La protéine chaperon HSP90 a de nombreux substrats par les voies de signalisations. Son inhibition engendre un effet tumoral. L'identification du complexe R2TP,un nouveau cochaperon d'HSP90, a mis en évidence de nouveaux substrats. Le système HSP90/R2TP est impliqué dans l'assemblage de complexes macromoléculaires ( PIKKs, Télomérase RNP, ARN polyméras, snoRNP) jouant un rôle clé dans la prolifération cellulaire et la tumorigénèse. Le R2TP est composé de 4 protéines : RUVBL1, RUVBL2, RPAP3 et PIH1D1 dont certains sont surexprimés dans les cancers hépatocellulaires ou colorectaux. Afin d'étudier le rôle du R2TP dans l'homéostasie intestinale et la carcinogénèse, nous avons généré un modèle d'invalidation de RPAP3 chez la souris. L'invalidation de RPAP3 dans l'organisme entier est létale. Son invalidation à l'âge adulte uniquement dans l'intestin entraine une phénotype sévère en 8jours aboutissant à la mort des individus à 10jours. En parallèle, nous avons étudié le rôle potentiel de RPAP3 dans la tumorigénèse intestinale et colorectale avec deux modèles d'induction , génétique avec l'invalidation du gène APC et chimique avec le protocole AOM/DSS. / Many substrates of HSP90 are involved in signal transduction pathways and related to tumour progression. Inhibition of HSP90 has anti-tumoral effects. Identification of the R2TP, a new HSP90 co-chaperon, allowed the identification of a new set of HSP90 substrates. HSP90/R2TP is involved in the assembly of snoRNPs, telomerase RNP, the nuclear RNA polymerases and PIKKs, which play key functions in cellular proliferation and tumorigenesis. R2TP is formed of four proteins: RUVBL1, RUVBL2, PIH1D1 and RPAP3, some of which are overexpressed in hepatocellular and colorectal cancer. We thus hypothesize that the co-chaperone R2TP could be involved in colorectal carcinogenesis.To study the role of R2TP in intestinal homeostasis and carcinogenesis, we generated a conditional knock-out murine model for RPAP3. We showed that RPAP3 invalidation in whole organism or only in colon is lethal at embryonic stage. The invalidation of RPAP3 in adult intestine, using an inducible recombinase (RPAP3 fl; Villin>Cre-ERT2), leads to a drastic phenotype as soon as 8 days post-induction, resulting in death after 10 days. This phenotype is reminiscent of proliferative defects.In parallel, we address the possibility of a therapeutic window to target RPAP3 during intestinal tumorigenesis by using heterozygous animals (RPAP3 fl/+; Villin>Cre-ERT2 ) in which tumorignesis is induced (i) either by a chemical treatment : for this, we take advantage of the established AOM /DSS protocol, or, (ii) by a genetic one ( Apc LoxP/+).These ongoing experiments will address the role of R2TP in a tissue with a constant turnover and the relevance of R2TP in tumorigenesis.

In this thesis, the relay communication between landed assets on the surface of Mars via a relay orbiter to Earth is studied for its latencies and the times which can be provided for the landed asset to work and for the ground to plan depending on the operational margins, the available ground station network and the available relay orbiters. In this context, an automated solver is developed to evaluate a locally optimal strategy of relay pass assignment respecting the different constraints and is proven to provide a solution close to the globally optimal one. The solver is determining the link opportunities and reasons on them, by minimising a cost function for each relay pass and choosing the cheapest ones in an iterative process. With this solver, it is shown that the best operational approach is to await commands confirmation and to provide the possibility of resending corrupted files. Moreover, it is shown that a 24/7 ground station coverage should be ideally provided, on which priority for booking should be given to relay missions since they depend on the actual timing of the orbiter overflights over the lander. Further- more, it is shown that adding additional relay orbiters increases the solution space drastically, making it desirable to use them. The possibility to restrict cross-agency support is assessed, showing that cross-support is still eligible. Finally, the data volume is shown to be sufficient to fulfil the ExoMars Rover and Surface Platform mission requirements, when using multiple orbiters, even though the solver itself would need extra capabilities to cope with allocating appropriate relay passes.

