Microwave spectrometry for the Evaluation of the structural integrity of metallic stents

Purpose: To assess the feasibility of a method based on microwave spectrometry to detect structural distortions of metallic stents in open air conditions and envisage the prospects of this approach toward possible medical applicability for the evaluation of implanted stents. Methods: Microwave absorbance spectra between 2.0 and 18.0 GHz were acquired in open air for the characterization of a set of commercial stents using a specifically design setup. Rotating each sample over 360º, 2D absorbance diagrams were generated as a function of frequency and rotation angle. To check our approach for detecting changes in stent length (fracture) and diameter (recoil), two specific tests were performed in open air. Finally, with a few adjustments, this same system provides 2D absorbance diagrams of stents immersed in a water-based phantom, this time over a bandwidth ranging from 0.2 to 1.8 GHz. Results: The authors show that metallic stents exhibit characteristic resonant frequencies in their microwave absorbance spectra in open air which depend on their length and, as a result, may reflect the occurrence of structural distortions. These resonances can be understood considering that such devices behave like dipole antennas in terms of microwave scattering. From fracture tests, the authors infer that microwave spectrometry provides signs of presence of Type I to Type IV stent fractures and allows in particular a quantitative evaluation of Type III and Type IV fractures. Recoil tests show that microwave spectrometry seems able to provide some quantitative assessment of diametrical shrinkage, but only if it involves longitudinal shortening. Finally, the authors observe that the resonant frequencies of stents placed inside the phantom shift down with respect to the corresponding open air frequencies, as it should be expected considering the increase of dielectric permittivity from air to water. Conclusions: The evaluation of stent resonant frequencies provided by microwave spectrometry allows detection and some quantitative assessment of stent fracture and recoil in open air conditions. Resonances of stents immersed in water can be also detected and their characteristic frequencies are in good agreement with theoretical estimates. Although these are promising results, further verifica tion in a more relevant phantom is required in order to foresee the real potential of this approach.

Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2.

TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.

CHARACTERIZING THE MECHANISM OF QUETIAPINE DISTRIBUTION IN LIPID-CORE NANOCAPSULES PSEUDO-PHASES USING A VALIDATED LC/UV METHOD

Quetiapine is an atypical antipsychotic used to treat schizophrenia. However, despite great interest for its chronic therapeutic use, quetiapine has some important side effects such as weight gain induction. The development of a quetiapine nanocarrier can potentially target the drug into central nervous system, resulting in a reduction of systemic side effects and improved patient treatment. In the present work, a simple liquid chromatography/ultraviolet detection (LC/UV) analytical method was developed and validated for quantification of total quetiapine content in lipid core nanocapsules as well as for determination of incorporation efficiency. An algorithm proposed by Oliveira et al. (2012) was applied to characterize the distribution of quetiapine in the pseudo-phases of the nanocarrier, leading to a better understanding of the quetiapine nanoparticles produced. The analytical methodology developed was specific, linear in the range of 0.5 to 100 µg mL−1 (r2 > 0,99), and accurate and precise (R.S.D < ±5%). The absolute recovery of quetiapine from the nanoparticles was approximately 98% with an incorporation efficiency of approximately 96%. The results indicated that quetiapine was present in a type III distribution according to the algorithm, and was mainly located in the core of the nanoparticle because of its logD in the formulation pH (6.86 ± 0.4).

Analyses of the development and glycoproteins present in the ovarian follicles of Poecilia vivipara (Cyprinodontiformes, Poeciliidae)

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

The effect of down-regulation of Smad3 by RNAi on hepatic stellate cells and a carbon tetrachloride-induced rat model of hepatic fibrosis

