The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Advanced image processing techniques for detection and quantification of drusen

Dissertation presented to obtain the degree of Doctor of Philosophy in Electrical Engineering, speciality on Perceptional Systems, by the Universidade Nova de Lisboa, Faculty of Sciences and Technology

Caracterização da aterosclerose sub-clínica e doença coronária em doentes com síndrome metabólica e diabetes mellitus

RESUMO- Introdução: A obesidade e a Síndrome Metabólica (SM) são atualmente um importante problema de saúde pública, com prevalências crescentes, que se acompanham também por aumento da prevalência de Diabetes Mellitus (DM).Estudos prévios demonstram associação destas entidades com o aumento de risco de eventos cardiovasculares, em particular a DM. A SM tem sido uma entidade muito debatida nos últimos anos, com aparecimento de diversas definições, contribuindo para resultados díspares no que diz respeito à influência da SM nas doenças cardiovasculares. Também têm sido descritas variações étnicas e regionais. Para além de alguns estudos epidemiológicos na população geral, a informação relativamente à sua influência na presença de doença cardiovascular é desconhecida em Portugal, em particular em populações com suspeita de doença coronária. Objetivos - Esclarecimento de questões relacionadas com a prevalência de SM e a sua influência na evolução de doença ateroclerótica arterial por avaliação de uma população com suspeita de doença coronária. População e Métodos - Estudo observacional, transversal, com inclusão prospetiva de indivíduos admitidos letivamente para realização de angiografia coronária por suspeita de doença coronária, tendo sido também efetuadas análises laboratoriais e ecografia carotidea para avaliação da espessura intima-média carotidea (EIMc) e da presença de placas carotídeas. Efetuou-se avaliação dos parâmetros demográficos, antropométricos, determinação do perfil lipídico, glicémia e insulinémia. Os exames angiográficos foram analisados por análise quantitativa semi-automática. Foram excluídos indivíduos com antecedentes conhecidos de doença cardíaca. Resultados - Incluíram-se 300 doentes, com idade média de 64 ± 9 anos, 59% do género masculino. A prevalência de SM de acordo com a definição da AHA/NHLBI foi 48,4% (ajustada para idade e género da população portuguesa) e a prevalência de DM foi 14,8% (ajustada). A concordância global das três definições mais recentes de SM foi de apenas 43%. A prevalência de SM aumenta com a idade e é também mais elevada no género feminino. O componente mais frequente foi a hipertensão arterial, seguido pela obesidade abdominal, a elevação da glicémia e por fim as alterações dos triglicéridos e do colesterol HDL. Por outro lado, a presença de doença coronária significativa (lesões ≥50%) ocorreu em apenas 51,3% dos doentes, sendo ainda mais baixa no género feminino. Demonstrou-se também uma baixa capacidade preditiva para doença coronária dos testes não invasivos clássicos, em particular no género feminino. A prevalência de doença coronária significativa foi idêntica nos indivíduos com SM comparativamente com indivíduos sem alterações metabólicas (46,3% vs. 48,2%, respectivamente), sendo mais elevado nos diabéticos (65,2%). Os fatores predizentes independentes de doença coronária significativa foram a idade, o género masculino, a elevação da glicémia e dos triglicéridos. Pelo contrário, o Índice de Massa Corporal (IMC) mostrou uma associação protetora relativamente à presença de doença coronária. A SM não é fator predizente de doença coronária. Relativamente às dimensões dos vasos coronários, o IMC correlaciona-se positivamente e a glicémia / DM correlacionam-se negativamente. A EIMc aumenta com o aumento da idade e no género masculino. A EIMc foi intermédia nos doentes com SM (0,88 ± 0,31 mm) comparativamente com os doentes diabéticos (0,97 ± 0,34 mm) e os indivíduos “Normais” (0,85 ± 0,34 mm). Os fatores predizentes independentes de