Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Localization in underground tunnels

Positioning technologies are becoming ubiquitous and are being used more and more frequently for supporting a large variety of applica- tions. For outdoor applications, global navigation satellite systems (GNSSs), such as the global positioning system (GPS), are the most common and popular choice because of their wide coverage. GPS is also augmented with network-based systems that exploit existing wireless and mobile networks for providing positioning functions where GPS is not available or to save energy in battery-powered devices. Indoors, GNSSs are not a viable solution, but many applications require very accurate, fast, and exible positioning, tracking, and navigation functions. These and other requirements have stim- ulated research activities, in both industry and academia, where a variety of fundamental principles, techniques, and sensors are being integrated to provide positioning functions to many applications. The large majority of positioning technologies is for indoor environments, and most of the existing commercial products have been developed for use in of ce buildings, airports, shopping malls, factory plants, and similar spaces. There are, however, other spaces where positioning, tracking, and navigation systems play a central role in safety and in rescue operations, as well as in supporting speci c activities or for scienti c research activities in other elds. Among those spaces are underground tunnels, mines, and even underwater wells and caves. This chapter describes the research efforts over the past few years that have been put into the development of positioning systems for underground tun- nels, with particular emphasis in the case of the Large Hadron Collider (LHC) at CERN (the European Organization for Nuclear Research), where localiza- tion aims at enabling more automatic and unmanned radiation surveys. Examples of positioning and localization systems that have been devel- oped in the past few years for underground facilities are presented in the fol- lowing section, together with a brief characterization of those spaces’ special conditions and the requirements of some of the most common applications. Section 5.2 provides a short overview of some of the most representative research efforts that are currently being carried out by many research teams around the world. In addition, some of the fundamental principles and tech- niques are identi ed, such as the use of leaky coaxial cables, as used at the LHC. In Section 5.3, we introduce the speci c environment of the LHC and de ne the positioning requirements for the envisaged application. This is followed by a detailed description of our approach and the results that have been achieved so far. Some last comments and remarks are presented in a nal section.

Triagem universal de alunos em risco de apresentarem dificuldades de aprendizagem específicas na leitura: um estudo quantitativo no 3º ano do 1º Ciclo do Ensino Básico

Doutoramento em Estudos da Criança (área de especialização em Educação Especial).

Prediction of drug targets in human pathogens

The identification of new and druggable targets in bacteria is a critical endeavour in pharmaceutical research of novel antibiotics to fight infectious agents. The rapid emergence of resistant bacteria makes today's antibiotics more and more ineffective, consequently increasing the need for new pharmacological targets and novel classes of antibacterial drugs. A new model that combines the singular value decomposition technique with biological filters comprised of a set of protein properties associated with bacterial drug targets and similarity to protein-coding essential genes of E. coli has been developed to predict potential drug targets in the Enterobacteriaceae family [1]. This model identified 99 potential target proteins amongst the studied bacterial family, exhibiting eight different functions that suggest that the disruption of the activities of these proteins is critical for cells. Out of these candidates, one was selected for target confirmation. To find target modulators, receptor-based pharmacophore hypotheses were built and used in the screening of a virtual library of compounds. Postscreening filters were based on physicochemical and topological similarity to known Gram-negative antibiotics and applied to the retrieved compounds. Screening hits passing all filters were docked into the proteins catalytic groove and 15 of the most promising compounds were purchased from their chemical vendors to be experimentally tested in vitro. To the best of our knowledge, this is the first attempt to rationalize the search of compounds to probe the relevance of this candidate as a new pharmacological target.

A Gß protein and the TupA Co-Regulator bind to protein kinase A Tpk2 to act as antagonistic molecular switches of fungal morphological changes

A Gß protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

Peptaibolin analogues by incorporation of α,α-dialkylglycines: synthesis and study of their membrane permeating ability

Analogues of Peptaibolin, a peptaibol with antibiotic activity, incorporating α,α-dialkylglycines (Deg, Dpg, and Ac6c) at selected positions were synthesised by MW-SPPS and fully characterized. A control analogue incorporating L-alanine was also prepared. The native peptide and the analogues were studied by fluorescence spectroscopy for their membrane permeating activity. Small unilamellar vesicles (SUVs) of egg phosphatidylcholine/ cholesterol (70:30) containing an encapsulated fluorescence probe (6-carboxyfluorescein) were used as membrane models. The assays of carboxyfluorescein release from SUVs upon peptide addition showed that Peptaibolin-Dpg and Peptaibolin-Ac6c are the most active peptides. These results indicate that the structure of the α,α-dialkylglycines is crucial for the membrane permeating ability of these Peptaibolin analogues.

