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Polymyxins are potent antibiotics that bind to the outer membrane of Gram-negative bacteria, entering the cell and disrupting the inner membrane, resulting in cell death. They were traditionally used as antibiotics of last resort, but the recent surge of multidrug resistant pathogens has renewed interest in these antibiotics. The emergence of polymyxin resistance determinants such as the recently discovered plasmid-mediated phosphoethanolamine transferase MCR-1 may put a strain on the future effectiveness of this antibiotic. One method to combat the rise in antibiotic resistant bacteria is through the identification and development of antibiotic adjuvants. These are small molecules that are able to inhibit the resistance mechanism, allowing previously ineffective antibiotics to once again become effective at treating bacterial infections. In this work, a high throughput cell-based screen was conducted using an in-house library of Actinomycete-derived crude cell extracts in order to search for a natural product inhibitor of an E. coli strain expressing mcr-1. In addition, the development of a new enzyme assay was attempted using purified MCR-1 C-terminal catalytic domain and a chromogenic substrate to test enzymatic activity in vitro, in hopes of establishing a simple means of studying inhibition of MCR-1. The structure-function relationship of MCR-1 was also explored by generating amino acid substitutions and studying their effect on the ability of the enzyme to confer resistance to colistin, as well as the generation of MCR-1 transmembrane truncation mutants to determine if it was possible to generate a shorter variant of MCR-1 that retained its enzymatic activity. This work furthers our understanding of the biochemistry and enzymology of MCR-1, and outlines attempts to identify inhibitors of MCR-1 in order to re-sensitize resistant bacteria to polymyxins. / Thesis / Master of Science (MSc) / Polymyxins are potent antibiotics that are threatened by the spread of multi-drug resistant bacteria. Resistance to these antibiotics is relatively rare, although the recent discovery of a mobile polymyxin resistance enzyme, MCR-1, threatens the future use of this antibiotic for treating infections, as it can readily transfer to other bacteria. The goal of this work was to search for a natural product inhibitor of MCR-1 in order to reverse its ability to confer resistance to polymyxins. A color-changing assay was conducted with MCR-1 in hopes of establishing a method to study the inhibition of MCR-1 in vitro. Additionally, amino acid substitutions were generated in MCR-1 to better understand how key amino acids affect enzyme function, as well as transmembrane domain truncations to determine if it was possible to create a shorter functioning variant of MCR-1.

The primary focus of my work is the application of informatics methods to the fields of materials science and analytical chemistry. The statistical analysis of data has become increasingly important in understanding the properties of materials and analytes. Statistical methods like principal component analysis (PCA) and multivariate curve resolution (MCR) are widely used for analysis in chemistry and other fields given their ability to categorize spectra in an unsupervised way. PCA is relatively easy to apply and has appealing mathematical properties. However, the results can be challenging to interpret, even for experienced users. In contrast, MCR results can be more interpretable, because the factors resemble real spectra and do not have negative scores or loadings. Nevertheless, the useful orthogonality properties of the scores and loadings in PCA are sacrificed in doing so. Other statistical analysis methods like cluster analysis and partial least squares regression (PLS-R) present their own challenges. Pattern recognition entropy (PRE) is a novel application of Shannon’s information theory for understanding the underlying complexity in spectra. Unlike PCA and MCR, PRE is a summary statistic that adopts the mathematical quantification of information and applies it for chemometric analysis. PRE values reflect the shape and complexity of spectra. Chapter 1 contains a description of the analytical methods/instruments that provided the data I analyzed by PRE and other informatics tools, including (i) X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) and (ii) liquid chromatography-mass spectrometry (LC-MS) and capillary electrophoresis (CE), (iii) a discussion of some of the commonly used statistical analysis tools like PCA, MCR, cluster analysis and PLS-R, and (iv) a description of PRE. Chapter 2 describes in much greater detail the theory associated with the statistical tools I used and PRE. Chapter 3 describes the PRE and informatics analysis of depth profiles through thin films by XPS and ToF-SIMS. Chapter 4 introduces the concept of the ‘reordered spectrum’ as an intuitive, visual representation of spectra to address the abstraction associated with PRE result. Total ion current chromatograms (TICCs) generated using LC-MS are often extremely complex and ‘noisy’. Chapter 5 describes the application of PRE as a variable reduction method for producing higher quality TICCs. Chapter 6 discusses the limitations associated with the application of PRE to TICCs and presents a new method using cross-correlation (CC) in conjunction with a PRE analysis. Chapter 7 discusses a new methodology that uses CE and PRE to detect autologous blood doping (ABD). Chapter 8 presents my conclusions of this present work and discusses the scope of future work on PRE. The thesis also contains several appendices. Appendix 1 introduces polyallylamine (PAAm) as a simple, easy-to-apply adhesion promoter for the widely used photoresist SU-8. Appendices 2, 3 and 4 contain articles I wrote that relate to trends in modern XPS instrumentation and 5-8 contain supplemental information relating to Chapters 3, 4, 5, and 7 respectively.

