Submitted by PPG Engenharia e Tecnologia de Materiais (engenharia.pg.materiais@pucrs.br) on 2018-05-17T18:27:28Z No. of bitstreams: 1 Tese - Elisa Magno Nunes de Oliveira.pdf: 7318970 bytes, checksum: a50f399a4afea119cd760c93cd71ff72 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-05-23T14:59:14Z (GMT) No. of bitstreams: 1 Tese - Elisa Magno Nunes de Oliveira.pdf: 7318970 bytes, checksum: a50f399a4afea119cd760c93cd71ff72 (MD5) / Made available in DSpace on 2018-05-23T15:10:26Z (GMT). No. of bitstreams: 1 Tese - Elisa Magno Nunes de Oliveira.pdf: 7318970 bytes, checksum: a50f399a4afea119cd760c93cd71ff72 (MD5) Previous issue date: 2018-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The present study focuses on the development of nanoparticles with an iron oxide magnetic core, with different biocompatible coatings, and on a comparative study of their toxicities. Uncoated and dextran-, chitosan-, polyethylene glycol- and silica-coated nanoparticles were synthesized. The addition of optical markers of the benzo-thiazoles class was also accomplished. The physico-chemical properties of the nano-particles were characterized, including their magnetic, optical and contrast properties in nuclear magnetic resonance imaging (relaxivities). In the particular case of nanopar-ticles functionalized with 6-OH-BTA-1 molecules, the affinity to the beta-amyloid pep-tide was also investigated. A second step was to evaluate the toxicological effects of these nanoparticles in vitro (using in VERO cells), and in vivo with zebrafish as an animal model. The size of the nanoparticles with the coatings ranged from 13 to 30 nm. Their crystalline structure was consistent with the ferrite spinel. The nanoparticles, independent of the coating, did not present residual magnetization and hysteresis, in-dicating superparamagnetic behaviour. For most nanoparticles, the r2 transverse re-laxivity values ranged from 76-64 mM-1.s-1, exceptfor uncoated and chitosan-coated nanoparticles, which present higher values, possibly due to the aggregation. The val-ues of r1 were similar for all nanoparticles (12.6 to 18 mM-1.s-1), with the exception of silica-coated nanoparticles (r1=2.1 mM-1.s-1). The r2/r1 ratios were between 4 and 17, typical of commercially available negative contrast-agents. The nanoparticles function-alized with benzothiazoles showed fluorescence with a Stokes shift of the emission peak of ~ 197 nm. The interaction of the beta-amyloid peptide with the 6-OH-BTA-1 molecule analyzed by fluorescence suppression is characterized by a static mecha-nism and Stern-Volmer constants of 1.53x104 mM-1 for the monomeric form, 1.40x104 mM -1 for oligomers) and 1.33x104 mM -1 for amyloid plaques.The in vitro toxicity assays indicated acceptable values of cell viability for iron concentration up to 2 mmol.L-1. The nanoparticles with the carboxysilane and polyethylene glycol showed higher biocom-patibility and silica-coated nanoparticles had the highest cytotoxicity. The in vivo as-says did not show significant changes in survival and hatchability rates, except for doses greater than 2 mmol.L-1 in the case of the chitosan-coated nanoparticles. The percentages of animals with anatomical alterations were similar between the treated and control groups. In the locomotion and exploration tests, only chitosan- and silica-coated nanoparticles induced significant changes. / O presente trabalho aborda o desenvolvimento de nanopart?culas compostas por um n?cleo magn?tico de ?xido de ferro com diferentes revestimentos biocompat?-veis e estudo comparativo de suas toxicidades. Foram sintetizadas nanopart?culas de ?xido de ferro sem revestimento e com revestimentos de dextrana, quitosana, polieti-lenoglicol e s?lica, e com a adi??o de marcadores ?pticos da classe dos benzotiaz?is. As propriedades f?sico-qu?micas das nanopart?culas foram caracterizadas, incluindo as suas propriedades magn?ticas, ?pticas e de contraste em imagens por resson?ncia magn?tica nuclear (relaxividades), bem como a afinidade ao pept?deo beta-amiloide, no caso particular de funcionaliza??o com a mol?cula 6-OH-BTA-1. Em uma segunda etapa, foram avaliados os efeitos toxicol?gicos dessas nanopart?culas em ensaios bi-ol?gicos