Codon ambiguities as a mechanism to alter the genetic code in Saccharomyces cerevisiae

Although the genetic code is generally viewed as immutable, alterations to its standard form occur in the three domains of life. A remarkable alteration to the standard genetic code occurs in many fungi of the Saccharomycotina CTG clade where the Leucine CUG codon has been reassigned to Serine by a novel transfer RNA (Ser-tRNACAG). The host laboratory made a major breakthrough by reversing this atypical genetic code alteration in the human pathogen Candida albicans using a combination of tRNA engineering, gene recombination and forced evolution. These results raised the hypothesis that synthetic codon ambiguities combined with experimental evolution may release codons from their frozen state. In this thesis we tested this hypothesis using S. cerevisiae as a model system. We generated ambiguity at specific codons in a two-step approach, involving deletion of tRNA genes followed by expression of non-cognate tRNAs that are able to compensate the deleted tRNA. Driven by the notion that rare codons are more susceptible to reassignment than those that are frequently used, we used two deletion strains where there is no cognate tRNA to decode the rare CUC-Leu codon and AGG-Arg codon. We exploited the vulnerability of the latter by engineering mutant tRNAs that misincorporate Ser at these sites. These recombinant strains were evolved over time using experimental evolution. Although there was a strong negative impact on the growth rate of strains expressing mutant tRNAs at high level, such expression at low level had little effect on cell fitness. We found that not only codon ambiguity, but also destabilization of the endogenous tRNA pool has a strong negative impact in growth rate. After evolution, strains expressing the mutant tRNA at high level recovered significantly in several growth parameters, showing that these strains adapt and exhibit higher tolerance to codon ambiguity. A fluorescent reporter system allowing the monitoring of Ser misincorporation showed that serine was indeed incorporated and possibly codon reassignment was achieved. Beside the overall negative consequences of codon ambiguity, we demonstrated that codons that tolerate the loss of their cognate tRNA can also tolerate high Ser misincorporation. This raises the hypothesis that these codons can be reassigned to standard and eventually to new amino acids for the production of proteins with novel properties, contributing to the field of synthetic biology and biotechnology.

Identification of molecules and pathways regulating mistranslation in Candida albicans

Candida albicans is the major fungal pathogen in humans, causing diseases ranging from mild skin infections to severe systemic infections in immunocompromised individuals. The pathogenic nature of this organism is mostly due to its capacity to proliferate in numerous body sites and to its ability to adapt to drastic changes in the environment. Candida albicans exhibit a unique translational system, decoding the leucine-CUG codon ambiguously as leucine (3% of codons) and serine (97%) using a hybrid serine tRNA (tRNACAGSer). This tRNACAGSer is aminoacylated by two aminoacyl tRNA synthetases (aaRSs): leucyl-tRNA synthetase (LeuRS) and seryl-tRNA synthetase (SerRS). Previous studies showed that exposure of C. albicans to macrophages, oxidative, pH stress and antifungals increases Leu misincorporation levels from 3% to 15%, suggesting that C. albicans has the ability to regulate mistranslation levels in response to host defenses, antifungals and environmental stresses. Therefore, the hypothesis tested in this work is that Leu and Ser misincorporation at CUG codons is dependent upon competition between the LeuRS and SerRS for the tRNACAGSer. To test this hypothesis, levels of the SerRS and LeuRS were indirectly quantified under different physiological conditions, using a fluorescent reporter system that measures the activity of the respective promoters. Results suggest that an increase in Leu misincorporation at CUG codons is associated with an increase in LeuRS expression, with levels of SerRS being maintained. In the second part of the work, the objective was to identify putative regulators of SerRS and LeuRS expression. To accomplish this goal, C. albicans strains from a transcription factor knock-out collection were transformed with the fluorescent reporter system and expression of both aaRSs was quantified. Alterations in the LeuRS/SerRS expression of mutant strains compared to wild type strain allowed the identification of 5 transcription factors as possible regulators of expression of LeuRS and SerRS: ASH1, HAP2, HAP3, RTG3 and STB5. Globally, this work provides the first step to elucidate the molecular mechanism of regulation of mistranslation in C. albicans.

Temperature, pH, and conductivity control of the nutrient solution of an hydroponic greenhouse

One of the aspects of modern agriculture is characterised by a culture without soil (hydroponic cultures). These culture techniques are identified by possessing automatic control systems to control the nutrient solution. In first hydroponic cultures this control was accomplished by “on- off” analog controllers that applied a single control law implemented in hardware. Therefore, the changes of the control law resulted in the change of all interface electronics. In digital control implemented by micro-controllers the alteration of such control law is easily performed by changing only a computer program, leaving untouched all the interface hardware. In this way, the use and substitution of the control strategy is improved, as well, the use of advanced control strategies.

Genesis of the brecciated rocks from mid atlantic ridge hydrothermal systems : Lucky Strike (37º50’N) and Menez Gwen (37º50’N)

Tese de doutoramento, Geologia (Metalogenia), Universidade de Lisboa, Faculdade de Ciências, 2014

Selection and combination of biomarkers to control and prevent the risk of occupational exposure to the mixture of lead, arsenic and manganese

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2014

A construção do espaço no desenho

Tese de doutoramento, Belas-Artes (Desenho), Universidade de Lisboa, Faculdade de Belas-Artes, 2015

Molecular features of glycine-mediated neurotransmission in rat brain

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2015

Contributo da geoquímica e da sedimentologia na caracterização de influências antropogénicas em ambientes estuarinos

Tese de doutoramento, Geologia (Geoquímica), Universidade de Lisboa, Faculdade de Ciências, 2016

Educational characteristics of adult students in portuguese technological schools

Despite a massive expansion of education in Portugal, since the 1970’s, educational attainment of the adult population in the country remains low. The numbers of working-age people in some form of continuing education are among the lowest, according to the OECD and EU-27 statistics. Technological Schools(TS), initially created in the 1990’s, under the umbrella of the Ministry of Economy in partnership with industry and industrial associations, aimed to prepare qualified staff for industries and services in the country, particularly in the engineering sector, through the provision of post secondary non-university programmes of studies, the CET (Technological Specialization Courses). Successful CET students are awarded a DET(Diploma of Technological Specialization), which corresponds to Vocational Qualification level IV of the EU, according to the latest alteration (2005) of the Education Systems Act (introduced in 1986). In this, CET’s are also clearly defined as one of the routes for access to Higher Education (HE), in Portugal. The PRILHE (Promoting Reflective and Independent Learning in Higher Education) multinational project, funded by the European Socrates Grundtvig Programme, aimed to identify the learning processes which enable adult students in higher education to become autonomous reflective learners and search best practices to support these learning processes. During this research, both quantitative and qualitative methods were used to determine how students organise their studies and develop their learning skills. The Portuguese partner in the project’ consortium used a two case studies approach, one with students of Higher Education Institutions and other with students of TS. This paper only applies to students of TS, as these have a predominant bias towards engineering. Results show that student motivation and professional teaching support contribute equally to the development of an autonomous and reflective approach to learning in adult students; this is essential for success in a knowledge economy, where lifelong learning is the key to continuous employment.

O tratamento dos activos biológicos nas microentidades

Dissertação para a obtenção do Grau de Mestre em Contabilidade e Finanças Orientador: Dr. Paulo Filipe Teixeira Aguiar

ß-Lactams: chemical structure, mode of action and mechanisms of resistance

This synopsis summarizes the key chemical and bacteriological characteristics of β-lactams, penicillins, cephalosporins, carbanpenems, monobactams and others. Particular notice is given to first-generation to fifth-generation cephalosporins. This review also summarizes the main resistance mechanism to antibiotics, focusing particular attention to those conferring resistance to broad-spectrum cephalosporins by means of production of emerging cephalosporinases (extended-spectrum β-lactamases and AmpC β-lactamases), target alteration (penicillin-binding proteins from methicillin-resistant Staphylococcus aureus) and membrane transporters that pump β-lactams out of the bacterial cell.

