Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a primary constituent of chronic, polymicrobial infections in the lungs of individuals with cystic fibrosis (CF). A significant consequence of CF is production of thick mucus along epithelial surfaces. In the lungs, this mucus collects and serves as an excellent growth substrate for a range of bacteria including. CF lung fluids (sputum) also enhance the virulence of P. aeruginosa, as production of a signaling molecule critical for virulence, the Pseudomonas quinolone signal (PQS), is enhanced in the presence of phenylalanine and tyrosine in CF sputum. The goal of this dissertation is to better understand how phenylalanine and tyrosine affect PQS production and ultimately P. aeruginosa virulence. To address this, I use transcriptome profiling to determine that genes for phenylalanine and tyrosine catabolism, PQS biosynthesis, and a transcriptional regulator called PhhR are up-regulated in the presence of phenylalanine and tyrosine. I determine that PhhR regulates genes for aromatic amino acid catabolism but not genes for PQS biosynthesis. The PhhR regulon is further characterized by mapping of PhhR-regulated promoters with primer extension, and evidence for direct regulation is presented. To explain enhanced production of PQS in CF sputum, I favor a model in which flux of a shared metabolic precursor, chorismate, toward PQS biosynthesis is enhanced when phenylalanine and tyrosine are present. I investigate this model by examining the first step in PQS biosynthesis, conversion of chorismate to anthranilate by an anthranilate synthase (AS). P. aeruginosa possesses two AS enzymes encoded by the trpEG and phnAB genes, with the former generating anthranilate specifically for tryptophan biosynthesis while the latter generates anthranilate for PQS biosynthesis. I investigate the evolutionary origins of these two enzymes and generate unmarked deletion mutants to dissect their roles in tryptophan and PQS biosynthesis. The ability of PhnAB to compensate for loss of TrpEG at high cell densities is documented, and a model explaining anthranilate sequestering is developed. Knowledge gained from these studies will be useful in developing novel therapeutic strategies. / text

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A gama-proteobactéria Pseudomonas aeruginosa é um patógeno oportunista humano frequentemente associado a pacientes com queimadura grave e aos portadores de fibrose cística. O estabelecimento de infecção depende de uma série de fatores que contribuem para a virulência deste patógeno, dentre eles a produção de sideróforos e outros sistemas de captação de ferro. Pioverdina é o principal sideróforo sintetizado por bactérias do gênero Pseudomonas e linhagens deficientes na sua produção são incapazes de estabelecer infecção em modelos animais. A regulação da biossíntese deste sideróforo envolve a agregação entre as células, indicando a dependência de contato para completa indução da sua produção. O contato com uma superfície altera o comportamento das células e diversos fenótipos são dependentes deste sinal mecânico. PrlC é uma oligopeptidase A putativamente envolvida na degradação de peptídeo-sinais e PA14_00800, uma pequena proteína com domínio de função desconhecida, codificada por um gene imediatamente à jusante de prlC. Existem poucos trabalhos na literatura sobre PrlC e seus homólogos e nenhuma informação sobre PA14_00800. Este trabalho teve como objetivo elucidar o envolvimento de PrlC e PA14_00800 na regulação da produção de pioverdina por células em contato com uma superfície. Para estabelecer uma correlação na expressão destes genes, um estudo da organização gênica foi realizado por RT-PCR, confirmando que eles fazem parte do mesmo operon e, portanto, que a expressão destes genes é regulada pelos mesmos fatores. Ensaios classicamente modulados pelo segundo mensageiro c-di-GMP, como formação de biofilme e motilidade, não apresentaram variações nas linhagens mutantes ΔprlC, ΔPA14_00800 ou Δoperon, indicando que a deleção destes genes não altera significativamente os níveis de c-di-GMP nas células. A motilidade do tipo swarming é, no entanto, severamente afetada na linhagem ΔPA14_00800 quando o meio de cultura não contém cloreto de cálcio e glicose, indicando um defeito na sinalização celular ou requerimento energértico desta linhagem nestas condições. PA14_00800 regula a fluorescência de P. aeruginosa em meio sólido e semissólido, mas não em meio líquido. Esta fluorescência depende tanto de pioverdina quanto de PQS, umamolécula de comunicação celular fluorescente, e a possibilidade de outros fatores estarem envolvidos neste fenótipo ainda está sob investigação. Análise do transcritoma por RNASeq com a linhagem ΔPA14_00800 comparada à linhagem parental foi realizada a partir de colônias destas linhagens crescidas em M9 modificado. Genes envolvidos no sistema de secreção do tipo III e do tipo VI e na biossíntese de PQS apareceram dentre os genes diferencialmente expressos, bem como genes para o catabolismo de glicose. Este trabalho foi o primeiro a investigar o papel de PA14_00800 na fisiologia de P. aeruginosa, e os conhecimentos adquiridos aqui podem ser