in vitro em c?lulas VERO, e in vivo tendo como animal modelo o peixe zebra. O tamanho das nanopart?culas com revestimentos variou entre 13 a 30 nm, e estrutura cristalina coerente com o espin?lio de ferrita. As nanopart?culas n?o apresentaram magnetiza??o residual e histerese, indicando superparamagnetismo, independente do revestimento. Para a maioria das nanopart?culas, os valores de relaxividade transver-sal r2 variaram de 76-64 mM-1.s-1, com exce??o das nanopart?culas sem revestimento e de quitosana, os quais foram mais elevados, possivelmente devido ao efeito de agrega??o. Os valores de r1 foram semelhantes para todas as nanopart?culas (12,6 a 18 mM-1.s-1), com exce??o das nanopart?culas de s?lica (r1 = 2,1 mM-1.s-1). As raz?es r2/r1 foram entre 4 e 17, valores t?picos de agentes de contraste negativos comercial-mente dispon?veis. As nanopart?culas funcionalizadas com os benzotiaz?is mantive-ram sua fluoresc?ncia com deslocamentos de Stokes na ordem de 197 nm para o pico de emiss?o. As an?lises da intera??o do pept?deo beta-amiloide com a mol?cula 6-OH-BTA-1, mostraram valores de constante de Stern-Volmer para supress?o de fluo-resc?ncia de 1,53x104 mM-1 (mon?mero), 1,40x104 mM-1 (olig?mero) e 1,33x104 mM-1 (placa), indicando quenching por um mecanismo est?tico. O pept?deo na forma mo-nom?rica demonstrou maior facilidade de acesso ?s mol?culas de 6-OH-BTA-1. Os resultados dos ensaios in vitro indicaram valores aceit?veis de viabilidade celular para concentra??o de ferro inferior a 2 mmol.L-1. As nanopart?culas com o carboxisilano e polietilenoglicol demostraram maior biocompatibilidade e as nanopart?culas de s?lica tiveram a maior citotoxicidade. Os resultados dos ensaios in vivo n?o mostraram alte-ra??es significativas na taxa de sobreviv?ncia e de eclos?o do corium, exceto para as doses maiores que 2 mmol.L-1 das nanopart?culas revestidas com quitosana. Os per-centuais de animais com altera??es anat?micas foram similares entre os grupos tra-tados e de controle. Nos ensaios de locomo??o e explora??o, apenas as nanopart?cu-las de quitosana e de s?lica induziram altera??es adversas significativas.

Cartilage tissue engineering has been emerging as a promising therapeutic approach, where engineered constructs or scaffolds are used as temporary supports to promote regeneration of functional cartilage tissue. Hybrid constructs fabricated from cells, hydrogels, and solid polymeric materials show the most potential for their enhanced biological and mechanical properties. However, fabrication of customized hybrid constructs with impregnated cells is still in its infancy and many issues related to their structural integrity and the cell functions need to be addressed by research. Meanwhile, it is noticed that nowadays monitoring the success of tissue engineered constructs must rely on animal models, which have to be sacrificed for subsequent examination based on histological techniques. This becomes a critical issue as tissue engineering advances from animal to human studies, thus raising a great need for non-invasive assessments of engineered constructs in situ. To address the aforementioned issues, this research is aimed to (1) develop novel fabrication processes to fabricate hybrid constructs incorporating living cells (hereafter referred as “construct biofabrication”) for cartilage tissue regeneration and (2) develop non-invasive monitoring methods based on synchrotron X-ray imaging techniques for examining cartilage tissue constructs in situ. Based on three-dimensional (3D) printing techniques, novel biofabrication processes were developed to create constructs from synthetic polycaprolactone (PCL) polymer framework and cell-impregnated alginate hydrogel, so as to provide both structural and biological properties as desired in cartilage tissue engineering. To ensure the structural integrity of the constructs, the influence of both PCL polymer and alginate was examined, thus forming a basis to prepare materials for subsequent construct biofabrication. To ensure the biological properties, three types of cells, i.e., two primary cell populations from embryonic chick sternum and an established chondrocyte cell line of ATDC5 were chosen to be incorporated