EIMc foram a idade, o género masculino, o colesterol HDL e a insulinémia. A EIMc permite predizer com uma acuidade moderada a presença de doença coronária significativa (AUC 0,638), em particular no género feminino, sendo um fator predizente independente de presença de doença coronária (OR 2,35, IC 95% 1,04-5,33. p=0,04). Apesar de não se correlacionar com o número de vasos coronários com doença, correlacionou-se com a gravidade da doença (pelo score de Gensini). A insulinémia e o índice HOMA aumentam diretamente com a idade e com o IMC, sendo contudo sobreponíveis em ambos os géneros. Os fatores predizentes de índice HOMA (resistência à insulina) foram o IMC, bem como os restantes componentes de SM, estando o índice HOMA relacionado com a presença de SM e o número dos seus componentes presentes. O limiar para resistência à insulina foi de 2,66 e para SM foi 2,41. Ao contrário das restantes definições de SM, a definição da AHA/NHLBI não é predizente da presença de DM no género masculino. A associação da resistência à insulina com doença coronária foi limiar (OR 1,13, IC 95% 1,00-1,28, p=0,045). Conclusões - Numa população com suspeita de doença coronária, a prevalência de SM é muito elevada (superior a 50%), sendo a prevalência de DM de 23%. Também a obesidade e o excesso de peso foram extremamente prevalentes nesta população. A concordância entre definições de SM é baixa. A hipertensão arterial e a obesidade abdominal são os componentes mais frequentes de SM, sendo menos prevalentes as alterações lipídicas. Pelo contrário, a presença de doença coronária significativa foi muito baixa, em particular nas mulheres. A SM não se associou à presença de doença coronária significativa, estando esta mais dependente das alterações do metabolismo glicídico e dos triglicéridos, bem como de outros fatores de risco não modificáveis, nomeadamente a idade e o género. A EIMc da carótida comum e a presença de placas carotídeas é mais elevada nos indivíduos diabéticos, estando também ligeiramente aumentada nos doentes com SM, sendo os fatores predizentes de EIMc apenas a idade, o género, a hiperinsulinémia bem como os níveis baixos de colesterol HDL. A utilização da avaliação da EIMc na estratificação de risco pré-angiografia coronária, poderá ser útil no género feminino. A hiperinsulinémia e o índice HOMA (índice de resistência à insulina), estão relacionados com o IMC e consequentemente com a presença de obesidade, embora também se correlacione de forma independente com os outros componentes de SM. A resistência à insulina associou-se à presença de SM. Relativamente à capacidade preditiva da coexistência com DM, verificou-se associação com a definição da NCEP-ATP III e da IDF, contudo, a definição da AHA/NHLBI só foi predizente de DMnas mulheres. -------------ABSTRACT - Introduction: Obesity and Metabolic Syndrome (MS) are a major public health problem, with increasing prevalence, that follows the increase in diabetes prevalence. Previous studies showed an association of both entities with increased cardiovascular risk, particularly diabetes. MS has been debated in the last few years, with several definitions and different results when analysed the influence of MS on cardiovascular diseases. There are also some regional and ethnical variations. Beyond general population epidemiological studies, information about the influence on cardiovascular disease in Portugal is unknown, particularly in patients with suspected coronary disease. Objectives- To clarify several questions regarding the prevalence of MS and the influence in arterial atherosclerotic disease by evaluation of a population with suspected coronary artery disease. Population and Methods- Observational, cross-sectional study with prospective inclusion of individuals admitted electively for coronary angiography with suspicion of coronary artery disease. All individuals also performed laboratorial evaluation and carotid ultrasound to evaluate carotid intima-media thickness (cIMT) and carotid plaques. We also evaluated demographic, anthropometric parameters, lipid profile, blood glucose and blood insulin. Angiographic data was obtained by semi-automated quantitation. Individuals with previously known cardiac history were excluded from the study. Results- We included 300 individuals with a mean age of 64 ± 9 years, 59% males. MS prevalence according to AHA/NHLBI definition was 48.4% (adjusted for age and gender of the Portuguese population) and the adjusted prevalence of diabetes was 14.8%. Global agreement between the more recent three definitions of MS was only 43%. MS prevalence increases with age and is also higher in women. The most frequent components were hypertension and abdominal obesity, followed by elevated glucose and triglicerides and low HDL-cholesterol. Significant coronary artery disease (stenosis ≥50%) was present in only 51.3% of patients, being lower in females. Non-invasive tests also had a low predictive capacity, particularly in females. The prevalence of significant coronary disease was identical in patients with MS compared with normal metabolism individuals (46.3% vs. 48.2%, respectively), being higher in diabetics (65.2%). Independent predictive factors for coronary disease were age, male gender, high blood glucose and triglycerides. On the contrary, Body Mass Index (BMI) was a protective factor for coronary disease. MS wasn’t a predictor of coronary disease. BMI showed a positive correlation with coronary vessel diameter and glucose /diabetes had a negative correlation. CIMT increased with age and was higher in males. CIMT was intermediate in patients with MS (0.88 ± 0.31 mm) when compared to diabetic patients (0.97 ± 0.34 mm) and “Normal” individuals (0.85 ± 0.34 mm). Independent predictors for cIMT were age, male gender, HDL-cholesterol and insulin. CIMT had a moderate predictive accuracy for coronary disease (AUC 0,638), particularly in females and is an independent predictor of the presence of significant coronary disease (OR 2.35, 95% CI 1.04-5.33. p=0.04). Although it did not correlate with the number of diseased coronary arteries, it correlated with coronary disease severity by the Gensini score. Insulin and HOMA index increase directly with age and BMI, but were identical in both genders. Predictive factors for HOMA index (insulin resistance) were BMI as well as the other MS components. HOMA index is related to MS and the number of its components. The cut-off for insulin resistance was 2.66 and for MS 2.41. Unlike other MS definitions, AHA/NHLBI definition is not a predictor of diabetes in males. There was a borderline association between insulin resistance and coronary disease (OR 1.13, 95% CI 1.00-1.28, p=0.045). Conclusions - In a population of patients with suspected coronary disease, MS prevalence is extremely high (above 50%) with a diabetes prevalence of 23%. Also obesity and overweight are very prevalent in this population. Global agreement between MS definitions is however low. Hypertension and abdominal obesity are the most frequent components, with a lower prevalence of lipid abnormalities. Coronary disease prevalence was low, particularly in women. MS wasn’t associated with coronary disease. Coronary disease was related to glucose and triglycerides, as well as with other non-modifiable factors such as age and gender. CIMT and carotid plaques are increased in diabetic patients, and also slightly elevated in patients with MS, but cIMT independent predictors were age, male gender, insulin and HDLcholesterol. CIMT can be useful in risk stratification before coronary angiography particularly in women. Elevated insulin and HOMA index (an insulin resistance index) are related with BMI and consequently with obesity, and it was also correlated with other MS components. Insulin resistance was associated with MS. The presence of diabetes was associated with the presence of MS by NCEP-ATP III and IDF definitions; however, AHA/NHLBI definition was only predictive of diabetes in females.