transpostos, com cautela, para compreensão da função dos homólogos de PA14_00800 em outras bactérias. / The gamma-proteobacterium Pseudomonas aeruginosa is a human opportunistic pathogen frequently associated with patients with severe burns and those with cystic fibrosis. The establishment of infection depends on several factors that contribute to the virulence of this pathogen, among them siderophore production and other iron uptake systems. Pyoverdine is the main siderophore synthesized by the bacteria of the genus Pseudômonas and pyoverdinedeficient strains are unable to establish infection in animal models. The regulation of biosynthesis of this siderophore involves cell aggregation, indicating contact dependency for complete induction of pyoverdine production. Surface contact alters cell behavior and several phenotypes are dependent on this mechanical cue. PrlC is an oligopeptidase A putatively involved in peptide-signals degradation and PA14_00800, a small protein with a domain of unknown function, encoded by a gene immediately downstream of prlC. There are few papers in the literature on PrlC and its homologues and no information on PA14_00800. This work aimed to elucidate the role of PrlC and PA14_00800 in surface-dependent regulation of pyoverdine production. To establish a correlation in the expression of these genes, a study of the gene organization was performed by RT-PCR, confirming that they are part of an operon and therefore the expression of these genes is regulated by the same factors. Traits classically modulated by the second messenger c-di-GMP, such as biofilm formation and motility, did not show variations in the ΔprlC, ΔPA14_00800 or Δoperon, indicating that the deletion of these genes does not significantly alter the levels of c-di-GMP within the cells. Swarming motility is, however, severely affected in the strain ΔPA14_00800 when the culture medium does not contain calcium chloride and glucose, indicating a cell signaling defect or energetic requirement under these conditions. PA14_00800 regulates surface-dependent fluorescence of P. aeruginosa, in solid and semi-solid medium. This fluorescence depends on both pyoverdine and PQS, a fluorescent cell-to-cell communication molecule, and the investigation of other putative factors involved in this phenotype is still under study. Transcriptomic analysis by RNASeq with the strain ΔPA14_00800 compared to PA14 was performed from colonies ofthese strains grown in modified M9 1% agar. Genes involved in the type III and type VI secretion systems, in PQS biosynthesis and glucose catabolism were differentially expressed. This work was the first to investigate the role of PA14_00800 in the physiology of P. aeruginosa, and the knowledge obtained here can be cautiously transposed to understanding the role of PA14_00800 homologues in other bactéria.

Submitted by NATHALIA CRISTINA RISSI null (nathaliarissi_farmacia@yahoo.com.br) on 2016-07-04T12:35:43Z No. of bitstreams: 1 Tese Nathalia Cristina Rissi 1.pdf: 3733548 bytes, checksum: bde3d4680fdf9def65256855510fa057 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-07T17:05:40Z (GMT) No. of bitstreams: 1 rissi_nc_dr_arafcf.pdf: 3733548 bytes, checksum: bde3d4680fdf9def65256855510fa057 (MD5) / Made available in DSpace on 2016-07-07T17:05:40Z (GMT). No. of bitstreams: 1 rissi_nc_dr_arafcf.pdf: 3733548 bytes, checksum: bde3d4680fdf9def65256855510fa057 (MD5) Previous issue date: 2016-06-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nos últimos anos, é possível observar um grande interesse no desenvolvimento de sistemas multifuncionais direcionados ao diagnóstico e tratamento do câncer. Estes sistemas também conhecidos como “teranósticos” têm se mostrado interessantes, pois ampliam a capacidade de liberação prolongada de fármacos anticancerígenos em células específicas, além de proporcionar um monitoramento ótico através da luminescência de pontos quânticos. Diante deste contexto, o presente estudo teve como objetivo estabilizar os pontos quânticos a base de óxido de zinco (ZnO) em meio aquoso através da modificação de superfície com o 3-(Glycidyloxypropyl)trimethoxysilane (GPTMS) e também em meio orgânico através da modificação com o Hexadecyltrimethoxysilane (HTMS), permitindo assim a veiculação do mesmo em micelas poliméricas A ligação entre os pontos quânticos e as moléculas dos modificadores ocorreu através das reações de hidrólise e condensação, utilizando como catalisador básico o hidróxido de lítio. Esta reação conduziu a formação de uma camada de siloxano ao redor das nanopartículas e resultou nas ligações covalentes do tipo ZnO-Si-O. Ainda com o objetivo de aumentar a estabilidade do ZnO em meio orgânico e consequentemente suas propriedades luminescentes, foi sintetizado uma bicamada formada entre o Ácido Oleico (AO) e o HTMS. As modificações na superfície do ZnO foram confirmadas pelas técnicas de espectroscopia vibracional na região do infravermelho e também pela espectroscopia de fotoelétrons induzidos por raios-X. O comportamento ótico foi feito através da espectroscopia de absorção na região do UV-Vis. Com está técnica, identificou-se o comprimento de onda limite associado ao pico excitônico do