in the construct biofabrication. The biological performance of the cells in the construct were examined along with the influence of the polymer melting temperature on them. The promising results of cell viability and proliferation as well as cartilage matrix production demonstrate that the developed processes are appropriate for fabricating hybrid constructs for cartilage tissue engineering. To develop non-invasive in situ assessment methods for cartilage and other soft tissue engineering applications, synchrotron phase-based X-ray imaging techniques of diffraction enhanced imaging (DEI), analyzer based imaging (ABI), and inline phase contrast imaging (PCI) were investigated, respectively, with samples prepared from pig knees implanted with low density scaffolds. The results from the computed-tomography (CT)-DEI, CT-ABI, and extended-distance CT-PCI showed the scaffold implanted in pig knee cartilage in situ with structural properties more clearly than conventional PCI and clinical MRI, thus providing information and means for tracking the success of scaffolds in tissue repair and remodeling. To optimize the methods for live animal and eventually for human patients, strategies with the aim to reduce the radiation dose during the imaging process were developed by reducing the number of CT projections, region of imaging, and imaging resolution. The results of the developed strategies illustrate that effective dose for CT-DEI, CT-ABI, and extended-distance CT-PCI could be reduced to 0.3-10 mSv, comparable to the dose for clinical X-ray scans, without compromising the image quality. Taken together, synchrotron X-ray imaging techniques were illustrated promising for developing non-invasive monitoring methods for examining cartilage tissue constructs in live animals and eventually in human patients.

Les nanocristaux à structure non-centrosymétrique présentent des propriétés optiques non linéaires prometteuses pour une utilisation en tant que marqueurs optiques en imagerie biomédicale, avec un intérêt significatif en termes de suivi sur de longues durées et de profondeur de pénétration dans les tissus biologiques. Le développement de ces marqueurs nécessite la détermination de leurs efficacités optiques non linéaires afin de pouvoir sélectionner les nanocristaux les plus prometteurs. Pour cela, la technique de diffusion Hyper-Rayleigh a été adaptée à la caractérisation de suspensions de nanoparticules (BaTiO3, KNbO3, KTP, LiNbO3 et ZnO, BiFeO3) pour lesquelles l’influence de paramètres comme la taille, la concentration ou l’état d’agrégation a été analysée et discutée. Les nanocristaux de BiFeO3 possèdent une efficacité optique non linéaire largement supérieure aux autres particules, démontrant leur potentiel pour la réalisation de nano-sondes optiques particulièrement performantes. Des mesures résolues en polarisation ont également été mises en œuvre pour déterminer les coefficients optiques non linéaires indépendants des particules étudiées. Dans ce cadre, une étude a permis de mettre en évidence l’influence de la forme des nanocristaux sur cette réponse. / Non-centrosymetric nanocrystals show promising nonlinear optical properties for being used as optical labels in bio-imaging applications, with significant interest for observations of long duration and for penetration depth into biological tissues. The development of such biomarkers requires the determination of their nonlinear optical properties to select the best potential markers. In this thesis, Hyper-Rayleigh Scattering (HRS) technique is used to determine nonlinear efficiencies of several nanocystals (BaTiO3, KNbO3, KTP, LiNbO3, ZnO and BiFeO3). These ensemble measurements have been performed on nanocrystals suspensions, for which the influence of parameters such as size, concentration and aggregation state was discussed. BiFeO3nanocrystals offer the best nonlinear optical efficiency compared to other particles, showing their potential as efficient optical biomarkers. Polarisation-resolved measurements have also been performed to retrieve individual coefficients of the nonlinear tensor of the investigated materials and influent parameters such as nanocrystals shape have been identified.

