The role of electron transfer in DNA building blocks: evaluation of strand breaks and their implications

Dissertação para obtenção do Grau de Doutor em Engenharia Física

Coherence and phase locking disruption in electromyograms of patients with amyotrophic lateral sclerosis

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica

Directed Giving: Evidence from an Inter-Household Transfer Experiment*

We investigate the determinants of giving in a lab-in-the-field experiment with large stakes. Study participants in urban Mozambique play dictator games where their counterpart is the closest person to them outside their household. Dictators share more with counterparts when they have the option of giving in kind (in the form of goods), compared to giving that must be in cash. Qualitative post-experiment responses suggest that this effect is driven by a desire to control how recipients use gifted resources. Standard economic determinants such as the rate of return to giving and the size of the endowment also affect giving, but the effects of even large changes in these determinants are significantly smaller than the effect of the in-kind option. Our results support theories of giving where the utility of givers depends on the composition (not just the level) of gift-recipient expenditures, and givers thus seek control over transferred resources.

Electron Transfer and Ligand Binding Properties of Cytochromes

Dissertation presented to obtain the Ph.D degree in Biochemistry

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.

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.

8-Phase Ring oscillator for modern receivers

The evolution of receiver architectures, built in modern CMOS technologies, allows the design of high efficient receivers. A key block in modern receivers is the oscillator. The main objective of this thesis is to design a very low power and low area 8-Phase Ring Oscillator for biomedical applications (ISM and WMTS bands). Oscillators with multiphase outputs and variable duty cycles are required. In this thesis we are focused in 12.5% and 50% duty-cycles approaches. The proposed circuit uses eight inverters in a ring structure, in order to generate the output duty cycle of 50%. The duty cycle of 1/8 is achieved through the combination of the longer duty cycle signals in pairs, using, for this purpose, NAND gates. Since the general application are not only the wireless communications context, as well as industrial, scientific and medical plans, the 8-Phase Oscillator is simulated to be wideband between 100 MHz and 1 GHz, and be able to operate in the ISM bands (447 MHz-930 MHz) and WMTS (600 MHz). The circuit prototype is designed in UMC 130 nm CMOS technology. The maximum value of current drawn from a DC power source of 1.2 V, at a maximum frequency of 930 MHz achieved, is 17.54 mA. After completion of the oscillator layout studied (occupied area is 165 μm x 83 μm). Measurement results confirm the expected operating range from the simulations, and therefore, that the oscillator fulfil effectively the goals initially proposed in order to be used as Local Oscillator in RF Modern Receivers.

Relatório de estágio na Federação Portuguesa de Futebol

This report aims to present and describe in detail the internship held in the Legal Division of the Portuguese Football Federation (FPF) from February, 15th to June, 15th 2015, in order to obtain a Master degree in Law. This internship had, as prior objective, to understand the work of the Legal Division and what its role in the organizational structure of FPF. Initially, and in order to acquaintance with the greatest diversity possible of topics on which the Legal Division has to pronounce, we proceed with the analysis and resolution of several questions that have been presented to us. In a second phase, and after deepen the study of a particular subject - the Intermediate – we sought to clarify the procedure that needed to be adopted after the entry into force of the two new Regulations on the matter - the FIFA Regulations and the FPF Regulations. The transfer of the responsibility for regulation of this matter from FIFA to the National Federations, has raised a new challenge, which this report aims help solving, becoming a quasi-instruction manual on the procedures involving intermediaries, in order to facilitate the role of those involved.

The role of crowdfunding in promoting entrepreneurship

Crowdfunding is a collaborative initiative, usually via internet, where people network to collectively raise funds in order to invest in and support projects delivered by other people or organizations. Tools such as crowdfunding are born and thrive in a grassroots environment, with a strong potential to positively disrupt the entrepreneurial generation setting and grow to a position of significant relevance in society, namely at a time when alternatives to traditional forms of finance are welcome and the technology to deliver them is abundant. Entrepreneurship is the act of transforming ideas and projects into economic products or services. Entrepreneurship related to starting new businesses is better known as start‐up ventures. Entrepreneurs face a series of challenges, from idea conception and business plan design, to obtaining finance, promoting new products and services, generating revenues and profits and generally growing and sustaining a business for the long‐run. These challenges can be overwhelming, namely in the start‐up phase of a new venture, leaving several ideas on paper without them having a chance to “grow legs and walk”. This paper and its analysis offer important insights about the contribution of crowdfunding to facilitate the attainment of critical factors for successful entrepreneurship. With extensive use of real practical examples, leveraging previous analytical studies of other crowdfunding implications and reviewing expert literature, by interviewing entrepreneurs, crowdfunding platform owners and by benefitting from hands on experience of working in such an organization, we intend to clarify the impact of crowdfunding in what we considered to be 7 key entrepreneurial requirements detailed further in the introduction section and later in the body of the paper. The findings have implications for entrepreneurs, naturally, and for business generation theory, extending current entrepreneurial guidelines with innovative tools and methodologies capable of sustaining successful ventures in a newly highlighted cooperative world. We live in innovative times where the channels for the transfer of funds and resources suffer disruptive changes with the potential to significantly improve the ability to generate new initiatives for the well‐being of entrepreneurs and all related communities.