Indiana University-Purdue University Indianapolis (IUPUI) / Giant axonal neuropathy (GAN) is a rare genetic disease that causes progressive damage to the nervous system. Neurons in GAN patients develop an abnormal organization of cytoskeletal proteins called intermediate filaments (IFs), which normally provide strength and support for the overall cell structure. The irregular IF structure in GAN patient neurons leads to a progressive loss of motor skills in children and subsequent death in adolescence. GAN is caused by reduced levels of the gigaxonin (Giga) protein. Giga functions to control the degradation of other cellular proteins, and the loss of Giga in GAN cells results in significantly elevated levels of the galectin-1 (Gal-1) protein. Gal-1 stabilizes the active form of the Ras signaling protein, which functions as a molecular switch to regulate the phosphorylation and subsequent organization of IFs. The connection between these pathways led us to propose that Giga regulates IF phosphorylation and structure by modulating Ras signaling through the degradation of Gal-1. Using GAN patient cells, we demonstrated that restoring Giga reduced Gal-1 protein levels, decreased IF phosphorylation, and reestablished normal IF organization. Similar effects of reduced IF phosphorylation and improved IF structure were also obtained in GAN cells by directly decreasing the protein levels of either Gal-1, or downstream Ras signaling proteins. Taken together, these results demonstrate that the loss of Giga induces Gal-1 mediated activation of Ras signaling, thereby leading to the increased IF phosphorylation and abnormal IF structure observed in GAN cells. Identification of aberrant Ras signaling is significant because it is the first to specify a mechanism by which the loss of Giga leads to the development of GAN and provides targets for novel drug therapies for the treatment of this currently immedicable genetic disease.

[pt] A informação sobre o posicionamento de objetos estranhos no interior do corpo humano é essencial para a sua eficiente remoção cirúrgica. Entretanto, os métodos convencionalmente utilizados não fornecem informação suficiente sobre a localização do objeto metálico para garantia de sucesso cirúrgico. No presente trabalho foi desenvolvido um sistema automatizado para mapear a densidade de fluxo magnético estático produzido por corpos ferromagnéticos posicionados em variados graus de liberdade 3D, utilizando um sensor de baixo custo, baseado no fenômeno da magnetoimpedância gigante (GMI - Giant Magnetoimpedance), que detecta somente campos magnéticos variantes no tempo. Assim, as medições automatizadas foram realizadas com a amostra em movimento a uma velocidade constante. Por meio de modelagens computacionais do campo magnético gerado foi possível reproduzir o comportamento da densidade de fluxo magnético gerado por uma fonte de campo magnético como a agulha retilínea utilizada nas medições in vitro. O software considerou as características do sensor GMI utilizado e a condição de medição com a fonte magnética em movimento. Os resultados da simulação foram validados por meio de comparações com os resultados experimentais, possibilitando a solução do problema direto com a caracterização da configuração espacial da densidade de fluxo magnético para variados posicionamentos da fonte magnética em relação ao sensor magnético GMI. Com a validação dos resultados simulados, os mesmos podem ser empregados no desenvolvimento de procedimento para solução do problema inverso de imageamentos clínicos utilizando o sensor GMI de baixo custo, limitado a medições magnéticas variantes no tempo, realizados para detecção e posicionamento de corpos estranhos que geram campos magnéticos estáticos. / [en] Information about the positioning of foreign objects inside the human body is essential for its efficient surgical removal. However, the methods conventionally used do not provide sufficient information on the location of the metallic object to guarantee surgical success. In the present work, an automated system was developed to map the static magnetic flux density produced by ferromagnetic bodies positioned in varying degrees of 3D freedom, using a low-cost sensor based on the giant magnetoimpedance phenomenon (GMI - Giant Magnetoimpedance), which detects only time-varying magnetic fields. Thus, automated measurements were performed with the sample moving at a constant speed. Through computational modeling of the generated magnetic field, it was possible to reproduce the behavior of the magnetic flux density generated by a magnetic field source, such as the straight needle used in in vitro measurements. The software considered the GMI sensor s characteristics and the measurement condition with the magnetic source in motion. The simulation results were validated through comparisons with the experimental results, enabling the solution of the direct problem with the characterization of the spatial configuration of the magnetic flux density for various magnetic source positions in relation to the GMI magnetic sensor. With the validation of the simulated results, they can be used in the development of a procedure to solve the inverse problem of clinical imaging using the low-cost GMI sensor, limited to time-varying magnetic measurements, performed for the detection and positioning of foreign bodies that generate static magnetic fields.