L’imagerie de filtrage en polarisation est une technique populaire largement utilisée en optique pour le biomédical pour le sondage des tissus superficiels, pour le sondage de volumes plus profonds, mais aussi pour l’examen sélectif de volumes sub-surfaciques. Du fait de l’effet de ’mémoire de polarisation’ de la lumière polarisée, l’imagerie de filtrage en polarisation elliptique est sensible á des épaisseurs de tissus différentes, depuis la surface, accessible avec la polarisation linéaire, jusqu’á une épaisseur critique accessible par la polarisation circulaire. Nous nous concentrons sur des méthodes utilisant des combinaisons de polarisations elliptiques afin de sélectionner la portion de lumière ayant maintenu son état de polarisation et éliminer le fond pour un meilleur contraste avec de plus une information sur la profondeur. Avec ce type de filtrage, il est possible d’accéder á des profondeurs de tissus biologiques bien définies selon l’ellipticité de polarisation. De plus, ces travaux ont permis d’étendre la méthode á la spectroscopie pour quantifier la concentration en chromophores á une profondeur spécifique. Les méthodes développées ont été validées in vivo á l’aide d’expériences réalisées sur des anomalies de la peau et aussi sur le cortex exposé d’un rat anesthésié. Enfin, une étude préliminaire a été réalisée pour examiner la possibilité d’étendre la méthode á l’imagerie de 'speckle'. Des tests préliminaires réalises sur fantômes montrent l’influence de l’ellipticité de polarisation sur la formation et le comportement du speckle, ce qui offre la possibilité d’accéder á des informations sur le flux sanguin á des profondeurs spécifiques dans les tissus. / Polarization gating imaging is a popular and widely used imaging technique in biomedical optics to sense tissues, deeper volumes, and also selectively probe sub-superficial volumes. Due to the ‘polarization memory’ effect of polarized light, elliptical polarization gating allows access to tissue layers between those of accessible by linear or circular polarizations. As opposed to the conventional linearly polarized illumination, we focus on polarization gating methods that combine the use of elliptically polarized light to select polarization-maintaining photons and eliminate the background while providing superior contrast and depth information. With gating, it has also become possible to access user-defined depths (dependent on optical properties) in biological tissues with the use of images at different ellipticities. Furthermore, this investigation allowed the application of polarization gating in spectroscopy to selectively quantify the concentration of tissue chromophores at user-desired depths. Polarization gating methods have been validated and demonstrated with in vivo experiments on abnormalities of human skin (nevus, burn scar) and also on the exposed cortex of an anaesthetized rat. Finally, as a first step towards the use of coherent illumination, adding the concept of polarimetry to laser-speckle imaging was demonstrated. Preliminary tests on phantoms (solid and liquid) suggested evidence of the influence of polarization ellipticity on the formation and behaviour of speckles, which could pave the way for more insight in the study of blood flow in tissues.

La technique de spectroscopie basée sur la diffusion Raman Stokes spontanée est un procédé standard employé dans de nombreux domaines allant de la thermodynamique à la médecine, en passant par la science des matériaux. À la faveur d'un échange d'énergie inélastique, elle permet de déterminer les fréquences des vibrations moléculaires présentes dans un objet. On peut ainsi remonter à l'identification des molécules et ainsi caractériser l'objet d'étude sans utiliser de marqueur spécifique. Cette méthode est néanmoins affligée de défauts. Outre la présence d'un signal de fluorescence qui peut submerger la réponse Raman, le désavantage majeur est le long temps d'exposition que requière cette technique. Dans le cas d'étude d'échantillon biologique, cela proscris son usage pour des mesures de microspectroscopie : la cartographie spectrale d'objet microscopique. Afin de pallier ce problème, de nouvelles techniques ont été développées. C'est le cas de la spectroscopie employant la diffusion Raman anti-Stokes Cohérente (ou CARS pour Coherent Anti-Stokes Raman Scattering). Du fait de sa cohérence et de sa directivité le signal anti-Stokes affiche une intensité 10^5 to 10^6 fois plus importante que dans le cas de la diffusion Raman spontanée, ce qui permet alors d'abaisser le temps d'exposition à un niveau tolérable pour les objets biologiques lors d'une mesure de microspectroscopie. De plus, le caractère anti-Stokes du signal l'épargne de la contribution de la fluorescence. Pourtant, un défaut majeur limite encore l'utilisation de cette technique : le bruit de fond non résonant. Ce phénomène peut diminuer, voir noyer la contribution résonante qui porte l'information. Cette thèse a permis le développement de techniques CARS autorisant une réduction du bruit de fond non résonant. Pour ce faire un dispositif de spectroscopie