A microextração líquido-líquido dispersiva baseada no uso de solventes orgânicos (OS-DLLME) vem recebendo grande destaque devido a facilidade de operação, baixo custo, rapidez e um consumo mínimo de solventes. Previamente a OS-DLLME um pré-tratamento do plasma se faz necessário a fim de propiciar a formação de uma adequada fase sedimentada. O levetiracetam (LEV) é um dos fármacos mais prescritos para o tratamento de crises epilépticas parciais e com generalizações secundárias. Assim, foi desenvolvido e validado um método bioanalítico para a quantificação do LEV em amostras de plasma por cromatografia em fase gasosa acoplada à espectrometria de massas (GC/MS). O pré-tratamento das amostras de plasma empregou ultrafiltros Amicon® (poros com tamanho de 10 kDa) que formavam um permeado adequado para a realização da OS-DLLME. A adição de 130 ?L de clorofórmio (solvente extrator) e 400 ?L de isopropanol (solvente dispersor), sem alteração na força iônica e agitação da amostra, proporcionaram 33 % de recuperação do LEV nas amostras de plasma. Tal análise foi realizada usando uma coluna de sílica fundida recoberta com Rtx-5MS (30 m × 0,25 mm x 0,25 ?m) empregando uma rampa de aquecimento. O tempo total de corrida cromatográfica foi de 9 minutos. O método apresentou-se linear no intervalo de concentração de 2 - 80 ?g mL-1 (r >= 0,99), onde os resultados foram ponderados (peso =1/x). A validade da regressão foi confirmada aplicando o teste da falta de ajuste linear. O limite inferior de quantificação (LIQ) foi de 2 ?g mL-1. Os parâmetros de precisão, exatidão, efeito matriz, efeito carry-over e estabilidade demonstraram-se em conformidade com o European Medicines Agency. Outro método foi desenvolvido e validado para a quantificação da risperidona (RSP) e seu metabólito 9-hidroxi-risperidona (9-OH-RSP) em amostras de plasma. A RSP e 9-OH-RSP correspondem a \"fração ativa\" do medicamento responsável pela atividade anticonvulsivante. As amostras de plasma foram pré-tratadas com ácido tricloroacético (TCA 30 %). O sobrenadante límpido foi diluído com solução de NaCl (10 %) e ajustado para o valor de pH 12, no qual foi desenvolvido a OS-DLLME. Foi adicionado um volume de 500 ?L de acetona (solvente dispersor) e 80 ?L de clorobenzeno (solvente extrator). Após otimização da extração, foi alcançado 89 % e 42 % de recuperação da RSP e 9-OH-RSP, respectivamente. Para tais análises foi empregada a cromatografia líquida acoplada à espectrometria de massas sequencial com ionização por eletrospray (LC-MS/MS). Uma coluna Ascentis® C18 (10 cm x 4,6 mm x 2,7 ?m), fase móvel composta por tampão acetato de amônio e acetonitrila, em modo de eluição gradiente, vazão de 500 ?L min-1 e temperatura da coluna de 40 ºC foram empregados. O tempo total da corrida cromatográfica foi de 8 minutos. O método foi linear na faixa de concentração de 5 - 80 ng mL-1 (r >= 0,99), para ambos os analitos. O LIQ foi de 5 ng mL-1. Todos os demais parâmetros estavam em conformidade com a agência regulatória. Ambos os métodos foram aplicados com sucesso em amostras de plasma de paciente em tratamento com