[pt] Esta Tese tem por objetivo o desenvolvimento de um sistema destinado à medição de campo magnético com alta sensibilidade e resolução, baseado nas características de fase da impedância em sensores que apresentam o efeito GMI, e a otimização das características de desempenho por meio do uso de configurações em malha fechada. A metodologia empregada inicia com a avaliação experimental das características de fase da impedância de amostras de diferente estrutura e composição química, em função do campo magnético externo, a fim de selecionar aquelas com alta sensibilidade, baixa histerese e maior homogeneidade. Na sequência, são realizadas avaliações teórico-computacionais dos transdutores magnéticos em malha aberta e fechada (magnetômetro e gradiômetro). Da mesma forma, as principais características dos circuitos e controladores software dos transdutores desenvolvidos são detalhadas ao longo do texto. Por sua vez, as principais figuras de mérito dos protótipos desenvolvidos são detalhadamente analisadas, tais como: sensibilidade, linearidade, resposta em frequência, densidade espectral de ruído e resolução. As caracterizações e ensaios experimentais realizados evidenciaram o grande potencial dos transdutores GMI em malha fechada para a atenuação da interferência 1/f, aprimoramento da linearidade e ampliação da faixa de operação. O magnetômetro GMI em malha fechada apresentou sensibilidade em torno de 75,8 mV/microteslas, fundo de escala maior que mais ou menos 40 microteslas, banda de passagem de 45 Hz e resolução na banda de passagem de 27,74 nT. Por outro lado, o gradiômetro GMI em malha fechada desenvolvido apresentou sensibilidade em torno de 102 mV/microteslas, fundo de escala maior que mais ou menos 40 microteslas, banda de passagem de 30 Hz e resolução na banda de passagem de 28,41 nT. / [en] This Thesis aims to develop a system for magnetic field measurement with high sensitivity and resolution, based on the impedance phase characteristics of sensors that have the GMI effect and the performance characteristics optimization through closed-loop configurations. The methodology starts with the experimental evaluation of the phase characteristics of the impedance in samples of different chemical composition and structure as a function of the external magnetic field in order to select those with high sensitivity, low hysteresis, and higher homogeneity. Subsequently, theoretical-computational assessments of magnetic transducers in open and closed-loop (magnetometer and gradiometer) are carried out. Likewise, the main characteristics of the circuits and software controllers of the developed transducers are detailed throughout the text. In turn, the main figures of merit of the developed prototypes are analyzed in detail, such as sensitivity, linearity, frequency response, noise spectral density, and resolution. The characterizations and experimental tests carried out showed the great potential of GMI transducers in a closed-loop configuration for attenuation of interference 1/f, improving linearity and expanding the operating range. The closed-loop GMI magnetometer showed a sensitivity of around 75.8 mV/microteslas, a full-scale range greater than plus or minus 40 microteslas, a pass band of 45 Hz and a resolution in the pass band of 27.74 nT. On the other hand, the GMI closed-loop gradiometer developed had a sensitivity of around 102 mV/microteslas, a full scale greater than plus or minus 40 microteslas, a passband of 30 Hz and a resolution in the pass band of 28.41 nT.

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This work presents the successful development of micro-origami sensorics by 3D self-assembling and reconfiguring in space integrated thin-film magnetic sensors, which rely on anisotropic (AMR) and giant magnetoresistance (GMR). Stimuli responsive polymeric materials able to reshape into mesoscale 3D “Swiss-roll” and polygonal architectures accomplish a strain driven parallel spatial realignment of magnetic sensors from the in-plane state. High performance 3D magnetic vector angular encoders demonstrates the successful realization of complex sensor configurations. The proposed concepts rely on parallel wafer scale processes, which allow for a monolithic fabrication of 3D sensor arrays and pave the way towards active sensory matrix circuits. As a proof of this concept, magneto-resistive Wheatstone bridge sensors are developed and integrated in the self-assembling platform at predefined rigid regions and integrated with an active matrix backplane circuit. This circuit, based on a-IGZO TFT technology, is specially designed for the operation with the Wheatstone bridge differential sensors and optimized to be compatible with the micro-origami self-folding technology. Such an active sensory matrix system with integrated 3D self-assembled pixels is called Integrated Micro-Origami Sensors or in short IMOS. IMOS is capable for static and dynamic mapping of magnetic fields enabling spatiotemporal mapping of artificial magnetic hair arrays embedded in an elastic skin layer. The presented results offer a fresh strategy for large area integration of microscale 3D electronic devices with various vector functionalities in active matrix circuits, which are of great interest in novel robotics, bioelectronics and diagnostic systems.

