Dissertationen zum Thema „Imaging methodologies“

Multispectral imaging is a method for acquiring image data over a series of wavelengths across the light spectrum. In combination with several image processing methods, it has become a valuable tool for the enhancement and recovery of information from cultural heritage documents. It enables the identification of distinct physical characteristics of the document and its material components by measuring their interaction with light. In this dissertation I describe an approach to evaluate multispectral imaging processing methods in the context of cultural heritage documents. I begin by framing a digital model, or phantom, of multispectral images of a document composed of interacting layers of text and other materials. Further, I apply a series of image processing methods to the phantom in order to extract and separate those interacting layers and I qualitatively assess the results of each of those methods. I proceed then to propose and apply a quantitative method, based on mutual information, to evaluate the efficacy of those image processing methods. Next, I describe the administration of a series of treatments that degrade the writing or otherwise degrade samples from a real document, and the acquisition of multispectral images from those samples. Finally, I report on the recovery of information via image processing of the acquired experimental data. The results of the image processing of the experimental data are then quantitatively evaluated using the proposed method, and qualitatively described. As multispectral imaging in combination with image processing methods become increasingly used in cultural heritage documents, we need a deeper understanding of how these methods perform in the analysis of this particular data. I contribute to this understanding in two important ways: I introduce a formal methodology to study the methods involved in processing and analysing the multispectral data, and provide a framework to continue testing and developing new methods.

The diverse applications of MALDI MSI are explored in this thesis with an emphasis on the sample preparation procedure and method development for small molecule analysis for a range of samples. The two main themes that have been focussed on are the pharmaceutical and metabolomic applications of this state of the art technique. MALDI MSI has been evaluated as a technique for the detection and imaging of antiasthmatic compounds in lung tissue. Four compounds were assessed initially with conventional MALDI MS experiments, followed by both direct and indirect tissue imaging experiments. Pharmaceutical tablet formulations have also been assessed using MALDI MSI to map the active component throughout the excipients contained within the tablet providing information that is critical to the manufacturing process such as the homogeneity of the active pharmaceutical ingredient (API) throughout the tablet. MALDI MSI has been applied to the relatively new addition to the 'omics sciences, metabolomics. A non-targeted metabolomics approach has been used to study both plant and animal tissue in an attempt to gain a greater understanding of the complex biological processes that occur within both types of tissue. Wheat grain was used as the model system to conduct the experiments and evaluate the application of both UV MALDI MS and IR LDI MS for plant metabolomics. These techniques provided complementary information to published literature, however the novel aspect of this study was the incorporation of imaging experiments for UV MALDI MS; this allowed the metabolites to be visualised in the wheat grain section. MALDI MSI was also used to explore the differences between mice with chronic relapsing experimental autoimmune encephalomyelitis; the animal model of multiple sclerosis alongside healthy controls. Spinal cord samples were analysed and the main difference was tentatively attributed to choline levels.

Fundamental level understandings on the processing behaviours of materials in granular and powder form is of high interest to number of engineering industries for example, mining, mineral, pharmaceutical, geotechnical and for advanced material processing applications. Handling and processing of pharmaceutical powders through confined geometries have very important role in pharmaceutical industry and many related powder process engineering sectors. Smooth flow of powders and granules mixtures from the feeding hopper to the compression chamber plays a very crucial role to achieve the integrity and quality of the final product. In this context, establishing clear understandings on the flow and compaction characteristics of particulates is vital. The mechanical behaviour of particulate materials such as powders and grains are different from the conventional states of matter. Depending on the loading levels and geometrical conditions, often they display combined features of solid, liquid and gaseous states. Though an extensive amount of studies are reported in the existing literatures on their mechanical response to loading, there are still a number of challenges to address: (i) Sensing stress distribution in particulate systems is not yet established especially when the size of the particulates are less than a millimetre (ii) Understanding is lacking on whether the stress distribution in initial static filling would influence the dynamic flow trajectories of the particulates when they are allowed to flow from the static state (iii) Micromechanical behaviour of particulates under low levels of external loading is still lacking and (iv) Interaction characteristics of stress and velocity distributions in particulate systems as a function of grain-scale properties and geometrical arrangements are still lacking. The present thesis addresses all of these important challenges in a systematic manner. The research is primarily based on the application of sensing stresses and displacements in particulates using advanced photo stress analysis tomography (PSAT), qualitative velocimetry using colour coding technique (CCT) and quantitative digital particle image velocimetry (DPIV). The required grain-scale properties are characterised comprehensively using a number of standard experimental methods. Where possible, experimental results on the stress and velocity distribution for particulate systems are compared with simulations using discrete element method (DEM) and analytical equations respectively, though the primary focus is on the experimental approaches. A number of outcomes from this research shed new lights and provide fundamental level understandings on the micromechanical properties of particulate systems with relevance to pharmaceutical granules processes.

Image classification is an active topic of computer vision research. This topic deals with the learning of patterns in order to allow efficient classification of visual information. However, most research efforts have focused on 2D image classification. In recent years, advances of 3D imaging enabled the development of applications and provided new research directions. In this thesis, we present methodologies and techniques for image classification using 3D image data. We conducted our research focusing on the attributes and limitations of depth information regarding possible uses. This research led us to the development of depth feature extraction methodologies that contribute to the representation of images thus enhancing the recognition efficiency. We proposed a new classification algorithm that adapts to the need of image representations by implementing a scale-based decision that exploits discriminant parts of representations. Learning from the design of image representation methods, we introduced our own which describes each image by its depicting content providing more discriminative image representation. We also propose a dictionary learning method that exploits the relation of training features by assessing the similarity of features originating from similar context regions. Finally, we present our research on deep learning algorithms combined with data and techniques used in 3D imaging. Our novel methods provide state-of-the-art results, thus contributing to the research of 3D image classification.