LEV ou RSP. Desta forma foi possível demonstrar aplicabilidade dos métodos bioanalíticos desenvolvidos e averiguar a concentração plasmática dos devidos fármacos no paciente / Dispersive liquid-liquid microextraction technique based on the use of organic solvents (OS-DLLME) has received highlighted due to the easily, rapidity, low cost and low consumption of organic solvent. Prior to OS-DLLME, plasma pretreatment is necessary to provide the formation of a suitable settled phase. Levetiracetam (LEV) is one of the most prescribed drugs for the treatment-refractory partial onset seizures with or without secondary generalization. So, it was developed and validated a bioanalytical method for the quantification of LEV in plasma samples by Gas Chromatography Mass Spectrometry (GC/MS). The pretreatment of the plasma samples employed tubes of ultrafiltration Amicon® (pore size 10 kDa) in order to form a suitable permeated to carry out the OS-DLLME. The addition of 130 ?L of chloroform (extraction solvent) and 400 ?L of isopropanol (disperser solvent), without ionic strength and agitation of samples, have reached 33 % of recovery of LEV from plasma samples. This analysis was carried out using a fused silica coated Rtx-5MS column (30 m × 0.25 mm × 0.25 ?m) and a heating ramp. The run time was 9 minutes. The method was linear in the concentration range of 2 - 80 ?g mL-1 (r >= 0.99) and the results were weighted (1/x). The linear regression was confirmed by applying the lack of fit test. The lower limit of quantitation (LLOQ) was 2 ?g mL-1. The precision, accuracy, matrix effect, carry-over and stability were in agreement with European Medicines Agency. Another method was developed and validated for the quantification of risperidone (RSP) and its 9-hydroxy-risperidone metabolite (9-OH-RSP) from plasma samples. The RSP and 9-OH-RSP constitutes the total active moiety responsible for the anticonvulsant activity. Plasma samples were pretreated with trichloroacetic acid (TCA 30%). The clear supernatant was diluted with NaCl solution (10%) and adjusted to pH 12, where was developed the OS-DLLME. It was added 500 ?L of acetone (dispersing solvent) and 80 ?L of chlorobenzene (extraction solvent). After the parameters optimization, the recovery was 89% and 42% for RSP and 9-OH-RSP, respectively. It was employed liquid chromatography-electrospray-tandem-mass spectrometry (LC/ MS/MS). Ascentis® C18 column (10 cm x 4.6 mm x 2.7 ?M), ammonium acetate buffer and acetonitrile as mobile phase in gradient elution mode, at flow rate 500 ?L min-1 and column temperature of 40 °C for this analysis. The run time was 8 minutes. The method was linear in the concentration range 5 - 80 ng mL-1 (r >= 0.99) for both analytes. The LLOQ was 5 ng mL-1. All other parameters were in agreement with the regulatory agency. Both methods have been successfully applied in plasma samples from patient that receive diary doses of LEV or RSP. In this way, it was possible to present the applicability of developed bioanalytical methods and the quantitation of plasmatic concentration of these studied drugs.

