Phylogeny and phenotypic characterization of pathogenic cryptococcus species and closely related saprobic taxa in the tremellales

Eukaryotic Cell, Vol.8, Nº3

Neural correlations during brain activation in arithmetical tasks – an approach using electroencephalographic data

Dissertação apresentada na Faculdade de Ciências e Tecnologiea da Universidade Nova de Lisboa, para obtenção do Grau de Mestre em Engenharia Biomédica

High resolution stratigraphy and miocene facies correlation in Lisbon and Setúbal Peninsula (Lower Tagus basin, Portugal)

Correlation between facies associations (marine, estuarine and distal fluviatile environments) and disconformities, observed between Foz da Fonte (SW of Setúbal Peninsula) and Santa Iria da Azóia (NE of Lisbon) are presented. The precise definition of the marine-continental facies relationships improved very much the chronology of the depositional sequence boundaries. Tectonic and eustatic controls are discussed on the basis of subsidence rates variation.

High-resolution correlation of coastal cliff sections in the Lagos- Portimao Formation (Lower - Middle Miocene, central Algarve, Portugal)

This paper describes a high-resolution stratigraphic correlation scheme for the early to middle Miocene Lagos-Portimão Formation of central Algarve, southern Portugal. The Lagos Portimão-Formation of central Algarve is a 60 m thick package of horizontally bedded siliciclastics and carbonates. The bryozoan and mollusc dominated biofacies is typical of a shallow marine, warm-temperate climatic environment. We define four stratigraphic marker beds based on biofacies, lithology, and gamma-ray signatures. Marker bed 1 is a reddish shell bed composed predominantly of bivalve shells in various stages of fragmentation. Marker bed 2 is a fossiliferous sandstone / sandy rudstone characterized by bryozoan masses. Marker bed 3 is also a fossiliferous sandstone with abundant larger foraminifers and foliate bryozoans. Marker bed 4 is composed of three distinct layers; two fossiliferous sandstones with an intercalated shell bed. The upper sandstone unit displays thickets of the bryozoan Celleporaria palmate associated with the coral Culizia parasitica. This stratigraphic framework allows to correlate isolated outcrops within the stratigraphic context of the Lagos-Portimão Formation and to establish high resolution chronostratigraphic Sr-isotopic dating.

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.

Design and performance evaluation of turbo FDE receivers

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

Role of ß-lactamase operon on mecA expression in Staphylococcus aureus

RESUMO: Os Staphylococcus aureus resistentes à meticilina (MRSA, do inglês “methicillin-resistant Staphylococcus aureus”) são um dos principais agentes responsáveis por infeções hospitalares. Os MRSA são resistentes a praticamente todos os antibióticos β-lactâmicos devido a dois mecanismos principais: produção de β-lactamase (bla), codificada pelo gene blaZ, e produção de uma proteína de ligação à penicilina (PBP2a, do inglês “penicillin binding protein 2”), codificada pelo gene mecA. Estes dois genes são regulados por sistemas homólogos, constituídos por um sensor-transdutor (BlaR1 e MecR1) e um repressor (BlaI e MecI), de tal modo que ambos os sistemas são capazes de co-regular os genes mecA e blaZ, embora com eficiências de indução muito diferentes. De facto, a indução mediada pelo sistema mecI-mecR1 é tão lenta que se acredita que este sistema não está funcional na maioria das estirpes MRSA. No entanto, dados recentes do nosso laboratório, demonstram a ausência de relação entre a presença do gene mecI e o nível de resistência à meticilina em estirpes MRSA epidémicas, e também que, o fenótipo de resistência da grande maioria das estirpes não é perturbado pela sobre-expressão em trans do repressor mecI. Curiosamente, as duas estirpes em que a expressão da resistência foi afetada pela sobre-expressão do mecI são negativas para o locus da β-lactamase, o que sugere que este locus pode interferir diretamente com a repressão do gene mecA mediada pelo MecI. Nesta tese de mestrado esta hipótese foi explorada usando estratégias de biologia molecular e ensaios fenotípicos da resistência aos -lactâmicos. Os resultados obtidos demonstram que a presença do plasmídeo nativo da β-lactamase não só anula a repressão mediada pelo MecI, como também aumenta o nível de resistência das estirpes parentais. Várias hipóteses foram então formuladas para explicar estas observações. Dados preliminares, em conjunto com evidências experimentais publicadas, sugerem que o BlaI forma hetero-dímeros com o MecI que, após a indução, são inativados eficientemente pelo BlaR1. Em conclusão, estes resultados apresentam novas perspetivas para o mecanismo de regulação do mecA e para uma nova importante função do operão da β-lactamase para o fenótipo das estirpes MRSA.-------------------ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and is also emerging in the community. MRSA is cross-resistant to virtually all β-lactam antibiotics and has acquired two main resistance mechanisms: production of β-lactamase (bla), coded by blaZ, and production of penicillin binding protein 2a (PBP2a), coded by mecA. Both genes are regulated by homologous sensor-transducers (BlaR1 and MecR1) and repressors (BlaI and MecI), and coregulation of mecA and blaZ by both systems has been demonstrated, although with remarkable different efficiencies. In fact, induction of mecA by mecI-mecR1 is so slow that it is believed it is not functional in most MRSA strains. However, recent data from our laboratory has unexpectedly demonstrated that not only there is no correlation between the presence of mecI gene and the resistance level in epidemic MRSA strains, but also that for most strains there were no significant changes on the resistance phenotype upon the mecI overexpression in trans. Interestingly, the two strains in which mecI overexpression affected the resistance expression were negative for the bla locus, suggesting that this locus may interfere directly with the MecI-mediated repression of mecA and account for those puzzling observations. In this master thesis we have explored this hypothesis using molecular biology strategies and phenotypic analysis of -lactam resistance. The data obtained demonstrate that the presence of a wild-type plasmid containing the bla locus not only disrupts the MecImediated repression, but also significantly enhances the expression of resistance. Several preliminary hypotheses were formulated to explain these observations and preliminary data, together with published evidence, support the working model that BlaI forms functional hetero-dimers with MecI, which upon induction are readily inactivated by BlaR1. These results provide new insights into the regulatory mechanism(s) of mecA and open new perspectives for the role of β-lactamase operon in the MRSA phenotype.