Engineered nanoparticles, such as super paramagnetic iron oxide nanoparticles (SPIONs) offer significant benefits for the development of various diagnostic and therapeutic strategies. Limitations of existing imaging methodologies in the study of NPs, such as the effects of fluorescent labelling and diffraction limited resolution, and the advantages that visualization of spatial localization can offer in studies, increases the demand for new and optimized imaging routines. Reflectance Confocal Microscopy (RCM) methods were optimized and Reflectance Structured Illumination Microscopy (R-SIM) was introduced, offering a two fold increase in resolution - particularly advantageous for NP quantification and localization studies. Analysis routines were developed to enable the automated quantification of NP presence within cells via the different methodologies. Correlative procedures were also established for imaging the same sample with different reflectance methods and TEM, maximizing the information attainable from a single sample and allowing comparisons between the techniques for specific applications. These aforementioned optimized techniques were then applied to the determination of NP uptake and trafficking in cancer cell lines, and, in combination with siRNA, to ascertain proteins that are involved in the uptake process. Studies were also performed to model the degradative process of SPIONs within cellular compartments. This thesis thus provided several important tools for the future assessment of the efficacy and safety of NPs for clinical use, enabling quantitative analysis of uptake route, sub-cellular localization and NP intracellular fate.

A crescente tendência de utilização de materiais austeníticos soldados e cladeados em componentes críticos em alguns setores industriais, como nas indústrias de óleo&gás e nuclear, leva a um aumento na demanda sobre ensaios não-destrutivos confiáveis na avaliação de sua integridade estrutural. Dentre os métodos utilizados na inspeção de soldas cladeados austeníticos estão os métodos de ultrassom por phased array, que são normalmente utilizados na detecção e localização de defeitos. No entanto, componentes com esse tipo de microestrutura são difíceis de inspecionar por phased array devido a anisotropia e inomogeneidade causadas pela microestrutura de grãos grosseiros que costumam levar ao aumento do nível de ruído, ao deslocamento de indicações e ao surgimento de indicações falsas. Sendo assim, a seleção de um método de phased array apropriado precisa levar em conta a habilidade do método em superar os problemas causados pela anisotropia e inomogeneidade. Esta tese apresenta dois métodos de imagem por phased array ultrassônico não-convencionais pensados como formas de ajudar na determinação da integridade de componentes onde soldas e cladeados austeníticos estão presentes. Ambos os métodos tem como base o método de foco total (TFM), sendo que o primeiro é uma extensão do método de leis de atraso adaptativas chamado Método de Foco Total de Atraso Adaptativo (ADTFM) e o segundo método usa fatores de coerência associado à imagens de TFM. A partir dos métodos de imagem aplicados é possível aumentar significativamente a qualidade das imagens por ultrassom em comparação com as imagens padrão obtidas por TFM, especialmente quando foi possível utilizar ambos os métodos combinados.
The increasing trend to use austenitic welded and cladded materials in critical components employed in some industrial sectors, such as the oil&gas and nuclear industries, leads to an increasing demand for their non-destructive assessment by reliable non-destructive methods. Among the methods used to access the integrity of austenitic welds and claddings are the Ultrasonic Phased Array methods, which are usually used to detect the presence and determine the position of defects. However, austenitic welds and claddings are challenging to inspect with Phased Array methods due to the anisotropy and inhomogeneity caused by their coarse grain microstructure, which is capable of increasing noise levels, misplace indications and create false indications. Therefore, the selection of an appropriate phased array method needs to take into account the method’s ability to overcome the impairment caused by anisotropy and inhomogeneity. This thesis presents two non-conventional methods based on ultrasonic phased array imaging techniques designed to assist the structural integrity assessment of components where austenitic welds and clads are present. Both proposed methods are based on the Total Focusing Method (TFM); the first approach is an expansion of the adaptive delay laws concept named Adaptive Delay Total Focusing Method (ADTFM), while the second method uses the coherence weights combined with the TFM images. From the imaging methods applied it was possible to significantly increase the quality of the ultrasonic images in comparison with the standard TFM, primarily when it was possible to combine both approaches.

Diffusion-weighted imaging of small animals at high field strengths is a challenging prospect due to its extreme sensitivity to motion. Periodically rotated overlapping parallel lines with enhanced reconstruction (PROPELLER) was introduced at 9.4T as an imaging method that is robust to motion and distortion. Proton density (PD)-weighted and T2-weighted PROPELLER data were generally superior to that acquired with single-shot, Cartesian and echo planar imaging-based methods in terms of signal-to-noise ratio (SNR), contrast-to-noise ratio and resistance to artifacts. Simulations and experiments revealed that PROPELLER image quality was dependent on the field strength and echo times specified. In particular, PD-weighted imaging at high field led to artifacts that reduced image contrast. In PROPELLER, data are acquired in progressively rotated blades in k-space and combined on a Cartesian grid. PROPELLER with echo truncation at low spatial frequencies (PETALS) was conceived as a post-processing method that improved contrast by reducing the overlap of k-space data from different blades with different echo times. Where the addition of diffusion weighting gradients typically leads to catastrophic motion artifacts in multi-shot sequences, diffusion-weighted PROPELLER enabled the acquisition of high quality, motion-robust data. Applications in the healthy mouse brain and abdomen at 9.4T and in stroke patients at 3T are presented. PROPELLER increases the minimum scan time by approximately 50%. Consequently, methods were explored to reduce the acquisition time. Two k-space undersampling regimes were investigated by examining image fidelity as a function of degree of undersampling. Undersampling by acquiring fewer k-space blades was shown to be more robust to motion and artifacts than undersampling by expanding the distance between successive phase encoding steps. To improve the consistency of undersampled data, the non-uniform fast Fourier transform was employed. It was found that acceleration factors of up to two could be used with minimal visual impact on image fidelity. To reduce the number of scans required for isotropic diffusion weighting, the use of rotating diffusion gradients was investigated, exploiting the rotational symmetry of the PROPELLER acquisition. Fixing the diffusion weighting direction to the individual rotating blades yielded geometry and anisotropy-dependent diffusion measurements. However, alternating the orientations of diffusion weighting with successive blades led to more accurate measurements of the apparent diffusion coefficient while halving the overall acquisition time. Optimized strategies are proposed for the use of PROPELLER in rapid high resolution imaging at high field strength.

