Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) places the fourth position in the ranking of most reported Salmonella serovars in Europe. During the last decade, a multi-drug resistant (MDR) S. Infantis population has rapidly increased and widespread in European and non-European broiler production. The study proposed here aimed to i) implement and evaluate the performance of a bioinformatics pipeline named WGSBAC for Salmonella in silico serotyping, and ii) identify the genetic determinants for the increased emergence of broiler-derived S. Infantis observed in Germany. First, we conducted an evaluation of WGSBAC and three bioinformatic tools (SISTR, SeqSero, and SeqSero2) for the characterization and genoserotyping of 43 Salmonella strains of 26 different serovars. Second, we performed sequencing of 30 broiler-derived S. Infantis isolates collected from two distant decades (the 1990s and the 2010s). We applied the WGSBAC pipeline and external bioinformatics software to i) assess and control the quality of the sequenced reads ii) assembly and quality control of assemblies, and iii) annotation, typing by classical MLST, cgMLST, genoserotyping, SNPs-based phylogenetic reconstruction and in silico phenotype prediction including antimicrobial resistance genes (AMR), virulence genes and plasmid replicons detection. To detect possible clonal relatedness with other S. Infantis clones from Europe, we performed a further comparative genome analysis using 17 public genomes of other S. Infantis clones circulating in Europe. WGSBAC was feasible for the serovar prediction of most of the 43 Salmonella strains. The tool SISTR reported the highest correlation (79.1%) followed by SeqSero2 (72.1%) and SeqSero (60.5%). The study of the S. Infantis strains revealed that in contrast to the isolates from the 1990s, the majority of the strains from the 2010s revealed the presence of a megaplasmid that carried a multidrug-resistant genes (MDR) pattern, a virulence genes pattern, and several fitness-associated determinants. We termed the MDR gene pattern “ESIr” and it coded for at least three antimicrobial families: ant(3”)-Ia (aminoglycosides), sul1 (sulfonamides), and tet(A) (tetracyclines). Besides, we termed the virulence pattern as “ESIv” which includes genes for fimbriae cluster, yersiniabactin siderophore, mercury resistance, and antitoxin/antitoxin systems. Furthermore, the genotyping analysis revealed the presence of a novel sequence type (ST2283) among the majority of the strains from the 2010s and ST32 and ST1032 within the strains from the 1990s. This genetic traits may promote the rapid incidence and dissemination of a novel MDR S. Infantis population. Following a WGS-based approach, this study evidences that MDR S. Infantis ST2283 strains carrying a pESI-like plasmid have emerged during the last decade and are currently circulating in the German poultry production chain. This event results in an urgent public health hazard, thus, control measures and the support of epidemiological studies are needed to prevent the entrance, transmission, and further dissemination of this clonal population in the food chain. / Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) nimmt die vierte Position in der Rangliste der am häufigsten gemeldeten Salmonella-Serovare in Europa ein. Während des letzten Jahrzehnts hat das Auftreten einer multiresistenten Salmonella enterica subspecies enterica serovar Infantis (S. Infantis)-Population rapide zugenommen und ist in der europäischen und außereuropäischen Broilerproduktion weit verbreitet. Die Studie zielte darauf ab, i) eine bioinformatische Pipeline für die in silico-Serotypisierung von Salmonellen zu implementieren und deren Leistungsfähigkeit zu bewerten, und ii) die genetischen Determinanten für das in der deutschen Broilerproduktion während des letzten Jahrzehnts beobachtete vermehrte Auftreten von S. Infantis zu identifizieren. Zunächst wurde eine Bioinformatik-Pipeline (WGSBAC) und drei bioinformatische Tools (SISTR, SeqSero und SeqSero2) zur Genoserotypisierung von 43 Salmonella spp.