[pt] Elementos sensores baseados no efeito GMI são uma nova família de sensores magnéticos que apresentam grande quando submetidos a campos magnéticos externos. Estes sensores têm sido utilizados no desenvolvimento de magnetômetros de alta sensibilidade, destinados à medição de campos ultra fracos. Por sua vez, a sensibilidade de um magnetômetro está diretamente associada à sensibilidade de seus elementos sensores. No caso de amostras GMI, esta sensibilidade é otimizada buscando-se a maximização da variação do módulo ou da fase da impedância em função do campo magnético ao qual a amostra é submetida. Estudos recentes mostram que transdutores GMI baseados na variação de fase podem exibir sensibilidades até 100 vezes superiores às apresentadas por transdutores baseados na leitura do módulo do elemento sensor, o que fez com que os trabalhos conduzidos nesta dissertação focassem na maximização da sensibilidade de fase, a qual é majoritariamente dependente de quatro fatores: o comprimento da amostra, o campo magnético externo, o nível DC e a frequência da corrente de excitação. Contudo, a busca do conjunto de parâmetros que otimiza a sensibilidade das amostras é geralmente empírica e muito demorada. Esta dissertação propõe uma nova técnica de otimização da sensibilidade, baseada no uso de algoritmos genéticos evoluindo em hardware, a fim de se definir qual o conjunto de parâmetros responsável pela maximização da sensibilidade das amostras. Ressalta-se que, além dos parâmetros de otimização anteriormente explicitados, também foram realizados testes considerando a amplitude da corrente de excitação como uma variável livre, sendo que os resultados obtidos são apresentados e discutidos. Foi implementada uma bancada de testes e desenvolvida uma interface gráfica em LabVIEW, para monitorar e medir o comportamento da impedância de amostras GMI em função de variações nos parâmetros de interesse. Por sua vez, implementou-se um módulo de otimização em Matlab, baseado em algoritmos genéticos, responsável por encontrar a combinação de parâmetros que maximiza a sensibilidade dos sensores GMI avaliados (ponto ótimo de operação). / [en] GMI sensors are a new family of magnetic sensors that exhibit a huge variation of their impedance when subjected to external magnetic fields. These sensors have been used in the development of high sensitivity magnetometers, aimed at measuring ultra-weak magnetic fields. In turn, the sensitivity of a magnetometer is directly associated with the sensitivity of their sensor elements. In the case of GMI samples, this sensitivity is optimized by maximizing the variation of the impedance magnitude or phase as a function of the magnetic field applied to the sample. Recent studies show that GMI transducers based on phase variation can exhibit sensitivities up to 100 times higher than those presented by transducers based on impedance magnitude readings. The results obtained in these previous studies made the current work focusing on the maximization of phase sensitivity, which is mostly dependent on four factors: sample length, external magnetic field, DC level and frequency of the excitation current. However, the search for the set of parameters that optimizes the sensitivity of the samples is usually empirical and very time consuming. Thus, this dissertation proposes a new optimization technique, based on the use of genetic algorithms evolving on hardware, in order to define which set of parameters is responsible for maximizing the sensitivity of the samples. It should be noted that in addition to the optimization parameters previously described, this work also carried out tests considering the amplitude of the excitation current as a free variable, and the results obtained are presented and discussed. A test bench was implemented and a graphical interface was developed in LabVIEW to monitor and measure the impedance behavior of GMI samples due to variations in the parameters of interest. In turn, a Matlab optimization module based on genetic algorithms was implemented, in order to find the combination of parameters that maximizes the impedance phase sensitivity of the evaluated GMI sensors (optimum operating point).

