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.

Digital Divide Across the European Union

Dissertação apresentada como requisito parcial para obtenção do grau de Mestre em Estatística e Gestão de Informação

Determinants of venture capital and private equity investments in renewable energy: A cross-country study

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

Management consulting labs-Galp social media

Social Media @ Galp Project had a very specific purpose – analyze the feasibility for Galp to enter in new Social Media platforms and, if appropriate, develop a short-term strategy for the entrance in which some guidelines are valid for the medium-long-term. As expected, the majority of the project was focused on the second part, which consists in an analysis of some aspects concerning the organization as well as in the relationship with customers and public in general

Construção de uma responsabilidade europeia além-fronteiras

The suppression of internal border controls has led the European Union to establish a mechanism for determining the Member State responsible for examining each asylum application, with the main intention of deterring asylum seekers from lodging multiple applications and guaranteeing that it will be assessed by one of the States – the Dublin System. Even though it holds on a variety of criteria, the most commonly used is the country of first entrance in the EU. The growing migrating flows coming mainly from Northern Africa have thus resulted in an incommensurable burden over the border countries. Gradually, countries like Greece, Bulgaria and Italy have lost capability of providing adequate relief to all asylum seekers and the records of fundamental rights violations related to the provision of housing and basic needs or inhuman detention conditions started piling up. To prevent asylum seekers who had already displaced themselves to other Member States from being transferred back to countries where their human dignity is questionable, the European Court of Human Rights and the Court of Justice have developed a solid jurisprudence determining that when there is a risk of serious breach of fundamental rights all transfers to that country must halt, especially when it is identified with systemic deficiencies in the asylum system and procedures. This reflexion will go through the jurisprudence that influenced very recent legislative amendments, in order to identify which elements form part of the obligation not to transfer under the Dublin System. At last, we will critically analyze the new rising obligation, that has clearly proven insufficient in light of the international fundamental rights framework that the Member States and the EU are bound to respect, proposing substantial amendments with a view to reach a future marked by high solidarity and global responsibility from the European Union.

O corpo da Guarda prisional como Órgão de Polícia Criminal e a sua importância na Segurança Interna

The study now presented a research study aimed at the exercise of security activity of the prison guard corps (CGP) in the specific context of the prison system (SP). The study also focused on the phenomenon of globalization and its influence on the current panorama of world crime and their relationship with the prison security object of study was the security activity of the CGP on the current inmate population, as well as the typology of crimes inherent to it and that requires the interpretation of the amendment to the correctional paradigm. In the preparation of the study we have tried to identify the legal framework of the profession based on the main laws and decree-laws governing the institution DGRSP and CGP's career. The difficulties and constraints were analyzed the performance of the security function of the CGP, resulting from the infrastructure, the interaction with the inmates and the need of assigning the status of Criminal Police (OPC). Really connected the relevant importance of OPC so the Corporation can establish a cooperation interaction and sharing of information with different security forces. We noted the importance of the CGP being OPC status before the framework for its action in work situations, specifically, on gatehouses, prison wings, area, and high-complexity operations such as escorts and riot control interventions.The explanation of this study enabled us to assess the importance of the actions of a police officer of the elements of the CGP for maintaining internal security. The analyses and studies were complemented by twenty years of career and exercise of the activity of the master's degree, during which time he served as guard and head of the CGP, the intervention Group and Prison Security, the Lisbon Prison, Prison, Prison of Caxias, Sintra and Monsanto.

Guarda prisional: força de segurança ou agente responsabilizador?

A permanently changing occidental society framework, simultaneously, to a world Globalization and a market liberalization, requires to know how important and which role the agents plays, in Estates representation, to guarantee their own intern security. Portugal is an example of that since has been integrated in European and world politics that allowed the borders opening, with all the negative consequences of that kind of measures. In way to struggle with those debilities emerge, in our Juridical Order, several security forces such as Prison Guards Corporation, whose contribute to intern security seems undefined and confuse, being urgent legislation in way to describe and clearly define their goals and functions. We begin with a brief history view to understand the evolution, focusing on the present moment, correlate several laws in way to clarify their juridical situation. Using a own critical sense, it draws attention to legislation lack problem in opposition to the conclusion that, Prison Guards Corporation is a security force with specialized expertise in matter and territory fields. Their activity occurs, generally, in penitentiaries where people see themselves without their freedom, legally determinated and confined to places as other individuals with deviant behaviors that deserve society refutation, establishing a separation period having rehabilitation as a goal – it is called general and special prevention. Penitentiaries specificities requires specially police force because penitentiaries are places where tensions are often, both between inmates and against employees, above all prison guards, the first to struggle inmates daily frustrations. In way that institutions achieve their purpose, it is necessary that citizens respect all the rules, although, to their efficacy is necessary to inflict punishment to those who did not respect the rules. Furthermore, it will be indispensable to act immediately in situations as impeding runaway helping, illegally standing in jail and to avoid violent acts against personal and patrimonial belongings. Juridical Order has a few security tools that are available to administration, in which is included coercive methods, that as damaging to citizens in whom they are use, are restricted, unavoidably, to inflexible control rules. Concluding, Prison Guards and Penitentiaries General Direction last goal is to give back recovered inmates to society, in a way to conduct their lives responsibly, without committing crimes.

Impactos territoriais de processos de cooperação transfronteiriça : caso de estudo : Elvas e Badajoz

A cooperação transfronteiriça é vista na União Europeia como um modelo de integração territorial e como uma alternativa para o desenvolvimento das regiões de fronteira, devido ao seu afastamento em relação aos grandes centros urbanos. No início da década de 90 foi lançado o programa do INTERREG tendo em vista a maior permeabilidade da fronteira e o desenvolvimento destes territórios, entre os quais o das regiões do Alto Alentejo e da Extremadura. A grande proximidade territorial entre Elvas e Badajoz permitiu desde sempre relações de cooperação mais ou menos intensas e consentidas, mesmo antes da abertura da fronteira, motivadas sobretudo pela diferença de valor entre as duas moedas. Contudo, com a entrada conjunta dos países ibéricos na CEE (1986), essas relações intensificaram-se ao nível do comércio, do emprego, do turismo, do lazer e da procura de serviços. Impactos territoriais como a procura e utilização conjunta de infraestruturas e de espaços públicos pelas populações das duas cidades, a promoção e realização de espetáculos culturais, a prática de cross-border shopping, a procura de uma segunda residência, em ambos os lados da fronteira, e a tendência para uma urbanização contínua no futuro, justificaram a criação da Eurocidade Elvas-Badajoz (2013). Esta iniciativa tem como principais objetivos promover a conceção, gestão e prestação conjunta de serviços, desenvolver projetos de cooperação em áreas de interesse comum, estimular a cooperação entre empresas e atrair investimentos geradores de novos postos de trabalho. No entanto os desafios para ambas as cidades prendem-se com a existência de um quadro jurídico e legal distinto que tem condicionado algumas das ações ou iniciativas e o desenvolvimento territorial conjunto ainda carece de uma proposta estratégica.

Jeronimo Martins in 2030 succession and strategy planning

The case outlines the history of Jerónimo Martins and the Dos Santos family in the context of a dialogue between the pater familias Alexandre and his successor Pedro. The case idea analyses the problems associated with next generation entrance in the family business and the outcomes on the different stakeholders and environment, with a particular focus on the evolution of the relationship between family, family holding and the JM group. The case is designed to assess the sustainability of a traditional succession strategy in the context of the challenges of globalization and growth of the company and family, analysing the reciprocal influence in a long-term strategy.