Electron Transfer and Ligand Binding Properties of Cytochromes

Dissertation presented to obtain the Ph.D degree in Biochemistry

Implementing the SC-FDMA transmission technique using the GNURadio platform

Dissertação apresentada para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Optimization of breast tomosynthesis image reconstruction using parallel computing

Breast cancer is the most common cancer among women, being a major public health problem. Worldwide, X-ray mammography is the current gold-standard for medical imaging of breast cancer. However, it has associated some well-known limitations. The false-negative rates, up to 66% in symptomatic women, and the false-positive rates, up to 60%, are a continued source of concern and debate. These drawbacks prompt the development of other imaging techniques for breast cancer detection, in which Digital Breast Tomosynthesis (DBT) is included. DBT is a 3D radiographic technique that reduces the obscuring effect of tissue overlap and appears to address both issues of false-negative and false-positive rates. The 3D images in DBT are only achieved through image reconstruction methods. These methods play an important role in a clinical setting since there is a need to implement a reconstruction process that is both accurate and fast. This dissertation deals with the optimization of iterative algorithms, with parallel computing through an implementation on Graphics Processing Units (GPUs) to make the 3D reconstruction faster using Compute Unified Device Architecture (CUDA). Iterative algorithms have shown to produce the highest quality DBT images, but since they are computationally intensive, their clinical use is currently rejected. These algorithms have the potential to reduce patient dose in DBT scans. A method of integrating CUDA in Interactive Data Language (IDL) is proposed in order to accelerate the DBT image reconstructions. This method has never been attempted before for DBT. In this work the system matrix calculation, the most computationally expensive part of iterative algorithms, is accelerated. A speedup of 1.6 is achieved proving the fact that GPUs can accelerate the IDL implementation.

State detection in a financial portfolio: a self-organizing maps approach for financial time series

This study analyses financial data using the result characterization of a self-organized neural network model. The goal was prototyping a tool that may help an economist or a market analyst to analyse stock market series. To reach this goal, the tool shows economic dependencies and statistics measures over stock market series. The neural network SOM (self-organizing maps) model was used to ex-tract behavioural patterns of the data analysed. Based on this model, it was de-veloped an application to analyse financial data. This application uses a portfo-lio of correlated markets or inverse-correlated markets as input. After the anal-ysis with SOM, the result is represented by micro clusters that are organized by its behaviour tendency. During the study appeared the need of a better analysis for SOM algo-rithm results. This problem was solved with a cluster solution technique, which groups the micro clusters from SOM U-Matrix analyses. The study showed that the correlation and inverse-correlation markets projects multiple clusters of data. These clusters represent multiple trend states that may be useful for technical professionals.

Cellulose nanorods in liquid crystalline elastomers for improved actuators

Nanotechnology plays a central role in ‘tailoring’ materials’ properties and thus improving its performances for a wide range of applications. Coupling nature nano-objects with nanotechnology results in materials with enhanced functionalities. The main objective of this master thesis was the synthesis of nanocrystalline cellulose (NCCs) and its further incorporation in a cellulosic matrix, in order to produce a stimuli-responsive material to moisture. The induced behaviour (bending/unbending) of the samples was deeply investigated, in order to determine relationships between structure/properties. Using microcrystalline cellulose as a starting material, acid hydrolysis was performed and the NCC was obtained. Anisotropic aqueous solutions of HPC and NCC were prepared and films with thicknesses ranging from 22μm to 61μm were achieved, by using a shear casting technique. Microscopic and spectroscopic techniques as well as mechanical and rheological essays were used to characterize the transparent and flexible films produced. Upon the application of a stimulus (moisture), the bending/unbending response times were measured. The use of NCC allowed obtaining films with response times in the order of 6 seconds for the bending and 5 seconds for the unbending, improving the results previously reported. These promising results open new horizons for building up improved soft steam engines.

