Metals are used as cofactors for enzymes, but are toxic in excess. In order to avoid the deleterious effects posed by metals, the cell must employ strict metal homeostasis systems. One such system is the Cue copper-resistance system in Salmonella enterica serovar Typhimurium (S. Typhimurium) which includes the periplasmic copper binding protein CueP. Previous studies have shown CueP to be a major periplasmic copper-sequestering protein that has a role in supplying copper to, and thus activating, the periplasmic Cu,Zn-superoxide dismutase enzyme SodCII (Osman et al., 2013). SodCII protects the cell from reactive oxygen species (ROS), due for example to the actions of the respiratory burst oxidase in host macrophages. However, despite its ability to sequester copper and activate SodCII, the precise physiological role of CueP in S. Typhimurium has remained unresolved since cueP mutants of S. Typhimurium strain SL1344 (the wild-type stain used in this study) do not exhibit a phenotype with respect to tolerance to copper or reactive oxygen species. In addition, the copper-binding mechanism of CueP and its interactions with other copper-binding proteins, including SodCII, have not been examined. An aim of this study was to establish a phenotype for a cueP mutant of S. Typhimurium with respect to copper and/or ROS tolerance. It was hypothesised that the possession of KatG (catalase) and multiple superoxide dismutases (SodCI, SodA and SodB), in addition to SodCII, by S. Typhimurium may confer functional redundancy with respect to copper and ROS tolerance. Hence mutants lacking katG (ΔkatG) or the various superoxide dismutase encoding genes (ΔsodA/ΔsodB/ΔsodCI/ΔsodCII) with and without functional cueP were generated. The ΔkatG mutants exhibited reduced catalase activity and reduced tolerance to hydrogen peroxide, consistent with the loss of KatG, however the additional loss of cueP did not reduce tolerance to hydrogen peroxide further. Similarly, tolerance to copper and extracellular superoxide was also unaltered in the ΔkatG/ΔcueP mutant. The tolerance of the various superoxide dismutase mutants to copper and various ROS was also unaffected by the presence or absence of CueP. To examine the role of CueP in SodCII activation in vivo, SodCII was over-expressed in S. Typhimurium (in a ΔsodA/ΔsodB/ΔsodCI/ΔsodCII background) with and without functional cueP and superoxide dismutase activity measured in both whole cells and periplasmic extracts. SodCII-dependent superoxide dismutase activity was successfully identified within the periplasmic extracts. However, surprisingly, the level of activity was unaffected by the presence 16 or absence of CueP and/or the addition of copper. It is possible that SodCII is thus able to scavenge sufficient copper for activity from the reagents used in these assays. Similarly, in an alternative approach to examine the role of CueP in vitro, both SodCII and CueP (WT and potential metal-binding residue mutant forms) were successfully over-expressed in E. coli and methods for their purification optimised (without the use of affinity tags). ICP-MS analysis indicated that a CuePC104S mutant contains > 18-fold less copper than the CueP WT protein. Furthermore, superoxide dismutase activity assays using purified proteins, indicated that the CuePC104S mutant was less able to activate SodCII than the WT CueP. Taken together, these results are consistent with a role for the Cys104 residue in copper-binding by CueP. Bioinformatics results suggest the presence of CueP or homologous genes in the presence of other bacteria, including pathogens such as Klebsiella, Yersinia and Shigella spp. Further understanding of the role of CueP and the systems used by S. Typhimurium to avoid both copper and ROS stress may inform the development of novel treatment strategies for bacterial diseases.