Correlation between the mechanical cycling behavior and microstructure in laser welded joints using NiTi memory shape alloys

Dissertação para obtenção do Grau de Mestre em Engenharia de Materiais

Spectral and coherence estimates on electroencephalogram recordings during arithmetical tasks

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

A solution for estimates in software development projects

The Corporate world is becoming more and more competitive. This leads organisations to adapt to this reality, by adopting more efficient processes, which result in a decrease in cost as well as an increase of product quality. One of these processes consists in making proposals to clients, which necessarily include a cost estimation of the project. This estimation is the main focus of this project. In particular, one of the goals is to evaluate which estimation models fit the Altran Portugal software factory the most, the organization where the fieldwork of this thesis will be carried out. There is no broad agreement about which is the type of estimation model more suitable to be used in software projects. Concerning contexts where there is plenty of objective information available to be used as input to an estimation model, model-based methods usually yield better results than the expert judgment. However, what happens more frequently is not having this volume and quality of information, which has a negative impact in the model-based methods performance, favouring the usage of expert judgement. In practice, most organisations use expert judgment, making themselves dependent on the expert. A common problem found is that the performance of the expert’s estimation depends on his previous experience with identical projects. This means that when new types of projects arrive, the estimation will have an unpredictable accuracy. Moreover, different experts will make different estimates, based on their individual experience. As a result, the company will not directly attain a continuous growing knowledge about how the estimate should be carried. Estimation models depend on the input information collected from previous projects, the size of the project database and the resources available. Altran currently does not store the input information from previous projects in a systematic way. It has a small project database and a team of experts. Our work is targeted to companies that operate in similar contexts. We start by gathering information from the organisation in order to identify which estimation approaches can be applied considering the organization’s context. A gap analysis is used to understand what type of information the company would have to collect so that other approaches would become available. Based on our assessment, in our opinion, expert judgment is the most adequate approach for Altran Portugal, in the current context. We analysed past development and evolution projects from Altran Portugal and assessed their estimates. This resulted in the identification of common estimation deviations, errors, and patterns, which lead to the proposal of metrics to help estimators produce estimates leveraging past projects quantitative and qualitative information in a convenient way. This dissertation aims to contribute to more realistic estimates, by identifying shortcomings in the current estimation process and supporting the self-improvement of the process, by gathering as much relevant information as possible from each finished project.