Searching for effective Smad3 gene-based gene therapies for hepatic fibrosis, we constructed siRNA expression plasmids targeting the rat Smad3 gene and then delivered these plasmids into hepatic stellate cells (HSCs). The effect of siRNAs on the mRNA levels of Smad2, Smad3, Smad4, and collagens I-α1, III-α1 and IV-α1 (Colα1, Col3α1, Col4α1, respectively) was determined by RT-PCR. Eighty adult male Sprague-Dawley rats were randomly divided into three groups. Twice a week for 8 weeks, the untreated hepatic fibrosis model (N = 30) and the treated group (N = 20) were injected subcutaneously with 40% (v/v) carbon tetrachloride (CCl4)-olive oil (3 mL/kg), and the normal control group (N = 30) was injected with olive oil (3 mL/kg). In the 4th week, the treated rats were injected subcutaneously with liposome-encapsulated plasmids (150 µg/kg) into the right liver lobe under general anesthesia once every 2 weeks, and the untreated rats were injected with the same volume of buffer. At the end of the 6th and 8th weeks, liver tissue and sera were collected. Pathological changes were assessed by a semi-quantitative scoring system (SSS), and a radioimmunoassay was used to establish a serum liver fibrosis index (type III procollagen, type IV collagen, laminin, and hyaluronic acid). The mRNA expression levels of the above cited genes were reduced in the HSCs transfected with the siRNA expression plasmids. Moreover, in the treated group, fibrosis evaluated by the SSS was significantly reduced (P < 0.05) and the serum indices were greatly improved (P < 0.01). These results suggest that Smad3 siRNA expression plasmids have an anti-fibrotic effect.

Investigations into the determinations of hydride-forming elements

Analytical methods for the determination of trace amounts of germanium, tin and arsenic were established using hydride generation coupled with direct current plasma atomic emission spectrometry. A continuous gas flowing batch system for the hydride generation was investigated and was applied to the determination of germanium(Ge), tin(Sn), antimony(Sb) and lead(Pb) (Preliminary results suggest that it is also applicable to arsenic)As) ). With this system, the reproducibility of signals was improved and the determination was speeded up, compared with the conventional batch type hydride generation system. Each determination was complete within one minute. Interferences from a number of transition metal ions, especially from Pd(II), Pt(IV), Ni(II), Cu(II), Co(II), and Fe(II, III), have proven to be very serious under normal conditions, in the determination of germanium, tin, and arsenic. These interference effects were eliminated or significantly reduced in the presence of L-cystine or L-cysteine. Thus, a 10-1000 fold excess of Ni(II), Cu(II), Co(II), Fe(II), Pt(IV), Pd(II), etc. can be tolerated without interference, In the presence of L-cystine or L-cysteine, compared with absence of interference reducing agent. The methods for the determination of Ge, Sn, and As were examined by the analyses of standard reference materials. Interference effects from the sample matrix, for example, in transition metal-rich samples, copper, iron and steel, were eliminated by L-cystine (for As and Sn) and by LI cysteine (for Ge). The analysis of a number of standard reference materials gave excellent results of As and Sn contents in agreement with the certified values, showing there was no systematic interference. The detection limits for both germanium and tin were 20 pg ml- I . Preliminary studies were carried out for the determination of antimony and lead. Antimony was found to react with NaBH4, remaInIng from the previous determinations, giving an analytical signal. A reversed injection manner, i.e., injection of the NaBH4 solution prior to the analyte solution was used to avoid uncertainty caused by residual NaBH4 present and to ensure that an excess of NaB H4 was available. A solution of 0.4% L-cysteine was found to reduce the interference from selected transition metal ions, Co(II), Cu(II), Ni(II) and Pt(IV). Hydrochloric acid - hydrogen peroxide, nitric acid - ammonium persulphate, and potassium dichromate malic acid reaction systems for lead hydride generation were compared. The potassium dichromate - malic acid reaction medium proved to be the best with respect to reproducibility and minimal interference. Cu(II), Ni(II), and Fe(II) caused strong interference In lead determinations, which was not reduced by L-cysteine or Lcystine. Sodium citrate, ascorbic acid, dithizone, thiosemicarbazide and penicillamine reduced interferences to some extent. Further interference reduction studies were carried out uSIng a number of amino acids, glycine, alanine, valine, leucine and histidine, as possible interference reducing agents in the determination of germanium. From glycine, alanine, valine to leucine, the interference reduction effect in germanium determinations decreased. Histidine II was found to be very promising In the reduction of interference. In fact, histidine proved more efficient than L-cystine and L-cysteine In the reduction of interference from Ni(II) in the determination of germanium. Signal enhancement by easily ionized elements (EIEs), usually regarded as an interference effect in analysis by DCP-AES, was studied and successfully applied to advantage in improving the sensitivity and detection limit in the determination of As, Ge, Sn, Sb, and Pb. The effect of alkali and alkaline-earth elements on the determination of the above five hydride forming elements was studied. With the appropriate EIE, a signal enhancement of 40-115% was achieved. Linear calibration and good reproducibility were also obtained in the presence of EIEs. III

