O conhecimento das propriedades dielétricas do osso tem se mostrado importante na medicina ortopédica para aplicações em diagnóstico e tratamento da osteoporose, de fraturas, controle da osteonecrose e, também, na área de cerâmicas industriais (porcelana de osso). Neste trabalho, apresenta-se uma metodologia de avaliação das permissividades complexas de osso cortical de fêmur bovino desidratado, utilizando-se o método de impedância em uma linha de transmissão. Esta se constitui de um condutor coaxial, do tipo N, de uma porta, e um terminal aberto, que é conectado a um analisador de impedância. A faixa de freqüência considerada está compreendida entre 100 Hz e 1 MHz. Em particular, apresenta-se uma metodologia de caracterização dielétrica de osso, com ênfase nas avaliações das suas características anisotrópicas. Alguns resultados das propriedades dielétricas são comparados com os valores experimentais e teóricos disponíveis na literatura. Este procedimento é justificado, haja vista que os valores experimentais encontrados na literatura são esparsos e pontuais, quando se considera toda a faixa do espectro eletromagnético avaliado. Ressalta-se que não existe um consenso entre especialistas da área sobre a precisão e metodologia de obtenção destes. O mesmo ocorre com relação aos protocolos de medição propostos nas normas internacionais, em particular, quando se buscam padrões de avaliações de materiais biológicos sólidos. Os valores da permissividade obtidos com as medidas realizadas entre 100 Hz e 1 MHz evidenciam a presença de uma anisotropia efetiva, sendo esta característica ressaltada em freqüências da ordem de algumas dezenas de kHz. Embora o fenômeno de anisotropia não se manifeste nas faixas superiores à anteriormente mencionada, algumas medidas foram realizadas, também, numa faixa de freqüências superiores, entre 50 MHz e 3 GHz. Utilizou-se um analisador de redes conectado a um dispositivo coaxial de duas portas, visando avaliar a adequabilidade dos procedimentos de preparação do tecido ósseo na avaliação das características de materiais biológicos em altas freqüências, já que se dispunha da instrumentação necessária e de alguns resultados, obtidos na literatura, para sua validação. / The knowledge of the dielectric properties of bones has become important to orthopedic therapy, for applications in the diagnosis and treatment of osteoporosis, fractures, and osteonecrosis, as well as is the industrial ceramics field (bone china). This work proposes a methodology for the evaluation of the complex permittivity of a dehydrated cortical bone of bovine femur, using an impedance method in a transmission line. The transmission line is composed by an one-port open-ended N-type coaxial conductor, connected to an impedance analyzer. The frequency range studied was 100 Hz - 1 MHz. The methodology proposed is proper for the dielectric characterization of bone and this work emphasizes the evaluation of its anisotropic characteristics. Some dielectric properties experimentally found in this work are compared to the experimental and theoretical results available in literature, which are sparse and punctual in the considered electromagnetic spectrum. It is pointed out that there is no agreement among the experts when the subject is the proper measuring methodology and its precision. The same occurs when the subject is the measuring protocols proposed by the international standards, especially for solid biological materials. The permittivity results obtained in frequencies between 100 Hz and 1 MHz evince an effective anisotropy, which stands out in frequencies of a few tens of kHz. Although the anisotropy phenomenon does not occur in a frequency range higher than the one previously mentioned, some measures were taken in the 50 MHz - 3 GHz range for validation, based on results from literature. A network analyzer and a two-port coaxial device were then used in such measures, which aimed the evaluation of the adequacy of the procedure used in the preparation of the bone tissue in high-frequency measurements.