-Stämmen von 26 verschiedenen Serovaren durchgeführt. Zweitens führten wir die Genomsequenzierung von 30 S. Infantis Broiler-Isolaten durch, die in zwei unterschiedlichen Jahrzehnten (den 1990er und den 2010er Jahren) gesammelt wurden. Wir setzen die WGSBAC-Pipeline und externe Bioinformatik-Software ein, um i) die Qualität der sequenzierten Reads zu bewerten und zu kontrollieren, ii) Assemblierungen und Qualitätskontrollen von und iii) Annotation, Typisierung durch klassischen MLST, cgMLST, Genoserotypisierung, phylogenetische Rekonstruktion mittels Einzelnukleotidänderungen (SNPs) und in-silico-Phänotyp-Vorhersage, einschließlich antimikrobieller Resistenzgene (AMR), Virulenzgene und Plasmid-Replikons zu erkennen. Die Bioinformatik-Pipeline WGSBAC ist geeignet, das antigene Profil der meisten der in der Studie verwendeten Salmonella-Stämme zu bestimmen. Das Tool SISTR zeigte dabei die höchste Übereinstimmung (79,1 %), gefolgt von SeqSero2 (72,1 %) und SeqSero (60,5 %). Die Untersuchung der S. Infantis-Stämme ergab, dass im Gegensatz zu den Isolaten aus den 1990er Jahren, die Mehrheit der Stämme aus den 2010er Jahren das Vorhandensein eines Megaplasmids zeigte, das homolog zu dem pESI-Plasmid aus Israel und anderen pESIähnlichen Plasmiden aus europäischen Isolaten ist. Das deutsche pESI-ähnliche Plasmid kodierte für ein Muster von multiresistenten Genen (MDR), ein Muster von Virulenzgenen und mehrere Fitness-assoziierte Determinanten, welches wir 'ESIr' nannten. Dieses korrelierte mit mindestens drei antimikrobiellen Familien: ant(3')-Ia (Aminoglykoside), sul1 (Sulfonamide) und tet(A) (Tetracycline). Der Genotyp korreliert hier vollständig mit dem antimikrobiellen Phänotyp. Außerdem bezeichneten wir das Virulenzmuster als 'ESIv', welches Gene für Fimbrien-Cluster, Yersiniabactin-Siderophore, Quecksilberresistenz und Antitoxin/Antitoxin- Systeme mit einschließt. Die Genotypisierungsanalyse ergab das Vorhandensein eines neuen Sequenztyps (ST2283) bei der Mehrzahl der Stämme aus den 2010er Jahren und ST32 und ST1032 bei den Stämmen aus den 1990er Jahren. Anhand eines auf Gesamtgenomsequenzierung-basierten Ansatzes zeigte diese Studie, dass MDR S. Infantis ST2283 Stämme, die ein pESI-ähnliches Plasmid tragen, während des letzten Jahrzehnts entstanden sind und derzeit in der deutschen Geflügelproduktionskette zirkulieren. Der Erwerb eines Megaplasmids, das für Resistenzen, Virulenz-assoziierte Determinanten und Fitnessmechanismen kodiert, könnte dieses schnelle und besorgniserregende epidemiologische Ereignis erklären. Dieses Ereignis stellt eine Gefahr für die öffentliche Gesundheit dar. Daher sind Kontrollmaßnahmen und die Unterstützung epidemiologischer Studien erforderlich, um den Eintritt, die Übertragung und die weitere Verbreitung dieser klonalen Population in der Lebensmittelkette zu verhindern.

Tuberculosis (TB) is the world’s most deadly infectious disease and causes enormous public health problems. The comorbidity with HIV and the rise of multidrug-resistant TB strains impede successful therapy through drug-drug interactions and the lack of efficient second-line treatments. The aim of this thesis was to support the improvement of anti-TB therapy through development of pharmacometric models, specifically focusing on the novel drug bedaquiline, pharmacokinetic interactions and methods for pooled population analyses. A population pharmacokinetic model of bedaquiline and its metabolite M2, linked to semi-mechanistic models of body weight and albumin concentrations, was developed and used for exposure-response analysis. Treatment response was quantified by measurements of mycobacterial load and early bedaquiline exposure was found to significantly impact the half-life of bacterial clearance. The analysis represents the first successful characterization of a concentration-effect relationship for bedaquiline. Single-dose Phase I studies investigating potential interactions between bedaquiline and efavirenz, nevirapine, ritonavir-boosted lopinavir, rifampicin and rifapentine were analyzed with a model-based approach. Substantial effects were detected in several cases and dose-adjustments mitigating the impact were suggested after simulations. The interaction effects of nevirapine and ritonavir-boosted lopinavir were also confirmed in patients with multidrug-resistant TB on long-term treatment combining the antiretrovirals and bedaquiline. Furthermore, the outcomes from model-based analysis were compared to results from conventional non-compartmental analysis in a simulation study. Non-compartmental analysis was found to consistently underpredict the interaction effect when most of the concentration-time profile was not observed, as commonly is the case for compounds with very long terminal half-life such as bedaquiline. To facilitate pooled analyses of individual patient data from multiple sources a structured development procedure was outlined and a fast diagnostic tool for extensions of the stochastic model components was developed. Pooled analyses of nevirapine and rifabutin pharmacokinetics were performed; the latter generating comprehensive dosing recommendations for combined administration of rifabutin and antiretroviral protease inhibitors. The work presented in this thesis demonstrates the usefulness of pharmacometric techniques to improve treatment of TB and especially contributes evidence to inform optimized dosing regimens of new and old anti-TB drugs in various clinical contexts.