Orientador: Laurence Duarte Colvara / Resumo: Este trabalho apresenta um estudo sobre o controle de Sistema de Energia Elétrica (SEE) com vistas à melhoria da estabilidade transitória por meio da atuação de dispositivos FACTS, neste momento considerando os compensadores em derivação (SVC) e em série (TCSC). Propõe-se a inclusão, em dispositivos pré-existentes, a incorporação de um controle adicional visando atuação em face de transitórios eletromecânicos. A influência dos dispositivos sobre a capacidade de sincronização entre os pares de máquinas, avaliada por meio do fator de efeito é inserida na atuação dinâmica/transitória do sistema. A evolução da trajetória pós-falta do sistema é considerada em relação às fronteiras da chamada Região de Sincronização Positiva (RSP) e simulações foram realizadas usando os sistemas-teste Simétrico de duas áreas e New England para Lei de Controle proposta e conclui-se que esta é efetiva na melhoria da estabilidade transitória do Sistema de Energia Elétrica e, com aprimoramentos, poderá ser incluída em procedimentos de operação em tempo real. / Abstract: The power system transient stability control is approached by means of FACTS devices, and at this first step the SVC and TCSC devices are considered. A certain device acts upon each pair of machines by means of their synchronization capability which is affected by the corresponding transfer admittance as repeated by the FACTS device. This influence is taken into account by means of a parameter named Effect Factor. The boundaries of a region surrounding the stable equilibrium point named Positive Stability Region are used as reference for critical trajectories and the control action intend to reverse the tendency of the trajectory of leaving this region. In view of the shown tested cases the results are very promising since the proposed control has conduced to improvements in fault critical clearing times and so it has potential to be included in online operation procedures. / Doutor

Submitted by MARCOS JOSÉ GONÇALVES null (marcosjg@ig.com.br) on 2018-05-22T03:01:06Z No. of bitstreams: 1 Tese Marcos Jose Goncalves FINAL.pdf: 1783203 bytes, checksum: d48ee12e54924878e9c6e28de57fb570 (MD5) / Approved for entry into archive by Cristina Alexandra de Godoy null (cristina@adm.feis.unesp.br) on 2018-05-22T12:09:34Z (GMT) No. of bitstreams: 1 goncalves_mj_dr_ilha.pdf: 1783203 bytes, checksum: d48ee12e54924878e9c6e28de57fb570 (MD5) / Made available in DSpace on 2018-05-22T12:09:34Z (GMT). No. of bitstreams: 1 goncalves_mj_dr_ilha.pdf: 1783203 bytes, checksum: d48ee12e54924878e9c6e28de57fb570 (MD5) Previous issue date: 2017-11-28 / Este trabalho apresenta um estudo sobre o controle de Sistema de Energia Elétrica (SEE) com vistas à melhoria da estabilidade transitória por meio da atuação de dispositivos FACTS, neste momento considerando os compensadores em derivação (SVC) e em série (TCSC). Propõe-se a inclusão, em dispositivos pré-existentes, a incorporação de um controle adicional visando atuação em face de transitórios eletromecânicos. A influência dos dispositivos sobre a capacidade de sincronização entre os pares de máquinas, avaliada por meio do fator de efeito é inserida na atuação dinâmica/transitória do sistema. A evolução da trajetória pós-falta do sistema é considerada em relação às fronteiras da chamada Região de Sincronização Positiva (RSP) e simulações foram realizadas usando os sistemas-teste Simétrico de duas áreas e New England para Lei de Controle proposta e conclui-se que esta é efetiva na melhoria da estabilidade transitória do Sistema de Energia Elétrica e, com aprimoramentos, poderá ser incluída em procedimentos de operação em tempo real. / The power system transient stability control is approached by means of FACTS devices, and at this first step the SVC and TCSC devices are considered. A certain device acts upon each pair of machines by means of their synchronization capability which is affected by the corresponding transfer admittance as repeated by the FACTS device. This influence is taken into account by means of a parameter named Effect Factor. The boundaries of a region surrounding the stable equilibrium point named Positive Stability Region are used as reference for critical trajectories and the control action intend to reverse the tendency of the trajectory of leaving this region. In view of the shown tested cases the results are very promising since the proposed control has conduced to improvements in fault critical clearing times and so it has potential to be included in online operation procedures.

Staphylococcus aureus remains an important pathogen, which, due to its capability to develop antimicrobial resistance, imposes an increasing threat to human health. Developing preventive means to decrease disease burden is a major aim. However, the development of an S. aureus vaccine, which would be one strategy to achieve such goals, has been complicated through limited understanding of the bacterium's pathogenic mechanisms. This work uses four approaches to address these limitations: Firstly, a reproducible RNA sequencing based method for the determination of gene transcription by S. aureus in vivo during mammalian infection. Secondly, examination of the impact of the bacterial transcription regulator 'Rsp' on the bacterium, which shows that mutations in this gene have profound functional and transcriptional impacts. Thirdly, by examining the in vivo transcription of multiple S. aureus strains during infection, proposing a 'core in vivo transcriptome' of induced genes under the conditions tested. Some of these genes are known to be involved in pathogenesis, others are not completely characterised and may represent suitable vaccine antigens. Finally, this work addresses limited understanding of S. aureus pathogenesis through defining transcriptional changes in vivo, which are induced by an altered immune response in immunised hosts. Together, this body of work contributes to the understanding of S. aureus pathogenesis and provides candidate antigens for future vaccine development.

