Resistance to salts of lime and pozzolan mortars

International RILEM Workshop on Repairs Mortars for Historic Masonry, Technical University of Delft, 2009

The impact of high involvement human resources practices on resistance to change: The mediator effect of commitment to change and the influence of ethical leadership

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Studies on resistance and response to vancomycin in Enterococcus faecalis: a last resort antibiotic

Dissertation presented to obtain a Ph.D. degree in Biochemistry by Instituto de Tecnologia Química e Biológica Universidade Nova de Lisboa.

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.

Positive Epistasis Drives the Acquisition of Multidrug Resistance

The evolution of multiple antibiotic resistance is an increasing global problem. Resistance mutations are known to impair fitness, and the evolution of resistance to multiple drugs depends both on their costs individually and on how they interact-epistasis. Information on the level of epistasis between antibiotic resistance mutations is of key importance to understanding epistasis amongst deleterious alleles, a key theoretical question, and to improving public health measures. Here we show that in an antibiotic-free environment the cost of multiple resistance is smaller than expected, a signature of pervasive positive epistasis among alleles that confer resistance to antibiotics. Competition assays reveal that the cost of resistance to a given antibiotic is dependent on the presence of resistance alleles for other antibiotics. Surprisingly we find that a significant fraction of resistant mutations can be beneficial in certain resistant genetic backgrounds, that some double resistances entail no measurable cost, and that some allelic combinations are hotspots for rapid compensation. These results provide additional insight as to why multi-resistant bacteria are so prevalent and reveal an extra layer of complexity on epistatic patterns previously unrecognized, since it is hidden in genome-wide studies of genetic interactions using gene knockouts.

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

Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Adjusting to external imbalances within the EMU, the case of Portugal

From 1995 to 2010 Portugal has accumulated a negative international asset position of 110 percent of GDP. In a developed and aging economy the number is astonishing and any argument to consider it sustainable must rely on extremely favorable forecasts on growth. Portuguese policy options are reduced in number: no autonomous monetary policy, no currency to devaluate, and limited discretion in changing fiscal deficits and government debt. To start the necessary deleveraging a remaining possible policy is a budget-neutral change of the tax structure that increases private saving and net exports. An increase in the VAT and a decrease in the employer’s social security contribution tax can achieve the desired outcome in the short run if they are complemented with wage moderation. To obtain a substantial improvement in competitiveness and a large decrease in consumption, the changes in the tax rates have to be large. While a precise quantitative assessment is difficult, the initial increase in the effective VAT rate needed to allow the social security tax to decrease by 16 percentage points (pp) is approximately 10 pp. Such a large increase in the effective VAT rate could be obtained by raising most of the reduced VAT rates to the new general VAT rate of 23 percent. The empirical analysis shows that over time the suggested tax swap could generate surpluses and improve the trade balance. A temporary version of the suggested tax-swap has the attractiveness to achieve a sharper increase in the private saving rate maintaining the short run gains in competitiveness. Finally, the temporary version of the fiscal devaluation could be the basis for an automatic stabilizer to external imbalances within a monetary union.Portugal has been running large current account deficits every year since 1995. These deficits have accumulated to an astonishing 110 percent of GDP negative external asset position. The sustainability of such a large external position is questionable and must rely on fantastic productivity growth expectations. The recent global financial crisis appears to have anticipated the international investors reality check on those future expectations with the result of a large increase in the cost of external financing. Today the rebalancing of the current account through an increase in national savings and an improvement in competitiveness must be at the top of the Portuguese authorities “to do” list as the cost of a pull out from international investors is of the order of 10% of GDP. The external rebalancing is difficult as the degrees of freedom of the Portuguese authorities are limited in number: they have no autonomous monetary policy, no currency to devaluate, and little discretion in fiscal policy as deficit limits and debt targets are set by the Stability Growth Pact and the postcrisis consensus on medium-term fiscal consolidation. One possibility that remains is to change the fiscal policy mix for a given budget deficit. The purpose of this paper is to explore the effects of a “fiscal devaluation”1 obtained through a tax swap between employers’ social security contributions and taxes on consumption2. The paper begins by illustrating Portugal’s current account evolution during the euro period. The second section section lays out a model to offer a qualitative assessment of the dynamic outcomes of the the tax swap. I show that the suggested tax swap can in theory achieve the desired outcomes in terms of competitiveness and consumption if complemented with moderation (stickiness) in wages. I also study the effects of a temporary version of the tax swap and show that it achieves a sharper improvement in the current account that accelerate the rebalancing. The third section moves to the empirical analysis and estimates the likely effects of the tax swap for the Portuguese economy. The fourth section concludes.

Lag-user method: Using laggards to leverage user innovation

Laggards are the last users to adopt a product. Prior literature on user-led innovation ignores laggards’ impact on innovation. In this paper, we develop the Lag-User Method, through which laggards can generate new ideas. Through six studies with 62 teams in three countries, we apply the method to different technologies and services and present our findings to executives to get managerial insights. Findings reveal that laggards who generate new ideas (lag-users) have different perceptions of user-friendly products and different unfulfilled needs. They prefer simple products. We propose that by involving lag-users in NPD, firms can improve the effectiveness of NPD.