Die Gentherapie hat in den letzten Jahren wesentliche Entwicklungen im Vektordesign, der kontrollierte Expression sowie der Sicherheit ihrer Anwendung durchgemacht. Die Erkenntnis, dass die Tumorgentherapie allein nur in begrenztem Maße zum erhofften therapeutischen Benefit für den Patienten beitragen kann, führte zum Konzept der lokalen Gentherapie als Teil anderer, etablierter Tumortherapien. In diesem Zusammenhang wird die Gentherapie als eine moderne Option zur Steigerung der Effizienz von Chemotherapie, Strahlentherapie oder Hyperthermie verstanden. Zum Erreichen dieses Zieles ist die Etablierung Therapie-regulierbarer Vektorsysteme von besonderer Attraktivität. Im Rahmen der Strategie des lokalen Transfers therapeutischer Gene bietet inzwischen die Anwendung nicht-viraler Transfersysteme, wie z.B. in vivo-Elektrotransfer, Gene-Gun oder Jet-Injection eine klinisch applikable Technologie. Die Etablierung einer effizienten, auf der Jet-Injection basierenden nicht-viralen Transfertechnologie und die Analyse ihres Potentials für eine klinische Anwendung in einem multimodalen Therapiekonzept war ein wesentliches Ziel der Arbeit. Es wurde gezeigt, dass die Jet-Injection in tierexperimentellen Tumormodellen zur effizienten Expression der Transgene führt, dass sowohl Eindringtiefen, als auch Verteilung der Jet-Injection optimal für einen effizienten Gentransfer sind und die Höhe der Genexpression mit etablierten Gentransfer-Technologien, wie z.B. der in vivo-Lipofektion, vergleichbar ist. Basierend auf der Strategie des Einsatzes der Gentherapie in Kombination mit anderen Therapien, bestand ein weiteres Ziel der Arbeit in der Charakterisierung und Anwendung konditioneller Vektorsysteme, mit denen die Expression therapeutischer Gene durch Chemotherapie oder Hyperthermie kontrollierbar ist. Derartige Vektoren, in denen der humane Multidrug Resistenzgen 1- (mdr1) Promotor genutzt wurde, exprimierten vor allem Zytokingene, die die therapeutische Effizienz von Zytostatika oder der Hyperthermie verbessern. Die Zytostatika-und auch Hitze-Induzierbarkeit der mdr1-Promotor gesteuerten Genexpression konnte in verschiedenen Tumormodellen in vitro und in vivo erfolgreich demonstriert werden Diese Untersuchungen zeigten, dass eine Zytostatika-induzierte Gentherapie zu einer besseren Tumortherapie beiträgt. Die Kombinations-Experimente der konditionellen Gentherapie im Kontext einer Hyperthermie geben erste Hinweise, dass auch hier die therapeutische Effektivität in vitro und in vivo gesteigert werden kann. Im Rahmen des Konzepts der kombinierten Gen- und Chemotherapie von Tumoren ist in der Arbeit vor allem auf das chemosensitivierende Potential von Zytokinen gesetzt worden. Besonders für TNF-a, IL-2 sowie IFN-g konnte gezeigt werden, dass diese Zytokine zu einer Modulation der Expression MDR-assoziierter Gene, wie dem mdr1, MVP/LRP und auch MRP1 in der Lage sind und dadurch zur Chemosensitivierung in verschiedenen Tumormodellen führt. Diese Befunde bildeten eine wichtige Rationale für den Einsatz von Zytokingenen im Rahmen der Tumorgentherapie zur Überwindung der MDR. Gentransferexperimente mit TNF-a- und IL-2-exprimierenden Vektoren konnten analog zur Applikation rekombinanter Zytokine die Modulation der Gene mdr1 und MVP/LRP zeigen, die mit der Erhöhung der Sensitivität gegenüber Zytostatika wie Vincristin oder Adriamycin assoziiert ist. / Gene therapy has made great achievements in vector design, controlled gene expression and in safety. The fact, that gene therapy as single therapy has only limited potential for the benefit in the therapy for cancer patients, has led to the concept of local gene therapy as part of other, established therapies. In this context, gene therapy serves as a modern option to improve the efficiency of chemotherapy, radiotherapy or hyperthermia. To achieve this goal, the establishment of therapy-regulatable vectors is of particular attractiveness. For the concept of local transfer of therapeutic genes non-viral transfer systems, such as in vivo electrotransfer, gene gun or jet-injection represent clinically applicable transfer technologies. One major issue of this work was the establishment of an efficient, jet-injection based non-viral transfer technology and the analysis of its potential for clinical application in a concept of multimodal therapy. It has been shown in vivo, that efficient transgene expression can be achieved by jet-injection, that penetration and distribution of the transgene are optimal for an efficient gene transfer and that the level of gene expression is comparable to established gene transfer technologies, sch as in vivo lipofection. Based on the strategy of combination of gene therapy with other therapies, another goal of this work aimed at the characterization and utilization of conditional vector systems, by which expression of therapeutic genes is controllable by chemotherapy or hyperthermia. By such vectors, in which the human multidrug resistance gene 1 (mdr1) promoter was employed, cytokine genes were expressed, which are capable to improve the therapeutic efficacy of cytostatic drugs or of hyperthermia. The drug- and heat-inducibility of mdr1 promoter-driven gene expression has successfully been demonstrated in in vitro and n vivo tumor models. The studies have also shown, that drug-induced gene therapy leads to improved tumor treatment. Combination experiments of conditional gene therapy in the context with hyperthermia give first indication of an increased therapeutic efficiency in vitro and in vivo. For the concept of combined gene- and chemotherapy the chemosensitizing potential of cytokines was exploited. It has been shown, particularly for TNF-a, IL-2 and IFN-g, that these cytokines are capable to modulate the expression of MDR-associated genes, such as mdr1, MVP/LRP or MRP1 leading to chemosensitization in different tumor models. These observations represent an important rationale for the use of cytokine genes in gene therapy for MDR-overcoming. Gene transfer experiments with TNF- or IL-2 expressing vectors showed the modulation of mdr1 or MVP/LRP expression, associated with increased sensitivity towards cytostatic drugs, such as vincristine or adriamycin.