Os fundamentos teóricos desenvolvidos neste trabalho e que dão suporte aos métodos propostos garantem que as estimativas obtidas sejam sempre conservadoras (no sentido de que elas são sempre subconjuntos da região de estabilidade verdadeira) e, portanto, possuam elevado grau de confiança ao concluir sobre a estabilidade do sistema. Os métodos apresentados consistem em extensões dos métodos Closest UEP e CUEP, utilizados na análise de estabilidade transitória de sistemas elétricos de potência, para sistemas que admitem FEG. Embora os métodos Closest UEP e CUEP forneçam estimativas de forma rápida e precisa, sua aplicação está limitada à existência de uma Função Energia (FE) para o sistema, o que consiste em uma forte limitação. Muitos sistemas não admitem FE e, mesmo quando se pode provar a existência de uma FE, a impossibilidade de exibi-la impede a aplicação dos métodos citados. Outra contribuição deste trabalho consiste em um método computacional que permite a obtenção de uma FEG para sistemas polinomiais. O método apresentado também é aplicado a uma classe de problemas não polinomiais, provenientes da modelagem de sistemas elétricos de potência, mediante uma mudança não linear de variáveis que permite a construção de um sistema polinomial equivalente. Através dos métodos apresentados, visa-se disponibilizar métodos computacionais que permitam a obtenção de estimativas rápidas e precisas e que possam ser aplicados a uma ampla classe de sistemas: aqueles que admitem FEG. Com isso, almeja-se não somente contribuir para o desenvolvimento de métodos para análise de estabilidade de sistemas elétricos de potência mas, também, disponibilizá-los a outras áreas do conhecimento. / In this work, we develop computational methods to estimate stability regions and the relevant part of stability boundary of attracting sets of nonlinear dynamical systems. Such methods are based on Generalized Energy Function (GEF) theory and, therefore, can be applied to a larger class of problems than those based on Energy Functions (EF). The theoretical foundations developed in this work, which support the proposed methods, ensure that the estimates are always conservative (in the sense that they are subsets of the true stability region), providing high confidence level when asserting the stability of a system. The presented methods are extensions of the Closest UEP and the CUEP methods, used in the assessment of stability of electrical power systems, to the systems that admit GEF. Even though the Closest UEP and CUEP methods provide estimates in a fast and accurate way, they are only applicable to systems that admit EFs, which consists in a strong limitation for their usage. Many systems do not admit EF and, even if it is possible to prove the existence of an EF, the impossibility to exhibit it in the form of elementary mathematical functions prevents the application of such methods. Other contribution of this work is a computational method to obtain a GEF for polinomial systems. We also applied the presented method to a class of non polinomial systems arising from electrical power system models, after a nonlinear change of variables that provides an equivalent polinomial system. By means of the proposed methods, we aim to offer computational methods to allow fast and accurate stability region estimates which could be used in a broad class of dynamical systems: those that admit GEF. This way, we plan to contribute for the development of methods used in the assessment of stability of electrical power systems and make such tools available to systems from other areas of science.

Os fundamentos teóricos desenvolvidos neste trabalho e que dão suporte aos métodos propostos garantem que as estimativas obtidas sejam sempre conservadoras (no sentido de que elas são sempre subconjuntos da região de estabilidade verdadeira) e, portanto, possuam elevado grau de confiança ao concluir sobre a estabilidade do sistema. Os métodos apresentados consistem em extensões dos métodos Closest UEP e CUEP, utilizados na análise de estabilidade transitória de sistemas elétricos de potência, para sistemas que admitem FEG. Embora os métodos Closest UEP e CUEP forneçam estimativas de forma rápida e precisa, sua aplicação está limitada à existência de uma Função Energia (FE) para o sistema, o que consiste em uma forte limitação. Muitos sistemas não admitem FE e, mesmo quando se pode provar a existência de uma FE, a impossibilidade de exibi-la impede a aplicação dos métodos citados. Outra contribuição deste trabalho consiste em um método computacional que permite a obtenção de uma FEG para sistemas polinomiais. O método apresentado também é aplicado a uma classe de problemas não polinomiais, provenientes da modelagem de sistemas elétricos de potência, mediante uma mudança não linear de variáveis que permite a construção de um sistema polinomial equivalente. Através dos métodos apresentados, visa-se disponibilizar métodos computacionais que permitam a obtenção de estimativas rápidas e precisas e que possam ser aplicados a uma ampla classe de sistemas: aqueles que admitem FEG. Com isso, almeja-se não somente contribuir para o desenvolvimento de métodos para análise de estabilidade de sistemas elétricos de potência mas, também, disponibilizá-los a outras áreas do conhecimento. / In this work, we develop computational methods to estimate stability regions and the relevant part of stability boundary of attracting sets of nonlinear dynamical systems. Such methods are based on Generalized Energy Function (GEF) theory and, therefore, can be applied to a larger class of problems than those based on Energy Functions (EF). The theoretical foundations developed in this work, which support the proposed methods, ensure that the estimates are always conservative (in the sense that they are subsets of the true stability region), providing high confidence level when asserting the stability of a system. The presented methods are extensions of the Closest UEP and the CUEP methods, used in the assessment of stability of electrical power systems, to the systems that admit GEF. Even though the Closest UEP and CUEP methods provide estimates in a fast and accurate way, they are only applicable to systems that admit EFs, which consists in a strong limitation for their usage. Many systems do not admit EF and, even if it is possible to prove the existence of an EF, the impossibility to exhibit it in the form of elementary mathematical functions prevents the application of such methods. Other contribution of this work is a computational method to obtain a GEF for polinomial systems. We also applied the presented method to a class of non polinomial systems arising from electrical power system models, after a nonlinear change of variables that provides an equivalent polinomial system. By means of the proposed methods, we aim to offer computational methods to allow fast and accurate stability region estimates which could be used in a broad class of dynamical systems: those that admit GEF. This way, we plan to contribute for the development of methods used in the assessment of stability of electrical power systems and make such tools available to systems from other areas of science.