Quantitative imaging in oncology aims at developing imaging biomarkers for diagnosis and prediction of cancer aggressiveness and therapy response before any morphological change become visible. This Thesis exploits Computed Tomography perfusion (CTp) and multiparametric Magnetic Resonance Imaging (mpMRI) for investigating diverse cancer features on different organs. I developed a voxel-based image analysis methodology in CTp and extended its use to mpMRI, for performing precise and accurate analyses at single-voxel level. This is expected to improve reproducibility of measurements and cancer mechanisms’ comprehension and clinical interpretability. CTp has not entered the clinical routine yet, although its usefulness in the monitoring of cancer angiogenesis, due to different perfusion computing methods yielding unreproducible results. Instead, machine learning applications in mpMRI, useful to detect imaging features representative of cancer heterogeneity, are mostly limited to clinical research, because of results’ variability and difficult interpretability, which make clinicians not confident in clinical applications. In hepatic CTp, I investigated whether, and under what conditions, two widely adopted perfusion methods, Maximum Slope (MS) and Deconvolution (DV), could yield reproducible parameters. To this end, I developed signal processing methods to model the first pass kinetics and remove any numerical cause hampering the reproducibility. In mpMRI, I proposed a new approach to extract local first-order features, aiming at preserving spatial reference and making their interpretation easier. In CTp, I found out the cause of MS and DV non-reproducibility: MS and DV represent two different states of the system. Transport delays invalidate MS assumptions and, by correcting MS formulation, I have obtained the voxel-based equivalence of the two methods. In mpMRI, the developed predictive models allowed (i) detecting rectal cancers responding to neoadjuvant chemoradiation showing, at pre-therapy, sparse coarse subregions with altered density, and (ii) predicting clinically significant prostate cancers stemming from the disproportion between high- and low- diffusivity gland components.

L’imagerie hyperspectrale est désormais considérée comme un outil analytique à part entière dans l’industrie pharmaceutique, aussi bien au cours du développement pour assurer la qualité d’un produit que pour résoudre des problématiques de production après la mise sur le marché du médicament. Dans ces travaux, la microscopie Raman est utilisée pour étudier la distribution en principes actifs et excipients au sein d’une forme pharmaceutique solide, en se focalisant tout particulièrement sur l’identification d’un composé faiblement dosé. Ce dernier est défini comme étant un produit ayant de faibles contributions spatiale et spectrale, signifiant qu’il est distribué dans quelques pixels de l’image avec une information spectrale peu présente dans un spectre de mélange. Alors que la plupart des algorithmes chimiométriques se basent sur la décomposition de moments statistiques, nécessitant une variation suffisante entre les échantillons (les pixels d’une image), les limites de ces outils pour résoudre ce cas spécifique sont rapidement atteintes.La première partie de la thèse met en évidence les difficultés de détection d’un composé faiblement dosé en utilisant l’analyse en composantes indépendantes et la résolution multivariée de courbes. Des méthodologies de travail sont proposées pour contourner ces limitations. Pour les deux techniques, les étapes de réduction de dimensions apparaissent comme des paramètres critiques de la méthode. La seconde partie de la thèse se focalise sur l’espace des signaux pour déterminer des cartes d’absence/présence de constituants ou pour détecter des constituants dans une formulation inconnue, en se basant sur des espaces spectraux portant une information relative aux constituants de la formulation. Les techniques proposées sont parfaitement adaptées à la détection d’un composé faiblement dosé et ces méthodes pourraient être adaptées à d’autres techniques de mesure ou d’autres domaines d’application
Hyperspectral imaging is now considered as a powerful analytical tool in the pharmaceutical environment, both during development to ensure the drug product quality and to solve production issues on commercialized products.In this thesis, Raman microscopy is used to study the distribution of actives and excipients in a pharmaceutical drug product, by especially focusing on the identification of a low dose compound. This latter product is defined as a compound which has low spatial and spectra contributions, meaning that it is scattered in a few pixels of the image and that its spectral response is mixed with the other compounds of the formulation. While most chemometric tools are based on the decomposition of statistical moments (requiring sufficient variations between samples or image pixels), some limitations have been rapidly reached. The first part of this thesis highlights the difficulty to detect a low dose compound in a product by using independent component analysis or multivariate curve resolution. Different methodologies are proposed to circumvent these limitations. For both techniques, reduction of dimensions and filtering steps appears as critical parameters of the method. The second part of the thesis focusses on the signal space to determine absence/presence compound maps or to detect the compounds in an unknown formulation. The proposed methods are only based on the spectral space of each formulation compound. There are perfectly suitable to a low dose compound and should be well-adapted to other analytical techniques or to other environments