Análise da relação entre intensidade da actividade física realiada com a sensação subjectiva de esforço na população com idade igual ou superior a 75 anos

RESUMO: Objectivo: O objectivo geral do estudo é analisar a relação entre intensidade da actividade física (AF) realizada pela população idosa com 75 anos ou mais, com a respectiva sensação subjectiva de esforço (SSE). Introdução: Em Portugal, e de acordo o Instituto Nacional de Estatística, em 2011 cerca de 19% da população tem mais de 65 anos. Em 2050, as estimativas apontam que esta percentagem seja de 25%, ou seja, um quarto do país. O envelhecimento propicía um declínio físico e funcional, e para o retardar estão recomendados 150 minutos por semana de AF moderada. A forma como tem sido avaliada a AF tem por base um pressuposto de comparação com valores de gasto energético de uma população com uma média de 40 anos. É fundamental a realização de estudos que analisem a associação entre o nível de intensidade de AF com a SSE, para aferir se esta comparação é transponível para esta faixa etária. Metodologia: Trata-se de um estudo observacional analítico transversal, com uma amostra constituída por 39 participantes, com uma mediana 77,0 anos de idade (percentil 25: 76,0; percentil 75: 80,0), sendo a mínima 75 e a máxima 86 anos. As variáveis são a SSE e a intensidade de cada actividade realizada. O protocolo de avaliação foi constituído pelo questionário de caracterização sóciodemográfica, Diário de AF e a Escala de Borg RPE 6-20. Resultados: Existe uma associação negativa significativa entre a idade e o gasto energético total (rs = -0,365, p=0,022) e uma associação negativa, moderada e significativa entre a idade e a SSE reportada média (rs = -0,521, p=0,001) e máxima (rs = -0,465, p=0,003). Observou-se uma associação positiva e significativa entre a SSE máxima diária e o tempo total dedicado a actividades físicas (r=0,367 e p=0,022) e o gasto energético total (r=0,569, p<0,001). Verificou-se igualmente que quanto maior o gasto energético total diário, maior a média da SSE (r=0,411; p=0,009). Existe uma concordância moderada e significativa (Kappa=0,475; p<0,001) entre a sensação reportada pelo participante e a classificação atribuída em cada actividade pelo Compêndio de Actividades Físicas (Ainsworth et al, 1993). No entanto, com uma tendência para a subestimação dos MET’s atribuídos a cada actividade Conclusão: Os resultados do estudo sugerem que existe uma associação positiva e significativa entre intensidade da AF realizada e a SSE, aquando da realização da mesma actividade. Apesar desta associação ser moderada, este estudo sugere que a classificação que o Compêndio utiliza não é directamente aplicável à população idosa com 75 anos ou mais.-------------ABSTRACT: Purpose: The overall objective of the study is to analyze the relationship between intensity of physical activity performed by the elderly population with 75 years or more with their subjective feeling of effort. Background: In Portugal, according with data from Instituto Português de Estatística, in 2011 aproximately 19% of the population has more than 65 years old. In 2050, estimates suggest that this percentage will be 25%, which means a quarter of the country. The way in which physical activity has been evaluated is based on a comparing assumption with values of energy expenditure in a population with an average of 40 years. It is critical to conduct studies to examine the association between the level of intensity of physical activity with the subjective feeling of effort, to assess whether this comparison is applied to this age group. Methods: This is a cross sectional observational study with a sample of 39 participants, with a median age of 77.0 years (25th percentile: 76.0; 75th percentile: 80.0), with the minimum age 75 years and maximum age 86 years. The variables are the subjective feeling of physical effort and intensity of each activity. It was applied one protocol evaluation consisting of a questionnaire of sociodemographic characteristics, A journal of physical activities and Borg RPE 6-20 scale. Results: There is a significant negative association between age and total energy expenditure (rs = -0.365, p = 0.022) and a negative, moderate and significant association between age and average (rs = -0.521, p = 0.001) and maximum subjective feeling of exertion reported (rs = -0.465, p = 0.003). There was a significant positive association between the subjective feeling of daily maximum effort and total time spent on physical activity (r = 0.367 and p = 0.022) and total energy expenditure (r = 0.569, p <0.001). It was also found that the higher the total daily energy expenditure, the higher the average of the subjective sense of effort (r = 0.411, p = 0.009). There is a moderate and significant correlation (kappa = 0.475, p <0.001) between the sensation reported by the participant and the rank assigned to each activity by Compendium of Physical Activities (Ainsworth et al, 1993). However, there is a tendency to over-estimate the subjective feeling of effort, or underestimation of METs assigned to each activity Conclusion: Study results suggest a positive and significant association between the level of intensity of physical activity and the subjective feeling of exertion, within the same activity. Even though this association is moderated, this study suggests that the Compendium of Physical Activities rating doesn’t suit for individuals with 75 years old or more.