ZnO e através do monitoramento deste pico, observou-se estabilidade em água por um período de 15 dias para a grande maioria das amostras de ZnO modificado pelo GPTMS e também para o ZnO modificado pelo HTMS e pela bicamada de AO/HTMS em clorofórmio. O tamanho final dos pontos quânticos foi analisado pela microscopia de transmissão eletrônica, apresentando um tamanho médio entre 3,5- 4nm para as amostras de ZnO modificado pelo GPTMS e de 4,5 nm para as amostras de ZnO modificado pelo HTMS e pela bicamada de AO/HTMS. Com a técnica de difração de raios-X, identificou-se os picos de difração correspondentes a uma estrutura cristalina do tipo Wurtzita. As medidas de fotoluminescência permitiu a obtenção de informações sobre a intensidade luminescente, assim como as cores emitidas dos pontos quânticos e demostraram a importância das modificações de superfície em relação ao aumento da intensidade luminescente. A veiculação do ZnO estáveis no meio orgânico em micelas poliméricas formadas pelo Pluronic F127 e Pluronic F68, foi realizada com sucesso. Este fato foi observado por meio dos espectros de absorção no UV-vis e também pelas medidas de fotoluminescência. Com a técnica de espalhamento de luz dinâmico, avaliou-se parâmetros relacionados com o tamanho e o índice de polidispersidade das micelas. O teste de citotoxicidade in vitro foi feita através do MTT e apresentou uma boa viabilidade celular para as linhagens celulares de queratinócito humano (HaCat) e de hepatoma humano (HepG2). A quantificação das células em que houve a internalização das micelas contendo os pontos quânticos hidrofóbicos como marcador foi feita através da citometria de fluxo. Diante dos resultados obtidos, conclui-se a existência de um potencial a ser explorado em relação ao ZnO modificado pelo GPTMS em aturar futuramente como sondas biológicas. Já as propriedades hidrofóbicas concedidas ao ZnO modificado pelo HTMS e pela bicamada formada entre o AO/HTMS permitiu a incorporação dos mesmos em sistemas micelares e tem como objetivo auxiliar na aplicação futura desses pontos quânticos através do desenvolvimento de sistemas teranósticos. / In recent years, it is possible to observe a growing interest in the development of multifunctional systems used, that can be used in the diagnosis and treatment of cancer These systems also known as " theranostic " and have gained considerable attention due to their capacity of release anticancer drugs into specific cells, besides to optical monitoring through quantum dots. In this context, the present study was aimed to stabilize the quantum dots of of ZnO in water by modifying with 3-(Glycidyloxypropyl)trimethoxysilane (GPTMS) with and organic medium.by modifying with Hexadecyltrimethoxysilane (HTMS), thus allowing them to be incorporated into polymeric micelles The binding between the quantum dots with the modifiers occurred by hydrolysis and condensation reaction under basic catalysis by lithium hydroxide, thus leading to the formation of siloxane layer and resulted in ZnO-Si-O covalent bond. In order to improve the stability of ZnO-QDs and consequently their photoluminescence properties, was synthesized a coating bilayer by OA and HTMS. The ZnO surface modification was confirmed by infrared spectroscopy and also by X-ray photoelectron spectroscopy. The optical behavior was performed by absorption spectroscopy in the UV-Vis region. With this technique was possible to identify the wavelength limit associated with the excitonic peak and by monitoring of this peak was observed a great stability during 15 days for almost all samples of ZnO modified by GPTMS in water and ZnO modified by HTMS and bilayer AO / HTMS in chloroform. The finale size of quantum dots was analyzed by transmission electron microscopy that showed an average size about 3,5- 4nm for the samples of znO modified by GPTMS, and 4,5 nm by HTMS and AO/HTMS bilayer. By X-ray diffraction, it was possible to identify through the diffraction peak a wurtzite structure. The photoluminescence measurements allowed to obtain information about luminescence intensity, as well as the emitted colors by quantum dots and demonstrated the importance of surface modifications in relation to increase of luminescence intensity. The placement of hydrophobic ZnO into polymeric micelles formed by Pluronic F127 and Pluronic F68 was successful. This fact can be observed by absorption spectroscopy in UV-vis and by photoluminescence measurements. With the dynamic light scattering, it was possible to observe the hydrodynamic size distribution and polydispersity index of the micelles. In vitro cytotoxicity assay was performed by MTT and showed a great cellular viability for human keratinocytes cells (HaCat) and for hepatocellular carcinoma cells (HepG2). The cellular internalization was performed by flow cytometry. Based on these results, it was concluded a potential to be explored in to ZnO modified by GPTMS to acting in the future as biological probes. The hydrophobic properties of ZnO modified by HTMS and the AO / HTMS bilayer allowed the incorporation in micellar systems and aims to assist in the implementation of these quantum dots through the development of systems theranostics.