Since the turn of the new millennium the UK construction industry has witnessed the naissance of a new paradigm known as ICT-enabled collaborative working. Advocates of this new approach believe that the adoption of ICTs will lead to better project information exchange, communications, integrated processes and therefore more efficient collaborative working. It is expected that ICT-enabled collaborative working will not only overcome the industry's many inherent problems, such as its adversarial and fragmented nature, but that it will also facilitate benefits for all involved. This supposition is based upon the recognition that its exploitation has proven successful at transforming other industries. The EngD thesis pertains to a four-year study on two methodologies currently applied under the concept of ICT-enabled collaborative working, 'Collaborative Prototyping' (CP) and the use of 'Construction Project Extranets' (CPE). The research project utilised literature reviews, case studies, project observations, active involvement, surveys, interviews and workshops to develop expertise and knowledge within the subject area. It shows that a large disparity exists between the use of a shared 3D model and the use of CPEs to promote collaborative working. Conversely, whilst CPEs have become the de facto ICT-enabled approach to seeking improvements in project efficiency (due to their potential to enhance communications, integration and collaboration) they are not being utilised effectively. This was attributed to the use of inadequate procedures that fail to provide proper consideration of all the necessary issues to ensure successful implementation, application and management of the CPE. As a result, project teams are failing to ascertain the full potential benefits offered by such collaboration tools. The research demonstrates that to overcome this, project teams require the development of an industry best-fit framework that defines proficient procedures. It highlights the main factors for inclusion within a protocol. Furthermore, it provides a simple form to promote greater awareness of the key factors that impinge on the successful application of CPE. The thesis concludes by outlining a number of recommendations for consideration by the industry, along with requirements for future work.

L'échocardiographie est la modalité d'imagerie la plus utilisée pour évaluer la morphologie et la fonction cardiaque. Il s'agit d'un outil non invasif pour le diagnostic et l'évaluation des maladies cardiaques et il permet en outre de surveiller la réponse au traitement. Cependant, la quantification des événements cardiaques rapides demeure un défi avec la cadence d’imagerie actuellement réalisable, en particulier dans des applications telles que l'échocardiographie d'effort. De plus, cette limitation devient plus prononcée en imagerie 3D conventionnelle focalisée en raison du temps nécessaire pour insonifier et acquérir un volume complet. Le fait que l’on puisse actuellement atteindre au mieux ~20 volumes par seconde est l'une des raisons qui limitent son utilisation courante dans la pratique clinique. Des améliorations dans ce domaine permettraient d'exploiter l'important potentiel de l'imagerie 3D pour la quantification complète de la déformation cardiaque.Dans ce contexte, l'objectif de cette thèse était de développer des méthodes à haute cadence d'images et de tester leur performance dans des conditions réalistes visant la prise de décision pour une transition vers la pratique clinique. Pour atteindre cet objectif, des expériences in vitro et in vivo ont été menées en utilisant l'imagerie 2D et 3D. Notre première contribution a été une comparaison 2D entre deux modalités à haute cadence d'images en termes de qualité d'image et de performance d'estimation de mouvement. Motivés par nos résultats 2D mais surtout par le défi d'implémenter le MLT dans la pratique, nous avons étendu cette approche en 3D. Nous avons étudié la faisabilité de la MLT 3D dans des conditions statiques et dynamiques. Enfin, comme l’évaluation de nouvelles approches dans des conditions physiologiques de flux complexes constitue un pas en avant vers la transition clinique, notre troisième contribution a consisté à valider des modalités 2D et 3D à haut cadence d'images sur un fantôme du vortex
Echocardiography is the most widely used imaging modality for assessing cardiac morphology and function. It does provide a non-invasive tool in diagnosis and assessment of heart diseases and it allows, in addition, monitoring the response to the treatment. However, quantifying fast cardiac events remains a challenge when using the current achievable frame rate, especially in applications such as stress-echocardiography. Moreover, this limitation becomes more pronounced in 3D conventional focused imaging due to the time needed to insonify and acquire a full volume. The fact that only ~20 volumes per second can currently be achieved is one of the reasons restricting its common usage in clinical practice. Improvements in this field would allow exploiting the important potential of 3D imaging in providing a full quantification of cardiac deformation.In this context, the aim of this thesis was to develop high frame rate methods and to test their performance in realistic conditions aiming decision making towards clinical translation. To achieve this objective, both in vitro and in vivo experiments were conducted using 2D and 3D imaging. Our first contribution was a 2D comparison between two high frame rate modalities in terms of image quality and motion estimation performance. Motivated by our 2D results but especially by the challenge of implementing MLT in practice, we extended this approach to 3D. We studied the feasibility of 3D MLT in both static and dynamic conditions. Finally, as testing novel approaches in physiological complex flows conditions is a step forward towards clinical translation, our third contribution was to validate 2D and 3D high frame rate modalities on a ring vortex phantom