[ES] El aumento de la esperanza de vida en la población con edad por encima de 50 años está generando un mayor número de casos detectados de cáncer de próstata (CaP). Por este motivo, los recursos se destinan al diagnóstico en etapas tempranas y al tratamiento efectivo. A pesar de la multitud de estudios basados en biomarcadores y discriminación histológica, es difícil diferenciar con efectividad los casos de CaP con baja agresividad de aquellos que progresarán y acabarán produciendo mortalidad o una disminución en la esperanza de vida del paciente. Con el objetivo de mejorar el diagnostico, localización y gradación de los tumores malignos, las técnicas de imagen por Resonancia Magnética (MRI) son las más adecuadas para el estudio del cáncer, proporcionando métodos de diagnóstico no-invasivos, sensibles y específicos, basados en secuencias morfológicas (T2w) y funcionales (perfusión de la sangre y difusión del agua). Las diferentes características y parámetros extraídos de estas secuencias, conocidos como biomarcadores de imagen, pueden evaluar las diferencias asociadas al desarrollo de los procesos tumorales, como los modelos farmacocinéticos para estudiar angiogénesis (perfusión) y los modelos mono- y bi-exponenciales para estudiar la caída de la señal en difusión con el objetivo de estudiar la celularización. Normalmente, estos biomarcadores de imagen se analizan de forma "univariante", sin aprovechar la información de las estructuras de correlación interna que existen entre ellos. Una manera de mejorar este análisis es mediante la aplicación de las técnicas estadísticas que ofrece el Análisis Multivariante de Imágenes (MIA), obteniendo estructuras (latentes) simplificadas que ayudan a entender la relación entre los parámetros (variables) y sus propios procesos fisiológicos, además de reducir la incertidumbre en la estimación de los biomarcadores. En esta tesis, se han desarrollado nuevos biomarcadores de imagen para perfusión y difusión con la aplicación de alguna de las herramientas de MIA como la Resolución Multivariante de Curvas con Mínimos Cuadrados Alternos (MCR-ALS), obteniendo parámetros que tienen interpretación clínica directa. A continuación, los métodos basados en mínimos cuadrados parciales (PLS) se aplicaron para estudiar la capacidad de clasificación de estos biomarcadores. En primer lugar, los biomarcadores de perfusión se utilizaron para la detección de tumores (control vs lesión). Posteriormente, la combinación de perfusión + difusión + T2 se empleó para estudiar agresividad tumoral con la aplicación de métodos PLS multibloque, en concreto (secuencial) SMB-PLS. Los resultados mostrados indican que los biomarcadores de perfusión obtenidos mediante MCR son mejores que los parámetros farmacocinéticos en la diferenciación de la lesión. Con lo que respecta al estudio de la agresividad tumoral, la combinación de los biomarcadores de difusión (empleando ambos métodos: modelos paramétricos y MCR) y los valores de T2w normalizados proporcionaron los mejores resultados. En conclusión, MIA se puede aplicar a las secuencias morfológicas y funcionales de resonancia magnética para mejorar el diagnóstico y el estudio de la agresividad de los tumores en próstata. Obteniendo nuevos parámetros cuantitativos y combinándolos con los biomarcadores más ampliamente utilizados en el ambiente clínico. / [CA] El increment de la esperança de vida en la població per damunt dels 50 anys està generant un major nombre de casos detectats de càncer de pròstata (CaP). Per aquest motiu, els recursos es destinen al diagnòstic en etapes primerenques i al tractament efectiu. Tot i la multitud de estudis basats en biomarcadors y discriminació histològica, es difícil diferenciar amb efectivitat els casos de CaP que tenen baixa agressivitat dels que progressaran y acabaran produint mortalitat o una disminució en la esperança de vida del pacient. Amb el objectiu de millorar el diagnòstic, localització y gradació dels tumors malignes, les tècniques de imatge per Ressonància Magnètica (MRI) son els mètodes més adequats per al estudi del càncer, proporcionant metodologies de diagnòstic no-invasius, sensibles y específiques basades en seqüències morfològiques (T2w) y funcionals (perfusió de la sang y difusió del aigua). Les diferents característiques i paràmetres extrets de aquestes seqüències, coneguts com biomarcadors d'imatge, poden avaluar les diferències associades al desenvolupament dels processos tumorals. Primer, amb els models farmacocinétics per a estudiar angiogènesis (perfusió) y segon, amb els models mono- i bi-exponencials per a estudiar