex velocity structures, due to the presence of rocks with different elastic properties. I introduce some modifications to the original method in order to make it suitable for small and highly complex media. In particular, for very complex media the single scattering approximation assumed by Nishigami (1991) is not applicable as the mean free path becomes short. The multiple scattering or diffusive approximation become closer to the reality. In this thesis, differently from the ordinary Nishigami’s method (Nishigami, 1991), I use the mean of the recorded coda envelope as reference curve and calculate the variations from this average envelope. In this way I implicitly do not assume any particular scattering regime for the "average" scattered radiation, whereas I consider the variations as due to waves that are singularly scattered from the strongest heterogeneities. The imaging method is applied to a relatively small area (20 x 20 km), this choice being justified by the small length of the analyzed codas of the low magnitude earthquakes. I apply the unmodified Nishigami’s method to the volcanic area of Campi Flegrei and compare the results with the other tomographies done in the same area. The scattering images, obtained with frequency waves around 18 Hz, show the presence of high scatterers in correspondence with the submerged caldera rim in the southern part of the Pozzuoli bay. Strong scattering is also found below the Solfatara crater, characterized by the presence of densely fractured, fluid-filled rocks and by a strong thermal anomaly. The modified Nishigami’s technique is applied to the Mt. Vesuvius area. Results show a low scattering area just below the central cone and a high scattering area around it. The high scattering zone seems to be due to the contrast between the high rigidity body located beneath the crater and the low rigidity materials located around it. The central low scattering area overlaps the hydrothermal reservoirs located below the central cone. An interpretation of the results in terms of geological properties of the medium is also supplied, aiming to find a correspondence of the scattering properties and the geological nature of the material. A complementary result reported in this thesis is that the strong heterogeneity of the volcanic medium create a phenomenon called "coda localization". It has been verified that the shape of the seismograms recorded from the stations located at the top of the volcanic edifice of Mt. Vesuvius is different from the shape of the seismograms recorded at the bottom. This behavior is justified by the consideration that the coda energy is not uniformly distributed within a region surrounding the source for great lapse time.

Hepatobiliary diseases in small animals: a comparison of ultrasonography and multidetector-row computed tomography

Ultrasonography (US) is an essential imaging tool for identifying abnormalities of the liver parenchyma, biliary tract and vascular system. US has replaced radiography as the initial imaging procedure in screening for liver disease in small animals. There are few reports of the use of conventional and helical computed tomography (CT) to assess canine or feline parenchymal and neoplastic liver disease and biliary disorders. In human medicine the development of multidetector- row helical computed tomography (MDCT), with its superior spatial and temporal resolution, has resulted in improved detection and characterization of diffuse and focal liver lesions. The increased availability of MDCT in veterinary practice provides incentive to develop MDCT protocols for liver imaging in small animals. The purpose of this study is to assess the rule of MDCT in the characterization of hepatobiliary diseases in small animals; and to compare this method with conventional US. Candidates for this prospective study were 175 consecutive patients (dogs and cats) referred for evaluation of hepatobiliary disease. The patients underwent liver US and MDCT. Percutaneous needle biopsy was performed on all liver lesions or alterations encountered. As for gallbladder, histopatological evaluation was obtained from cholecystectomy specimens. Ultrasonographic findings in this study agreed well with those of previous reports. A protocol for dual-phase liver MDCT in small animals has been described. MDCT findings in parenchymal disorders of the liver, hepatic neoplasia and biliary disorders are here first described in dogs and cats and compared with the corresponding features in human medicine. The ability of MDCT in detection and characterization of hepatobiliary diseases in small animals is overall superior to conventional US. Ultrasonography and MDCT scanning, however, play complementary rules in the evaluation of these diseases. Many conditions have distinctive imaging features that may permit diagnosis. In most instances biopsy is required for definitive diagnosis.

