Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

How to improve urban mobility in Lisbon: intermodality and information and ticketing systems

Urban mobility is one of the main challenges facing urban areas due to the growing population and to traffic congestion, resulting in environmental pressures. The pathway to urban sustainable mobility involves strengthening of intermodal mobility. The integrated use of different transport modes is getting more and more important and intermodality has been mentioned as a way for public transport compete with private cars. The aim of the current dissertation is to define a set of strategies to improve urban mobility in Lisbon and by consequence reduce the environmental impacts of transports. In order to do that several intermodal practices over Europe were analysed and the transport systems of Brussels and Lisbon were studied and compared, giving special attention to intermodal systems. In the case study was gathered data from both cities in the field, by using and observing the different transport modes, and two surveys were done to the cities users. As concluded by the study, Brussels and Lisbon present significant differences. In Brussels the measures to promote intermodality are evident, while in Lisbon a lot still needs to be done. It also made clear the necessity for improvements in Lisbon’s public transports to a more intermodal passenger transport system, through integration of different transport modes and better information and ticketing system. Some of the points requiring developments are: interchanges’ waiting areas; integration of bicycle in public transport; information about correspondences with other transport modes; real-time information to passengers pre-trip and on-trip, especially in buses and trams. After the identification of the best practices in Brussels and the weaknesses in Lisbon the possibility of applying some of the practices in Brussels to Lisbon was evaluated. Brussels demonstrated to be a good example of intermodality and for that reason some of the recommendations to improve intermodal mobility in Lisbon can follow the practices in place in Brussels.

Human mobility patterns at the smallest scales

We present a study on human mobility at small spatial scales. Differently from large scale mobility, recently studied through dollar-bill tracking and mobile phone data sets within one big country or continent, we report Brownian features of human mobility at smaller scales. In particular, the scaling exponents found at the smallest scales is typically close to one-half, differently from the larger values for the exponent characterizing mobility at larger scales. We carefully analyze 12-month data of the Eduroam database within the Portuguese university of Minho. A full procedure is introduced with the aim of properly characterizing the human mobility within the network of access points composing the wireless system of the university. In particular, measures of flux are introduced for estimating a distance between access points. This distance is typically non-Euclidean, since the spatial constraints at such small scales distort the continuum space on which human mobility occurs. Since two different ex- ponents are found depending on the scale human motion takes place, we raise the question at which scale the transition from Brownian to non-Brownian motion takes place. In this context, we discuss how the numerical approach can be extended to larger scales, using the full Eduroam in Europe and in Asia, for uncovering the transi- tion between both dynamical regimes.

Aging and Alzheimer's disease: Searching for novel molecular cues

Tese de Doutoramento em Ciências da Saúde

The effects of solvent composition on the affinity of a peptide towards hair keratin: experimental and molecular dynamics data

The study of the interaction between hair filaments and formulations or peptides is of utmost importance in fields like cosmetic research. Keratin intermediate filaments structure is not fully described, limiting the molecular dynamics (MD) studies in this field although its high potential to improve the area. We developed a computational model of a truncated protofibril, simulated its behavior in alcoholic based formulations and with one peptide. The simulations showed a strong interaction between the benzyl alcohol molecules of the formulations and the model, leading to the disorganization of the keratin chains, which regress with the removal of the alcohol molecules. This behavior can explain the increase of peptide uptake in hair shafts evidenced in fluorescence microscopy pictures. The model developed is valid to computationally reproduce the interaction between hair and alcoholic formulations and provide a robust base for new MD studies about hair properties. It is shown that the MD simulations can improve hair cosmetic research, improving the uptake of a compound of interest.

On line pre-concentration for simultaneous determination of low molecular weight organic acids and inorganic anions in Amazonian river water samples employing ion chromatography with conductivity detection

An ion chromatography procedure, employing an IonPac AC15 concentrator column was used to investigate on line preconcentration for the simultaneous determination of inorganic anions and organic acids in river water. Twelve organic acids and nine inorganic anions were separated without any interference from other compounds and carry-over problems between samples. The injection loop was replaced by a Dionex AC15 concentrator column. The proposed procedure employed an auto-sampler that injected 1.5 ml of sample into a KOH mobile phase, generated by an Eluent Generator, at 1.5 mL min-1, which carried the sample to the chromatographic columns (one guard column, model AG-15, and one analytical column, model AS15, with 250 x 4mm i.d.). The gradient elution concentrations consisted of a 10.0 mmol l-1 KOH solution from 0 to 6.5 min, gradually increased to 45.0 mmol l-1 KOH at 21 min., and immediatelly returned and maintained at the initial concentrations until 24 min. of total run. The compounds were eluted and transported to an electro-conductivity detection cell that was attached to an electrochemical detector. The advantage of using concentrator column was the capability of performing routine simultaneous determinations for ions from 0.01 to 1.0 mg l-1 organic acids (acetate, propionic acid, formic acid, butyric acid, glycolic acid, pyruvate, tartaric acid, phthalic acid, methanesulfonic acid, valeric acid, maleic acid, oxalic acid, chlorate and citric acid) and 0.01 to 5.0 mg l-1 inorganic anions (fluoride, chloride, nitrite, nitrate, bromide, sulfate and phosphate), without extensive sample pretreatment and with an analysis time of only 24 minutes.