la caiguda de la senyal en difusió amb el objectiu de estudiar la cel·lularització. Normalment, aquests biomarcadors d'imatge s'analitzen de forma "univariant", sense aprofitar la informació de las estructures de correlació interna que existeixen entre ells. Una forma de millorar aquest anàlisis es mitjançant la aplicació de las tècniques estadístiques aportades pel Anàlisis Multivariant de Imatges (MIA), obtenint estructures (latents) simplificades què ajuden a entendre la relació entre els paràmetres (variables) i els seus processos fisiològics, a més de reduir la incertesa en la estimació dels biomarcadors. En aquesta tesis, s'han desenvolupat nous biomarcadors d'imatge per a perfusió i difusió amb la aplicació de alguna de las ferramentes de MIA com la Resolució Multivariant de Corbes i Mínims Quadrats Alterns (MCR-ALS), obtenint paràmetres què tenen interpretació clínica directa. A continuació, els mètodes basats en mínims quadrats parcials (PLS) s'han aplicat per a estudiar la capacitat de classificació d'aquests biomarcadors. En primer lloc, els biomarcadors de perfusió s'han utilitzat per a la detecció de tumors (control contra lesió). Posteriorment, la combinació de perfusió + difusió + T2 s'ha utilitzat per a estudiar agressivitat tumoral amb la aplicació de mètodes PLS multi-bloc, en concret (seqüencial) SMB-PLS. Els resultats mostren què els biomarcadors de perfusió obtinguts mitjançant MCR són millors què els paràmetres farmacocinètics en la diferenciació de la lesió. En lo què es refereix al estudi de la agressivitat tumoral, la combinació dels biomarcadors de difusió (utilitzant els dos mètodes: models paramètrics i MCR) i els valors de T2w normalitzats proporcionaren els millors resultats. En conclusió, MIA es pot aplicar a les seqüències morfològiques i funcionals de ressonància magnètica per a millorar el diagnòstic i el estudi de l'agressivitat dels tumors en pròstata. Obtenint nous paràmetres quantitatius y combinant-los amb els biomarcadors més utilitzats en el ambient clínic. / [EN] The increase in life expectancy and population with age higher than 50 years is producing a major number of detected cases of prostate cancer (PCa). For this reason, the resources are focused in the early diagnosis and effective treatment. In spite of multiple studies with histologic discriminant biomarkers, it is hard to clearly differentiate the low aggressiveness PCa cases from those that will progress and produce mortality or rather a decrease in the life expectancy. With the objective of improving the diagnosis, location and gradation of the malignant tumors, Magnetic Resonance Imaging (MRI) has come up as the most appropriate image acquisition technique for cancer studies, which provides a non-invasive, sensitive and specific diagnosis, based on morphological and functional (blood perfusion and water diffusion) sequences. The different characteristics and parameters extracted from these sequences, known as imaging biomarkers, can evaluate the different processes associated to tumor development, like pharmacokinetic modeling for angiogenesis assessment (perfusion) or mono- and bi-exponential signal decay modeling for cellularization (diffusion). Normally, these imaging biomarkers are analyzed in a "univariate" way, without taking advantage of the internal correlation structures among them. One way to improve this analysis is by applying Multivariate Image Analysis (MIA) statistical techniques, obtaining simplified (latent) structures that help to understand the relation between parameters (variables) and the inner physiological processes, moreover reducing the uncertainty in the estimation of the biomarkers. In this thesis, new imaging biomarkers are developed for perfusion and diffusion by applying MIA tools like Multivariate Curve Resolution Alternating Least Squares (MCR-ALS), obtaining parameters with direct clinical interpretation. Partial Least Squares (PLS) based methods are then used for studying the classification capability of these biomarkers. First, perfusion imaging biomarkers have been tested for tumor detection (control vs lesion). Then, diffusion + perfusion have been combined to study tumor aggressiveness by applying PLS-multiblock methods (SMB-PLS). The results showed that MCR-based perfusion biomarkers performed better than state-of-the-art pharmacokinetic parameters for lesion differentiation. Regarding the assessment of tumor aggressiveness, the combination of diffusion-based imaging biomarkers (using both the parametric models and MCR) and normalized T2-weighted measurements provided the best discriminating outcome, while perfusion was not needed as it did not supply additional information. In conclusion, MIA can be applied to morphologic and functional MRI to improve the diagnosis and aggressiveness assessment of prostate tumors by obtaining new quantitative parameters and combining them with state-of-the-art imaging biomarkers. / Aguado Sarrió, E. (2019). Application of multivariate image analysis to prostate cancer for improving the comprehension of the related physiological phenomena and the development and validation of new imaging biomarkers [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/134023