2009/2010
Neuroimaging plays a critically important role in neuroscience research and management of neurological and mental disorders. Modern neuroimaging techniques rely on various “source” signals that change across different spatial and temporal scales in accompany with neuronal activity. Nowadays, several types of noninvasive neuroimaging modalities are available based on biophysical signals related to either brain electrophysiology or hemodynamics/metabolism. In this dissertation, advanced model-based neuroimaging methods for the estimation of cortical brain activity from combined high-resolution electroencephalography (EEG), multimodal Magnetic Resonance Imaging (MRI) and functional Magnetic Resonance Imaging (fMRI) data are presented. The present dissertation begins with a review of the current state-of-the-art in the major neuroimaging techniques. Particular attention has been devoted to EEG modelling since such signals propagate (virtually) instantaneously from the activated neuronal tissues via volume conduction to the recording sites on/above the scalp surface. The instantaneous nature of EEG indicates an intrinsically high temporal resolution and precision, which make it well suited for studying brain functions on the neuronal time scale. The collective nature suggests low spatial resolution and specificity, which impede mapping brain functions in great regional details. However, this is regardless of recent advancements in electromagnetic source imaging, which has led to great strides in improving the EEG/MEG spatial resolution to a centimetre scale or even smaller. These methods entail: 1) modeling the brain electrical activity; 2) modeling the head volume conduction process so as to link the modeled electrical activity to EEG; and 3) reconstructing the brain electrical activity from recorded EEG data. For this aim, a subject's multicompartment head model (scalp, skull, CSF, brain cortex, white matter) is constructed from either individual magnetic resonance images or approximated geometry models. We compared different spherical and realistic head modelling techniques in estimating EEG forward solutions from current dipole sources distributed on a standard cortical space reconstructed from Montreal Neurological Institute (MNI) MRI data. Computer simulations are presented for three different four-shell head models, two with realistic geometry, either surface-based (BEM) or volume-based (FDM), and the corresponding sensor-fitted spherical-shaped model. Point Spread Function (PSF) and Lead Field (LF) cross-correlation analyses were performed for 26 symmetric dipole sources to quantitatively assess models’ accuracy in EEG source reconstruction. Both statistical and imaging analysis point to the realistic geometry as a relevant factor of improvement, particularly important when considering sources placed in the temporal or in the occipital cortex. In these situations, using a realistic head model will allow a better spatial discrimination of neural sources when compared to the spherical model. Moreover a brief overview of Diffusion Weighted Imaging and Diffusion Tensor Imaging is also given, as their application in modelling refinement is increasing the accuracy and the complexity of the brain models. Both fMRI and EEG represent brain activity in terms of a reliable anatomical localization and a detailed temporal evolution of neural signals. Simultaneous EEG-fMRI recordings offer the possibility to greatly enrich the significance and the interpretation of the single modality results because the same neural processes are observed from the same brain at the same time. Nonetheless, the different physical nature of the measured signals by the two techniques renders the coupling not always straightforward, especially in cognitive experiments where spatially localized and distributed effects coexist and evolve temporally at different temporal scales. The purpose of the last chapter is to illustrate the combination of simultaneously recorded EEG and fMRI signals exploiting the principles of EEG distributed source modelling. We define a common source space for fMRI and EEG signal projection and novel framework for the spatial and temporal comparative analysis. We use simultaneous EEG-fMRI in order to explore the relationship between the envelope of spontaneous neuronal oscillations in the alpha frequency band (8-13 Hz) recorded with EEG during eyes closed rest and spontaneous fluctuations of the fMRI BOLD signal. We showed on a single-subject analysis how the presented approach, when combined to an accurate realistic head modelling, is able to localize the alpha rhythmic modulation in the occipital visual area and the parieto-occipital sulcus. This finding is in line with recent studies, asserting that, within these regions, time-frequency analysis and phase-synchronization analysis indicated increased alpha power and alpha-band phase-synchronization in eyes-closed condition versus eyes-open condition. Given the lack in the scientific literature of group-analysis experimental studies performed with realistic modelling approach in this field, this topic will be further investigated in future work.
XXII Ciclo
1980

In this thesis, the detection of global proteomics alteration and changes in neuropeptide distribution caused by neuropathic pain in rat spinal cord tissue was the main focus. Neuropathic pain (NP) is a major clinical syndrome caused by disease or dysfunction of the nervous system and often mediated by neuronal networks in the spinal cord. The estimated prevalence of NP is 6-8% in general population. Only in the United States, the indirect cost associated with chronic pain has been estimated to 100 billion dollars each year and NP substantially contributes to this cost. So far, the underlying mechanisms of NP are not well understood. Proteomics techniques are commonly used in biology system studies, due to its high throughput, capability of unbiased analysis and sensitivity. It builds up a bridge to link genes, peptides, proteins, and the disease. Two proteomic/peptidomic approaches were developed, evaluated and discussed in this thesis. Both of them were further applied in the studies of neuropathic pain. First approach is a quantitative proteomic approach using liquid chromatography combined with Fourier transform mass spectrometry (LC-FTMS), which is developed for quantitative analysis of proteins originated from small central nervous system (CNS) samples. This approach was successfully applied in the study of the rat spinal cord tissue proteome in a neuropathic pain model. Another approach is using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) for the visualization of the distribution of neuropeptides in rat spinal cord, which in the future will be applied in investigating the ongoing signal transmission under neuropathic pain conditions. Results provided by these two methods are of high importance for the general understanding of the underlying pathophysiological mechanisms and potential identification of new targets for novel treatment of neuropathic pain.

Biofilms are microbial communities developing at the interface between two phases, usually solidliquid, where the micro-organisms are nested in a self-secreted polymer matrix. The biofilm mode of growth is predominant in nature (for e.g. the slimy matter forming on rocks at river bottoms, the viscous deposit in water pipes or even dental plaque) and confers a suitable environment for the development of the micro-organisms. This is particularly the case for porous media which provide favourable substrates given their significant surface to volume ratio. The multi-physical framework of biofilms in porous media is highly complex where the mechanical, chemical and biological aspects interacting at different scales are poorly understood and very partially controlled. An example is the feedback mechanism between flow, spatial distribution of the micro-organisms and the transport of nutrient (by diffusion and advection). Biofilms developing in porous media are a key process of many engineering applications, for example biofilters, soil bio-remediation, CO2 storage and medical issues like infections. Progress in this domain is substantially hindered by the limitations of experimental techniques in metrology and imaging in opaques structures. The main objective of this thesis is to propose robust and reproducible experimental methodologies for the investigation of biofilms in porous media. An experimental workbench under controlled physical and biological conditions is proposed along with a validated 3D imaging protocol based on X-ray micro-tomography (XR MT) using a novel contrast agent (barium sulfate and agarose gel) to quantify the spatial distribution of the biofilm. At first, the XR MT-based methodology is compared to a commonly used techniques for biofilm observation: one or multiple photon excitation fluorescence microscopy, here two-photon laser scanning microscopy (TPLSM). This comparison is performed on Pseudomonas Aeruginosa biofilms grown in transparent glass capillaries which allows for the use of both imaging modalities. Then, the study of uncertainty associated to different metrics namely volume, 3D surface area and thickness, is achieved via an imaging phantom and three different segmentation algorithms. The quantitative analysis show that the protocol enables a visualisation of the biofilm with an uncertainty of approximately 17% which is comparable to TPLSM (14%). The reproducibility and robustness of the XR MT-based methodology is demonstrated. The last step of this work is the achievement of a novel bioreactor elaborated by additive manufacturing and controlled by a high-performance micro-fluidic system. The experimental workbench that we have designed enables to monitor in real-time the evolution of transport properties (effective permeability), O2 concentrations and biofilm detachment by spectrophotometry, all under controlled hydrodynamical conditions. Our methodology allows to investigate the influence of biophysical parameters on the colonisation of the porous medium, for example, the influence of flow rate or nutrient concentration on the temporal development of the biofilm. In conclusion, the thesis work proposes a robust and reproducible experimental methodology for the controlled growth and 3D imaging of biofilms in porous media; while providing versatility in the control of the substrate’s micro-architecture as well as on the flow and biochemical culture conditions. To our knowledge, the scientific approach followed, along with the experimental apparatus, form the most complete methodology, at this time, for the study of biofilms in porous media.

Les hominidés sont dotés d'un cerveau volumineux et énergivore, ce qui nécessite des réseaux vasculaires efficaces dans la tête pour assurer le soutien métabolique. Le système veineux diploïque qui traverse les os du crâne est important pour la circulation sanguine dans la tête, car il facilite les échanges sanguins entre les réseaux vasculaires extracrâniens et intracrâniens. Compte tenu de son importance physiologique, le système veineux diploïque pourrait avoir influencé l'évolution du cerveau. Cependant, son anatomie et son évolution restent méconnues chez la plupart des hominidae, rendant difficile la vérification de sa relation avec l'évolution cérébrale. De plus, les structures internes du crâne peuvent servir de signaux taxonomiques, il est donc pertinent d'estimer la signification taxonomique du système veineux diploïque en cartographiant ses caractéristiques dans chaque taxon.Cette étude a analysé les grands singes actuels, les humains modernes et les fossiles d'Homo du Pléistocène précoce, moyen et tardif. Un nouveau protocole a été développé pour identifier, reconstruire et analyser le système veineux diploïque à partir d'images Micro-CT, permettant une reconstruction non invasive des structures diploïques. Les canaux osseux diploïques ont été utilisés comme indicateurs des veines, et des analyses qualitatives ont décrit leur distribution, drainage et interactions spatiales avec les endocasts et l'épaisseur osseuse. Des méthodes quantitatives, incluant l'échelle d'intensité, l'analyse fractale et l'analyse du volume relatif, ont été employées pour évaluer leur complexité, taille et intensité générale. Cette étude présente le système veineux diploïque chez des spécimens fossiles de plusieurs espèces humaines, a découvert des structures non documentées chez les grands singes et a affiné et corrigé les descriptions classiques des humains actuels. Les résultats ont montré que le système veineux diploïque des grands singes est beaucoup moins développé que celui des hominines. Un système hautement développé n'était pas propre à Homo sapiens, mais est apparu chez les hominines dès le Pléistocène précoce et était largement partagé parmi les Homo. Les différences taxonomiques dans les modèles de drainage veineux diploïque entre hominidés apparaissent principalement dans la grande aile du sphénoïde, la région astérionnale, les sinus paranasaux et les foramina pariétaux. La taille du cerveau ne déterminait pas l'intensité du réseau veineux diploïque, car les espèces d'hominines à petit cerveau avaient aussi des réseaux veineux diploïques denses et larges, comparables à ceux des grands cerveaux. La forme du cerveau pouvait également affecter les vaisseaux diploïques, la compression des gyrus cérébraux correspondant aux zones minces du crâne, souvent contournées par les vaisseaux diploïques. Comme la forme du cerveau variait parmi les hominidés, elle contribuait aux différences taxonomiques dans la distribution des vaisseaux diploïques. De plus, les structures veineuses diploïques pouvaient faciliter les échanges thermiques, mais aucune preuve claire n'indique qu'elles jouent un rôle clé dans la thermorégulation cérébrale. Enfin, le système veineux diploïque pourrait avoir coévolué avec d'autres réseaux vasculaires et les sinus paranasaux. Ce travail est la première comparaison exhaustive des systèmes veineux diploïques chez les grands singes, les hominines fossiles et les humains actuels. Les résultats enrichissent notre connaissance du système veineux diploïque et cartographient sa trajectoire évolutive générale dans la lignée humaine. Cette étude éclaire aussi l'histoire évolutive des cerveaux humains, révélant l'interaction spatiale entre les vaisseaux, les os crâniens et les cerveaux. Le système veineux diploïque a des valeurs potentielles pour les futures discussions taxonomiques et phylogénétiques
Hominidae are featured with large and energy-consuming brains, which necessitates effective vascular networks providing metabolic support. The diploic venous system is important to cranial blood circulation, since it facilitates the exchanges between extra-and intra-cranial blood. Considering the physiological significance of the diploic venous system, it may have been involved in brain evolution. However, the anatomy and evolution of the diploic venous system remain unknown in most Hominidae members, hindering us from verifying its relationship with brain evolution. Moreover, as the cranial inner structures can potentially function as taxonomic signals, it is meaningful to estimate the taxonomic significance of the diploic venous system, of which the precondition is mapping the characteristics in each taxon.This study investigated extant great apes, extant humans, and Homo fossils from the Early, Middle, and Late Pleistocene. The diploic venous systems of most fossil specimens were unexplored in previous studies. Many among them are critical to understanding human evolution. This study developed a new protocol for identifying, reconstructing, and analyzing the diploic venous system. With Micro-CT images, this study non-invasively detected and digitally reconstructed the diploic structures. As vascular soft tissues were not preserved, the bony diploic channels were used as proxies of veins. Through qualitative inspections, this study described their distribution, drainage, and their spatial interactions with endocasts and bone thickness. Through quantitative methods, including the intensity scale, fractal analysis, and relative volume analysis, this study evaluated their degrees of complexity, sizes, and general intensity levels.This study presents the diploic venous system in extinct hominins, and it discovered non-documented structures in great apes and corrected the classic description of extant humans. The results showed that great apes had less developed diploic venous systems than hominins. A highly developed system was not an autapomorphy of Homo sapiens, instead, it emerged in hominins since the Early Pleistocene and was widely shared among Homo. The taxonomic differences in the diploic venous drainage patterns between hominids mainly appeared in the sphenoid greater wing, asterional region, paranasal sinuses, and parietal foramina. Brain size did not determine the intensity of diploic venous network, as small-brained hominin species also had dense and large diploic venous networks comparable to those of large-brained taxa. Brain shape could also affect diploic vessels, as the compression from pronounced brain gyri corresponded to the areas with evident thinning in cranium, which were usually bypassed by diploic vessels. As brain shape varied among hominids, it contributed to the taxonomic differences in diploic vessel distribution. Additionally, the diploic venous structures could facilitate heat exchange, but no clear evidence indicated that it had a key role in brain thermoregulation. Finally, the diploic venous system might have co-evolved with other vascular networks and paranasal sinuses.In conclusion, this work is the first comprehensive comparison of the diploic venous systems among great apes, fossil hominins, and extant humans. The findings contribute to our knowledge of the diploic venous system, preliminarily mapping its general evolutionary trajectory in human lineage. This study also sheds light on the evolutionary history of human brains, revealing the spatial interaction between vessels, cranial bones, and brains. The diploic venous system has potential application values in future taxonomic and phylogenetic discussions

This dissertation was divided into two parts: Part 1 A novel methodology of aminomethylation of alkenes and alkynes was studied. The methodology developed involved the use of TFA to promote iminium formation from tetramethyldiaminomethane, which in turn reacts with a nucleophilic double bond to deliver an amine. A very well-rounded scope was accomplished with examples that range from aliphatic, cyclic and aromatic alkenes. Functional group tolerance was also pursuit with the method showing a great tolerance towards commonly used groups such as halides and esters. A total number of 25 boc-protected amines were synthesized using alkenes with yields up 82 %. Part 2 The second part consisted on the development of a fluorescence probe using a fla-vylium ion for in vivo imaging. In order to develop a fluorescent system that could be used in in vivo imaging for detection cancer cells, the prepared flavilyum was coupled with the theraupeutical relevant Celastrol. A new flavilyum ion was synthesized via condensation of a 7-diethylaminophenol with acetophenone possessing an halogenated alkylic linker at the 4’ position. Different pathways were investigated in order to reach and optimize the synthesis of a C4 functionalized flavylium salt that was prepared in three steps. Attempts were performed to couple the prepared flavylium to Celastrol.

In frequency-domain photothermal radiometry (FD-PTR) a low-power intensity-modulated optical excitation generates thermal-wave field inside the sample and the subsequent infrared radiation from the sample is analyzed to detect material’s inhomogeneities. The non-contact nature of FD-PTR makes it very suitable for non-destructive evaluation of broad range of materials. Moreover, the methodology is based on intrinsic contrast of light absorption which can be used as a diagnostic tool for inspection of malignancy in biological tissues. Nevertheless, the bottom line is that the physics of heat diffusion allows for a highly damped and dispersive propagation of thermal-waves. As a result, the current FD-PTR modalities suffer from limited inspection depth and poor axial/depth resolution. The main objective of this thesis is to show that using alternative types of modulation schemes (such as linear frequency modulation and binary phase coding) and radar matched filter signal processing, one can obtain localized responses from inherently diffuse thermal wave fields. In this thesis, the photothermal responses of turbid, transparent, and opaque media to linear frequency modulated and binary phase coded excitations are analytically derived. Theoretical simulations suggest that matched-filtering in diffusion-wave field acts as constructive interferometry, localizing the energy of the long-duty excitation under a narrow peak and allowing one to construct depth resolved images. The developed technique is the diffusion equivalent of optical coherence tomography and is named thermal coherence tomography. It was found that the narrow-band binary phase coded matched filtering yields optimal depth resolution, while the broad-band linear frequency modulation can be used to quantify material properties through the multi-parameter fitting of the experimental data to the developed theory. Thermophotonic detection of early dental caries is discussed in detail as a potential diagnostic application of the proposed methodologies. The performance of the diagnostic system is verified through a controlled demineralization protocol as well as in teeth with natural caries.