Microscopy image segmentation by active contour models

In this thesis a semi-automated cell analysis system is described through image processing. To achieve this, an image processing algorithm was studied in order to segment cells in a semi-automatic way. The main goal of this analysis is to increase the performance of cell image segmentation process, without affecting the results in a significant way. Even though, a totally manual system has the ability of producing the best results, it has the disadvantage of taking too long and being repetitive, when a large number of images need to be processed. An active contour algorithm was tested in a sequence of images taken by a microscope. This algorithm, more commonly known as snakes, allowed the user to define an initial region in which the cell was incorporated. Then, the algorithm would run several times, making the initial region contours to converge to the cell boundaries. With the final contour, it was possible to extract region properties and produce statistical data. This data allowed to say that this algorithm produces similar results to a purely manual system but at a faster rate. On the other hand, it is slower than a purely automatic way but it allows the user to adjust the contour, making it more versatile and tolerant to image variations.

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.

Automated image tagging through tag propagation

Trabalho apresentado no âmbito do Mestrado em Engenharia Informática, como requisito parcial Para obtenção do grau de Mestre em Engenharia Informática

Declarative approach to data extraction of web pages

Thesis submitted to Faculdade de Ciências e Tecnologia of the Universidade Nova de Lisboa, in partial fulfilment of the requirements for the degree of Master in Computer Science

Automated Generation of Indoor Accessibility Information for Mobility-Impaired Individuals

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

A semi-automatic approach to code smells detection

Eradication of code smells is often pointed out as a way to improve readability, extensibility and design in existing software. However, code smell detection remains time consuming and error-prone, partly due to the inherent subjectivity of the detection processes presently available. In view of mitigating the subjectivity problem, this dissertation presents a tool that automates a technique for the detection and assessment of code smells in Java source code, developed as an Eclipse plugin. The technique is based upon a Binary Logistic Regression model that uses complexity metrics as independent variables and is calibrated by expert‟s knowledge. An overview of the technique is provided, the tool is described and validated by an example case study.

Semantic relations extraction from unstructured information for domain ontologies enrichment

Based in internet growth, through semantic web, together with communication speed improvement and fast development of storage device sizes, data and information volume rises considerably every day. Because of this, in the last few years there has been a growing interest in structures for formal representation with suitable characteristics, such as the possibility to organize data and information, as well as the reuse of its contents aimed for the generation of new knowledge. Controlled Vocabulary, specifically Ontologies, present themselves in the lead as one of such structures of representation with high potential. Not only allow for data representation, as well as the reuse of such data for knowledge extraction, coupled with its subsequent storage through not so complex formalisms. However, for the purpose of assuring that ontology knowledge is always up to date, they need maintenance. Ontology Learning is an area which studies the details of update and maintenance of ontologies. It is worth noting that relevant literature already presents first results on automatic maintenance of ontologies, but still in a very early stage. Human-based processes are still the current way to update and maintain an ontology, which turns this into a cumbersome task. The generation of new knowledge aimed for ontology growth can be done based in Data Mining techniques, which is an area that studies techniques for data processing, pattern discovery and knowledge extraction in IT systems. This work aims at proposing a novel semi-automatic method for knowledge extraction from unstructured data sources, using Data Mining techniques, namely through pattern discovery, focused in improving the precision of concept and its semantic relations present in an ontology. In order to verify the applicability of the proposed method, a proof of concept was developed, presenting its results, which were applied in building and construction sector.

Extraction automatique d´associations lexicales à partir de corpora

The automatic acquisition of lexical associations from corpora is a crucial issue for Natural Language Processing. A lexical association is a recurrent combination of words that co-occur together more often than expected by chance in a given domain. In fact, lexical associations define linguistic phenomena such as idiomes, collocations or compound words. Due to the fact that the sense of a lexical association is not compositionnal, their identification is fundamental for the realization of analysis and synthesis that take into account all the subtilities of the language. In this report, we introduce a new statistically-based architecture that extracts from naturally occurring texts contiguous and non contiguous. For that purpose, three new concepts have been defined : the positional N-gram models, the Mutual Expectation and the GenLocalMaxs algorithm. Thus, the initial text is fisrtly transformed in a set of positionnal N-grams i.e ordered vectors of simple lexical units. Then, an association measure, the Mutual Expectation, evaluates the degree of cohesion of each positional N-grams based on the identification of local maximum values of Mutual Expectation. Great efforts have also been carried out to evaluate our metodology. For that purpose, we have proposed the normalisation of five well-known association measures and shown that both the Mutual Expectation and the GenLocalMaxs algorithm evidence significant improvements comparing to existent metodologies.

Optimização da estrutura de um semi-reboque com vista à flexibilidade na sua adequação funcional

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Mecânica

Water treatments in semi-closed cooling circuits and their impact on the quality of effluents discharged by CERN

Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia do Ambiente, Gestão de Sistemas Ambientais

Water treatments in semi-closed cooling circuits and their impact on the quality of effluents discharged by CERN

Mestrado integrado em Engenharia do Ambiente, perfil: Gestão de Sistemas Ambientais

Células solares semi-transparentes de silício amorfo micro/nanocristalino

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Engenharia dos Materiais