Sleep is well-established to become more superficial and fragmented as we age, with deficits in cognitive processing also commonly observed. While effects have been identified in both humans and mice (used in this thesis), there are important species differences in these findings and importantly, very little is known about the neural dynamics underlying these changes. By integrating several state-of-the-art approaches from putative single unit electrophysiological recordings to behavioural and pharmacological assessments, this thesis aimed to provide novel insights into the neural mechanisms involved in the age-dependent changes in sleep and cognition in mice. Firstly, this thesis investigated the neural activity underpinning the known global sleep changes that occur with ageing. Surprisingly, the majority of neuronal measures quantified in this study were resilient to the effects of ageing. Therefore the global sleep disruptions identified with ageing are unlikely to arise from changes in local cortical activity. Secondly, diazepam injection was found to suppress neural activity, in addition to previously reported effects on electroencephalography (EEG). Subtle differences in the effects of diazepam were identified across age groups, which may account for the variability seen in the efficacy of benzodiazepines in older individuals. Thirdly, ageing and sleep deprivation were found to have only a few effects on performance in a spatial learning task, the Morris water maze (MWM). Suggesting that spatial learning may be fairly resilient to the effects of ageing and sleep deprivation. Finally, this thesis presents preliminary analyses that showed mice were able to perform two novel paradigms of the visual discrimination task, suggesting their suitability in studying the link between ageing, sleep and cognition. Together the studies presented in this thesis provide insights into the differences between global and local mechanisms affected by ageing. Only by understanding local mechanisms will we be able improve on current treatments aimed at helping with the unwanted effects of healthy ageing, such as cognitive decline and sleep disruptions.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-07-10T22:49:41Z No. of bitstreams: 1 472135 - Texto Completo.pdf: 929985 bytes, checksum: 28553b1c8275c157e5128080cce87fe9 (MD5) / Made available in DSpace on 2015-07-10T22:49:41Z (GMT). No. of bitstreams: 1 472135 - Texto Completo.pdf: 929985 bytes, checksum: 28553b1c8275c157e5128080cce87fe9 (MD5) Previous issue date: 2015-02-04 / The Cortical dysplasia is one of the most frequent forms of malformations of cortical development, with a condition that causes a large portion of refractory partial epilepsy to drug treatment. This condition is characterized by a heterogeneous group of cortical lesions and can also be described as cortical dysgenesis or neuronal migration disorder. The genetic factors involved in cortical dysplasia, such as the expression of the telomerase enzyme, are not commonly investigated, mainly due to the limited number of cases is the lack of an experimental model. Objective: This study aimed to analyze the activity and the expression of the enzyme telomerase in patients with cortical dysplasia of Taylor, normal fibroblasts, stem cells from umbilical cord tissue and human tumor. Methodology: the activity and expression of hTERT portion of telomerase were evaluated in patient fibroblasts with Cortical Dysplasia of Taylor (n = 1) and compared with control patients fibroblasts (n = 3) and umbilical cord stem cells (n = 3) and human tumor cell line A549 were maintained in culture. The analysis of the hTERT gene was done by PCR and the telomerase activity was measured by TRAP assay that uses real-time PCR. Results: telomerase activity was higher in tumor cells (Ct = 28.32), while in other cell types and other types studied values were similar: control (Ct = 33.73) Umbilical Cord (Ct = 32.88) and dysplasia (Ct = 33.56). The expression of hTERT portion of telomerase was the control group was 2,58x more expressed, the Dysplasia and tumor groups were 61,25x and 635,48x more expressed respectively as the control. Conclusion: The telomerase appeared to be more active and higher expression in the tumor cell line when compared to other cell types surveyed. The hTERT gene was more expressed in the patient Taylor cortical dysplasia than in the other groups and control umbilical cord indicating that telomerase is expressed in most patients with this disease. / A Displasia Cortical ? uma das formas mais frequentes de malforma??es do desenvolvimento cortical, sendo uma patologia que causa uma grande parcela de epilepsias parciais refrat?rias ao tratamento medicamentoso. Essa patologia ? caracterizada por um grupo heterog?nio de les?es corticais, podendo tamb?m ser descrita como disgenesia cortical ou desordem de migra??o neuronal. Os aspectos gen?ticos envolvidos na displasia cortical, como a express?o da enzima telomerase, n?o s?o comumente investigados, principalmente devido ao n?mero limitados de casos e a pela falta de um modelo experimental. Objetivo: este estudo teve como objetivo analisar a atividade e a express?o da enzima telomerase em pacientes com displasia cortical de Taylor, fibroblastos normais, c?lulas-tronco de tecido de cord?o umbilical e linhagem tumoral humana. Metodologia: a atividade e express?o da por??o hTERT da telomerase foram avaliadas em fibroblastos de paciente com Displasia Cortical de Taylor (n=1) e comparada com fibroblastos de pacientes do grupo controle (n=3) e c?lulas-tronco de cord?o umbilical (n=3) e a linhagem tumoral A549 humanos que foram mantidas em cultura. A an?lise do gene hTERT foi feita por PCR e a atividade da telomerase foi medida pelo ensaio TRAP que utiliza a PCR em tempo real. Resultados: a atividade da telomerase foi maior nas c?lulas tumorais (Ct=28,32), ao passo que nos demais tipos celulares e nos demais tipos estudados os valores foram semelhantes: Controle (Ct=33,73) Cord?o Umbilical (Ct=32,88) e Displasia (Ct=33,56). A express?o da por??o hTERT da telomerase foi do grupo Controle foi 2,58x mais expresso, os grupos Dsiplasia e Tumor estavam 61,25x e 635,48x mais expressos, respectivamente que o Controle. Conclus?o: a telomerase se mostrou mais ativa e com maior express?o na linhagem tumoral do que quando comparada com os outros tipos celulares pesquisados. O gene hTERT e mostrou mais expresso, no paciente com Displasia Cortical de Taylor do que nos outros grupos Controle e Cord?o Umbilical, indicando que a telomerase ? mais expressa em pacientes com esta patologia.