CARS multiplex (M-CARS) en configuration copropagative a été construit. Ses capacités sont illustrées par des mesures spectrales d'échantillons minéral, végétal et biologique. À partir de ce système, il a été établi une méthode innovante permettant de discriminer le signal résonant du bruit non résonant en utilisant un champ électrique continu. Il est aussi démontré la mise en place d'un procédé qui a permis de mener la première mesure de microspectroscopie M-CARS en configuration contrapropagative sur un échantillon biologique. Cette configuration limite la collecte du signal à l'objet d'étude, empêchant ainsi l'acquisition du signal résonant et non résonant issu du solvant, principal responsable du bruit de fond non résonant lors d'une mesure CARS en configuration copropagative. / The spectroscopy technique based on spontanée Raman Stokes scattering is a standard process used in many fields spanning from thermodynamic and medicine, to materials sciences. An inelastic energy exchange permits to determinate the frequency of the molecular vibrations in an object. One can identify the molecules and thus, can characterize the object of study in a label-free way. Nevertheless, this method is afflicted with faults. Beside the presence of fluorecence that can drown the Raman answer, the main drawback is the long exposition time required. In the case of biological sample, this can prohibit the use of spontaneous Raman scattering for microspectroscopy measures: the spectral mapping of microscopic objects. To avoid this problem, new techniques have been developed. It is the case of Coherent anti-Stokes Raman scattering (CARS) spectroscopy. Due to its coherence and its directivity, the anti-Stokes signal has an intensity 105 to 106 times greater than the spontaneous Raman scattering one. The exposition time is then reduced to a tolerable level for biological objects during microspectroscopy measures. Moreover, the anti-Stokes characteristic of the signal prevents the fluorescence contribution. However, a major fault still limits the use of this technique: the nonresonant background. This phenomenon can diminish, even overwhelm the resonant contribution carrying the information. This thesis permitted the development of CARS approaches that allow the reduction of the nonresonant background. To do so, a multiplex CARS (M-CARS) spectroscopy apparatus in a forward configuration has been built. Its abilities are illustrated with spectral measures of mineral, vegetal and biological samples. Based on this system, it has been established an innovative method that can discriminate the resonant signal from the nonresonant one thanks to a static electric field. It has been also been demonstrated the development of a process that has allowed the first M-CARS microspectroscopy measure of a biological sample in a contrapropagative configuration. This setup limits the collect of the signal to the object of study, avoiding the acquisition of the resonant and resonant signals coming from the solvent, responsible for the major part of non resonant background during a CARS measure in a forward configuration.

No description available.

<p>Magnetic resonance spectroscopy (MRS) is a non-invasive diagnostic technique that provides unique information about the biochemical composition of the human body. By excluding the overwhelming signals from water and fat, clinically relevant biomarkers such as lactate, N-acetyl aspartate, choline, creatine, glutamate/glutamine (Glx), gamma-aminobutyric acid (GABA), glutathione, and myoinositol can be reliably quantified. MRS has diverse applications in investigating the metabolic window of a wide range of biochemical processes. </p> <p>Here, we have utilized MRS to better understand chemical changes associated with neurological disorders and treatment response. We have investigated neurometabolic imbalances in brain regions related to post-traumatic stress disorder (PTSD). MRS was applied to better understand the neurobiological processes of hyperbaric oxygen therapy in military veterans with clinically diagnosed traumatic brain injury and/or PTSD.</p>

<p>  </p> <p>In this study, we present advancements in smart contact lenses, highlighting their potential as minimally or non-invasive diagnostic and drug delivery platforms. The eyes, rich in physiological and diagnostic data, make contact lens sensors an effective tool for disease diagnosis. These sensors, particularly smart contact lenses, can measure various biomolecules like glucose, urea, ascorbate, and electrolytes (Na+, K+, Cl-, HCO3-) in ocular fluids, along with physical biomarkers such as movement of the eye, intraocular pressure (IOP) and ocular surface temperature (OST).</p> <p>The study explores the use of continuous, non-invasive contact lens sensors in clinical or point-of-care settings. Although promising, their practical application is hindered by the developmental stage of the field. This thesis addresses these challenges by examining the integration of contact lens sensors, covering their working principle, fabrication, sensitivity, and readout mechanisms, with a focus on monitoring glaucoma and eye health conditions like dry eye syndrome and inflammation.