Problème centre-foyer et application

Dans ce mémoire, nous étudions le problème centre-foyer sur un système polynomial. Nous développons ainsi deux mécanismes permettant de conclure qu’un point singulier monodromique dans ce système non-linéaire polynomial est un centre. Le premier mécanisme est la méthode de Darboux. Cette méthode utilise des courbes algébriques invariantes dans la construction d’une intégrale première. La deuxième méthode analyse la réversibilité algébrique ou analytique du système. Un système possédant une singularité monodromique et étant algébriquement ou analytiquement réversible à ce point sera nécessairement un centre. Comme application, dans le dernier chapitre, nous considérons le modèle de Gauss généralisé avec récolte de proies.

Role of CD4+ T cells in the regulation of the immune response against encapsulated Group B Streptococcus

Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1. En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène.

The study of RNA tertiary interactions in tRNA structure and function

Le rôle des deux paires de bases universelles inverse Hoogsteen U : A ( RHUAs ) présentent chez les ARNt standards , une dans la boucle T et l'autre dans le noyau de la forme en L , a été étudiée. Pour chacun des RHUAs , un criblage génétique spécialisé in vivo chez les bactéries , le système suppresseur ambre ( pour l'étude de la RHUA dans la boucle T ) et le système d'ARNt de la sélénocystéine ( tRNASec ) ( pour l'étude de la RHUA dans le noyau ) , ont été utilisé pour générer des variants fonctionnels à partir de multiples librairies combinatoires . Ces variants ont ensuite été séquencé et soumis à une analyse systématique qui comprend la modélisation informatique et un type d'analyse phylogénétique. Les résultats du système suppresseur ambre ont montré un ensemble de variants fonctionnels qui ne nécessitent pas le motif RHUA dans la boucle T et qui ont remplacé la méthode standard de l'interaction entre les boucles D et T avec une double hélice interboucle , ILDH . D'autres études ont abouti à la détermination d'un modèle In silico de l'alternative à la norme standard de la boucle T, sous le nom de type III . Les résultats du système tRNASec ont révélé que pour cette ARNt exceptionnel, l'absence de RHUA ( dans le noyau ) assure une flexibilité accrue qui est spécifiquement nécessaire pour la fonction de tRNASec . Ainsi, les ARNt standards , à la différence de tRNASec , avec la présence universelle de RHUA dans le noyau , a été naturellement sélectionnée pour être rigide . Pris ensemble, la RHUA joue un rôle essentiel dans la stabilisation des interactions tertiaires.

Etude du développement de la réponse humorale dirigée contre la capsule polysaccharidique de Streptococcus suis et Streptococcus du groupe B