The aim of this thesis was to isolate the compounds responsible for the antibacterial activity of cell extracts of the marine diatom, Phaeodactylum tricornutum. Marine microalgae are not only important primary producers but, due to their phylogenetic diversity, they are also a potential source of novel bioactive compounds. The marine diatom, P. tricornutum, was selected for study because its cell extracts are known to be antibacterial but the compounds responsible have not been isolated. In this thesis, the compounds responsible for the antibacterial activity are isolated from aqueous methanol P. tricornutum cell extracts by column chromatography and reverse phase high-performance liquid chromatography using a bioassay-guided approach. The compounds in three active fractions were identified by mass spectrometry and nuclear magnetic resonance spectroscopy as the unsaturated fatty acids (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, (9Z)-hexadecenoic acid and (6Z, 9Z, 12Z)-hexadecatrienoic acid. The fatty acids were found to be antibacterial against Staphylococcus aureus at micromolar concentrations. P. tricornutum exists in different cell morphs and, interestingly, extracts prepared from cultures in the fusiform morph were found to have greater antibacterial activity than extracts from oval cultures. This is explained by greater levels of the three antibacterial fatty acids in the fusiform cell extracts. The antibacterial fatty acids are proposed to be released by enzyme action when the diatom cells lose their integrity. The release of free fatty acids by diatoms is suggested to be a simple, very low cost population-level activated defence mechanism against potential pathogenic bacteria triggered when the cell loses its integrity. Further, this pathway may act against multiple threats to the microalga, including grazers, as fatty acids exhibit activity in diverse biological assays. Finally, whilst two of the fatty acids, (9Z)-hexadecenoic acid and (5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid, inhibited the growth of MRSA their usefulness as therapeutic compounds may be limited due to their instability and their broad biological activity.

<p>In this case study research project I have determined that multidrug sedation in children younger than eight years are possible.<br /> Conscious sedation [or sedation where verbal contact with the patient is possible] can be used successfully to decrease anxiety and fear for unpleasant experiences, like dental procedures.</p> <p><br /> Behaviour therapy in conjunction with one or more drugs can be used to depress the central nervous system in order to decrease the patient&rsquo / s awareness of unpleasant stimuli. This enables treatment to be carried out without patient interference. Extensive literature surveys were done to determine the ideal drugs as well as the ideal route for conscious sedation in dental treatment for children. In this study project drugs like midazolam, propofol, alfentanyl and ketamine were titrated intravenously to achieve conscious sedation.</p>

El objetivo central del presente trabajo de Tesis Doctoral fue restablecer in vitro la susceptibilidad antimicrobiana de cepas comensales de Escherichia coli (con fenotipo multirresistente-MDR-) aisladas de explotaciones pecuarias, mediante la asociación de diferentes antimicrobianos con el inhibidor de bombas de eflujo 1-(1-naphthylmethyl)-piperazine (NMP). De esta manera, se pretende contribuir al desarrollo de planes de dosificación de máxima eficacia antimicrobiana minimizando el riesgo de emergencia y diseminación de resistencia bacteriana; permitiendo así la obtención de productos de origen animal de excelencia sanitaria, en función de la correcta interpretación de los parámetros farmacocinéticos/farmacodinámicos (modelización PK/PD) para una correcta dosificación, del mecanismo de resistencia y del eventual bloqueo de éste último. Se obtuvieron muestras de materia fecal mediante hisopado rectal de vacas en ordeñe, terneros y animales de compañía, pertenecientes a tambos de la provincia de Buenos Aires; así como también de pozos sépticos, agua de consumo y bombas estercoleras. Se determinaron los perfiles de sensibilidad, mediante el método estandarizado de Kirby-Bauer de difusión en agar, frente a ocho antimicrobianos a todas las cepas de E.coli aisladas. Se hallaron diez cepas de E. coli multirresistentes a partir de los animales muestreados en el estudio de tipo transversal. Se emplearon cepas isogénicas de E. coli con diferentes grados de expresión de bombas de eflujo como control de calidad (AG100A con deleción total de bombas de eflujo, AG100 como cepa “normal” y AG112 con sobreexpresión de bombas). Se determinó la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) de florfenicol, ciprofloxacina, tetraciclina y ampicilina por el método de microdilución seriada en caldo Luria-Bertani (LB) con y sin NMP. Para la determinación de las interacciones entre los tres antimicrobianos seleccionados (florfenicol, ciprofloxacina y tetracilina) y el inhibidor de bombas se calculó el Índice de Concentración Inhibitoria Fraccionaria (CIF) a fin de evaluar la manifestación o no de efecto sinérgico. Para evaluar la cinética de muerte bacteriana se realizaron curvas de muerte bacteriana de las cepas de referencia y las cepas de campo MDR con y sin NMP. Con los datos obtenidos se realizó el análisis estadístico correspondiente. Por último, a partir de la información farmacodinámica obtenida se realizó una modelización farmacocinética/farmacodinámica (PK/PD), para lo cual se utilizaron datos obtenidos de estudios farmacocinéticos de florfenicol, danofloxacina y oxitetraciclina realizados previamente por el grupo de investigación de la Cátedra de Farmacología. Se aislaron cepas resistentes y multirresistentes con altos niveles de resistencia frente a tetraciclina y ampicilina. Todas las combinaciones de resistencia múltiple siempre incluyeron en su perfil a la tetraciclina. El NMP asociado a ampicilina tuvo un efecto nulo tanto en las cepas isogénicas como en las de campo. Sin embargo, ciprofloxacina, florfenicol y tetraciclina, demostraron ser claros sustratos de las bombas. Se evidenció la ocurrencia de sinergismo de potenciación ya que con una concentración de antimicrobiano varias veces inferior a la de su CIM, se logró un efecto antibacteriano mejorado con la incorporación de NMP. Se comprobó que es posible disminuir la concentración de los antimicrobianos -florfenicol, ciprofloxacina y tetraciclina- con la incorporación de NMP, sin modificar de manera importante la “cinética de muerte bacteriana”, tanto en las curvas de muerte de las cepas de referencia como en las cepas problema. En cuanto a la relación PK/PD, en la cepa AG112 se mejoraron notablemente los parámetros predictores de eficacia con la adición del inhibidor, con la consecuente disminución de la concentración de los antimicrobianos. Resulta prometedor el efecto de la combinación de un fármaco inhibidor de bombas de eflujo como coadyuvante de aquellos antimicrobianos sustratos de las bombas de eflujo sobreexpresadas como mecanismo inespecífico de resistenca bacteriana.

A signifficant obstacle facing the healthcare industry is the phenomenon of multidrug resistance (MDR) in which a cell acquires simultaneous resistance to many unrelated drugs that it has never been exposed to. At the molecular level, MDR can be characterised by a reduction of intracellular drug levels due to their active efflux by multidrug transporters such as P-glycoprotein (Pgp). Pgp is able to efflux a phenomenally wide variety of chemically unrelated drugs and causal relationships have been established between its expression and the acquisition of MDR to numerous anticancer and central nervous system (CNS) drugs. There has thus been much effort to understand the molecular biology of Pgp and how it functions. However, many aspects of its functioning remain unclear. From a drug discovery viewpoint, we have yet to fully understand what features make some drugs susceptible to Pgp-mediated efflux (substrates) and what makes others able to inhibit Pgp function (inhibitors). From a mechanistic viewpoint, it is still uncertain what the exact nature of Pgp's binding site is, the role of ATP binding and hydrolysis in transport and how both of these interplay with ligand binding. The work presented in this thesis attempts to answer these questions from two perspectives. Firstly the mouse Pgp crystal structure [PDB 3G60] was used as a unique starting point for molecular dynamics (MD) simulations to characterise the dynamics and conformational exibility of Pgp, properties believed to be integral to its function. The simulations revealed Pgp to be a highly dynamic molecule at both its transmembrane (TM) and nucleotide binding domains (NBDs). The latter exhibited a conformational asymmetry that supports the Constant Contact model of ATPase activity. In the presence of the Pgp substrate, daunorubicin, the NBDs exhibited tighter asymmetric dimerisation leading to increased affinity for ATP. In contrast, the presence of the Pgp inhibitor, QZ59-RRR led to NBD conformational changes that reduced their affinity for ATP. Thus providing an appealing mechanism for how QZ59-RRR inhibits Pgp ATPase activity. MD simulation was also used to provide atomic-detail interpretations of multiple binding stoichiometries of drug and lipid molecules observed by collaborator-led mass spectrometry experiments. This also provided opportunity to validate the Pgp simulations against novel experimental data. The second strand of the thesis explored the membrane permeation dynamics of CNS therapeutics in order to identify differences in protonation states, conformations, orientations and membrane localisation that might distinguish those that are Pgp substrates and from those that are not. These properties were studied using complementary MD simulation and nuclear magnetic resonance (NMR) techniques. The simulations revealed a novel set of criteria that in uence the likelihoodof a drug to 'flip-flop' across a membrane, a behaviour that may make drugs more susceptible to Pgp efflux. These observations were broadly consistent with the NMR experiments. However, the NMR data also highlighted limitations in the simulation approaches used in this thesis and emphasised the need to also consider the kinetics of permeation in addition to its thermodynamics.

P. aeruginosa e espécies de Acinetobacter são causas comuns de diversas infecções em pacientes hospitalizados, principalmente nos internados em centros de tratamento intensivo. Além disso, esses microrganismos se destacam por apresentarem resistência, intrínseca e adquirida, a várias classes de antibióticos, conferindo à bactéria fenótipos de multirresistência e panresistência. O objetivo deste estudo foi avaliar a participação de integrons (elementos genéticos que carreiam genes de resistência), de genes codificadores de metalo--lactamases, da perda de porinas (canais protéicos da membrana externa), e da atividade de efluxo aumentada, como determinantes do fenótipo de multirresistência e panresistência. Foram estudadas 147 P. aeruginosa e 57 Acinetobacter spp. isolados de pacientes hospitalizados no Hospital Universitário da Universidade Federal de Juiz de Fora, no período de 2003 a 2006. O perfil de sensibilidade destes isolados foi determinado por disco de difusão e utilizado para classificá-las como multirresistentes (MDR) e não multirresistentes (n-MDR). A variabilidade clonal dos isolados foi investigada por PFGE. Os isolados pertencentes aos grupos MDR e n-MDR foram investigados quanto a presença de integrons de classe 1, 2 e 3, por PCR e análise de RFLP. Os cassetes gênicos contidos nestes integrons, assim como genes codificadores de carbapenemases (ex. IMP, VIM e SPM), foram detectados por PCR e identificados por seqüenciamento. Avaliação da expressão gênica de bombas de efluxo (mexB, mexY, mexD e adeB) e de porina (OprD) foi conduzida por real-time RT-PCR. Os dados apresentados para os isolados do grupo MDR foram comparados àqueles do grupo n-MDR e a associação entre os determinantes de resistência e o fenótipo MDR foi calculada estatisticamente. Fenótipo de multiresistência foi observado em 42,2% e 84,2% das P. aeruginosa e Acinetobacter spp. estudadas. Nenhum isolado bacteriano apresentou fenótipo panresistente. Em 65 (44,2%) dos isolados de P. aeruginosa, foram detectados integrons de classe 1. Esses elementos apresentaram relação estatisticamente significativa com fenótipos MDR em P. aeruginosa. Entretanto, a maioria desses integrons não carreava nenhum cassete gênico (43/65) ou continham apenas cassetes gênicos de resistência a aminoglicosídeos (19/65). Entre os isolados de Acinetobacter spp., 11 (17,5%) apresentaram integrons de classe 1 e 30 (47,6%) integrons de classe 2. Apenas os últimos foram estatisticamente associados com fenótipos MDR. A pesquisa de metalo--lactamase (MBL) revelou a produção de enzimas SPM em 24 isolados de P. aeruginosa. Os estudos de expresão gênica demonstraram que, entre os sistemas de efluxo mais relatados para P. aeruginosa, MexXY-OprM foi o que mostrou maior diferença entre o nível de expressão dos grupos MDR e n-MDR, sugerindo que este sistema de efluxo desempenha importante papel no fenótipo MDR. Diminuição, em média de 66,4%, da produçãode OprD também foi um padrão encontrado nos isolados MDRem relação aos n-MDR. Dois grupos clonais de P. aeruginosa e dois de Acinetobacter spp. foram predominantes e tiveram relação com presença de integrons, produção de SPM-1 e com fenótipo MDR. Portanto, esse fenótipo pode ser consequência de acúmulo de determinantes de resistência em clones específicos. / The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii.

A tuberculose multirresistente (TBMR) - resistência simultânea a rifampicina e isoniazida, principais fármacos do esquema de tratamento da tuberculose (TB), gera mais ônus aos doentes e aos Serviços de Saúde, pois acarreta maiores custos, aumenta o tempo de tratamento e relaciona-se a prognósticos desfavoráveis. A TBMR ocorre devido à falha em algum princípio do tratamento, seja por parte dos profissionais e serviços de saúde, seja por questões ligadas ao doente. Frente a isso, o presente estudo objetivou identificar e analisar as condições de produção da TBMR relacionadas ao doente e seu entorno. Esta investigação foi realizada junto aos sujeitos em seguimento em um hospital de referência do interior paulista, entre janeiro de 2010 a janeiro de 2012. Foi utilizada a abordagem qualitativa. Por meio da análise dos prontuários médicos do serviço terciário, caracterizamos o universo do estudo - composto por todos os doentes já seguidos pela instituição, descrevendo e analisando dados sociodemográficos e clínicos correspondentes. Caracterizamos, também, a amostra estudada, formada por oito doentes de TBMR em seguimento, detalhando seu contexto de vida e trajetória com a doença, assim como a estruturação municipal para seu acompanhamento. A segunda etapa do trabalho constituiu na análise das percepções dos sujeitos a cerca do adoecimento por TB e pela forma MR. Os dados foram coletados por meio de entrevistas semi-estruturadas, gravadas e transcritas na íntegra. Os textos resultantes constituíram o corpus do estudo, organizado com recurso do software Atlas. Ti versão 7.0 e analisado sob o referencial teórico da Análise de Discurso, de matriz francesa. Os resultados da investigação baseiam-se na análise de três aspectos: percurso diagnóstico - em que é apontadas a percepção da doença e dos sintomas, as histórias pessoais e familiares do adoecimento por TB, o desenvolver até a obtenção do diagnóstico e as histórias de fracasso dos tratamentos convencionais; tratamento e acompanhamento dos casos MR - momento em que são discutidas questões ligadas a percepção do tratamento pelos doentes, as modalidades de supervisão empreendidas, os instrumentos e insumos fornecidos, além do apoio da rede familiar; coordenação da assistência - em que são analisadas as nuances da relação entre os diferentes serviços envolvidos na atenção ao doente, tanto dentro do mesmo nível assistencial quanto em sua intersecção com a atenção terciária, a fim de compreender suas fragilidades e promover as potencialidades para o tratamento dos sujeitos. Essas condições de produção mostraram-se complexas, ao passo que sofrem influência das peculiaridades da forma como os serviços locais de atenção organizando-se para atender esses doentes, além de questões relacionadas à trajetória de vida e doença desses sujeitos, apontando para a necessidade de ampliação do espaço de negociação dentro do sistema de saúde. / Multidrug-resistant tuberculosis - simultaneous resistance to rifampicin and isoniazid, the main drugs in the treatment for tuberculosis (TB), generates more onuses to patients and Health Services because it involves higher costs, increases the treatment time and relates to the unfavorable outcomes. MDR-TB occurs due to failure in some treatment principles, either by professionals and health services, or by patient issues. Concerning this, the present study aimed to identify and analyze the conditions of MDR-TB production related to the patients and their surroundings. This investigation was conducted with the subjects followed up in a referral hospital in São Paulo State, between January 2010 and January 2012. It was used a qualitative approach. By analyzing the medical records of the tertiary service, we characterized the universe of the study - consisting of all patients who were already followed by the institution, describing and analyzing the demographic and clinical data matching. We also characterized the sample, formed by eight MDR-TB patients in follow-up, detailing the context of their lives and the disease trajectory, as well as the municipal structure to monitor them. The second stage of the work consisted of analyzing the subjects\' perceptions about TB development and MR strain. Data was collected through semi-structured interviews which were recorded and fully transcribed. The resulting texts constituted the corpus of the study, organized using the Atlas.Ti software version 7.0 and analyzed from the theoretical framework of Discourse Analysis of French matrix. The results are based on analysis of three aspects: The first is diagnosis route - which shows the disease and symptoms perception of the patient, personal and family histories of TB development, the disease development until the diagnosis, and the failed conventional treatment stories. The second aspect was treatment and monitoring of MDR cases - when cases are discussed we focused on the following aspects: perceptions of treatment by patients, undertaken methods of supervision, instruments and inputs provided, and the familiar support network. The third is assistance coordination - where the nuances of the relationship between several departments involved in the patient care are analyzed, within the same care level as well as its intersection with tertiary care in order to understand their weaknesses and promote the potential treatments for the subjects. These production conditions proved to be complex whereas the local care services peculiarities influence the way they are organized to assist these patients, as well as aspects related to life and illness trajectory of these subjects, indicating the need of expanding the negotiation space within the health system

Blood-brain barrier (BBB) is a unique membranous barrier, which segregates brain from the circulating blood. It works as a physical and metabolic barrier between the central nervous system (CNS) and periphery. In mammals, endothelial cells were shown to be of BBB and are characterized by the tight junctions along with efflux system which are responsible for the restriction of movement of molecules within the cells. Efflux system consists of multidrug resistance proteins such as P-glycoprotein (P-gp). P-gp removes substances out back from the brain to the blood before they reach to the brain. So the barrier is impermeable to many compounds such as amino acids, ions, small peptides and proteins, making it the most challenging factor for the development of new drugs for targeting CNS. Curcumin is a bioactive compound that has a number of health promoting benefits such as anti-inflammatory, anticancer, anti-oxidant agent; as well as a role in neurodegenerative diseases, but low oral bioavailability is the major limiting factor. Low water solubility and rapid metabolism are the two important factors responsible for poor bioavailability of curcumin. Galaxolide is a musk compound and previously known for the bioaccumulation of toxic components in the aquatic animals by interference with the activity of multidrug/multixenobiotic resistance efflux transporters (MDR/MXR). The bioavailability of curcumin can be enhanced when administered with galaxolide. This study was carried out to investigate the effect of galaxolide on the permeation of curcumin through the epithelial cell monolayers. MDCKII-MDR1 cell monolayer is used an in vitro blood-brain barrier model while Caco-2 monolayer is used as an in vitro intestinal model, which also expresses the P-glycoprotein. The curcumin and galaxolide were separately solubilised in the DMSO and used in combination to perform permeation study, to determine the effect of galaxolide on curcumin permeation through epithelial cell monolayers. The galaxolide shows an efflux protein inhibition activity and this activity was used to enhance permeation of curcumin through the Caco-2 monolayer. In summary, galaxolide is a novel permeation enhancer molecule, which can be used for the improvement of drug delivery of other bioactive compounds in future.

Leung, Winnie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 155-164). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / Abbreviations --- p.xii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Clinical management of Cancer --- p.2 / Chapter 1.2 --- Multidrug resistance --- p.8 / Chapter 1.3 --- Aim of study --- p.14 / Chapter Chapter 2 --- Identification of gene contributing to multidrug resistance in human uterine sarcoma cells --- p.16 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Material and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Cell lines --- p.20 / Chapter 2.