Les cellules épithéliales sont des cellules polarisées qui forment l’un des types cellulaires le plus abondant dans le corps humain. Leur polarité apico-basale (A/B) est établie et maintenue par la ségrégation asymétrique de protéines adaptatrices hautement conservées. Cette polarité est essentielle pour de nombreuses fonctions cellulaires clés comme l’adhésion (jonctions intercellulaires) ou la signalisation et la prolifération par la localisation et la concentration des complexes de signalisation. Durant la cancérogénèse, un grand nombre de ces processus est dérégulé aboutissant à la sur-prolifération, la migration et/ou l’invasion des cellules cancéreuses. Une meilleure compréhension des mécanismes à l’origine de ces processus est indispensable pour trouver de nouvelles cibles thérapeutiques pour le traitement du cancer. Dans l’équipe, nous sommes particulièrement intéressés par les protéines adaptatrices à domaines PDZ (domaine de liaison protéine-protéine). De par leur structure modulaire et la diversité de leurs partenaires, ces protéines adaptatrices sont impliquées dans la régulation de très nombreuses fonctions et fournissent des plateformes où différents processus peuvent être intégrés. Durant mon doctorat, j’ai étudié deux protéines adaptatrices dans le système animal modèle Drosophila melanogaster, Bbg et Magi, impliquées dans deux processus cellulaires essentiels : la dynamique des jonctions et la prolifération. Grâce aux molécules d’adhésion, les cellules non seulement restent cohésives dans un tissu, mais c’est aussi à ce niveau qu’elles peuvent obtenir une information concernant la densité cellulaire d'un tissu. Cette information est alors relayée au cytosquelette d’actine via des protéines adaptatrices spécialisées dans le but de réguler la prolifération et la voie Hippo. Cependant, le contrôle de la voie de signalisation Hippo par certaines protéines adaptatrices et par le cytosquelette d’actine n'est que partiellement compris à ce jour. Dans le laboratoire, nous étudions notamment le rôle d'une nouvelle protéine adaptatrice apicale nommée Big Bang (Bbg) dans le disque d’aile de la drosophile. Nous nous sommes intéressés à Bbg car c’est une cible de la voie Notch chez la drosophile et son homologue humain PDZD2 (pour PDZ domain-containing 2 protein) est sur-exprimé dans plusieurs cancers (sein et prostate).Mes résultats montrent que Bbg est un nouveau régulateur du cytosquelette d’actine et de la voie Hippo. Une étude détaillée de la fonction de Bbg et de ses partenaires permet de mieux comprendre les relations existantes entre dynamique de l’actine et prolifération. Bbg induit une accumulation d’actine filamenteuse en augmentant l’activité d’Enabled et la phosphorylation de Myosin Light Chain (MLC). Cette régulation résulte en une augmentation de l’activité de Yorkie, effecteur final de la voie Hippo, pour soutenir la prolifération cellulaire.La régulation des jonctions adhérentes est une étape cruciale lors de l’évolution d'une tumeur solide. Malgré les récentes avancées dans le domaine, de nombreux aspects clés de la dynamique des jonctions restent peu caractérisés.Dans le laboratoire, nous recherchons de nouveaux régulateurs de jonctions et grâce au modèle de remodelage des AJs lors du développement de l’œil de pupe de drosophile. Nous avons identifié Magi en tant que protéine adaptatrice recrutant le complexe formé de RASSF8 et ASPP. Magi régule le recrutement de Bazooka à la membrane, le dépôt d’E-Cadhérine et des Caténines et finalement le remodelage des jonctions pendant la morphogénèse. J’ai identifié Echinoid, une protéine de type immunoglobuline impliquée dans l’adhésion cellulaire et la régulation de la voie Hippo, comme un nouveau partenaire responsable du recrutement de Magi aux futures zones de jonctions. / Epithelial cells are polarised cells that form one of the most abundant cell types in the human body. Their apico-basal (A/B) polarity is established and maintained by the asymmetric segregation of highly conserved scaffold proteins. Proper A/B polarity is critical for many key cellular functions such as intercellular junctions and therefore adhesion, or signalling and proliferation by localising and concentrating signalling complexes. During carcinogenesis, many of these processes are mis-regulated leading to the over-proliferation, migration and/or invasion of cancer cells. A better understanding of the mechanisms underlying these processes is really needed to find new therapeutic targets in cancer treatment.In the team, we are particularly interested in scaffold proteins with PDZ domains (protein-protein interaction domains). Due to their modular structure, the high number of interactions they engage in, and the variety of their