Streptococcus suis et Streptococcus du groupe B (GBS) sont deux bactéries encapsulées qui induisent des pathologies similaires chez l’homme et/ou l’animal, incluant septicémies et méningites. La capsule polysaccharidique (CPS) est un facteur de virulence clé de ces deux pathogènes et les anticorps (Ac) anti-CPS présentent un bon potentiel protecteur. Néanmoins, ces molécules sont faiblement immunogéniques et les mécanismes de la génération de la réponse humorale anti-CPS demeurent méconnus. L’objectif principal de cette thèse était d’évaluer les caractéristiques et les mécanismes du développement de la réponse Ac dirigée spécifiquement contre les CPS de S. suis et GBS, ainsi que l’effet de la biochimie de la CPS dans cette réponse. Nous avons étudié S. suis types 2 et 14 et GBS types III et V, dont les CPS présentent plusieurs similarités dans leurs compositions et leurs structures, incluant la présence d’acide sialique, un sucre potentiellement immunosuppresseur, tout en possédant une antigénicité propre. Nous avons tout d’abord analysé la nature de la réponse Ac anti-CPS sérique face à la bactérie entière. Les souris infectées par S. suis développent une réponse très faible (S. suis type 2) voire insignifiante (S. suis type 14) de profil isotypique restreint à l’IgM et sont incapables de monter une réponse mémoire efficace face à une seconde infection. Un profil similaire est obtenu chez le porc infecté par S. suis type 2. On détecte des titres d’IgM anti-CPS significatifs chez les souris infectées par GBS (type III ou V). Toutefois, la magnitude de la réponse reste globalement faible et aucune commutation de classe n’est observée. Nous avons ensuite examiné l’influence de la biochimie de la CPS sur ces profils de réponse en conduisant des expériences avec la CPS hautement purifiée de ces pathogènes. Tandis que la CPS de GBS type III administrée aux souris conserve des propriétés immunogéniques similaires à celles observées durant l’infection par la bactérie intacte, les CPS de S. suis type 2 et GBS type V perdent toute capacité à induire une réponse Ac spécifique. L’analyse de l’interaction in vitro des CPS avec les cellules dendritiques (DC) murines, des acteurs clés dans la détection des pathogènes et l’orchestration des réponses immunitaires subséquentes, révèle que ces molécules stimulent la production de niveaux conséquents de chémokines via différents récepteurs. Néanmoins, les CPS sont inaptes à induire la sécrétion de cytokines et elles interfèrent avec la capacité des DC à exprimer BAFF, une cytokine clé dans la différenciation des lymphocytes B en plasmocytes. L’utilisation de CPS chimiquement désialylées démontre que l’acide sialique ne joue aucun rôle immunosuppresseur majeur dans le développement de la réponse Ac dirigée contre les CPS purifiées de S. suis ou GBS, ni sur l’interaction des CPS avec les DC in vitro, ni sur profil de la réponse in vivo. D’autres propriétés biochimiques intrinsèques à ces CPS seraient responsables de l’inaptitude de l’hôte infecté à monter une réponse Ac adéquate et les identifier constituera un outil précieux pour une meilleure compréhension de l’immunopathogénèse de S. suis et GBS ainsi que pour développer des moyens de lutte efficaces contre ces bactéries.

Factores asociados a infección de fracturas abiertas de extremidades por accidentes de tránsito. Bogotá, 2012-2013

Los traumatismos por accidentes de tránsito, constituyen un problema de salud pública, a nivel mundial. Las lesiones más frecuentes son las fracturas de extremidades (84.3%). Las fracturas tienen un elevado riesgo de presentar infecciones, secuelas e incapacidades permanentes. Objetivo : Determinar si los factores asociados con la patología (lugar de fractura, clasificación de fractura, comorbilidades del paciente) y/o los factores relacionados con la atención médica (uso de profilaxis antibiótica diferente al protocolo institucional, tiempo prolongado para remisión, demoras en manejo quirúrgico) se asocian a mayor probabilidad de presentar infección de fracturas abiertas, en población mayor a 15 años, atendidos por accidente de tránsito, en una clínica de Bogotá de tercer nivel especializada en atención de SOAT, durante el período Octubre de 2012 a Octubre de 2013. Metodología: Estudio de casos y controles no apareado, relación 1:3, conformado por 43 casos (fracturas abiertas infectadas) y 129 controles (fracturas abiertas no infectadas). Resultados: La edad media de los casos fue de 39.42 +/- 16.82 años (med=36 años) y la edad media de los controles fue de 33.15 +/- 11.78 años (med=30 años). El 83.7% de los casos y el 78.3% de los controles corresponden al sexo masculino. Predominaron los accidentes en motocicleta en el 81.4% de los casos y el 86% de los controles. En el análisis bivariado se encuentra que la edad mayor a 50 años (p=0.042), una clasificación de la fractura grado IIIB o IIIC (p=0.02), cumplimiento del protocolo antibiótico institucional según el grado de fractura (p=0.014) y un tiempo mayor a 24 horas desde el momento del accidente al centro especializado en trauma (p=0.035) se asociaron significativamente con infección de la fractura abierta. En el análisis multivariado se encuentra únicamente que la clasificación de la fractura grado IIIB o IIIC se asocia con infección de la fractura OR 2.6 IC95% (1.187 – 5.781) (p=0.017). La duración de hospitalización fue mayor en los casos (32.37+/- 22.92 días, med=26 días) que en los controles (8.81 +/- 7.52 días, med=6 días) (p<0.001). El promedio de lavados quirúrgicos fue mayor en los casos (4.85±4.1, med=4.0) que en el grupo control (1.94±1.26, med=2) (p<0.001). Conclusiones: La infección posterior a una fractura abierta, implica costos elevados de atención con hospitalizaciones prolongadas y mayor frecuencia de intervenciones quirúrgicas como se evidencia en el presente estudio. Se debe fortalecer el sistema de remisión y contra remisión para acortar los tiempos de inicio de manejo especializado de los pacientes con fracturas abiertas. Se debe incentivar dentro de las instituciones, el cumplimiento de protocolos de profilaxis antibiótica según el grado de la fractura para disminuir el riesgo de complicación infecciosa.