<p>The objective of this work was to investigate reflective-functioning (RF) as a measure of process in two independent studies that included three types of brief psychotherapy. RF is defined as the ability to recognize the existence and nature of mental processes taking place in the self and in others (e.g., intentions, beliefs, desires, and wishes). Theorists have suggested the ability for RF is crucial for predicting social causality and low RF has been found related to mental disorders. It has recently been suggested in the literature that improved ability for RF might be an important component of successful psychotherapy outcome, especially with respect to achieving structuralchange. RF was in this work investigated during the process through discourse analysis of the patients’ narratives of self-other interactions in the treatment sessions. The Psychotherapy Process Q-set (PQS) was implemented in order to isolate specific components of the process (process correlates) that identified high and low RF and to investigate the links between the process correlates and outcome. The first study investigated 29 cases of cognitive-behavioral therapy(CBT) and 35 cases of interpersonal psychotherapy (IPT) with an average treatment length of 16.2 sessions in a sample from the National Institute of Mental Health (NIMH) randomized clinical trial Treatment of Depression Collaborative Research Program (TDCRP). The sample in the first study consisted of 128 sessions in total, were one session from the early part (on average the 4th session) and one session in the later part of the treatment (on average the 12th session) were rated for RF. The second study investigated a sample of 30 cases of brief psychodynamic psychotherapy (BPDT) with an average treatment length of 15.8 sessions in a naturalistic designand obtained from the Mount Zion Psychotherapy Research Group. In total, the second study included 90 sessions of BPDT, and RF was assessed during the 1st, the 5th, and the 14th session of each treatment. The results from these two studies suggested that the patients’ ability for RF, as measured through the discourse from therapy sessions, is stable (in CBT and BPDT) or decreased(IPT) during the treatments. Furthermore, the process correlates defining high RF had a relation with good outcome, and process correlates defining low RF had a relation with poor outcome.The process correlates identified during the PQS-analysis suggested that both high and low RF was linked with personality characteristics in the patients. For example, high RF was linked to patients’ ability for introspection, expression of negative emotions, and commitment to treatment.Low RF was linked to patients’ expression of passivity, defensiveness, and suspiciousness. This work supported theorists’ suggestions that brief treatments are supportive in their nature and therefore do not promote structural changes (e.g., changes in RF). It is suggested that the abilityfor RF as assessed pre-treatment might be a useful predictor for success in brief psychotherapy and could therefore be used as a patient inclusion criteria for such treatments.</p>

The objective of this work was to investigate reflective-functioning (RF) as a measure of process in two independent studies that included three types of brief psychotherapy. RF is defined as the ability to recognize the existence and nature of mental processes taking place in the self and in others (e.g., intentions, beliefs, desires, and wishes). Theorists have suggested the ability for RF is crucial for predicting social causality and low RF has been found related to mental disorders. It has recently been suggested in the literature that improved ability for RF might be an important component of successful psychotherapy outcome, especially with respect to achieving structuralchange. RF was in this work investigated during the process through discourse analysis of the patients’ narratives of self-other interactions in the treatment sessions. The Psychotherapy Process Q-set (PQS) was implemented in order to isolate specific components of the process (process correlates) that identified high and low RF and to investigate the links between the process correlates and outcome. The first study investigated 29 cases of cognitive-behavioral therapy(CBT) and 35 cases of interpersonal psychotherapy (IPT) with an average treatment length of 16.2 sessions in a sample from the National Institute of Mental Health (NIMH) randomized clinical trial Treatment of Depression Collaborative Research Program (TDCRP). The sample in the first study consisted of 128 sessions in total, were one session from the early part (on average the 4th session) and one session in the later part of the treatment (on