The aim of the present thesis is to understand the phase transformation behavior of embedded alloy nanoparticles embedded in immiscible matrices. Embedded alloy inclusions have been dispersed in immiscible matrix via rapid solidification method. The present work deals with synthesis of embedded particles, evolution of microstructure, morphology and crystallographic orientation relation relationships among different phases, phase transformation and phase stability behavior of embedded alloy inclusions in different matrices. In the present investigation the systems chosen are Bi-Sn and Bi-Pb in Zn matrix and Cd-Sn in Al matrix. Chapter 1 gives the brief introduction of present work Chapter 2 gives a brief review of nanoscale materials, various synthesis techniques, microstructure evolution, solidification and melting theories. Chapter 3 discusses the processing and experimental techniques used for characterization of the different samples in the present work. Melt-spinning technique used to synthesize the rapidly solidified ribbons. The structural characterization is carried out using X-ray diffraction and transmission electron microscopy. Chapter 4 illustrates the size dependent solubility and phase transformation behavior of Sn-Cd alloy nanoparticles embedded in aluminum matrix. X-ray diffraction study shows the presence of fcc Al, bct Sn, hcp Cd solid solution and hcp Cd phases. Based on Zen’s law, the amount of Sn present Cd solid solution is estimated. Using overlapped sterograms, the orientational relationships among various phases are found. Microscopy studies reveal that majority of the alloy nano inclusions exhibit a cuboctahedral shape with 111 and 100 facets and they are bicrystalline. STEM-EDS analysis shows that both phases exhibit size dependent solubility behavior and for particles size smaller than 18 nm, single phase solid solution could only be observed. Calorimetric studies reveal a depression in eutectic melting point of bimetallic particles. In situ heating studies show that melting initiates at triple line junction corner and melt first grows into the interior of the Sn rich phase of the particle and then later the melt grows into the interior of the Cd phase of the particle. During cooling first Cd phase solidifies later Sn phase solidifies and on further cooling at low temperatures entire particle transforming into complete solid solution phase particle. Size dependent melting studies show that during heating smaller particles melted first, later bigger particles melted. During cooling first bigger particle solidified later smaller particles solidified. High resolution imaging indicates presence of steps across particle-matrix interface that may get annihilated during heating. During cooling, molten particles in the size range of 16-30 nm solidify as solid solution which for molten particles greater than 30 nm solidify as biphasic particle. Insitu heating studies indicates that for solid particles less than 15 nm get dissolved in the Al matrix at temperatures at around 135°C. Differential scanning calorimetry (DSC) studies show in the first heating cycle most of the particles melt with an onset of melting of at 166.8°C which is close to the bulk eutectic temperature of Sn-Cd alooy. The heating cycle reveals that the melting event is not sharp which can be understood from in-situ microscopy heating studies. In the second and the third cycles, the onset of melting observed at still lower temperatures 164.3°C and 158.5°C .The decrease in onset melting point in subsequent heating cycles is attributed to solid solution formation of all small particles whose size range below 30 nm during cooling. cooling cycles exhibit an undercooling of 90°C with respect to Cd liquidus temperature. Thermal cycling experiments using DSC were carried out by arresting the run at certain pre-determined temperatures during cooling and reheating the sample to observe the change in the melting peak position and area under the peak. The areas of these endothermic peaks give us an estimate of the fraction of the particles solidified upto the temperature when the cycling is reversed. Based on experimental observations, a thermodynamic model is developed, to understand the solubility behavior and to describe the eutectic melting