Genetic diversity and epidemiology of antimicrobial resistant Streptococcus pneumoniae isolates

Dissertation presented to obtain a Ph.D. degree in Biology, speciality in Microbiology, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Heat stress adaptation in hyperthermophiles: bosynthesis of inositol-containing compatible solutes

Dissertation presented to obtain the Ph.D. degree in Biochemistry

The inhibitory activity of synthetic compounds and ions against transporters of multi-drug resistant bacteria

RESUMO: Sessenta e três derivados de hidantoína foram utilizados para avaliar possíveis efeitos de modulação na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um método fluorimétrico semi-automático. Nenhum dos compostos apresentaram actividade anti-bacteriana até concentrações de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modulação de effluxo na presença de glucose. Para testar esta actividade, estirpes de Salmonella resistentes à ciprofloxacina, induzidas a elevados níveis de resistência com sobre-expressão de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes à ciprofloxacina. Uma vez que os 63 compostos estudados apresentaram pouco efeito inibitório /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de iões em eucariotas, foi questionado o papel dos iões na regulação de BE bacterianas, que poderão influenciar a eficácia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE. Devido à importância de iões de cálcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modulação do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo é independente de energia metabólica; contudo, a pH 8 é absolutamente dependente, sendo que o Ca2+ é indispensável para manter a actividade das ATPases bacterianas. A acumulação/effluxo de EtBr pela E. coli AG100 foi determinada na presença/ausência de Ca2+, clorpromazina (inibidor de ligação de Ca2+ a proteínas), e ácido etilenodiamino tetra-acético (quelante de Ca2+). Acumulação/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua importância no funcionamento das BE bacterianas. Em resumo este trabalho colocou em evidência que muitos aspectos bioquímicos e bioenergéticos devem ser tomados em consideração no estudo da resistência bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains. Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity. Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases. Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems. Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.

Bioactive beads for local sensing of proteases in 3D engineered tissues

Dissertação para obtenção do Grau de Doutor em Bioengenharia (MIT)

Heat recovery from wastewater: numerical modelling of sewer systems

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente, perfil Engenharia Sanitária

On the track of β-lactam resistance: Studies on the regulation of methicillinresistance in Staphylococcus aureus

Dissertação para obtenção do Grau de Doutor em Biologia

Development of biocolonization resistant mortars: preliminary results

Restoration of Buildings and Monuments, vol. 13, nº 6 (2007), p.389-400

Evaluation of salt resistant mortars

Restoration of Buildings and Monuments, vol.11, nº 2 (2005), p.105-110

The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus

Dissertation for the Master’s Degree in Structural and Functional Biochemistry

Lime mortars with heat treated clays and ceramic waste: A review

The formulation and use of lime mortars with ceramic particles has, in the past, been a very common technique. Knowledge of such used techniques and materials is fundamental for the successful rehabilitation and conservation of the built heritage. The durability that these mortars have shown encourages the study of the involved mechanisms, so that they may be adapted to the current reality. The considerable amount of waste from old ceramics factories which is sent for disposal might present an opportunity for the production of reliable improved lime mortars. In this paper a number of studies that characterize old building mortars containing ceramic fragments are reviewed. The most important research undertaken on laboratory prepared mortars with several heat treated clays types is presented, specifically with incorporated ceramic waste. Some studies on the pozzolanicity of heat treated clays are examined and the heating temperatures that seem most likely to achieve pozzolanicity are presented. It was verified that some heating temperatures currently used by ceramic industries might correspond to the temperatures that will achieve pozzolanicity.