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas. / Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

Parameter estimation techniques have been successfully and extensively applied to deterministic models based on ordinary differential equations but are in early development for stochastic models. In this thesis, we first investigate using parameter estimation techniques for a deterministic model to approximate parameters in a corresponding stochastic model. The basis behind this approach lies in the Kurtz limit theorem which implies that for large populations, the realizations of the stochastic model converge to the deterministic model. We show for two example models that this approach often fails to estimate parameters well when the population size is small. We then develop a new method, the MCR method, which is unique to stochastic models and provides significantly better estimates and smaller confidence intervals for parameter values. Initial analysis of the new MCR method indicates that this method might be a viable method for parameter estimation for continuous time Markov chain models.

L’objectif de ce travail a consisté en le développement de nouveaux processus multi-composant radicalaires et leur application en synthèse organique. Des carbo-alcénylation d'oléfines ont ainsi été réalisées avec de nouveaux précurseurs de radicaux, des oléfines diverses, en présence de Z-diphénylsulfonyléthylène comme accepteur terminal. Les conditions de la réaction ont été optimisées, en introduisant notamment la diphénylsulfonylhydrazine comme amorceur de radicaux sous irradiation UV, et substitut au couteux DTBHN. Des conditions sans étain ont également été étudiées avec l’objectif de remplacer le réactif (Bu3Sn)2 par des radicaux silylés. Le tris (triméthylsilyl)silylthiopropene a ainsi été testé avec succès en tant qu'agent de propagation des chaînes radicalaires. A l’issue de ce travail méthodologique, nous avons développé une stratégie de synthèse de l'Eucophylline, un alcaloïde isolé de Leuconotis griffithii, dont le squelette tétracyclique a été élaboré sur la base d’une réaction de carbo-oximation radicalaire d’oléfine. Ce processus multicomposant, suivi d’une réduction de la fonction oxime et d’une lactamisation offre une voie d’accès rapide au motif bicyclo[3.3.1]lactame, intermédiaire-clé de la synthèse. Une réaction de type Friedländer entre ce lactame et un ortho-aminobenzonitrile a permis d’accéder au squelette tetrahydrobenzo[1,8]naphthyridine de l'Eucophylline. La synthèse du composé modèle a enfin été complétée par l’introduction du substituant vinylique par un couplage de Heck. / The aim of this work was to develop new radical multi-component processes and their application in organic synthesis. Carbo-alkenylation processes were thus performed with new radical precursors, different olefins, in the presence of Z-diphenylsulfonylethylene as a terminal acceptor. Reaction conditions have also been optimized, including the diphenylsulfonylhydrazine as a radical initiator under U.V. irradiation, and substitute to the costly DTBHN. Tin-free conditions were also screened with the goal of replacing (Bu3Sn)2 with silyl radicals. Tris(trimethylsilyl)silylthiopropene was tested with success as a radical chain carrier. After this methodology studies, we developed a strategy toward the synthesis of Eucophylline, an alcaloid isolated from Leuconotis griffithii, which tetracyclic skeleton was elaborated based on a carbo-oximation of olefin. This multicomponent process, followed by a reduction of the oxime function and a lactamization offered a fast access to the bicyclo[3.3.1]lactam, a key-intermediate in the synthesis. A Friedländer-type reaction between this lactam and an ortho-aminobenzonitrile allowed an access to the Eucophylline tetrahydrobenzo[1,8]naphthyridine skeleton. The synthesis of the model compound was finally completed with the introduction of the vinylic substituent through a Heck coupling.