binding partners, these scaffold proteins are implicated in the regulation of many key cellular functions and processes. During my PhD, I have studied two scaffold proteins in the Drosophila melanogaster animal model, Bbg and Magi, which are involved in two important cell processes: adherens junctions (AJs) dynamic and cell proliferation.Through adhesion molecules, epithelial cells not only remain cohesive, but can also sense cellular density in a tissue and relay this information through dedicated scaffolds to the actin cytoskeleton to ultimately regulate the Hippo pathway and proliferation. However, many aspects of the control of Hippo signalling by apical scaffolds and the actin cytoskeleton are still poorly understood. In the laboratory, we are interested in the study of a new conserved apical scaffold, Big Bang (Bbg). Bbg is a new and quite unknown protein expressed in a variety of Drosophila epithelia, and appears as a potential Notch target in Drosophila. Its’ human homolog called PDZD2 (PDZ domain-containing 2 protein) has been shown to be over-expressed in several types of cancers (breast and prostate cancers).My results show that Bbg is a new regulator of the actin cytoskeleton and of the Hippo pathway in Drosophila. A detailed study of Bbg function and of its associated partners have helped to better understand the intricate relationships between actin dynamics and proliferation. My results suggest that Bbg promotes accumulation of filamentous actin (F-Actin) through the increase of the activity of Enabled (Ena) and the phosphorylation of the molecular motor Myosin Light Chain (MLC). This regulation leads to the increase of Yorkie activity, the final effector of the Hippo pathway, to promote cell proliferation.The regulation of adhesion, and in particular of Adherens Junctions (AJs), is a critical step during the evolution of solid tumours. A better understanding of how these structures are regulated will provide valuable insights into different phases of the disease. Despite the recent advances, many key aspects of AJ dynamics remain poorly understood. In the laboratory, we are interested in the identification of new AJs regulators. Using the remodelling of AJs during the development of the Drosophila pupal eye as a model, we have identified Magi as a scaffold recruiting a complex formed by RASSF8 and ASPP, regulating Bazooka membrane recruitment, E-Cadherin and catenins deposition, and ultimately AJs remodelling during morphogenesis. I uncovered Echinoid, an immunoglobulin-like protein involved in cell adhesion and in Hippo pathway regulation, as a new binding partner responsible for the recruitment of Magi at future AJ sites.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-06-22T19:22:17Z No. of bitstreams: 1 TES_DANIEL_RODRIGO_MARINOWIC_COMPLETO.pdf: 16202491 bytes, checksum: 5bb7aaf0de3a700a163c7437ecf2adef (MD5) / Made available in DSpace on 2016-06-22T19:22:17Z (GMT). No. of bitstreams: 1 TES_DANIEL_RODRIGO_MARINOWIC_COMPLETO.pdf: 16202491 bytes, checksum: 5bb7aaf0de3a700a163c7437ecf2adef (MD5) Previous issue date: 2016-03-03 / Changes in the cerebral cortex development are presented as a group of distinct defects with a not well-defined pathogenesis. The focal cortical dysplasia (FCD) is one of the most frequent form of cortical development malformation, that encompasses multiple types of changes both in the cortical architecture and cytological abnormalities. It is an underlying pathology of a significant proportion of partial epilepsy refractory to drug treatment. Especially the limited number of cases and the lack of suitable experimental models rarely document the mechanisms involved with the genesis of FCD. The scarse predictive preclinical models that can be used to study the pathophysiology and to translate the therapeutic discovery from animal models to human use, strengthens the need to study the brain development from cells originated from patients that harbor these central nervous systems diseases. The generation of iPSC cells and the differentiation into specific cells and tissues will provide important testing and an unique ability to study the development and progress of CNS diseases. The objective of this study was to establish a cellular model of focal cortical dysplasia through the generation of induced pluripotent stem cells (iPSC) from fibroblasts derived from affected patients. Human fibroblasts were obtained from skin biopsies from two patients and cultured up to the fifth passage. Immunofluorescence analysis of AKT/mTOR signaling pathways was performed in the dysplastic brain tissue. iPSC cells were generated from fibroblasts through transfection with a viral vector containing the genes OCT4, KLF4, SOX2, and C-MYC and characterized