Gas gap heat switch for CryoFree project

Cryogen-free superconducting magnet systems have become popular during the last two decades for the simple reason that with the use of liquid helium is rather cumbersome and is a scarce resource. Some available CFMS uses a mechanical cryocooler as cold source of the superconductor magnet. However, the cooling of the sample holder is still made through an open circuit of helium. A thermal management of a completely cryogen-free system is possible to be implemented by using a controlled gas gap heat switch (GGHS) between the cryocooler and the variable temperature insert (VTI). This way it would eliminate the helium open circuit. Heat switches are devices that allow to toggle between two distinct thermal states (ON and OFF state). Several cryogenic applications need good thermal contact and a good thermal insulation at different stages of operation. A versatile GGHS was designed and built with a 100 mm gap and tested with helium as exchange gas. An analytic thermal model was developed and a good agreement with the experimental data was obtained. The device was tested on a crycooler at 4 to 80 K ranges. A 285 mW/K thermal conductance was measured at ON state and 0.09 mW/K at OFF. 3000 ON/OFF thermal conductance ratio was obtained at 4 K with helium.

Insights into the epidemiology of drug-resistant Streptococcus pneumoniae and closely-related streptococci in the era of conjugate vaccines

In Portugal, the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) has led to significant changes in the population structure of Streptococcus pneumoniae. However, the levels of antimicrobial resistance have not decreased and have been a matter of concern. (...)

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Portugal:

Methicillin-resistant Staphylococcus aureus (MRSA), a human pathogen confined to hospitals (HAMRSA) for over 30 years have been emerging worldwide in the last two decades as a leading cause of severe infections in healthy individuals in the community (CA-MRSA). Despite its clinical significance, in the beginning of our studies no information existed on the prevalence, and population structure of CA-MRSA in Portugal. Moreover, it remained to be clarified how CA-MRSA emerged in our country. In particular, it was not known if CA-MRSA emerged locally by acquisition of the staphylococcal cassette chromosome mec (SCCmec) by established methicillin-susceptible S. aureus (MSSA) in the community, if they were imported from abroad or have escaped from the hospital.(...)

Functional genomic analysis of heat stress in Vitis vinifera

Grapevine (Vitis vinifera) is one of most agro-economically important fruit crops worldwide, with a special relevance in Portugal where over 300 varieties are used for wine production. Due to global warming, temperature stress is currently a serious issue affecting crop production especially in temperate climates. Mobile genetic elements such as retrotransposons have been shown to be involved in environmental stress induced genetic and epigenetic modifications. In this study, sequences related to Grapevine Retrotransposon 1 (Gret1) were utilized to determine heat induced genomic and transcriptomic modifications in Touriga Nacional, a traditional Portuguese grapevine variety. For this purpose, growing canes were treated to 42 oC for four hours and leaf genomic DNA and RNA was utilized for various techniques to observe possible genomic alterations and variation in transcription levels of coding and non-coding sequences between non-treated plants and treated plants immediately after heat stress (HS-0 h) or after a 24 hour recovery period (HS-24 h). Heat stress was found to induce a significant decrease in Gret1 related sequences in HS-24 h leaves, indicating an effect of heat stress on genomic structure. In order to identify putative heat induced DNA modifications, genome wide approaches such as Amplified Fragment Length Polymorphism were utilized. This resulted in the identification of a polymorphic DNA fragment in HS-0 h and HS-24 h leaves whose sequence mapped to a genomic region flanking a house keeping gene (NADH) that is represented in multiple copies in the Vitis vinifera genome. Heat stress was also found to affect the transcript levels of various non-coding and gene coding sequences. Accordingly, quantitative real time PCR results established that Gret1 related sequences are up regulated immediately after heat stress whereas the level of transcript of genes involved in identification and repair of double strand breaks are significantly down regulated in HS-0 h plants. Taken together, the results of this work demonstrated heat stress affects both genomic integrity and transcription levels.

Insights into the biochemical strategies of adaptation to heat stress in the hyperthermophilic archaeon Pyrococcus furiosus

Organisms that thrive optimally at temperatures above 80°C are called hyperthermophiles. These prokaryotes have been isolated from a variety of hot environments, such as marine geothermal areas, hence they are usually slightly halophilic. Like other halophiles, marine hyperthermophiles have to cope with fluctuations in the salinity of the external medium and generally use low-molecular mass organic compounds to adjust cell turgor pressure. These compounds can accumulate to high levels without interfering with cell metabolism, thereby deserving the designation of compatible solutes. Curiously, the accumulation of compatible solutes also occurs in response to supraoptimal temperatures.(...)