Made available in DSpace on 2014-12-17T15:32:29Z (GMT). No. of bitstreams: 1 KaruzaMAP_Tese.pdf: 829792 bytes, checksum: 60cd145c0f060b54f7dfbb5463648200 (MD5) Previous issue date: 2010-02-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The Giant Cell Lesions, both the Central Giant Cells Lesions (CGCL) as the Peripheral Giant Cells Lesions (PGCL), correspond to a group of oral lesions that are histologically similar entities; however they show a variable clinical behaviour. The purpose of this study was to compare the immunohistochemical expression of bone resorption factors RANK (Receptor Activator of Nuclear Factor kappa B), RANKL (Receptor Activator of Nuclear Factor kappa B Ligand) and OPG (Osteoprotegerin) between CGCL and PGCL. Additionally, these bone resorption factors were examined in terms of aggressiveness of these lesions. The sample consisted of 61 cases, 30 cases of PGCL and 31 CGCL (16 non-aggressive and 15 aggressive). The analysis was performed by quantification of mononuclear cells (MO) and giant multinucleated cells (CG) immunopositive to anti-RANK, anti-RANKL and anti-OPG antibodies in 10 fields. Moreover, according to the proportion between the amount of cells positive for RANKL and OPG, the cases were categorized into: RANKL>OPG, OPG>RANKL e RANKL=OPG. CGCL showed a higher amount of MO (p=0.002) and total cells (p=0.003) both positives to RANKL compared with the PGCL. Additionally, the CGCL revealed a significant association with the ratio of RANKL>OPG (p=0.001). Analysis of the bone resorption factors revealed no significant differences between aggressive and non-aggressive CGCL (p>0.05). It was observed a positive correlation between the markers themselves, and a negative correlation between lesion size and quantity of OPG positive MO cells (p=0,004) and total cells (p=0,009). Through these results, we suggest that the greatest CGCL resorptive potential compared to the PGCL, may have occurred to the high expression of RANKL. Furthermore differences in the biological behavior of aggressive and non-aggressive CGCL appear to be related to the expression of these bone resorption factors / As Les?es de C?lulas Gigantes, tanto as Les?es Centrais (LCCG) quanto as Perif?ricas (LPCG), correspondem a um grupo de les?es orais que apresentam-se histologicamente semelhantes, por?m demonstram um comportamento cl?nico vari?vel. O prop?sito deste estudo foi comparar a express?o imuno-histoqu?mica dos fatores de reabsor??o ?ssea RANK (Receptor Ativador do Fator Nuclear kappa B), RANKL (Ligante do Receptor Ativador do Fator Nuclear kappa B) e OPG (Osteoprotegerina) entre LCCG e LPCG. Adicionalmente, esses fatores foram analisados nas LCCG quanto ? agressividade destas. A amostra consistiu de 61 casos, sendo 30 casos de LPCG e 31 de LCCG (16 n?o-agressivos e 15 agressivos). A an?lise foi realizada por meio da quantifica??o das c?lulas mononucleadas (MO) e c?lulas gigantes multinucleadas (CG) imunopositivas aos anticorpos anti-RANK, anti-RANKL e anti-OPG, em 10 campos. Al?m disso, de acordo com a propor??o entre quantidade total de c?lulas positivas para RANKL e para OPG, os casos foram categorizados em: RANKL>OPG, OPG>RANKL e RANKL=OPG. As LCCG apresentaram maior quantidade de MO (p=0,002) e c?lulas totais (p=0,003) positivas para RANKL, em compara??o com as LPCG. As LCCG ainda revelaram uma associa??o significativa com a propor??o de RANKL>OPG (p=0,001). A an?lise dos fatores de reabsor??o ?ssea n?o revelou diferen?as significativas entre LCCG agressivas e n?o-agressivas (p>0,05). Foi constatada correla??o positiva dos marcadores entre si, bem como uma correla??o negativa entre o tamanho das les?es e a quantidade de MO (p=0,004) e c?lulas totais (p=0,009) positivas para OPG. Diante desses resultados, concluise que o maior potencial reabsortivo das LCCG frente ?s LPCG pode ser decorrente da elevada express?o de RANKL. Al?m disso, as diferen?as nos comportamentos biol?gicos de LCCG agressivas e n?o-agressivas parecem n?o estar relacionadas com a express?o desses fatores de reabsor??o ?ssea.