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

Radio mobility in the digital era: interactivity, participation and content share possibilities in Iberian broadcasters

In the context of the scientific research into radio, recent years have encouraged many theories about the meaning of a post-radio (Oliveira & Portela, 2011), thus enlisting several parameters regarding the inclusion of contemporary radio in the digital and online environments. This digital migration has led to the development of mobile applications for radio, broadening the communicative potential of audiences (Aguado, Feijoo & Martínez, 2013), as well as promoting convergence of interactive content among listeners-users. Aware of this opportunity, the main broadcasters in Spain and Portugal have broadened their radiophonic scope to the mobile platform, especially geared towards smartphones through the development of mobile applications, commonly known as apps (Cerezo, 2010). As a symbol of a culture in permanent changing, smartphones not only provide greater easiness in terms of access and interaction, but also afford larger opportunities for disseminating content among audiences, a phenomenon that some studies have labelled as user distributed content (Villi, 2012). This article presents an exploratory analysis of the current policies of the main Spanish and Portuguese radio broadcasters regarding mobile applications, evaluating the different levels of interaction and participation in these platforms. This observation led to the conclusion, among other findings, that the mobile platform represents a supplementary channel for traditional FM radio, rather than a new medium with its own language and expression.

To be or not to be a pseudogene: a molecular epidemiological approach to the mclx genes and its impact in tuberculosis

Tuberculosis presents a myriad of symptoms, progression routes and propagation patterns not yet fully understood. Whereas for a long time research has focused solely on the patient immunity and overall susceptibility, it is nowadays widely accepted that the genetic diversity of its causative agent, Mycobacterium tuberculosis, plays a key role in this dynamic. This study focuses on a particular family of genes, the mclxs (Mycobacterium cyclase/LuxR-like genes), which codify for a particular and nearly mycobacterial-exclusive combination of protein domains. mclxs genes were found to be pseudogenized by frameshift-causing insertion(s)/deletion(s) in a considerable number of M. tuberculosis complex strains and clinical isolates. To discern the functional implications of the pseudogenization, we have analysed the pattern of frameshift-causing mutations in a group of M. tuberculosis isolates while taking into account their microbial-, patient- and disease-related traits. Our logistic regression-based analyses have revealed disparate effects associated with the transcriptional inactivation of two mclx genes. In fact, mclx2 (Rv1358) pseudogenization appears to be primarily driven by the microbial phylogenetic background, being mainly related to the Euro-American (EAm) lineage; on the other hand, mclx3 (Rv2488c) presents a higher tendency for pseudogenization among isolates from patients born on the Western Pacific area, and from isolates causing extra-pulmonary infections. These results contribute to the overall knowledge on the biology of M. tuberculosis infection, whereas at the same time launch the necessary basis for the functional assessment of these so far overlooked genes.

Widespread environmental contamination with Mycobacterium tuberculosis complex revealed by a molecular detection protocol

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

Molecular regulation of flowering induction in Quercus suber

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepeneurship

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Ionic, paramagnetic and photophysical properties of a new biohybrid material incorporating copper perchlorate

The sol-gel method was employed in the synthesis of di-urethane cross-linked poly(-caprolactone) (d-PCL(530)/siloxane biohybrid ormolytes incorporating copper perchlorate, (Cu(ClO4)2). The highest ionic conductivity of the d PCL(530)/siloxanenCu(ClO4)2 system is that with n = 10 (1.4 x 10-7 and 1.4 x 10-5 S cm-1, at 25 and 100 ºC, respectively). In an attempt to understand the ionic conductivity/ionic association relationship, we decide to inspect the chemical environment experienced by the Cu2+ ions in the d-PCL(530)/siloxane medium. The observed EPR spectra are typical of isolated monomeric Cu2+ ions in axially distorted sites. The molecular orbital coefficients obtained from the EPR spin Hamiltonian parameters and the optical absorption band suggests that bonding between the Cu2+ and its ligand in the ormolytes are moderately ionic. Investigation by photoluminescence spectroscopy did not evidence or allow selective excitation of transitions corresponding to complexed Cu2+ species.

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-Aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC50 10-28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 microM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.