by immunohistochemistry. Cell migration co-cultured with fibroblast and iPSC was investigated. Morphological and molecular characterization of neurodifferentiated iPSC were performed by immunohistochemistry and PI3K/ATK/mTOR signaling pathway analysis. Both patients were diagnosed with FCD type IIb. The AKT and mTOR phosphorylated and non-phosphorylated was higher in brain sections from patient 01. iPSC clones from patients and controls were generated from skin fibroblasts. Both iPSC from patients and controls showed polarization and morphology similar to nerve cells. In the cell migration assay, fibroblasts derived from FCD migrate with greater intensity within 24 hours (p<0,0001) and 48 hours (p<0,001), and iPSC cells did not present difference in cell migration. During neurodifferentiation, iPSC cells from patients with FCD showed lower values of 4EBP-1, ?-catenin, CIAP-1, CIAP-2 and PI3K, and higher values of MCL 1 gene expression. Changes in cell migration in adult tissue, uncontrolled cell proliferation, cell adhesion protein deficiency, and alterations in the expression of genes responsible for apoptosis and PI3K pathway that are implicated with an complete and well successful CNS formation. Alterations in some of these were detected in cells and iPSC from patients with FCD and may be related to the ethiopathology of the disease. / As altera??es do desenvolvimento do c?rtex cerebral apresentam-se como um grupo de malforma??es com uma distin??o e patog?nse ainda n?o bem definidas. A displasia cortical focal (DCF) ? uma das formas mais frequentes de malforma??es do desenvolvimento cortical, sendo a patologia subjacente a uma parcela significativa de epilepsias parciais refrat?rias ao tratamento medicamentoso. A displasia cortical focal engloba m?ltiplos tipos de altera??es tanto na arquitetura cortical quanto em anormalidades citol?gicas. Palmini e colaboradores classificaram as displasias corticais focais de acordo com observa??es na subst?ncia branca e na arquitetura da camada cortical. Os mecanismos envolvidos na g?nese da DCF s?o pouco investigados, principalmente pelo n?mero limitado de casos e a falta de modelos experimentais adequados e suas causas provavelmente est?o relacionadas a muta??es som?ticas. A falta de modelos pr?-cl?nicos preditivos que possam ser utilizados no estudo da fisiopatologia e o hist?rico enfraquecido quando se trata em traduzir a descoberta terap?utica de modelos animais para o uso humano, fortalece a necessidade de estudar o desenvolvimento cerebral a partir de c?lulas originadas do pr?prio paciente. A gera??o de c?lulas iPSC e diferencia??o tecidual espec?fica de c?lulas de pacientes acometidos por doen?as neurol?gicas relevantes possui um valor inestim?vel para a realiza??o de testes al?m de fornecerem uma capacidade adicional e ?nica para estudar o desenvolvimento inicial e a progress?o das patologias associadas ao SNC. O objetivo do presente trabalho ? estabelecer um modelo celular de Displasia Cortical Focal atrav?s da gera??o de c?lulas-tronco pluripotentes induzidas (iPSC) a partir de fibroblastos de pacientes afetados. Fibroblastos humanos foram obtidos de bi?psias de pele de dois pacientes com DCF e foram cultivados at? a quinta passagem. Foi realizada an?lise imunopatol?gica e das vias de sinaliza??o AKT/mTOR no tecido cerebral displ?sico. C?lulas iPSC foram geradas a partir dos fibroblastos atrav?s da exposi??o a vetores virais contendo os genes OCT4, KLF4, SOX2, e C-MYC e caracterizadas por imunohistoqu?mica. Foi realizado ensaio de migra??o celular dos fibroblastos (24h, 48h e 72h) e das iPSC (3 dias e 7 dias). As c?lulas iPSC foram neurodiferenciadas e analisadas nos per?odos de 14 dias, 22 dias e 35 dias. Nesses per?odos, foram realizadas an?lises morfol?gicas, imunohistoqu?mica (polariza??o) e moleculares (genes relacionados a via PI3K/ATK/mTOR). Ambos os pacientes foram diagnosticados com DCF tipo IIb. O paciente 01 apresentou valores maiores que o paciente 02 em rela??o ? ?rea marcada das vias AKT e mTOR, tanto na via fosforilada como n?o fosforilada no tecido cerebral, com diferen?as estatisticamente significativas. Clones iPSC dos pacientes e controles foram gerados e caracterizados a partir dos fibroblastos de pele. Tanto as iPSC dos pacientes quanto do controle apresentaram morfologia e polariza??o semelhante a c?lulas nervosas ap?s protocolo de neurodiferencia??o. No ensaio de migra??o celular, fibroblastos com DCF migraram com mais intensidade em 24 horas (p<0,0001) e 48 horas (p<0,001). As c?lulas iPSC n?o apresentaram diferen?a no potencial de migra??o celular nos per?odos analisados. Durante o protocolo de neurodiferencia??o, as c?lulas iPSC dos pacientes com DCF apresentaram valores menores de express?o dos genes 4EBP-1, ?-Catenina, CIAP-1, CIAP-2 e PI3K, e valores mais elevados da express?o de MCL 1. Altera??es na migra??o celular no tecido adulto e em processos como de prolifera??o celular acentuada, defici?ncia de prote?na de ades?o celular, altera??o na express?o de genes respons?veis pelo controle de apoptose e altera??o na via PI3K respons?vel no sistema nervoso central pela sobreviv?ncia celular, controle de apoptose, migra??o neuronal, desenvolvimento morfol?gico dos neur?nios e forma??o das neurotransmiss?es, puderam ser evidenciadas nas c?lulas dos pacientes com DCF em rela??o aos pacientes controle e podem estar relacionadas com a forma??o do c?rebro com displasia.