average the 12th session) were rated for RF. The second study investigated a sample of 30 cases of brief psychodynamic psychotherapy (BPDT) with an average treatment length of 15.8 sessions in a naturalistic designand obtained from the Mount Zion Psychotherapy Research Group. In total, the second study included 90 sessions of BPDT, and RF was assessed during the 1st, the 5th, and the 14th session of each treatment. The results from these two studies suggested that the patients’ ability for RF, as measured through the discourse from therapy sessions, is stable (in CBT and BPDT) or decreased(IPT) during the treatments. Furthermore, the process correlates defining high RF had a relation with good outcome, and process correlates defining low RF had a relation with poor outcome.The process correlates identified during the PQS-analysis suggested that both high and low RF was linked with personality characteristics in the patients. For example, high RF was linked to patients’ ability for introspection, expression of negative emotions, and commitment to treatment.Low RF was linked to patients’ expression of passivity, defensiveness, and suspiciousness. This work supported theorists’ suggestions that brief treatments are supportive in their nature and therefore do not promote structural changes (e.g., changes in RF). It is suggested that the abilityfor RF as assessed pre-treatment might be a useful predictor for success in brief psychotherapy and could therefore be used as a patient inclusion criteria for such treatments.

Le but de cette thèse a été d'étudier les propriétés structurales et optiques de puits quantiques (PQs) d’InGaN/(Al)GaN obtenus par épitaxie en phase vapeur d’organométalliques. Différentes approches ont été mises en œuvre pour atteindre une émission dans le vert-jaune: la première utilisant une concentration d'indium ≥ 20% avec un PQ d’InGaN d’épaisseur <3.0 nm et vice versa. L'effet d'une couche d’encapsulation a également été étudié. Les techniques de microscopie à force atomique, de diffraction des rayons X, de photoluminescence (PL) et surtout de microscopie électronique à transmission (MET) ont été utilisées pour caractériser ces structures. Les épaisseurs des PQs et les compositions en indium ont été déterminées par le traitement numérique des franges de réseau dans les images MET haute résolution en section transverse. Un traitement original a été développé pour analyser quantitativement les fluctuations de l’épaisseur des PQs. L'analyse structurale des PQs ayant une composition en In élevé a montré que les défauts structuraux sont créés dans les PQs. La nature et la densité de ces défauts ont été déterminées et différents mécanismes pour leur formation ont été proposés. Il a également été montré que quelques monocouches d’encapsulation de GaN ou d’AlGaN déposées à la température de croissance des PQs limitent l’évaporation et/ou la diffusion d’indium. Ce procédé permet d’étendre la longueur d'onde d'émission avec une réduction de la dégradation de l'efficacité de la PL. Mon travail propose quelques pistes afin d'obtenir un bon compromis entre les paramètres contradictoires qui régissent l'efficacité des PQs émettant dans le vert-jaune. / The goal of this thesis was to study the structural and optical properties of InGaN/(Al)GaN multiple QWs grown by metal organic chemical vapor deposition. Different approaches have been implemented to achieve green-yellow emission: high indium concentration (≥ 20%) with low InGaN QW thickness (< 3 nm) or vice versa. Moreover, the effect of a capping layer on top of the QWs has also been investigated. Atomic force microscopy, X-ray diffraction, room temperature photoluminescence (RTPL) and mainly transmission electron microscopy (TEM) techniques have been used to characterize these structures. The QW thicknesses and indium compositions have been determined by digital processing of lattice fringes in cross-sectional high resolution TEM images. An original treatment has been developed to analyze quantitatively InGaN QW thickness fluctuations. The structural analysis of multiple QWs with high indium composition has shown that structural defects are created in the QWs. The nature and the density of these defects have been determined and different mechanisms for their formation have been proposed. It has also been shown that a few monolayers of AlGaN or GaN capping layers deposited at the InGaN QW growth temperature prohibited indium evaporation and/or diffusion. It therefore helps to extend the emission wavelength with a reduced degradation of the RTPL efficiency. My work offers a few ways to obtain a good compromise between the conflicting parameters that govern the efficiency of QWs emitting in the green-yellow spectrum range.