Melanosome transfer between melanocytes and keratinocytes

RESUMO: A pele é o maior órgão do corpo humano e a sua pigmentação é essencial para a sua coloração e proteção contra os efeitos nocivos da radiação ultravioleta (UV). A pigmentação da pele resulta essencialmente de três processos: a síntese e o armazenamento de melanina pelos melanócitos, em organelos especializados denominados melanossomas; o transporte dos melanossomas dentro dos melanócitos; e finalmente, a transferência dos melanossomas para os queratinócitos adjacentes. Nos queratinócitos, a melanina migra para a região perinuclear apical da célula para formar um escudo protetor,responsável pela proteção do DNA dos danos causados pela radiação UV. Os melanócitos estão localizados na camada basal da epiderme e contactam com 30-40 queratinócitos. Em conjunto, estas células formam a “unidade melano-epidérmica”. Apesar dos processos de síntese e transporte de melanina nos melanócitos estarem bastante bem caracterizados, os mecanismos moleculares subjacentes à transferência inter-celular de melanina são menos conhecidos e ainda controversos. Dados preliminares obtidos pelo nosso grupo, que se basearam na observação de amostras de pele humana por microscopia electrónica, indicam que a forma predominante de transferência de melanina na epiderme consiste na exocitose dos melanossomas pelos melanócitos e subsequente endocitose da melanina por queratinócitos. Para além disso sabe-se que as proteínas Rab, que controlam o tráfego membranar, estão envolvidas em várias etapas de pigmentação da pele, nomeadamente na biogénese e no transporte de melanina. Assim, dado o seu papel fundamental nestes processos, questionámo-nos sobre o seu envolvimento na transferência de melanina. Com este trabalho, propomo-nos a expandir o conhecimento atual sobre a transferência de melanina na pele, através do estudo detalhado dos seus mecanismos moleculares, identificando as proteínas Rab que regulam o processo. Pretendemos também confirmar o modelo de exo/endocitose como sendo o mecanismo principal de transferência de melanina. Primeiro, explorámos a regulação da secreção de melanina pelos melanócitos e analisámos o papel de proteínas Rab neste processo. Os resultados foram obtidos recorrendo a um método in vitro, desenvolvido previamente no laboratório, que avalia a quantidade de melanina segregada para o meio de cultura por espectrofotometria, e ainda por microscopia, contando o número de melanossomas transferidos para os queratinócitos. Através de co-culturas de melanócitos e queratinócitos, verificou-se que os queratinócitos estimulam a libertação de melanina dos melanócitos para o meio extra-celular, bem como a sua transferência para os queratinócitos. Além disso, a proteína Rab11b foi identificada como um regulador da exocitose de melanina e da sua transferência para os queratinócitos. De facto, a diminuição da expressão de Rab11b em melanócitos provocou a redução da secreção de melanina estimulada por queratinócitos, bem como da transferência desta. Em segundo lugar, para complementar o nosso estudo, centrámos a nossa investigação na internalização de melanina por queratinócitos. Especificamente, usando uma biblioteca de siRNA, explorámos o envolvimento de proteínas Rab na captação de melanina por queratinócitos. Como primeira abordagem, usámos esferas fluorescentes como substituto de melanina, avaliando os resultados por citometria de fluxo. No entanto, este método revelou-se ineficaz uma vez que a internalização destas esferas é independente do recetor PAR-2 (recetor 2 ativado por protease), que foi previamente descrito como essencial na captação de melanina por queratinócitos Posteriormente, foi desenvolvido um novo protocolo de endocitose baseado em microscopia, usando melanossomas sem a membrana envolvente (melanocores) purificados do meio de cultura de melanócitos, incluindo um programa informático especialmente desenhado para realizar uma análise semi-automatizada. Após internalização, os melanocores acumulam-se na região perinuclear dos queratinócitos, em estruturas que se assemelham ao escudo supranuclear observado na pele humana. Seguidamente, o envolvimento do recetor PAR-2 na captação de melanocores por queratinócitos foi confirmado, utilizando o novo protocolo de endocitose desenvolvido. Para além disso, a necessidade de quatro proteínas Rab foi identificada na internalização de melanocores por queratinócitos. A redução da expressão de Rab1a ou Rab5b em queratinócitos diminuiu significativamente o nível de internalização de melanocores, enquanto o silenciamento da expressão de Rab2a ou Rab14 aumentou a quantidade de melanocores internalizados por estas células. Em conclusão, os resultados apresentados corroboram as observações anteriores, obtidas em amostras de pele humana, e sugerem que o mecanismo de transferência predominante é a exocitose de melanina pelos melanócitos, induzida por queratinócitos, seguida por endocitose pelos queratinócitos. A pigmentação da pele tem implicações tanto ao nível da cosmética, como ao nível médico, relacionadas com foto-envelhecimento e com doenças pigmentares. Assim sendo, ao esclarecer quais os mecanismos moleculares que regulam a transferência de melanina na pele, este trabalho pode conduzir ao desenvolvimento de novas estratégias para modular a pigmentação da pele.