Imaging of crustal scatterers using multiple seismic arrays

Array seismology is an useful tool to perform a detailed investigation of the Earth’s interior. Seismic arrays by using the coherence properties of the wavefield are able to extract directivity information and to increase the ratio of the coherent signal amplitude relative to the amplitude of incoherent noise. The Double Beam Method (DBM), developed by Krüger et al. (1993, 1996), is one of the possible applications to perform a refined seismic investigation of the crust and mantle by using seismic arrays. The DBM is based on a combination of source and receiver arrays leading to a further improvement of the signal-to-noise ratio by reducing the error in the location of coherent phases. Previous DBM works have been performed for mantle and core/mantle resolution (Krüger et al., 1993; Scherbaum et al., 1997; Krüger et al., 2001). An implementation of the DBM has been presented at 2D large-scale (Italian data-set for Mw=9.3, Sumatra earthquake) and at 3D crustal-scale as proposed by Rietbrock & Scherbaum (1999), by applying the revised version of Source Scanning Algorithm (SSA; Kao & Shan, 2004). In the 2D application, the rupture front propagation in time has been computed. In 3D application, the study area (20x20x33 km3), the data-set and the source-receiver configurations are related to the KTB-1994 seismic experiment (Jost et al., 1998). We used 60 short-period seismic stations (200-Hz sampling rate, 1-Hz sensors) arranged in 9 small arrays deployed in 2 concentric rings about 1 km (A-arrays) and 5 km (B-array) radius. The coherence values of the scattering points have been computed in the crustal volume, for a finite time-window along all array stations given the hypothesized origin time and source location. The resulting images can be seen as a (relative) joint log-likelihood of any point in the subsurface that have contributed to the full set of observed seismograms.

Three dimensional seismic imaging and earthquake locations in a complex, segmented fault region in Southern Apennines (Italy)

The southern Apennines of Italy have been experienced several destructive earthquakes both in historic and recent times. The present day seismicity, characterized by small-to-moderate magnitude earthquakes, was used like a probe to obatin a deeper knowledge of the fault structures where the largest earthquakes occurred in the past. With the aim to infer a three dimensional seismic image both the problem of data quality and the selection of a reliable and robust tomographic inversion strategy have been faced. The data quality has been obtained to develop optimized procedures for the measurements of P- and S-wave arrival times, through the use of polarization filtering and to the application of a refined re-picking technique based on cross-correlation of waveforms. A technique of iterative tomographic inversion, linearized, damped combined with a strategy of multiscale inversion type has been adopted. The retrieved P-wave velocity model indicates the presence of a strong velocity variation along a direction orthogonal to the Apenninic chain. This variation defines two domains which are characterized by a relatively low and high velocity values. From the comparison between the inferred P-wave velocity model with a portion of a structural section available in literature, the high velocity body was correlated with the Apulia carbonatic platforms whereas the low velocity bodies was associated to the basinal deposits. The deduced Vp/Vs ratio shows that the ratio is lower than 1.8 in the shallower part of the model, while for depths ranging between 5 km and 12 km the ratio increases up to 2.1 in correspondence to the area of higher seismicity. This confirms that areas characterized by higher values are more prone to generate earthquakes as a response to the presence of fluids and higher pore-pressures.

In vivo evaluation of the translations of the gleno-humeral joint using Magnetic resonance imaging

Gleno-humeral joint (GHJ) is the most mobile joint of the human body. This is related to theincongr uence between the large humeral head articulating with the much smaller glenoid (ratio 3:1). The GHJ laxity is the ability of the humeral head to be passively translated on the glenoid fossa and, when physiological, it guarantees the normal range of motion of the joint. Three-dimensional GHJ linear displacements have been measured, both in vivo and in vitro by means of different instrumental techniques. In vivo gleno-humeral displacements have been assessed by means of stereophotogrammetry, electromagnetic tracking sensors, and bio-imaging techniques. Both stereophotogrammetric systems and electromagnetic tracking devices, due to the deformation of the soft tissues surrounding the bones, are not capable to accurately assess small displacements, such as gleno-humeral joint translations. The bio-imaging techniques can ensure for an accurate joint kinematic (linear and angular displacement) description, but, due to the radiation exposure, most of these techniques, such as computer tomography or fluoroscopy, are invasive for patients. Among the bioimaging techniques, an alternative which could provide an acceptable level of accuracy and that is innocuous for patients is represented by magnetic resonance imaging (MRI). Unfortunately, only few studies have been conducted for three-dimensional analysis and very limited data is available in situations where preset loads are being applied. The general aim of this doctoral thesis is to develop a non-invasive methodology based on open-MRI for in-vivo evaluation of the gleno-humeral translation components in healthy subjects under the application of external loads.