Les lipides « switch » - bascules - appartiennent à la famille des matériaux sensibles à un stimulus. Quand ces lipides bascules sont incorporés aux nanoparticules lipidiques (LNP), ils permettent la délivrance contrôlée grâce à un changement de conformation activé par une baisse de pH. Des expériences précédentes avaient démontré que les LNP bascules ont transfecté le petits ARN interférents (siRNA) in vitro et in vivo, silençant la protéine fluorescente verte (GFP) et la protéine hépatique Facteur VII, respectivement. La double administration de micro ARN (miRNA) et d'agent anticancéreux melphalan a également été réalisée par les LNP bascule sur un modèle de rétinoblastome murin. Ces résultats prometteurs nous ont encouragé à élargir les applications de LNP bascules en tant que vecteur de siRNA. De plus, le mécanisme par lequel les LNP bascules induisent la déstabilisation de la membrane et la libération de matériaux encapsulé au milleu acide reste obscur. La compréhension de ce mécanisme est cruciale pour cerner les avantages et les limites des LNP bascules, pour proposer des futures applications et pour prévenir leur toxicité. Dans ce mémoire, nous avons comme objectif d’évaluer le potentiel des LNP bascules pour le traitement du cancer. Nous avons évalué les LNP bascules comme vecteur de livraison du siRNA ciblant l'une des protéines cancéreuses les plus spécifiques découvertes à ce jour, la survivine. En parallèle, nous avons étudié le comportement biophysique des membranes contenant des lipides bascules dans des vésicules de taille micromètrique. Dans la première étude, nous avons démontré que les LNP bascules ont permis le silençage de la survivine dans une gamme de lignées cellulaires cancéreuses (poumon, cervical, ovaire, sein, côlon, rétinoblastome). Dans les cellules du rétinoblastome humain (Y79), nous avons examiné plusieurs agents cytotoxiques utilisés en clinique quant à leur synergie avec le silençage de la survivine: melphalan, topotécan, téniposide et carboplatine. Le prétraitement avec les LNP chargées de siRNA-survivine a amélioré de manière synergique la cytotoxicité du carboplatine et du melphalan mais dans une moindre mesure celle du topotécan et du téniposide. Cet effet était spécifique aux cellules cancéreuses car les cellules saines (ARPE.19) n'exprimaient pas de survivine. L'inhibition de la survivine par silençage de siRNA s'est révélée plus spécifique et moins dommageable pour les cellules saines (ARPE.19) que le YM155, un inhibiteur moléculaire de la survivine. Dans la deuxième étude, nous avons observé par microscopie confocale que les lipides bascules induisaient rapidement le stress, la fission et une courbure positive dans les membranes des vésicules unilamellaires géantes lorsqu'elles étaient exposées à des conditions acides. La dynamique de la membrane a été confirmée par des expériences de diffusion dynamique de la lumière (DLS) et de fuite de calcéine. Ces phénomènes ont également été observés lorsque des lipides bascules ont été incorporés dans une membrane hybride polymère/lipide, fournissant des propriétés sensibles au pH aux vésicules hybrides. À notre connaissance, c'est la première fois qu'une vésicule hybride sensible au pH est reportée. Nos résultats corroborent l'applicabilité des LNP bascules en tant qu'agents de vectorisation des siRNA pour le traitement du cancer grâce au silençage de la survivine, en particulier comme adjuvant à la chimiothérapie. L'investigation biophysique a révélé que les lipides bascules agissent sur la fluidité de la membrane, en particulier à pH acide. Cette sélectivité en pH garantit leur biocompatibilité à pH neutre ainsi que la libération efficace et rapide de leur cargo à pH acide. La compatibilité avec les vésicules hybrides polymère/lipide ouvre de nouvelles applications au niveau de vésicules biomimétiques et l'administration de médicaments. / Cationic switchable lipids belong to the class of stimuli-responsive materials. When incorporated in lipid nanoparticles (LNP), switchable LNP promote pH-triggered delivery of payload based on a molecular switch mechanism. Previous studies have demonstrated that switchable LNP successfully delivered small interferring RNA (siRNA) in vitro and in vivo, promoting the silencing of a reporter Green Fluorescencen Protein (GFP) protein and liver-produced factor VII, respectively. Dual delivery of micro RNA (miRNA) and anticancer agent melphalan was also achieved through switchable LNP in a