Modelling, control and supervision for a class of hybrid systems

The aim of this thesis is to narrow the gap between two different control techniques: the continuous control and the discrete event control techniques DES. This gap can be reduced by the study of Hybrid systems, and by interpreting as Hybrid systems the majority of large-scale systems. In particular, when looking deeply into a process, it is often possible to identify interaction between discrete and continuous signals. Hybrid systems are systems that have both continuous, and discrete signals. Continuous signals are generally supposed continuous and differentiable in time, since discrete signals are neither continuous nor differentiable in time due to their abrupt changes in time. Continuous signals often represent the measure of natural physical magnitudes such as temperature, pressure etc. The discrete signals are normally artificial signals, operated by human artefacts as current, voltage, light etc. Typical processes modelled as Hybrid systems are production systems, chemical process, or continuos production when time and continuous measures interacts with the transport, and stock inventory system. Complex systems as manufacturing lines are hybrid in a global sense. They can be decomposed into several subsystems, and their links. Another motivation for the study of Hybrid systems is the tools developed by other research domains. These tools benefit from the use of temporal logic for the analysis of several properties of Hybrid systems model, and use it to design systems and controllers, which satisfies physical or imposed restrictions. This thesis is focused in particular types of systems with discrete and continuous signals in interaction. That can be modelled hard non-linealities, such as hysteresis, jumps in the state, limit cycles, etc. and their possible non-deterministic future behaviour expressed by an interpretable model description. The Hybrid systems treated in this work are systems with several discrete states, always less than thirty states (it can arrive to NP hard problem), and continuous dynamics evolving with expression: with Ki ¡ Rn constant vectors or matrices for X components vector. In several states the continuous evolution can be several of them Ki = 0. In this formulation, the mathematics can express Time invariant linear system. By the use of this expression for a local part, the combination of several local linear models is possible to represent non-linear systems. And with the interaction with discrete events of the system the model can compose non-linear Hybrid systems. Especially multistage processes with high continuous dynamics are well represented by the proposed methodology. Sate vectors with more than two components, as third order models or higher is well approximated by the proposed approximation. Flexible belt transmission, chemical reactions with initial start-up and mobile robots with important friction are several physical systems, which profits from the benefits of proposed methodology (accuracy). The motivation of this thesis is to obtain a solution that can control and drive the Hybrid systems from the origin or starting point to the goal. How to obtain this solution, and which is the best solution in terms of one cost function subject to the physical restrictions and control actions is analysed. Hybrid systems that have several possible states, different ways to drive the system to the goal and different continuous control signals are problems that motivate this research. The requirements of the system on which we work is: a model that can represent the behaviour of the non-linear systems, and that possibilities the prediction of possible future behaviour for the model, in order to apply an supervisor which decides the optimal and secure action to drive the system toward the goal. Specific problems can be determined by the use of this kind of hybrid models are: - The unity of order. - Control the system along a reachable path. - Control the system in a safe path. - Optimise the cost function. - Modularity of control The proposed model solves the specified problems in the switching models problem, the initial condition calculus and the unity of the order models. Continuous and discrete phenomena are represented in Linear hybrid models, defined with defined eighth-tuple parameters to model different types of hybrid phenomena. Applying a transformation over the state vector : for LTI system we obtain from a two-dimensional SS a single parameter, alpha, which still maintains the dynamical information. Combining this parameter with the system output, a complete description of the system is obtained in a form of a graph in polar representation. Using Tagaki-Sugeno type III is a fuzzy model which include linear time invariant LTI models for each local model, the fuzzyfication of different LTI local model gives as a result a non-linear time invariant model. In our case the output and the alpha measure govern the membership function. Hybrid systems control is a huge task, the processes need to be guided from the Starting point to the