----------------ABSTRACT: Skin pigmentation is achieved through the highly regulated production of the pigment melanin in specialized organelles, termed melanosomes within melanocytes. These are transported from their site of synthesis to the melanocyte periphery before being transferred to keratinocytes where melanin forms a supra-nuclear cap to protect the DNA from UVinduced damage. Together, melanocytes and keratinocytes form a functional complex, termed “epidermal-melanin unit”, that confers color and photoprotective properties to the skin. Skin pigmentation requires three processes: the biogenesis of melanin; its intracelular transport within the melanocyte to the cell periphery; and the melanin transfer to keratinocytes. The first two processes have been extensively characterized. However, despite significant advances that have been made over the past few years, the mechanisms underlying inter-cellular transfer of pigment from melanocytes to keratinocytes remain controversial.Preliminary studies from our group using electron microscopy and human skin samples found evidence for a mechanism of coupled exocytosis-endocytosis. Rab GTPases are master regulators of intracellular trafficking and have already been implicated in several steps of skin pigmentation. Thus, we proposed to explore and characterize the molecular mechanisms of melanin transfer and the role of Rab GTPases in this process. Moreover, we investigated whether the exo/endocytosis model is the main mechanism of melanin transfer. We first focused on melanin exocytosis by melanocytes. Then, we started to investigate the key regulatory Rab proteins involved in this step by establishing an in vitro tissue culture model of melanin secretion. Using co-cultures of melanocytes and keratinocytes, we found that keratinocytes stimulate melanin release and transfer. Moreover, depletion of Rab11b decreases keratinocyte-induced melanin exocytosis by melanocytes. In order to determine whether melanin exocytosis is a predominant mechanism of melanin transfer, the amount of melanin transferred to keratinocytes was then assayed in conditions where melanin exocytosis was inhibited. Indeed, Rab11b depletion resulted in a significant decrease in melanin uptake by keratinocytes. Taken together, these observations suggest that Rab11b mediates melanosome exocytosis from melanocytes and transfer to keratinocytes. To complement and extend our study, we of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptakecentred our attention in the internalization of melanin by keratinocytes. Thus, we aimed to explore the effect of depleting Rab GTPases on melanin uptake and trafficking within keratinocytes. As a first approach, we used fluorescent microspheres as a melanin surrogate. However, the uptake of microspheres was observed to be independent of PAR-2, a receptor that is required for melanin uptake.Therefore, we concluded that microspheres were uptaken by keratinocytes through a different pathway than melanin. Subsequently, we developed a microscopy-based endocytosis assay using purified melanocores (melanosomes lacking the limiting membrane) from melanocytes, including a program to perform a semi-automated analysis. Melanocores are taken up by keratinocytes and accumulate in structures in the perinuclear area that resemble the physiological supranuclear cap observed in human skin. We then confirmed the involvement of PAR-2 receptor in the uptake of melanocores by keratinocytes, using the newly developed assay. Furthermore, we identified the role of four Rab GTPases on the uptake of melanocores by keratinocytes. Depletion of Rab1a and Rab5b from keratinocytes significantly reduced the uptake of melanocores, whereas Rab2a, and Rab14 silencing increased the amount the melanocores internalized by XB2 keratinocytes. In conclusion, we present evidence supporting keratinocyte-inducedmelanosome exocytosis from melanocytes, followed by endocytosis of the melanin core by keratinocytes as the predominant mechanism of melanin transfer in skin. Although advances have been made, there is a need for more effective and safer therapies directed at pigmentation disorders and also treatments for cosmetic applications. Hence, the understanding of the above mechanisms of skin pigmentation will lead to a greater appreciation of the molecular machinery underlying human skin pigmentation and could interest the pharmaceutical and cosmetic industries.

A built-in self-test technique for high speed analog-to-digital converters

Fundação para a Ciência e a Tecnologia (FCT) - PhD grant (SFRH/BD/62568/2009)

The system of Transfer Pricing in a multinational

Transfer prices are used by the majority of firms worldwide when intermediate products or services are transferred within the same organization. These prices are reported as revenue for the selling entity (division, unit, department etc.) and as cost for the buying entity. Nevertheless, transfer prices lead to many disputes among managers in the same organization as transfer prices influence the performance of their entities. In cross-border transactions, transfer prices can be used by firms to reduce corporate taxes and thus, increase total firm profits. In order to fight against this firms’ practice, tax authorities require firms to establish a transfer pricing system in accordance with OECD1 Transfer Pricing Guidelines.