transition of a binary Sn-Cd alloy particle embedded in Al matrix. Chapter 5 discusses the phase stability and phase transformation behavior of nanoscaled Bi-Sn alloys in Zn matrix. Bi-Sn alloys with eutectic composition embedded in Zn matrix using melt spinning technique. X-ray diffraction study shows the presence of rhombohedral Bi, pure BCT Sn and hcp Zn phases. In X-ray diffractogram, there are also other new peaks observed, whose peak positions (interplanar spacings) do not coincide either with rhombohedral Bi or bct Sn or hcp Zn. Assuming these new phase peaks belong to bct Sn rich solid solution(based on earlier work on Bi-Sn rapidly solidified metastable alloys) whole pattern fitting done on x-ray diffractogram using Lebail method. The new phase peaks indicated as bct M1(metastable phase1), bct M2(metastable phase2) phases. The amount of Bi present in M1, M2 solid solution is estimated using Zens law. Two sets of inclusions were found, one contains equilibrium bismuth and tin phases and the other set contains equilibrium bismuth and a metastable phase. In-situ TEM experiments suggest that as temperature increases bismuth diffuses into tin and becomes complete solid solution. Melting intiates along the matrix–particle interface leading to a core shell microstructure. During cooling the entire inclusion solidify as solid solution and decomposes at lower temperatures. High temperature XRD studies show that as temperature increases M1, M2 phases peaks merge with Sn phase peaks and Bi phase peak intensities slowly disappear and on further increasing temperature Sn solid solution phase peaks also disappear. During cooling diffraction studies show that first Sn solid solution phase peaks appear and later Bi phase peaks appear. But, the peaks belong to metstable phases not appeared while cooling. Chapter 6 presents morphology and phase transformation of nanoscaled bismuth-lead alloys with eutectic (Pb44.5-Bi55.5) and peritectic (Pb70-Bi30) compositions embedded in zinc matrix. using melt spinning technique. In alloy1[ Zn-2at%(Pb44.5-Bi55.5)] inclusions were found to be phase separated into two parts one is rhombohedral Bi and the other is hcp Pb7Bi3 phase. X-ray diffraction study shows the presence of rhombohedral Bi, hcp Pb7Bi3 and hcp Zn phases in Zn-2at%(Pb44.5-Bi55.5) melt spun sample. The morphology and orientation relationships among various phases have been found. In-situ microscpy heating studies show that melt initially spreads along the matrix–particle interface leading to a core-shell microstructure. And in the core of the core-sell particles, first Bi phase melts later Pb7Bi3 phase will melt and during cooling the whole particle solidify as biphase particle with large undercooling. In-situ heating studies carried out to study the size dependent melting and solidification behavior of biphase particles. During heating smaller particles melt melt first later bigger particle will melt. In contrast, while cooling smaller particles solidifies first, later bigger particles will solidify. Detailed high temperature x-ray diffraction studies indicate there increases first Bi phase peaks disappear later Pb7Bi3 phase peaks disappear and during cooling first Pb7Bi3 phase peaks appear and later Bi phase peaks appear. In alloy2[ Zn-2at%(Pb70-Bi30)] inclusions were found to be single phase particles. X-ray diffraction study shows the presence of hcp Pb7Bi3 and hcp Zn phases in Zn-2at%(Pb70-Bi30) melt spun sample. The crystallographic orientation relationship between hcp Pb7Bi3 and hcp Zn phases. In-situ microscpy heating studies show that melting initiates across the matrix–particle interface grows gradually into the interior of the particle. Three phase equilibrium at peritectic reaction temperature is not observed during insitu heating TEM studies. Size dependent melting point depression of single phase particles is not observed from in-situ heating studies. Detailed high temperature x-ray diffraction studies show that while heating the Pb7Bi3 phase peak intensities start decreasing after 170°C and become zero at 234°C. And during cooling Pb7Bi3 phase peaks starts appearing at 200°C and on further cooling the Pb7Bi3 phase peak intensities increase upto 150°C, below this temperature peak intensities remain constant.