</p> <p>Our design adapts these sensors to fit various corneal curvatures and thicknesses. The lenses can visually indicate IOP through microfluidic channels' mechanical deformation under ambulatory conditions. We also introduce a colorimetric hydrogel tear fluid sensor that detects pH, electrolytes, and ocular surface temperature, indicating conditions like dry eye disease and inflammation.</p> <p>The evaluation of these contact lens sensors includes in vivo/vitro biocompatibility, ex vivo functionality studies, and in vivo safety assessments. Our comprehensive analysis aims to enhance the practicality and effectiveness of smart contact lenses in ophthalmic diagnostics and therapeutics.</p>

<p>  </p> <p>Heart disease is the leading cause of death for individuals with Duchenne muscular dystrophy (DMD). DMD is a devastating and progressive neuromuscular disease with no known cure. This X-linked genetic disorder affects nearly 1 in 5000 boys and manifests as debilitating muscle weakness and progressive cardiomyopathy (CM). While CM in some individuals with DMD progresses rapidly and fatally in their teenage years, others can live relatively symptom-free into their thirties or forties. Early identification and treatment can improve quality and length of life, but currently, there are no standard imaging biomarkers that can detect early onset or rapidly progressing DMD CM. Addressing this gap, we describe here a novel cardiac image analysis paradigm using 4D cardiac magnetic resonance imaging (CMR) to map left-ventricular kinematics comprehensively in DMD CM. The primary goal of this dissertation work is to introduce novel imaging biomarkers and computational methods to enable earlier diagnosis and precise prognosis for cardiac function in DMD. Central to this goal, we identified myocardial strain biomarkers that predict the early onset and rapid progression of cardiac disease in vulnerable patients. These findings bridge clinical gaps and pave the way for multi-center studies to characterize DMD CM progression and assessment of individual patient risk profiles for improved treatment and outcomes in DMD.</p>

Computed tomography has been widely used in medical diagnosis to generate accurate images of the body's internal organs. However, cancer risk is associated with high X-ray dose CT scans, limiting its applicability in medical diagnosis and telemedicine applications. CT scans acquired at low X-ray dose generate low-quality images with noise and streaking artifacts. Therefore we develop a deep learning-based CT image enhancement algorithm for improving the quality of low-dose CT images. Our algorithm uses a convolution neural network called DenseNet and Deconvolution network (DDnet) to remove noise and artifacts from the input image. To evaluate its advantages in medical diagnosis, we use DDnet to enhance chest CT scans of COVID-19 patients. We show that image enhancement can improve the accuracy of COVID-19 diagnosis (~5% improvement), using a framework consisting of AI-based tools. For training and inference of the image enhancement AI model, we use heterogeneous computing platform for accelerating the execution and decreasing the turnaround time. Specifically, we use multiple GPUs in distributed setup to exploit batch-level parallelism during training. We achieve approximately 7x speedup with 8 GPUs running in parallel compared to training DDnet on a single GPU. For inference, we implement DDnet using OpenCL and evaluate its performance on multi-core CPU, many-core GPU, and FPGA. Our OpenCL implementation is at least 2x faster than analogous PyTorch implementation on each platform and achieves comparable performance between CPU and FPGA, while FPGA operated at a much lower frequency. / Master of Science / Computed tomography has been widely used in the medical diagnosis of diseases, such as cancer/tumor, viral pneumonia, and more recently, COVID-19. However, the risk of cancer associated with X-ray dose in CT scans limits the use of computed tomography in biomedical imaging. Therefore we develop a deep learning-based image enhancement algorithm that can be used with low X-ray dose computed tomography scanners to generate high-quality CT images. The algorithm uses a state-of-the-art convolution neural network for increased performance and computational efficiency. Further, we use image enhancement algorithm to develop a framework of AI-based tools to improve the accuracy of COVID-19 diagnosis. We test and validate the framework with clinical COVID-19 data. Our framework applies to the diagnosis of COVID-19 and its variants, and other diseases that can be diagnosed via computed tomography. We utilize high-performance computing techniques to reduce the execution time of training and testing AI models in our framework. We also evaluate the efficacy of training and inference of the neural network on heterogeneous computing platforms, including multi-core CPU, many-core GPU, and field-programmable gate arrays (FPGA), in terms of speed and power consumption.