This thesis documents the development and application of a unique database orientated software package, BugsCEP, for environmental and climatic reconstruction from fossil beetle (Coleoptera) assemblages. The software tools are described, and the incorporated statistical methods discussed and evaluated with respect to both published modern and fossil data, as well as the author’s own investigations. BugsCEP consists of a reference database of ecology and distribution data for over 5 800 taxa, and includes temperature tolerance data for 436 species. It also contains abundance and summary data for almost 700 sites - the majority of the known Quaternary fossil coleopteran record of Europe. Sample based dating evidence is stored for a large number of these sites, and the data are supported by a bibliography of over 3 300 sources. Through the use of built in statistical methods, employing a specially developed habitat classification system (Bugs EcoCodes), semi-quantitative environmental reconstructions can be undertaken, and output graphically, to aid in the interpretation of sites. A number of built in searching and reporting functions also increase the efficiency with which analyses can be undertaken, including the facility to list the fossil record of species found by searching the ecology and distribution data. The existing Mutual Climatic Range (MCR) climate reconstruction method is implemented and improved upon in BugsCEP, as BugsMCR, which includes predictive modelling and the output of graphs and climate space maps. The evaluation of the software demonstrates good performance when compared to existing interpretations. The standardization method employed in habitat reconstructions, designed to enable the inter-comparison of samples and sites without the interference of differing numbers of species and individuals, also appears to be robust and effective. Quantitative climate reconstructions can be easily undertaken from within the software, as well as an amount of predictive modelling. The use of jackknifing variants as an aid to the interpretation of climate reconstructions is discussed, and suggested as a potential indicator of reliability. The combination of the BugStats statistical system with an enhanced MCR facility could be extremely useful in increasing our understanding of not only past environmental and climate change, but also the biogeography and ecology of insect populations in general. BugsCEP is the only available software package integrating modern and fossil coleopteran data, and the included reconstruction and analysis tools provide a powerful resource for research and teaching in palaeo-environmental science. The use of modern reference data also makes the package potentially useful in the study of present day insect faunas, and the effects of climate and environmental change on their distributions. The reconstruction methods could thus be inverted, and used as predictive tools in the study of biodiversity and the implications of sustainable development policies on present day habitats. BugsCEP can be downloaded from http://www.bugscep.com

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas. / Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

La fitorremediación es una técnica de descontaminación de ecosistemas que aprovecha la capacidad de las plantas para acumular sustancias tóxicas en su interior sin que afecten severamente a su ciclo vital. Dicha técnica presenta un bajo impacto medioambiental, convirtiéndose en una alternativa a los métodos clásicos, más agresivos y costosos. En lo referente a metales pesados, las plantas inducen la síntesis intracelular de fitoquelatinas (PC), ligandos tiólicos que complejan los metales y los almacenan en orgánulos celulares de bajo metabolismo como las vacuolas. A pesar de las investigaciones realizadas hasta la fecha, el mecanismo de actuación de las fitoquelatinas no está totalmente establecido, incluida la secuencia de formación y la estequiometria final de los complejos PC-M. Por esta razón, resulta de gran interés estudiar estos procesos de complejación, pues sus conclusiones pueden ayudar a entender la fitorremediación y a optimizar su aplicación. Las investigaciones recogidas en esta tesis se sustentan -como metodología básica- en el análisis quimiométrico mediante MCR-ALS de los datos obtenidos de valoraciones complexométricas registradas por técnicas electroanalíticas, concluyendo con la proposición de modelos de complejación para el sistema en estudio. La elección del plomo como metal complejante se debe a su alta toxicidad y dispersión en el medio ambiente, así como por suponer un paso más en el uso de dicha metodología, pues el estudio de los sistemas PC/Pb(II) presentan problemas que la dificultan. Estos problemas son, principalmente, el