desired End point, passing a through of different specific states and points in the trajectory. The system can be structured in different levels of abstraction and the control in three layers for the Hybrid systems from planning the process to produce the actions, these are the planning, the process and control layer. In this case the algorithms will be applied to robotics ¡V a domain where improvements are well accepted ¡V it is expected to find a simple repetitive processes for which the extra effort in complexity can be compensated by some cost reductions. It may be also interesting to implement some control optimisation to processes such as fuel injection, DC-DC converters etc. In order to apply the RW theory of discrete event systems on a Hybrid system, we must abstract the continuous signals and to project the events generated for these signals, to obtain new sets of observable and controllable events. Ramadge & Wonham¡¦s theory along with the TCT software give a Controllable Sublanguage of the legal language generated for a Discrete Event System (DES). Continuous abstraction transforms predicates over continuous variables into controllable or uncontrollable events, and modifies the set of uncontrollable, controllable observable and unobservable events. Continuous signals produce into the system virtual events, when this crosses the bound limits. If this event is deterministic, they can be projected. It is necessary to determine the controllability of this event, in order to assign this to the corresponding set, , controllable, uncontrollable, observable and unobservable set of events. Find optimal trajectories in order to minimise some cost function is the goal of the modelling procedure. Mathematical model for the system allows the user to apply mathematical techniques over this expression. These possibilities are, to minimise a specific cost function, to obtain optimal controllers and to approximate a specific trajectory. The combination of the Dynamic Programming with Bellman Principle of optimality, give us the procedure to solve the minimum time trajectory for Hybrid systems. The problem is greater when there exists interaction between adjacent states. In Hybrid systems the problem is to determine the partial set points to be applied at the local models. Optimal controller can be implemented in each local model in order to assure the minimisation of the local costs. The solution of this problem needs to give us the trajectory to follow the system. Trajectory marked by a set of set points to force the system to passing over them. Several ways are possible to drive the system from the Starting point Xi to the End point Xf. Different ways are interesting in: dynamic sense, minimum states, approximation at set points, etc. These ways need to be safe and viable and RchW. And only one of them must to be applied, normally the best, which minimises the proposed cost function. A Reachable Way, this means the controllable way and safe, will be evaluated in order to obtain which one minimises the cost function. Contribution of this work is a complete framework to work with the majority Hybrid systems, the procedures to model, control and supervise are defined and explained and its use is demonstrated. Also explained is the procedure to model the systems to be analysed for automatic verification. Great improvements were obtained by using this methodology in comparison to using other piecewise linear approximations. It is demonstrated in particular cases this methodology can provide best approximation. The most important contribution of this work, is the Alpha approximation for non-linear systems with high dynamics While this kind of process is not typical, but in this case the Alpha approximation is the best linear approximation to use, and give a compact representation.

Pathogenicity and other genomic islands in plant pathogenic bacteria

Pathogenicity islands (PAIs) were first described in uropathogenic E. coli. They are now defined as regions of DNA that contain virulence genes and are present in the genome of pathogenic strains, but absent from or only rarely present in non-pathogenic variants of the same or related strains. Other features include a variable G+C content, distinct boundaries from the rest of the genome and the presence of genes related to mobile elements such as insertion sequences, integrases and transposases. Although PAIs have now been described in a wide range of both plant and animal pathogens it has become evident that the general features of PAIs are displayed by a number of regions of DNA with functions other than pathogenicity, such as symbiosis and antibiotic resistance, and the general term genomic islands has been adopted. This review will describe a range of genomic islands in plant pathogenic bacteria including those that carry effector genes, phytotoxins and the type III protein secretion cluster. The review will also consider some medically important bacteria in order to discuss the range, acquisition and stabilization of genomic islands.

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution.