<p>Processes where a molecule absorbs visible light and then disposes of the excess energy via electron/energy transfer reactions have an important role both in nature (e.g. in photosynthesis) and in many technical applications (e.g. in photography and photovoltaics). This thesis uses different spectroscopical techniques, mainly ultrafast transient absorption, to study such processes. The thesis can roughly be divided into three parts.</p><p>In the first part, donor-acceptor systems linked by different conjugated bridges are studied. The objective was to see to what extent the conjugated link could enhance excited state energy or electron transfer, via so-called superexchange processes. The studied links do enhance the electron/energy transfer but in the electron transfer study the resulting charge separated state was very short lived.</p><p>The second part explores the possibility of constructing acceptor-donor-acceptor triads where the direction of electron transfer is determined by the electronic state of the donor. Direct evidence of electron transfer in the form of radical absorption was found from both the first and the second excited states of the donor.</p><p>In the last part, two common chromophores were investigated by transient absorption anisotropy. In the case of Ru(bpy)₃²⁺, it was found that the complex lost all memory of the polarization of the exciting light much faster than what was previously thought. This means that electron transfer between ligands is normally not the rate limiting step in electron transfer reactions involving this complex. In the case of zinc porphyrin, it was seen that the measured anisotropy differed depending on which electronic state was excited suggesting differences in the degree of coherence.</p>

Processes where a molecule absorbs visible light and then disposes of the excess energy via electron/energy transfer reactions have an important role both in nature (e.g. in photosynthesis) and in many technical applications (e.g. in photography and photovoltaics). This thesis uses different spectroscopical techniques, mainly ultrafast transient absorption, to study such processes. The thesis can roughly be divided into three parts. In the first part, donor-acceptor systems linked by different conjugated bridges are studied. The objective was to see to what extent the conjugated link could enhance excited state energy or electron transfer, via so-called superexchange processes. The studied links do enhance the electron/energy transfer but in the electron transfer study the resulting charge separated state was very short lived. The second part explores the possibility of constructing acceptor-donor-acceptor triads where the direction of electron transfer is determined by the electronic state of the donor. Direct evidence of electron transfer in the form of radical absorption was found from both the first and the second excited states of the donor. In the last part, two common chromophores were investigated by transient absorption anisotropy. In the case of Ru(bpy)32+, it was found that the complex lost all memory of the polarization of the exciting light much faster than what was previously thought. This means that electron transfer between ligands is normally not the rate limiting step in electron transfer reactions involving this complex. In the case of zinc porphyrin, it was seen that the measured anisotropy differed depending on which electronic state was excited suggesting differences in the degree of coherence.

Magnetic resonance imaging (MRI) has long become one of the most powerful and accurate tools of medical diagnostic imaging. Central to the diagnostic capabilities of MRI is the notion of contrast, which is determined by the biochemical composition of examined tissue as well as by its morphology. Despite the importance of the prevalent T₁, T₂, and proton density contrast mechanisms to clinical diagnosis, none of them has demonstrated effectiveness in delineating the morphological structure of the white matter - the information which is known to be related to a wide spectrum of brain-related disorders. It is only with the recent advent of diffusion-weighted MRI that scientists have been able to perform quantitative measurements of the diffusivity of white matter, making possible the structural delineation of neural fibre tracts in the human brain. One diffusion imaging technique in particular, namely high angular resolution diffusion imaging (HARDI), has inspired a substantial number of processing methods capable of obtaining the orientational information of multiple fibres within a single voxel while boasting minimal acquisition requirements. HARDI characterization of fibre morphology can be enhanced by increasing spatial and angular resolutions. However, doing so drastically reduces the signal-to-noise ratio. Since pronounced measurement noise tends to obscure and distort diagnostically relevant details of diffusion-weighted MR signals, increasing spatial or angular resolution necessitates application of the efficient and reliable tools of image denoising. The aim of this work is to develop an effective framework for the filtering of HARDI measurement noise which takes into account both the manifold to which the HARDI signal belongs and the statistical nature of MRI noise. These goals are accomplished using an approach rooted in non-local means (NLM) weighted averaging. The average includes samples, and therefore dependencies, from the entire manifold and the result of the average is used to deduce an estimate of the original signal value in accordance with MRI statistics. NLM averaging weights are determined adaptively based on a neighbourhood similarity measure. The novel neighbourhood comparison proposed in this thesis is one of spherical neighbourhoods, which assigns large weights to samples with similar local orientational diffusion characteristics. Moreover, the weights are designed to be invariant to both spatial rotations as well as to the particular sampling scheme in use. This thesis provides a detailed description of the proposed filtering procedure as well as experimental results with synthetic and real-life data. It is demonstrated that the proposed filter has substantially better denoising capabilities as compared to a number of alternative methods.

Nos travaux de thèse ont pour objectif l'adaptation de la couche transport des systèmes de communications hybrides satellite/terrestre. Les réseaux satellites DVB-S2/RCS représente une solution alternative dans les zones reculés ou non couvertes par les réseaux filaires. Cependant, vu leur coût élevé et les caractéristiques qu'ils présentent, ils doivent être utilisés en complémentarité avec les solutions terrestres, constituants des réseaux intégrées ou hybrides (satellite/terrestre), en support du multiservice, avec ce que cela entraine en termes d'exigences liées aux nouveaux usages (accès généralisé et transparent). La première contribution consiste à l'analyse d'un système réel afin de caractériser le lien satellite. Cette démarche permet une éventuelle configuration d'un système émulé et s'avère le meilleur moyen pour bien paramétrer ce dernier. Par la suite, nous avons procédé à un ensemble de tests d'évaluation de plusieurs versions de TCP en utilisant l'émulateur SATEM. Vu les caractéristiques que représentent ces nouvelles versions de TCP, nous avons abouti à une proposition originale consistant à utiliser sur le segment satellite ces versions sans autre artifice architectural. Nous avons aussi proposé une solution qui permet d'améliorer le contrôle de congestion sur la voie aller DVB-S2 avec l'ACM pour les applications streaming, en utilisant le DCCP. Enfin, nous proposons une solution d'adaptation au niveau transport, qui s'avère efficace pour les handovers dans les réseaux hybrides satellites/terrestres et qui vient étayer nos résultats obtenus sur le segment satellite.

One of the most common risk factors for stroke is diabetes. Diabetics are 2 to 4 times more likely to have a stroke and are also significantly more likely to show poor functional recovery. In order to determine why diabetes is associated with poor stroke recovery, we tested the hypotheses that diabetes either exacerbates initial stroke damage, or inhibits neuronal circuit plasticity in surviving brain regions that is crucial for successful recovery. Type 1 diabetes was chemically induced in mice four weeks before receiving a targeted photothrombotic stroke in the right forelimb somatosensory cortex to model a chronic diabetic condition. Following stroke, a subset of diabetic mice were treated with insulin to determine if controlling blood glucose levels could improve stroke recovery. Consistent with previous studies, one behavioural test revealed a progressive improvement in sensory function of the forepaw in non-diabetic mice after stroke. By contrast, diabetic mice treated with and without insulin showed persistent deficits in sensori-motor forepaw function. To determine whether these different patterns of stroke recovery correlated with changes in functional brain activation, forepaw evoked responses in the somatosensory cortex were imaged using voltage sensitive dyes at 1 and 14 weeks after stroke. In both diabetic and non-diabetic mice that did not have a stroke, brief mechanical stimulation of the forepaw evoked a robust and near simultaneous depolarization in the primary (FLS1) and secondary somatosensory (FLS2) cortex. One week after stroke, forepaw-evoked responses had not been remapped in the peri-infarct cortex in both diabetic and non-diabetic mice. Fourteen weeks after stroke, forepaw evoked responses in non-diabetic mice re-emerged in the peri-infarct cortex whereas diabetic mice showed very little activation, reminiscent of the 1 week recovery group. Moreover, controlling hyperglycemia using insulin therapy failed to restore sensory evoked responses in the peri-infarct cortex. In addition to these differences in peri-infarct responsiveness, we discovered that stroke was associated with increased responsiveness in FLS2 of non-diabetic, but not diabetic or insulin treated mice. To determine the importance of FLS2 in stroke recovery, we silenced the FLS2 cortex and found that it re-instated behavioural impairments in stroke recovered mice, significantly more so than naïve mice that still had a functioning FLS1. Collectively, these results indicate that both diabetes and the secondary somatosensory cortex play an important role in determining the extent of functional recovery after ischemic cortical stroke. Furthermore, the fact that insulin therapy after stroke did not normalize functional recovery, suggests that prolonged hyperglycemia (before stroke) may induce pathological changes in the brain’s circulation or nervous system that cannot be easily reversed. / Graduate

Magnetic resonance imaging (MRI) has long become one of the most powerful and accurate tools of medical diagnostic imaging. Central to the diagnostic capabilities of MRI is the notion of contrast, which is determined by the biochemical composition of examined tissue as well as by its morphology. Despite the importance of the prevalent T₁, T₂, and proton density contrast mechanisms to clinical diagnosis, none of them has demonstrated effectiveness in delineating the morphological structure of the white matter - the information which is known to be related to a wide spectrum of brain-related disorders. It is only with the recent advent of diffusion-weighted MRI that scientists have been able to perform quantitative measurements of the diffusivity of white matter, making possible the structural delineation of neural fibre tracts in the human brain. One diffusion imaging technique in particular, namely high angular resolution diffusion imaging (HARDI), has inspired a substantial number of processing methods capable of obtaining the orientational information of multiple fibres within a single voxel while boasting minimal acquisition requirements. HARDI characterization of fibre morphology can be enhanced by increasing spatial and angular resolutions. However, doing so drastically reduces the signal-to-noise ratio. Since pronounced measurement noise tends to obscure and distort diagnostically relevant details of diffusion-weighted MR signals, increasing spatial or angular resolution necessitates application of the efficient and reliable tools of image denoising. The aim of this work is to develop an effective framework for the filtering of HARDI measurement noise which takes into account both the manifold to which the HARDI signal belongs and the statistical nature of MRI noise. These goals are accomplished using an approach rooted in non-local means (NLM) weighted averaging. The average includes samples, and therefore dependencies, from the entire manifold and the result of the average is used to deduce an estimate of the original signal value in accordance with MRI statistics. NLM averaging weights are determined adaptively based on a neighbourhood similarity measure. The novel neighbourhood comparison proposed in this thesis is one of spherical neighbourhoods, which assigns large weights to samples with similar local orientational diffusion characteristics. Moreover, the weights are designed to be invariant to both spatial rotations as well as to the particular sampling scheme in use. This thesis provides a detailed description of the proposed filtering procedure as well as experimental results with synthetic and real-life data. It is demonstrated that the proposed filter has substantially better denoising capabilities as compared to a number of alternative methods.

Made available in DSpace on 2016-08-17T18:39:44Z (GMT). No. of bitstreams: 1 4615.pdf: 2688296 bytes, checksum: 18c8fbe6fb83004856a32c02827d70f5 (MD5) Previous issue date: 2011-11-22 / Universidade Federal de Sao Carlos / Rabies is a zoonotic viral disease caused by a virus of the genus Lyssavirus that affects several species of mammals. Rabies remains a global public health threat that kills more than 55,000 people per year mainly in developing countries, this disease once established do not have a specific treatment. The RV envelope is composed of a glycoprotein, known as a unique antigen capable of conferring immune response against the rabies, and therefore, is the focus of research for development an efficient and safe recombinant vaccine based on this viral antigen. Cell line stably transfected S2 Drosophila melanogaster have been used in the production of many heterologous proteins and has been studied for the production of the rabies virus glycoprotein (RVGP) in our laboratory. This approach involves the selection of high producing cell populations; procedure that requires considerable periods of time (months), increasing management and costs of production. In this sense, in recent decades, many systems focused on the expression of heterologous proteins by transient expression of genes, were analyzed because they allow obtaining significant quantities of recombinant protein in a short period of time (weeks). For the use of transient transfection technology can be found a variety of methods and available agents, such as electroporation, cationic lipids, cationic polymers and calcium phosphate precipitated. The choice and optimization of each of them depends mainly on the cell type and protein being expressed. Thus, the objective of this study was to evaluate the transient expression of the glycoprotein gene of rabies virus (RVGP) in insect cells of Drosophila melanogaster S2 lineage, evaluating the vehicles transfection: calcium phosphate, cationic lipid (Cellfectin) and cationic polymer (ExGen500 and JetPEI). In order to determine the most efficient transfection agent, experiments were performed in 6 well plate and bottle of 100 mL of culture, which analyzed the influence of cell density, the concentration of DNA and transfection reagent volume on the expression of RVGP assessed by ELISA and fluorescence microscopy. Yields ranging from 50-90 ng/107cel were obtained in different experiments on multiwell plate, suggesting strong effect of ratio DNA: transfection agent used. Comparison of transfection agents showed no significant differences. In transfections made in suspension culture was analyzed the effect of the plasmid (whether or not the signal of BiP cell secretion) on the expression RVGP. When we used the plasmid containing the signal BiP (pMTiRVGP) were obtained 160 ng/107cel of RVGP production, and 200 ng/mL of volumetric production without significant differences between the different transfection agents. However, significant differences were found when we used the plasmid not containing the signal BiP (pMTRVGP), with the RVGP production was 60 ng/107cells in cells transfected with Cellfectin, ExGen500 and calcium phosphate, except in cells transfected with JetPEI was reached a production of 120 ng/107cells. In preliminary experiment bottle type "spinner" with a working volume of 60 mL were achieved expressions of 140 ng/107cel of RVGP in cells transfected with JetPEI and calcium phosphate. This suggests that optimization of culture conditions and transfection are possible to increase recombinant protein expression in cultured on a large scale. / A raiva é uma enfermidade causada por um vírus do gênero Lyssavirus que afeta várias espécies de mamíferos. Esta doença apresenta um alto custo social e econômico principalmente em países em desenvolvimento. Na superfície do vírus da raiva está localizada a glicoproteína do vírus, reconhecida como antígeno capaz de conferir resposta imunológica contra a raiva, sendo, o foco de pesquisas no desenvolvimento de uma vacina recombinante. Células da linhagem Drosophila melanogaster S2 estavelmente transfectadas têm sido usadas na produção de muitas proteínas heterólogas e tem sido estudada para a produção da glicoproteína do vírus da raiva (RVGP) em nosso laboratório. A abordagem para a obtenção de linhagens recombinantes estáveis envolve a seleção de populações celulares altamente produtoras; sendo um processo que requer consideráveis períodos de tempo (meses), uma elevada manipulação e altos custos de produção. Neste sentido, nas últimas décadas, muitos sistemas focados na expressão de proteínas heterólogas através da expressão transiente de genes foram analisados, porque eles permitem a obtenção de quantidades consideráveis de proteína recombinante em um curto período de tempo (semanas). Para o uso da tecnologia de transfecção transiente pode ser encontrada uma variedade de métodos e agentes disponíveis, tais como eletroporação, lipídeos catiônicos, polímeros catiônicos e fosfato de cálcio. Assim, o objetivo deste trabalho foi avaliar a expressão transiente do gene da glicoproteína do vírus da raiva (RVGP) em células de inseto da linhagem Drosophila melanogaster S2, avaliando os veículos de transfecção fosfato de cálcio, lipídeo catiônico (Cellfectin) e polímero catiônico (ExGen500 e JetPEI). A fim de determinar o agente de transfecção mais eficiente, foram feitos experimentos em placa de 6 poços e frasco de cultivo de 100mL, onde foram analisados a influência da densidade celular; a concentração de DNA, o volume do reagente de transfecção sobre a expressão da RVGP analisada através do método de ELISA. Quantidades de RVGP que variaram entre 50-90 ng/107cel foram obtidas nos diferentes experimentos feitos em placa, sugerindo um efeito da relação DNA: agente de transfecção. A comparação entre os agentes de transfecção não mostrou diferenças significativas. Nas transfecções feitas em cultura em suspensão foi analisado o efeito de transfectar o plasmídeo para expressão de RVGP contendo ou não o sinal de secreção celular BiP. Quando foi usado o plasmídeo contendo o sinal BiP (pMTiRVGP) foram atingidas valores de RVGP de 160 ng/107cel e produções volumétricas de 200 ng/mL, porém sem diferenças significativas entre os diferentes agentes de transfecção. Entretanto, foram encontradas diferenças quando foi usado o plasmídeo não contendo o sinal BiP (pMTRVGP), onde a produção de RVGP foi de 60 ng/107cells nas células transfectadas com Cellfectin, ExGen500 e fosfato de cálcio, porém as células transfectadas com JetPEI obtiveram uma produção de 120 ng/107cels de RVGP. Em experimento em frasco de cultivo tipo spinner com volume de trabalho de 60 mL, foram atingidas expressões de RVGP de 140 ng/107cel para células transfectadas com JetPEI e fosfato de cálcio, sugerindo que otimizações nas condições de cultivo e transfecção ainda podem ser testadas visando aumentar a expressão da proteína recombinante em cultivos em larga escala.

Made available in DSpace on 2016-06-02T19:56:29Z (GMT). No. of bitstreams: 1 1471.pdf: 1734309 bytes, checksum: e1a25902d99a75515b696dcd901f26a8 (MD5) Previous issue date: 2007-03-28 / Universidade Federal de Sao Carlos / In animal cell cultures there are two kinds of cell death: apoptosis (programmed cell death) and necrosis (cell death due to external sources). The inovative method of cell death identification that have attracted interest is the use of fluorescence microscopy and fluorescent dye (to bind DNA and RNA). Image analysis techniques has become a useful tool in cell death quantification, once they are non destructive for the culture and have easy application with using adjusted softwares. Based in these new technological trends, the present work considers the development of a methodology for quantification of apoptotic and necrotic cell death in cultures of insect cells Drosophila melanogaster (S2). This methodology involved the development of a experimental procedure using new dyes (YO-PRO-1 and Propidium Iodide), that have presented advantages over the others because they are non destructive to the culture an are able to clearly discriminate between apoptotic and necrotic cell death. The use of this dyes was compared with the method of exclusion of Trypan blue and fluorescent dyes Acridine Orange and Ethidium Bromide. Besides being less toxic to cells S2, YO-PRO-1 and Propidium Iodide showed more sensitivity in the identification of death and in the calculation of cell viability. It also envolved the development of an algorithm based on image analysis techniques for quantification of the two kinds of cell death, less subjective than the manual methods currently used. The algorithm showed to be efficient, fast an of easy application with a error around 2% when compared to manual methodology. The calculated cell concentration by the algorithm was very close to experimental. / Em cultivos de células animais existem dois tipos de morte celular: apoptose (morte celular programada) e necrose (morte devido a lesões causadas por fontes externas). O método inovador de identificação dessas mortes de células que tem atraído muito interesse é o uso de microscopia de fluorescência e corantes celulares fluorescentes capazes de tingir moléculas de DNA e RNA. Técnicas de análise de imagem têm se tornado uma ferramenta útil nessa quantificação de morte celular, uma vez que é não destrutiva para a cultura e de fácil aplicação com o uso de softwares adequados. Baseado nessas novas tendências tecnológicas, o presente trabalho teve como meta o desenvolvimento de uma metodologia para quantificação de morte apoptótica e necrótica no cultivo de células de inseto Drosophila melanogaster (S2). Essa metodologia envolveu o desenvolvimento de um procedimento experimental utilizando corantes novos no mercado: o YO-PRO-1 e o Iodeto de Propídio, que têm apresentado vantagens sobre os outros corantes por não serem destrutivos para a cultura e discriminarem claramente a morte apoptótica da necrótica. A metodologia que faz uso destes corantes foi comparada com o método de exclusão por Azul de Tripan e com os corantes fluorescentes Laranja de Acridina e Brometo de Etídio. Além de serem menos tóxicos as células, o YO-PRO-1 e o Iodeto de Propído mostraram mais sensibilidade na identificação da morte e posterior cálculo da viabilidade celular. A metodologia também envolveu o desenvolvimento de um algoritmo baseado em técnicas de análise de imagens para quantificação dos dois tipos de morte celular, menos subjetiva que os métodos manuais atualmente utilizados. O algoritmo se mostrou eficiente, rápido e de fácil aplicação, gerando um erro de 2% quando comparado com a metodologia manual. Também realizou-se o cálculo da concentração celular por método computacional, ficando os valores bem próximos dos experimentais.

Wood is a complex organic orthotropic viscoelastic material with acellular structure. When stressed, wood will deform over timethrough a process called creep. Creep affects all wooden structureand can be difficult, time-consuming and expensive to measure. For this thesis, a simple computer model of the woodenmicrostructure was developed. The hypothesis was that the modelledmicrostructure would display similar elastic and viscoelasticproperties as the macroscopic material. The model was designed by finding research with cell geometries ofconiferous trees measured. The model considered late- and earlywoodgeometries as well as growth rings. Rays were ignored as they onlycomposed 5-10% of the material. By applying a finite element method, the heterogeneous late- andearlywood cells could be homogenized by sequentially loading thestrain vector and calculating the average stress. The computer model produced stiff but acceptable values for theelastic properties. Using the standard linear solid method to modelviscoelasticity, the computer model assembled creep curvescomparable to experimental results. With the model sufficiently validated, parametric studies on thecell geometry showed that the elastic and viscoelastic propertieschanged greatly with cell shape. An unconventional RVE was alsotested and shown to give identical result to the standard RVE. Although not perfect, the model can to a certain degree predict theelastic and viscoelastic characteristics for wood given itscellular geometry. Inaccuracies were thought to be caused byassumptions and approximations when building the model.

This work aims at the creation of a~system of massive recording of multimedia data, especially speech data in various languages. The first issue is to find out high quality data source, the second is to build the system for managing and storing received data in the digital form. A digital satellite transmission is chosen as a signal source (DVB-S system). Main system features include recording of multiple streams in parallel, support of multiple cards, retrieving and storing of additional information (from Internet) and scheduling of recordings. The system will provide massive amounts of data for training of a language identification system.