desplazamiento lateral de las señales voltamperométricas que produce un decrecimiento en la linealidad de los datos obtenidos, y la presencia de señales anódicas que favorece un fuerte solapamiento entre los picos de las diferentes especies químicas; ambos problemas comprometen la aplicación del método MCR-ALS y el correcto examen de los resultados. El objetivo de esta tesis adquiere así una doble vertiente. Por un lado, para solucionar dichos problemas y, de alguna forma, ampliar la aplicabilidad de MCR-ALS a eventuales sistemas más complejos, se estudian nuevas adaptaciones metodológicas (análisis MCR-ALS de matrices espectro-voltamperométricas), herramientas (programa shiftfit, que corrige el desplazamiento de potencial de las señales voltamperométricas) y metodologías experimentales (uso de electrodos alternativos al de mercurio). Por otro lado, se procura la consecución de resultados para los propios sistemas PC/Pb(II) estudiados, es decir, la determinación de modelos de complejación lo bastante completos y sólidos para servir de apoyo a los resultados de los estudios in vivo o in vitro. Los trabajos publicados y la explicación de los resultados están organizados en tres bloques: • El primer bloque (artículo 11.1) recoge la aproximación inicial al estudio de la complejación de fitoquelatinas y ligandos relacionados con plomo utilizando la metodología básica. Los resultados ponen de relieve la relativa solvencia del procedimiento, así como el verdadero alcance de los problemas que se describen en el capítulo 5. • El segundo bloque recopila los resultados de la aplicación de las nuevas metodologías y herramientas propuestas para solucionar las insuficiencias o ambigüedades de los modelos de complejación obtenidos en el artículo anterior. Estas metodologías son el análisis quimiométrico conjunto de valoraciones registradas por polarografía y dicroísmo circular (11.2), la formulación del programa shiftfit que corrige el movimiento lateral de señales polarográficas, es decir, la falta de un valor fijo del potencial de pico (11.3) y, finalmente, el uso del electrodo de película de bismuto (BiFE) con la intención de minimizar el solapamiento que producen las señales anódicas (11.4). • Tras la aplicación de estas metodologías a sistemas sencillos, en el tercer bloque se aplican a sistemas con plomo (11.5 y 11.6), incluyendo la comparación de los diferentes modelos de complejación obtenidos en los artículos 11.1 y 11.5 para el sistema PC3/Pb(II). / Phytoremediation is a decontamination technique that takes profit by the plants ability to accumulate toxic substances without affecting severely their vital cycles. This technique presents the advantatges of being cheap and non-destructive to ecological systems. Regarding heavy metals, plants induce the intracellular synthesis of phytochelatins (PC), Cys-rich polypeptides that complex metals and storage them in cellular organelles of limited metabolism as vacuoles. Despite the research achieved to date, the mechanism of actuation of phytochelatins is not entirely established, including the sequence of formation and the final stoichiometry of the PC-M complexes. By this reason, it is of great interest to study these complexation processes, reaching conclusions that would be able to help the optimization of phytoremediation. The investigations collected in this doctoral thesis are held -as basic methodology- in the chemometric analysis through MCR-ALS of the data obtained from complexometric titrations carried out by voltammetric techniques. This methodology concludes with the proposition of complexation models for the systems under study, but when lead is used as complexing metal, PC/Pb(II) systems present problems that make it more difficult. Mainly, these problems are the lateral movement of the voltammetric signals (producing a decrease in the linearity of the data), and the presence of anodic signals that propitiate a strong overlapping between peaks of different chemical species; both troubles compromise the MCR-ALS application and the correct investigation of the results. To solve the aforementioned problems -and that way to increase the applicability of MCR-ALS to more complex systems-, some new tools have been applied for the first time: • Methodologic adaptations as the MCR-ALS simultaneous analysis of spectro- and electrochemical data (row-wise CD-DPP augmented matrices) to differenciate real chemical species from physicochemical or kinetic phenomena of the diffusion layer. • Chemometric programs as shiftfit that corrects the potential shift of the polarographic signals to obtain a matrix of corrected voltammograms with an increased linearity. • The use of the bismuth film electrode (BiFE) for complexation studies, to minimize the overlapping produced by the anodic signals.