The porcine animal model goes through the 3Rs: development of in vitro and ex vivo system to study vascular biology

Since the publication of the book of Russell and Burch in 1959, scientific research has never stopped improving itself with regard to the important issue of animal experimentation. The European Directive 2010/63/EU “On the protection of animals used for scientific purposes” focuses mainly on the animal welfare, fixing the Russell and Burch’s 3Rs principles as the foundations of the document. In particular, the legislator clearly states the responsibility of the scientific community to improve the number of alternative methods to animal experimentation. The swine is considered a species of relevant interest for translational research and medicine due to its biological similarities with humans. The surgical community has, in fact, recognized the swine as an excellent model replicating the human cardiovascular system. There have been several wild-type and transgenic porcine models which were produced for biomedicine and translational research. Among these, the cardiovascular ones are the most represented. The continuous involvement of the porcine animal model in the biomedical research, as the continuous advances achieved using swine in translational medicine, support the need for alternative methods to animal experimentation involving pigs. The main purpose of the present work was to develop and characterize novel porcine alternative methods for cardiovascular translational biology/medicine. The work was mainly based on two different models: the first consisted in an ex vivo culture of porcine aortic cylinders and the second consisted in an in vitro culture of porcine aortic derived progenitor cells. Both the models were properly characterized and results indicated that they could be useful to the study of vascular biology. Nevertheless, both the models aim to reduce the use of experimental animals and to refine animal based-trials. In conclusion, the present research aims to be a small, but significant, contribution to the important and necessary field of study of alternative methods to animal experimentation.

The foodomics approach to investigate the impact of the matrix structure on food quality: applications of magnetic resonance spectroscopy, relaxometry and imaging

The aim of this thesis is to explore the possible influence of the food matrix on food quality attributes. Using nuclear magnetic resonance techniques, the matrix-dependent properties of different foods were studied and some useful indices were defined to classify food products based on the matrix behaviour when responding to processing phenomena. Correlations were found between fish freshness indices, assessed by certain geometric parameters linked to the morphology of the animal, i.e. a macroscopic structure, and the degradation of the product structure. The same foodomics approach was also applied to explore the protective effect of modified atmospheres on the stability of fish fillets, which are typically susceptible to oxidation of the polyunsaturated fatty acids incorporated in the meat matrix. Here, freshness is assessed by evaluating the time-dependent change in the fish metabolome, providing an established freshness index, and its relationship to lipid oxidation. In vitro digestion studies, focusing on food products with different matrixes, alone and in combination with other meal components (e.g. seasoning), were conducted to investigate possible interactions between enzymes and food, modulated by matrix structure, which influence digestibility. The interaction between water and the gelatinous matrix of the food, consisting of a network of protein gels incorporating fat droplets, was also studied by means of nuclear magnetic relaxometry, in order to create a prediction tool for the correct classification of authentic and counterfeit food products protected by a quality label. This is one of the first applications of an NMR method focusing on the supramolecular structure of the matrix, rather than the chemical composition, to assess food authenticity. The effect of innovative processing technologies, such as PEF applied to fruit products, has been assessed by magnetic resonance imaging, exploiting information associated with the rehydration kinetics exerted by a modified food structure.

Can gut microbiota analysis be beneficial for ex-situ and in-situ conservation of threatened animal species?