O presente trabalho tem como objetivo investigar a presença de diferentes escalas de tempo nos modelos matemáticos que descrevem a dinâmica dos sistemas elétricos de potência (SEPs), em particular a existência de duas escalas de tempo distintas: lenta e rápida, e explorá-las no estudo de estabilidade transitória destes sistemas através da utilização de métodos diretos (funções energia). Em particular, o método do Ponto de Equilíbrio Instável de Controle (CUEP) para modelos com duas escalas de tempo será estudado e aplicado na análise de estabilidade transitória de SEPs. As bases teóricas para a análise de estabilidade transitória, de sistemas com duas escalas de tempo, serão apresentadas, assim como funções energia e novos algoritmos numéricos para o cálculo do CUEP nestes sistemas, a fim de evidenciar as melhorias e possíveis limitações deste novo método CUEP em duas escalas de tempo quando comparado ao método CUEP tradicional. Explorando as escalas de tempo lenta e rápida na análise de estabilidade transitória, espera-se que novos algoritmos numéricos mais robustos para o cálculo do CUEP sejam obtidos, assim como a diminuição do conservadorismo dos resultados. / The main objective of this work is to investigate the existence of several time-scales in the mathematical models of electric power systems, in particular the existence of two-time scales: slow and fast, and exploit these features in the direct transient stability assessment. In particular, the Controlling Unstable Equilibrium Point (CUEP) method is studied for two-time scale models of power systems and applied to transient stability analysis. In order to accomplish this aim, a sound theoretical basis for two-time scale transient stability analysis of electric power system models will be provided, as well as energy functions and new numerical algorithms for proper two-time scale CUEP calculations, with the purpose of investigating improvements and possible limitations of this method when compared with the traditional CUEP method. Exploiting the two-time scale features of power system models, it is intended to obtain new robust numerical algorithms for transient stability analysis, as well as to diminish the conservativeness of the results.

O presente trabalho tem como objetivo investigar a presença de diferentes escalas de tempo nos modelos matemáticos que descrevem a dinâmica dos sistemas elétricos de potência (SEPs), em particular a existência de duas escalas de tempo distintas: lenta e rápida, e explorá-las no estudo de estabilidade transitória destes sistemas através da utilização de métodos diretos (funções energia). Em particular, o método do Ponto de Equilíbrio Instável de Controle (CUEP) para modelos com duas escalas de tempo será estudado e aplicado na análise de estabilidade transitória de SEPs. As bases teóricas para a análise de estabilidade transitória, de sistemas com duas escalas de tempo, serão apresentadas, assim como funções energia e novos algoritmos numéricos para o cálculo do CUEP nestes sistemas, a fim de evidenciar as melhorias e possíveis limitações deste novo método CUEP em duas escalas de tempo quando comparado ao método CUEP tradicional. Explorando as escalas de tempo lenta e rápida na análise de estabilidade transitória, espera-se que novos algoritmos numéricos mais robustos para o cálculo do CUEP sejam obtidos, assim como a diminuição do conservadorismo dos resultados. / The main objective of this work is to investigate the existence of several time-scales in the mathematical models of electric power systems, in particular the existence of two-time scales: slow and fast, and exploit these features in the direct transient stability assessment. In particular, the Controlling Unstable Equilibrium Point (CUEP) method is studied for two-time scale models of power systems and applied to transient stability analysis. In order to accomplish this aim, a sound theoretical basis for two-time scale transient stability analysis of electric power system models will be provided, as well as energy functions and new numerical algorithms for proper two-time scale CUEP calculations, with the purpose of investigating improvements and possible limitations of this method when compared with the traditional CUEP method. Exploiting the two-time scale features of power system models, it is intended to obtain new robust numerical algorithms for transient stability analysis, as well as to diminish the conservativeness of the results.