retinoblastoma rat model. These promising results encouraged us to enlarge the applications of switchable LNP as siRNA carrier. Moreover, the mechanism whereby switchable LNP mediate acid-triggered membrane destabilization and, thus, payload release remains elusive. Understanding this mechanism is crucial to draw the advantages and limitations of switchable LNP, and to tailor their future applications and prevent their potential toxicity. In this dissertation, we aimed to further understand the potential of switchable LNP for cancer treatment. We assessed switchable LNP as a siRNA delivery carrier by targeting one of the most specific cancer protein discovered to date, survivin. Meanwhile, we investigated the biophysical behavior of switchable-lipid containing membranes in micron-sized vesicles. In the first study, we demonstrated that switchable LNP efficiently silenced survivin in a range of cancer cell line models (lung, cervical, ovary, breast, colon, retinoblastoma). In retinoblastoma (RB) cells (Y79), several clinically used cytotoxic agents were screened for their synergy with survivin silencing: melphalan, topotecan, Teniposide, and carboplatin. Pretreatment with LNP loaded with siRNA targeted against survivin synergistically enhanced the cytotoxicity of carboplatin and melphalan but in lesser extent topotecan and teniposide. This effect was specific to cancer cells since healthy cells (ARPE.19) did not express survivin. Survivin inhibition through siRNA silencing revealed more specific and less damageable for healthy cells (ARPE.19) than a molecular approach, such as YM155. In the second study, we observed by confocal microscopy that switchable lipids rapidly induced stress, fission, and positive curvature in giant unilamellar vesicles’ membranes when submitted to acidic conditions. The membrane dynamics was confirmed by dynamic light scattering and calcein leakage experiments. Remarkably, these phenomena were also observed when switchable lipids were embedded into a hybrid polymer/lipid membrane, providing pH-sensitive properties to hybrid vesicles. To the best of our knowledge, this is the first time a pH-sensitive hybrid vesicle is reported. Our findings corroborate with the applicability of switchable LNP as siRNA delivery agents for cancer treatment through survivin silencing, especially as an adjuvant to chemotherapy. The biophysical investigation revealed that the switchable lipids act on the membrane fluidity, specifically at acidic pH. This pH selectivity guarantees their biocompatibility at neutral pH as well as its efficient and quick release of their cargo at acidic pH. Their compatibility with hybrid polymer/lipid vesicles opens new applications in biomimetic vesicles and drug delivery.

Dans le cadre d'une approche Higher Tamm Dancoff Approximation notée HTDA, nous avons étudié les corrélations vibrationnelles de type quadrupole, avec et sans appariement. Le champ moyen a été déterminé dans le cadre d'une approche microscopique utilisant l'interaction effective de Skyrme. Une interaction résiduelle schématique de type delta plus quadrupole-quadrupole, tenant compte en particulier de l'appariement neutron-proton T=0 et T=1 a été utilisé. Les résultats obtenus pour la résonance géante quadrupolaire isoscalaire du noyau Ca40 ont été comparés aux données expérimentales et à d'autres résultats théoriques. Nous avons également étudié un gaz de fermions piégés dans un potentiel d'oscillateur harmonique à 2D, et à température nulle, en présence d'un champ magnétique uniforme. Les expressions exactes des quelques grandeurs thermodynamiques ont été dérivées à partir de la matrice densité de Bloch. / Within the framework of the so-called Higher Tamm Dancoff Approxiamtion (HTDA), we have studied the quadrupole vibrational correlations with and without pairing correlations. The mean field has been determined within a microscopic approach using the Skyrme effective interaction. A schematic residual interaction of the delta plus quadrupole-quadrupole type, allowing in particular neutron-proton T=0 and T=1 pairing, has been used. The results which have been obtained for the isoscalar quadrupole giant resonance of the Ca40 have been compared with the experimental data. A fermion gaz trapped in a 2D harmonic oscillator well at zero temperature and in the presence of a uniform magnetic field has been investigated. Exact expressions of some thermodynamic quantities have been derived from the Bloch density matrix.