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

Constraints on new phenomena via Higgs boson couplings and invisible decays with the ATLAS detector

The ATLAS experiment at the LHC has measured the Higgs boson couplings and mass, and searched for invisible Higgs boson decays, using multiple production and decay channels with up to 4.7 fb−1 of pp collision data at √s=7 TeV and 20.3 fb−1 at √s=8 TeV. In the current study, the measured production and decay rates of the observed Higgs boson in the γγ, ZZ, W W , Zγ, bb, τ τ , and μμ decay channels, along with results from the associated production of a Higgs boson with a top-quark pair, are used to probe the scaling of the couplings with mass. Limits are set on parameters in extensions of the Standard Model including a composite Higgs boson, an additional electroweak singlet, and two-Higgs-doublet models. Together with the measured mass of the scalar Higgs boson in the γγ and ZZ decay modes, a lower limit is set on the pseudoscalar Higgs boson mass of m A > 370 GeV in the “hMSSM” simplified Minimal Supersymmetric Standard Model. Results from direct searches for heavy Higgs bosons are also interpreted in the hMSSM. Direct searches for invisible Higgs boson decays in the vector-boson fusion and associated production of a Higgs boson with W/Z (Z → ℓℓ, W/Z → jj) modes are statistically combined to set an upper limit on the Higgs boson invisible branching ratio of 0.25. The use of the measured visible decay rates in a more general coupling fit improves the upper limit to 0.23, constraining a Higgs portal model of dark matter.

Production and characterization of ZrCN-Ag coatings deposited by mganetron sputtering

Tese de Doutoramento (Programa doutoral em Engenharia de Materiais)

Identificação e caracterização de alunos em risco de desenvolverem dislexia: um estudo sobre o uso da Monitorização com base no currículo no terceiro ano de escolaridade

Dissertação de mestrado em Educação Especial (área de especialização em Dificuldades de Aprendizagem Específicas)

Monitorização com base no currículo na identificação de alunos em risco de dislexia: estudo quantitativo no 4º ano do 1º ciclo do ensino básico no concelho de Braga

Dissertação de mestrado em Educação Especial (área de especialização em Dificuldades de Aprendizagem Específicas)

Toxicity induced by a metal mixture (Cd, Pb and Zn) in the yeast Pichia kudriavzevii: The role of oxidative stress

The present work aims to contribute for the elucidation of the role of oxidative stress in the toxicity associated with the exposure of Pichia kudriavzevii to multi-metals (Cd, Pb and Zn). Cells of the non-conventional yeast P. kudriavzevii exposed for 6 h to the action of multi-metals accumulated intracellular reactive oxygen species (ROS), evaluated through the oxidation of the probe 2,7-dichlorodihydrofluorescein diacetate. A progressive loss of membrane integrity (monitored using propidium iodide) was observed in multi-metal-treated cells. The triggering of intracellular ROS accumulation preceded the loss of membrane integrity. These results suggest that the disruption of membrane integrity can be attributed to the oxidative stress. The exposure of yeast cells to single metal showed that, under the concentrations tested, Pb was the metal responsible for the induction of the oxidative stress. Yeast cells coexposed to an antioxidant (ascorbic acid) and multi-metals did not accumulate intracellular ROS, but loss proliferation capacity. Together, the data obtained indicated that intracellular ROS accumulation contributed to metal toxicity, namely for the disruption of membrane integrity of the yeast P. kudriavzevii. It was proposed that Pb toxicity (the metal responsible for the toxic symptoms under the conditions tested) result from the combination of an ionic mechanism and the intracellular ROS accumulation.