O conhecimento das propriedades dielétricas do osso tem se mostrado importante na medicina ortopédica para aplicações em diagnóstico e tratamento da osteoporose, de fraturas, controle da osteonecrose e, também, na área de cerâmicas industriais (porcelana de osso). Neste trabalho, apresenta-se uma metodologia de avaliação das permissividades complexas de osso cortical de fêmur bovino desidratado, utilizando-se o método de impedância em uma linha de transmissão. Esta se constitui de um condutor coaxial, do tipo N, de uma porta, e um terminal aberto, que é conectado a um analisador de impedância. A faixa de freqüência considerada está compreendida entre 100 Hz e 1 MHz. Em particular, apresenta-se uma metodologia de caracterização dielétrica de osso, com ênfase nas avaliações das suas características anisotrópicas. Alguns resultados das propriedades dielétricas são comparados com os valores experimentais e teóricos disponíveis na literatura. Este procedimento é justificado, haja vista que os valores experimentais encontrados na literatura são esparsos e pontuais, quando se considera toda a faixa do espectro eletromagnético avaliado. Ressalta-se que não existe um consenso entre especialistas da área sobre a precisão e metodologia de obtenção destes. O mesmo ocorre com relação aos protocolos de medição propostos nas normas internacionais, em particular, quando se buscam padrões de avaliações de materiais biológicos sólidos. Os valores da permissividade obtidos com as medidas realizadas entre 100 Hz e 1 MHz evidenciam a presença de uma anisotropia efetiva, sendo esta característica ressaltada em freqüências da ordem de algumas dezenas de kHz. Embora o fenômeno de anisotropia não se manifeste nas faixas superiores à anteriormente mencionada, algumas medidas foram realizadas, também, numa faixa de freqüências superiores, entre 50 MHz e 3 GHz. Utilizou-se um analisador de redes conectado a um dispositivo coaxial de duas portas, visando avaliar a adequabilidade dos procedimentos de preparação do tecido ósseo na avaliação das características de materiais biológicos em altas freqüências, já que se dispunha da instrumentação necessária e de alguns resultados, obtidos na literatura, para sua validação. / The knowledge of the dielectric properties of bones has become important to orthopedic therapy, for applications in the diagnosis and treatment of osteoporosis, fractures, and osteonecrosis, as well as is the industrial ceramics field (bone china). This work proposes a methodology for the evaluation of the complex permittivity of a dehydrated cortical bone of bovine femur, using an impedance method in a transmission line. The transmission line is composed by an one-port open-ended N-type coaxial conductor, connected to an impedance analyzer. The frequency range studied was 100 Hz - 1 MHz. The methodology proposed is proper for the dielectric characterization of bone and this work emphasizes the evaluation of its anisotropic characteristics. Some dielectric properties experimentally found in this work are compared to the experimental and theoretical results available in literature, which are sparse and punctual in the considered electromagnetic spectrum. It is pointed out that there is no agreement among the experts when the subject is the proper measuring methodology and its precision. The same occurs when the subject is the measuring protocols proposed by the international standards, especially for solid biological materials. The permittivity results obtained in frequencies between 100 Hz and 1 MHz evince an effective anisotropy, which stands out in frequencies of a few tens of kHz. Although the anisotropy phenomenon does not occur in a frequency range higher than the one previously mentioned, some measures were taken in the 50 MHz - 3 GHz range for validation, based on results from literature. A network analyzer and a two-port coaxial device were then used in such measures, which aimed the evaluation of the adequacy of the procedure used in the preparation of the bone tissue in high-frequency measurements.