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.20 / Chapter 2.2.1.3 --- Gene expression assay reagents --- p.22 / Chapter 2.2.1.4 --- Western blotting reagents --- p.24 / Chapter 2.2.1.5 --- MTT assay reagents --- p.29 / Chapter 2.2.1.6 --- Apoptosis analysis by flow cytometry reagents --- p.29 / Chapter 2.2.2 --- Metho --- p.ds / Chapter 2.2.2.1 --- Cell Culture --- p.31 / Chapter 2.2.2.2 --- MTT assay --- p.32 / Chapter 2.2.2.3 --- Gene expression essay (RT-PCR) --- p.33 / Chapter 2.2.2.4 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.37 / Chapter 2.2.2.5 --- Quantification of doxorubicin uptake by flow cytometry --- p.40 / Chapter 2.2.2.6 --- Apoptosis analysis by flow cytometry --- p.41 / Chapter 2.3 --- Results --- p.4 / Chapter 2.3.1 --- Cytotoxicity of doxorubicin on SA and DX5 cells --- p.43 / Chapter 2.3.2 --- mRNA expression of multidrug resistance related genes in SA and DX5 cells --- p.46 / Chapter 2.3.3 --- P-glycoprotein expression in SA and DX5 cells --- p.49 / Chapter 2.3.4 --- Doxorubicin (Dox) uptake by SA and DX5 cells --- p.51 / Chapter 2.3.5 --- Doxorubicin induced Apoptosis in SA and DX5 cells --- p.54 / Chapter 2.4 --- Discussion --- p.61 / Chapter 2.5 --- Conclusion --- p.65 / Chapter Chapter 3 --- Alternation in P-glycoprotein expression in DX5_Ninjl cells --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials / Chapter 3.2.1.1 --- Cell lines --- p.70 / Chapter 3.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.70 / Chapter 3.2.1.3 --- Gene expression assay reagents --- p.70 / Chapter 3.2.1.4 --- Western blotting reagents --- p.72 / Chapter 3.2.1.5 --- Plasmid DNA extraction --- p.75 / Chapter 3.2.1.6 --- Transient transfection --- p.76 / Chapter 3.2.1.7 --- MTT reagents --- p.76 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.78 / Chapter 3.2.2.2 --- Gene expression essay (RT-PCR) --- p.79 / Chapter 3.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.81 / Chapter 3.2.2.4 --- DNA plasmid extraction --- p.83 / Chapter 3.2.2.5 --- Transient transfection --- p.84 / Chapter 3.2.2.6 --- MTT assay --- p.85 / Chapter 3.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.86 / Chapter 3.3 --- Results / Chapter 3.3.1 --- mRNA expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.87 / Chapter 3.3.2 --- The protein expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.89 / Chapter 3.3.3 --- Ninjurin1 (Ninj1) cDNA transfection in DX5 cells --- p.91 / Chapter 3.3.4 --- mRNA expression of MDR1 in Ninjurin1-transfected DX5 cells (DX5_Ninjl) --- p.93 / Chapter 3.3.5 --- P-glycoprotein expression in Ninjurin1-transfected DX5 cells --- p.95 / Chapter 3.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5_Ninjl cells" --- p.97 / Chapter 3.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_Ninjl cells" --- p.99 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.5 --- Conclusion --- p.105 / Chapter Chapter 4 --- Alternation in MDR1 expression in DX5一THBS1 cells --- p.106 / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials / Chapter 4.2.1.1 --- Cell lines --- p.109 / Chapter 4.2.1.2 --- Cell culture medium； supplements and buffers --- p.109 / Chapter 4.2.1.3 --- Gene expression assay reagents --- p.109 / Chapter 4.2.1.4 --- Western blotting reagents --- p.111 / Chapter 4.2.1.5 --- Plasmid DNA extraction --- p.114 / Chapter 4.2.1.6 --- Transient transfection --- p.115 / Chapter 4.2.1.7 --- MTT reagents --- p.115 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Cell culture --- p.117 / Chapter 4.2.2.2 --- Gene expression essay (RT-PCR) --- p.118 / Chapter 4.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.120 / Chapter 4.2.2.4 --- DNA plasmid extraction --- p.123 / Chapter 4.2.2.5 --- Transient transfection --- p.123 / Chapter 4.2.2.6 --- MTT assay --- p.124 / Chapter 4.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1 --- mRNA expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.126 / Chapter 4.3.2 --- The protein expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.128 / Chapter 4.3.3 --- Thrombospondinl (THBS1) cDNA transfection in DX5 cells --- p.130 / Chapter 4.3.4 --- mRNA expression of MDR1 in Thrombospondinl-transfected DX5 cells (DX5_THBS1) --- p.132 / Chapter 4.3.5 --- P-glycoprotein expression in Thrombospondinl-transfected DX5 cells --- p.134 / Chapter 4.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5一THBS1 cells" --- p.136 / Chapter 4.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_THBS1 cells" --- p.138 / Chapter 4.4 --- Discussion --- p.141 / Chapter 4.5 --- Conclusion --- p.145 / Chapter Chapter 5 --- General discussion --- p.146 / Chapter 5.1 --- Doxorubicin induced multidrug resistance in human uterin sarcoma cells via upregulation of P-glycoprotein --- p.147 / Chapter 5.2 --- The down-regulation of Ninjurin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.148 / Chapter 5.3 --- The down-regulation of Thrombospondin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.150 / Chapter 5.4 --- Conclusion and Future Perspective --- p.153 / Reference --- p.155