Acompanhamento da III fase do aterro sanitário da Meia Serra

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Civil na Área de Especialização de Hidráulica

Contribuição para o conhecimento das propriedades pozolânicas da fracção fina resultante da produção de areia de origem granítica

O presente estudo diz respeito a um trabalho de pesquisa no âmbito de uma Tese de Mestrado incluída no segundo ciclo de estudos do curso de Engenharia Geotécnica e Geoambiente, realizado sobre as condições de desidroxilação para a obtenção de metacaulino com propriedades cimentíceas, a partir da fracção argilosa proveniente dos finos residuais da produção de areias de natureza granítica. O produto resultante da alteração e desintegração dos feldspatos constituintes dos granitos são ricos em caulinite. Na natureza e em particular no Norte de Portugal, existem significativos depósitos cauliníticos com características potenciadoras para a produção de metacaulino. O metacaulino utilizado neste estudo foi obtido de uma amostra de argila submetida a 750oC, por um período de tempo de 30 minutos, processo que permitiu a desidroxilação quase total da matéria-prima, transformando esta numa fase amorfa e irreversível, com propriedades pozolânicas. Os metacaulinos, também conhecidos por geopolímeros, são produtos de fácil produção utilizando uma matéria-prima abundante e proporcionam a obtenção de novos produtos que permitem a substituição parcial do cimento Portland normal na composição das pastas de betão, com vantagens significativas no comportamento mecânico e na resistência aos agentes atmosféricos. Neste estudo são apresentados os resultados dos ensaios de caracterização da matéria-prima, das condições de calcinação e do produto resultante da desidroxilação, nomeadamente a determinação da pozolanicidade e das características fundamentais para a aplicabilidade do produto. No âmbito da especialidade de Georrecursos, consideramos que este trabalho está perfeitamente adequado, já que, para além do estudo para o conhecimento das propriedades da matéria-prima, foi possível, através das alterações introduzidas com o tratamento térmico, obter um novo produto, cuja utilização terá importantes reflexos na sustentabilidade dos recursos naturais e sua utilização.

As características geotécnicas de um maciço como factores preponderantes na planificação e organização de obras de escavação subterrâneas - uma perspectiva sobre a produção

Com o presente documento pretende-se abordar e identificar os diferentes factores que influenciam directamente a produção e execução de uma escavação subterrânea, com especial relevo sobre a influência exercida pela geotecnia do maciço intersectado. Inicialmente são focados os principais aspectos a ter em conta na caracterização geotécnica de um maciço, seguindo-se uma introdução a diferentes métodos de escavação actuais e metodologias de suporte de uma obra subterrânea, com particular realce para os utilizados em maciços brandos. Depois de tratados estes conceitos, é apresentada uma obra subterrânea em execução que foi acompanhada durante 4 meses para efeitos de desenvolvimento deste estudo. Assim, são abordados neste documento diferentes aspectos construtivos, no que diz respeito à mão-de-obra utilizada, metodologias e técnicas aplicadas, redes técnicas auxiliares instaladas, produções e rendimentos verificados. De seguida e de modo a atestar a importância da caracterização geotécnica ao longo da obra, foi feito um estudo do maciço intersectado, relativamente às descontinuidades que o intersectam, litologia, alteração, e resistência à compressão. Para este último parâmetro foram utilizadas técnicas distintas mas complementares, nomeadamente o ensaio de carga pontual (em laboratório), e o esclerómetro portátil (in situ). Por último, tendo em conta os parâmetros e características presentes e as implicações que uma obra do género acarreta, são propostas de modo sucinto, técnicas alternativas de escavação do maciço cuja viabilidade de implementação seja possível no contexto em questão.

β-Lactams: chemical structure, mode of action and mechanisms of resistance

This synopsis summarizes the key chemical and bacteriological characteristics of β-lactams, penicillins, cephalosporins, carbanpenems, monobactams and others. Particular notice is given to first-generation to fifth-generation cephalosporins. This reviewalso summarizes the main resistancemechanism to antibiotics, focusing particular attention to those conferring resistance to broad-spectrum cephalosporins by means of production of emerging cephalosporinases (extended-spectrum β-lactamases and AmpC β-lactamases), target alteration (penicillin-binding proteins from methicillin-resistant Staphylococcus aureus) and membrane transporters that pump β-lactams out of the bacterial cell.