It is essential to characterize and quantify naturally occurring morphometric changes in the human brain when investigating the onset or progression of neurodegenerative disorders. The aim of this thesis is to characterize the properties and measure the performance of several popular automated magnetic resonance image analysis tools dedicated to brain morphometry. The thesis begins with an overview of morphometric analysis methods, followed by a literature review focusing on cortical parcellation protocols. Our work identified unanimous protocol weaknesses across all packages in particular issues when addressing cortical variability. The next chapters present a ground truth dataset and a dedicated software to analyse manually parcellated data. The dataset (https://datashare.is.ed.ac.uk/handle/10283/2936) includes 10 healthy middle-aged subjects, whose metrics we used as reference against automated tools. To develop the ground truth dataset, we also present a manual parcellation protocol (https://datashare.is.ed.ac.uk/handle/10283/3148) providing step-by-step instructions for outlining three cortical gyri known to vary with ageing and dementia: the superior frontal gyrus, the cingulate gyrus and the supramarginal gyrus. The software, Masks2Metrics (https://datashare.is.ed.ac.uk/handle/10283/3018), was built in Matlab to calculate cortical thickness, white matter surface area, and grey matter volume from 3D binary masks. Characterizing these metrics allowed further understanding of the assumptions made by software when creating and measuring anatomical parcels. Next, we present results from processing the raw T1-weighted volumes in the latest versions of several automated image analysis tools-FreeSurfer (versions 5.1 and 6.0), BrainGyrusMapping, and BrainSuite (version 13a)- against our ground truth. Tool repeatability for the same system was confirmed as multiple runs yielded identical results. Compared to our ground truth, the closest results were generated by BrainGyrusMapping for volume metrics and by FreeSurfer 6.0 for thickness and surface area metrics. In conclusion, our work sheds light on the significance of clearly detailed parcellation protocols and accurate morphometric tools due to the implications that they both will have. We therefore recommend extra caution when selecting image analysis tools for a study, and the use of independent publicly available ground truth datasets and metrics tools to assist with the selection process.

The collecting duct of the distal nephron marks the final location where adjustments to Na+ excretion can be made, therefore determining the final concentration of Na+ conserved in the extracellular fluid which plays a role in governing overall blood volume and pressure. This transport of Na+ is subject to hormonal regulation but the signalling pathways underpinning this regulation however, are not fully understood. In this thesis the signalling pathways allowing both basal and insulin-stimulated Na+ absorption were explored in the murine collecting duct cell line, mpkCCDcl4. The effects of two insulin-sensitizing drugs, TZDs, on ENaC-mediated Na+ transport were investigated and the signalling pathways underlying two other hormonal regulators of ENaC, dexamethasone and vasopressin, were also examined. Unstimulated monolayers of mpkCCDcl4 cells generated spontaneous Na+ absorption which was quantified by measuring equivalent short circuit current (Ieq). Selective inhibition of PI3-kinase, mTORC2 and SGK1 left ~80 % of the current intact, indicating these signalling molecules are not required for basal Na+ transport. Acute addition of insulin stimulated Ieq and this occurred with a concomitant increase in mTORC2, SGK1 and Akt activity. Inhibition of PI3-kinase abolished the insulin-stimulated response as well as phosphorylation of downstream substrates, indicating a crucial role of PI3-kinase. Inhibition of mTORC1 with rapamycin did not alter basal or insulin-stimulated Na+ transport. The mTOR inhibitors TORIN1 and PP242 could therefore be used to evaluate the role of mTORC2. These inhibitors greatly reduced insulin-stimulated ENaC-mediated Na+ transport and also abolished SGK1 and mTORC2 activity, indicating a novel role of mTORC2. An inhibitor of SGK1, GSK650394A abolished insulin-stimulated Na+ transport and specifically inhibited SGK1 acitivty demonstrating the importance of SGK1 in insulin signalling. The inhibitor Akti-1/2 also abolished insulin-mediated Na+ transport but this compound inhibited both Akt and SGK1 activity. The TZDs pioglitazone and rosiglitazone did not alter basal or insulin-stimulated Na+ transport and had no effect on SGK1 activity indicating these drugs do not alter Na+ absorption in this cell line. Dexamethasone stimulated ENaC-mediated Na+ transport in a similar manner to insulin and this could be blocked with rapamycin. This drug did not alter phosphorylation of NDRG1 indicating that dexamethasone stimulates Na+ transport in an mTORC1-dependent manner but without altering SGK1 activity. Arginine vasopressin also stimulated Ieq but did so by reducing Rt with an associated depolarisation of Vt. Ieq could be blocked with amiloride and vasopressin-stimulated Ieq was insensitive to TORIN1 and PP242. Vasopressin suppressed SGK1 phosphorylation of NDRG1 but did stimulate protein kinase A (PKA) activity. Therefore vasopressin stimulates Ieq via a PKA-dependent but mTOR- and SGK1-independent pathway.