Low-cost unmanned aerial vehicles (UAVs) utilizing push-broom hyperspectral scanners are poised to become a popular alternative to conventional remote sensing platforms such as manned aircraft and satellites. In order to employ this emerging technology in fields such as high-throughput phenotyping and precision agriculture, direct georeferencing of hyperspectral data using onboard integrated global navigation satellite systems (GNSS) and inertial navigation systems (INS) is required. Directly deriving the scanner position and orientation requires the spatial and rotational relationship between the coordinate systems of the GNSS/INS unit and hyperspectral scanner to be evaluated. The spatial offset (lever arm) between the scanner and GNSS/INS unit can be measured manually. However, the angular relationship (boresight angles) between the scanner and GNSS/INS coordinate systems, which is more critical for accurate generation of georeferenced products, is difficult to establish. This research presents three alternative calibration approaches to estimate the boresight angles relating hyperspectral push-broom scanner and GNSS/INS coordinate systems. For reliable/practical estimation of the boresight angles, the thesis starts with establishing the optimal/minimal flight and control/tie point configuration through a bias impact analysis starting from the point positioning equation. Then, an approximate calibration procedure utilizing tie points in overlapping scenes is presented after making some assumptions about the flight trajectory and topography of covered terrain. Next, two rigorous approaches are introduced – one using Ground Control Points (GCPs) and one using tie points. The approximate/rigorous approaches are based on enforcing the collinearity and coplanarity of the light rays connecting the perspective centers of the imaging scanner, object point, and the respective image points. To evaluate the accuracy of the proposed approaches, estimated boresight angles are used for ortho-rectification of six hyperspectral UAV datasets acquired over an agricultural field. Qualitative and quantitative evaluations of the results have shown significant improvement in the derived orthophotos to a level equivalent to the Ground Sampling Distance (GSD) of the used scanner (namely, 3-5 cm when flying at 60 m).

Num mundo VICA (volátil, incerto, complexo e ambíguo), é necessário que o ensino e a aprendizagem acompanhem o ritmo acelerado imposto pela sociedade. O acesso à informação para criação de conhecimento é cada vez mais rápido e está relacionado com a evolução tecnológica. Esta evolução é acompanhada pelos alunos que vêem nas metodologias mais inovadoras um fator de motivação adicional para a sua aprendizagem. Neste contexto, a introdução de metodologias ativas facilita a integração dos alunos no processo de ensino e aprendizagem. Neste relatório pretende-se desenvolver a utilização da imagem associada às aplicações móveis (apps) como forma de desenvolvimento de competências geográficas (científicas), técnicas e tecnológicas, numa turma de 8ª ano. Os alunos têm a oportunidade de construir o seu conhecimento, em oposição à exposição de conteúdos feita pelo professor e associado à construção do saber memorista, de uma forma inovadora e com recurso a novas tecnologias. Pretende-se analisar a eficácia e a eficiência da utilização destas metodologias, com recurso à imagem nas apps, no desenvolvimento das competências expressas nas Aprendizagens Essenciais (AE) de Geografia, no desenvolvimento das diferentes áreas de competências do Perfil dos Alunos à Saída da Escolaridade Obrigatória (PASEO) e integrando, sempre que possível, a Estratégia Nacional de Educação para a Cidadania (ENEC). Para tal, foram utilizadas, entre outras, as apps Thinglink e Actionbound, para potenciar a aprendizagem dos alunos e torná-la mais dinâmica, interativa e inovadora. A partir da análise de resultados e do feedback dos alunos foi possível aferir que a imagem aliada às apps constitui uma mais-valia para o processo de ensino do professor e de aprendizagem dos alunos. Estas metodologias apresentam-se como um fator de motivação para a aprendizagem e contribuem para desenvolvimento das AE de Geografia e das competências inscritas no PASEO, tornando os alunos mais despertos e capazes para o mundo que os rodeia, do ponto de vista de uma cidadania territorial mais ativa inscrita na ENEC, pelo que não devem ser descuradas da prática letiva dos professores.
In a VUCA (volatile, uncertain, complex, and ambiguous) world, it is necessary that teaching, and learning keep up with the fast pace imposed by society. Access to information for the creation of knowledge is increasingly faster and is related to technological evolution. This evolution is accompanied by the students who see in the most innovative methodologies an additional motivation factor for their learning. In this context, the introduction of active methodologies facilitates the integration of students in the teaching and learning process. In this report we intend to develop the use of image associated to mobile applications (apps) to develop geographic (scientific), technical and technological skills, in an 8th grade class. Students have the opportunity to build their knowledge, as opposed to the exposure of content made by the teacher and associated with the construction of memoristic knowledge, in an innovative way and using new technologies. We intend to analyse the effectiveness and efficiency of the use of these methodologies, using the image in the apps, in the development of the competences expressed in the Essential Learning (EA) of Geography, in the development of the different areas of competences of the Profile of Students Leaving Compulsory Education (PASEO) and integrating, whenever possible, the National Strategy of Education for Citizenship (ENEC). To this end, the apps Thinglink and Actionbound were used, among others, to enhance students' learning and make it more dynamic, interactive, and innovative. Based on the analysis of the results and the students' feedback, it was possible to conclude that the image combined with the apps is an added value to the teacher's teaching and students' learning process. These methodologies are a motivating factor for learning and contribute to the development of Geography AE and the skills included in PASEO, making students more aware and able to the world around them, from the point of view of a more active territorial citizenship included in ENEC, so they should not be neglected in the teaching practice of teachers.