Ex-situ conservation and the in-situ conservation of natural habitats are the tools to conserve biodiversity. Habitats and ecosystems have been becoming altered by human activities and a growing number of species requires form of management to ensure their survival. Conservation queries become more complex and urgent. Developing scientifically based and innovative approaches to ex-situ conservation is necessary. Recent studies underline importance of gut microbiome in animal health with implications for animal conservation and management. Animal and human studies have demonstrated that environmental factors can impact gut microbiome composition. Within this scenario, the present work focused on species belonging to different taxa, reptiles and mammals: Aldabrachelys gigantea, the giant tortoise of the Seychelles islands and Indri indri, the greatest leaving lemur of Madagascar. The Seychelles giant tortoise is vulnerable species with declining population, whereas the indri is a critically endangered species that could reach the extinction within 25 years. Both need research to help them to survive. Tortoises live for very long time and to observe how they can afford the environmental changes is very difficult. Indris, instead, are able to survive only in a small area of the Madagascar forest, with a very strong link between the species’ survival and the environment. The obtained results underline importance of environmental factors, both in-situ and ex-situ, for species conservation. Microbiome could help the organisms to respond on a short timescale and cope with, environmental changes. However, species with long generation time might not be able to adapt to fast changes but bacteria with a short generation time can adapt on a shorter timescale allowing the host to cope with fluctuating environment. Gut microbiome plays an important role in an animal’s health and has the potential to improve the management of individuals under human care for conservation purposes.

Advanced studies on two animal models, murine and fish. RadKI21A626T mouse model: new insights into molecular mechanisms of enteric neuro-epithelial pathology. European sea bass: study of the effects of essential oils in different diets on the gastric system.

My PhD research period was focused on the anatomical, physiological and functional study of the gastrointestinal system on two different animal models. In two different contexts, the purpose of these two lines of research was contribute to understand how a specific genetic mutation or the adoption of a particular dietary supplement can affect gastrointestinal function. Functional gastrointestinal disorders are chronic conditions characterized by symptoms for which no organic cause can be found. Although symptoms are generally mild, a small subset of cases shows severe manifestations. This subset of patients may also have recurrent intestinal sub-occlusive episodes, but in absence of mechanical causes. This condition is referred to as chronic intestinal pseudo-obstruction, a rare, intractable chronic disease. Some mutations have been associated with CIPO. A novel causative RAD21 missense mutation was identified in a large consanguineous family, segregating a recessive form of CIPO. The present thesis was aimed to elucidate the mechanisms leading to neuropathy underlying CIPO via a recently developed conditional KI mouse carrying the RAD21 mutation. The experimental studies are based on the characterization and functional analysis of the conditional KI Rad21A626T mouse model. On the other hand aquaculture is increasing the global supply of foods. The species selected and feeds used affects the nutrients available from aquaculture, with a need to improve feed efficiency, both for economic and environmental reasons, but this will require novel innovative approaches. Nutritional strategies focused on the use of botanicals have attracted interest in animal production. Previous research indicates the positive results of using essential oils (EOs) as natural feed additives for several farmed animals. Therefore, the present study was designed to compare the effects of feed EO supplementation in two different forms (natural and composed of active ingredients obtained by synthesis) on the gastric mucosa in European sea bass.

Inside standard and hyperspectral low-dose CT variational imaging: parameter identification and material decomposition

The main contribution of this thesis is the proposal of novel strategies for the selection of parameters arising in variational models employed for the solution of inverse problems with data corrupted by Poisson noise. In light of the importance of using a significantly small dose of X-rays in Computed Tomography (CT), and its need of using advanced techniques to reconstruct the objects due to the high level of noise in the data, we will focus on parameter selection principles especially for low photon-counts, i.e. low dose Computed Tomography. For completeness, since such strategies can be adopted for various scenarios where the noise in the data typically follows a Poisson distribution, we will show their performance for other applications such as photography, astronomical and microscopy imaging. More specifically, in the first part of the thesis we will focus on low dose CT data corrupted only by Poisson noise by extending automatic selection strategies designed for Gaussian noise and improving the few existing ones for Poisson. The new approaches will show to outperform the state-of-the-art competitors especially in the low-counting regime. Moreover, we will propose to extend the best performing strategy to the hard task of multi-parameter selection showing promising results. Finally, in the last part of the thesis, we will introduce the problem of material decomposition for hyperspectral CT, which data encodes information of how different materials in the target attenuate X-rays in different ways according to the specific energy. We will conduct a preliminary comparative study to obtain accurate material decomposition starting from few noisy projection data.