PURPOSE: Extremely low gestation age newborns (ELGANs) are at high risk for developmental brain abnormalities. This study is to determine deep and superficial gray matter volumetric abnormalities of ELGAN children and full term children at 9 to 11 years of age. METHODS: High-resolution magnetic resonance imaging (MRI) scans were obtained from 160 ELGAN children (70 males and 90 females) and 30 full term children (15 males and 15 females) using a dual-echo turbo spin-echo (DE-TSE) pulse sequence at 3.0T (or 1.5T at only one site). The DICOM MR images were processed with quantitative MRI algorithms programmed in Mathcad. The brain deep gray matter (dGM) was manually segmented; dGM and cortical gray matter (cGM) volumes were quantified using semi-automated clustering segmentation algorithms. RESULTS: ELGAN children had smaller deep gray matter volume (41.86 ± 7.42 ml) than full term children (49.24 ± 10.91 ml). Deep gray matter volumes of ELGAN children showed similar distribution range (SD = 7.42 ml) with the full term children (SD = 10.91 ml). About 83% of the ELGAN children had smaller deep gray matter volumes compared to the average volume of full term children at the same ages. Male children had smaller deep gray matter volumes in ELGAN (42.77 ± 7.09 ml) than in full term (51.74 ± 9.76 ml), but female children had similar deep gray matter volumes in ELGAN (41.14 ± 7.62 ml) with full term (44.27 ± 7.56 ml). Additionally, smaller deep gray matter volumes were observed more often in males (90%) than in females (65%). Cortical gray matter volumes of ELGAN children distributed from 345.60 to 1177.50ml. Moreover, female ELGAN children had smaller cortical gray matter volumes (828.14 ± 147.61 ml) than males (883.13 ± 151.34 ml). Correlation analysis revealed a positive correlation between cerebral deep gray matter volumes and total gray matter volumes (total: r = 0.57, p<0.0001; male: r = 0.542, p < 0.0001; female: r = 0.587, p < 0.0001). CONCLUSION: Male ELGAN children had smaller brain deep gray matter volumes than full term children at ages of 9 to 11 years, but not females. Cortical gray matter volumes of female ELGAN were smaller than male ELGAN. Smaller deep gray matter volumes were associated with smaller total gray matter volumes in ELGAN children.

Analysis of surface models reconstructed from human MR images gives re- searchers the ability to quantify the shape and size of the cerebral cortex. Increasing the reliability of automatic reconstructions would increase the precision and, therefore, power of studies utilizing cortical surface models. We looked at four different workflows for reconstructing cortical surfaces: 1) BAW + LOGIMSOS- B; 2) FreeSurfer + LOGISMOS-B; 3) BAW + FreeSurfer + Machine Learning + LOGISMOS-B; 4) Standard FreeSurfer(Dale et al. 1999). Workflows 1-3 were developed in this project. Workflow 1 utilized both BRAINSAutoWorkup(BAW)(Kim et al. 2015) and a surface reconstruction tool called LOGISMOS-B(Oguz et al. 2014). Workflow 2 added LOGISMOS-B to a custom built FreeSurfer workflow that was highly optimized for parallel processing. Workflow 3 combined workflows 1 and 2 and added random forest classifiers for predicting the edges of the cerebral cortex. These predictions were then fed into LOGISMOS-B as the cost function for graph segmentation. To compare these work- flows, a dataset of 578 simulated cortical volume changes was created from 20 different sets of MR scans. The workflow utilizing machine learning (workflow 3) produced cortical volume changes with the least amount of error when compared to the known volume changes from the simulations. Machine learning can be effectively used to help reconstruct cortical surfaces that more precisely track changes in the cerebral cortex. This research could be used to increase the power of future projects studying correlations between cortical morphometrics and neurological health.