Bactérias multirresistentes (MRs) pertencentes ao grupo ESKAPE (i.e., Enterococcus faecium resistente à vancomicina, VRE; Staphylococcus aureus resistente à meticilina, MRSA; Klebsiella spp., e Escherichia coli produtoras de β-lactamases de amplo espectro; Acinetobacter baumannii, Pseudomonas aeruginosa e Enterobacter spp. resistentes aos carbapenêmicos) são importantes patógenos de infecções relacionadas à assistência à saúde (IRAS), onde a sua endemicidade e prevalência tem sido decorrente da seleção de linhagens clonais. Embora, o fenótipo MR decorra da expressão de mecanismos mediados por genes intrínsecos e/ou adquiridos, o crescimento bacteriano na forma de biofilme contribui para um importante fenômeno fisiológico de resistência, o qual é inespecífico quanto ao substrato antimicrobiano. O presente estudo teve como objetivo avaliar a eficácia de agentes físicos e químicos contra biofilmes produzidos por clones de bactérias MRs de importância clínica e epidemiológica no Brasil. Cerdas de poliamida foram utilizadas como modelo de superfície de adesão para o crescimento de biofilmes, os quais foram monitorados por microscopia eletrônica de varredura (MEV). In vivo, o modelo de biofilme foi avaliado pela inserção das cerdas na proleg de larvas de Galleria mellonella, enquanto que, diferentes tratamentos foram aplicados para inibir a formação do biofilme. Adicionalmente, mediante ao ensaio de bioluminescência, o modelo de biofilme produzido pela cepa de P. aeruginosa PAO1/lecA::lux foi avaliado na presença de soluções hipertônicas de cloreto de sódio (NaCl). In vitro, soluções hipertônicas de cloreto de sódio (> 6%) utilizadas de maneira profilática, apresentaram efeito bacteriostático (CIM90= 1,7 M) contra biofilmes produzidos por todos os isolados analisados. Além disso, através do uso profilático de soluções hipertônicas de NaCl, foi possível visualizar a inibição da motilidade dos isolados. Por outro lado, os compostos quaternários de amônio (CQAs) cloreto de benzalcônio (CBA) e cloreto de cetilpiridínio (CCP) apresentaram efeito bactericida (CBM90= 256 µg/mL) contra biofilmes previamente formados em 24h. A atividade de ambos os CQAs foi potencializada na presença de soluções salinas hipertônicas, como avaliado pela metodologia de checkerboard, tendo um efeito sinérgico contra E. coli (ST10, ST101) MCR-1 (∑FIC= 0,5); parcialmente sinérgico contra A. baumannii OXA-23 (ST79), E. cloacae CTX-M-8 (ST131), E. faecium VRE (ST478) e K. pneumoniae KPC-2 (ST340) (∑FIC= 0,75); e indiferente contra cepas de P. aeruginosa SPM-1 (ST277) e S. aureus MRSA (ST5). Adicionalmente, a CIM de carbapenêmicos, fluoroquinolonas e aminoglicosídeos contra biofilmes de bactérias Gram-negativas MRs foi potencializada na presença de solução salina hipertônica resultando em uma queda da CIM >=2. Finalmente, in vivo, para todas as espécies MRs estudadas, biofilmes formados em 08, 12 e 24h resultaram em 100% de morte das larvas de G. mellonella em até 96 horas pós-infecção. O mesmo comportamento foi observado para a cepa PAO1/lecA::lux, sendo possível detectar sinais intensos de bioluminescência nas larvas infectadas com os biofilmes. Entretanto, para os biofilmes previamente tratados com solução salina hipertônica, observou-se a diminuição dos sinais de bioluminescência em até 60%. Já para biofilmes formados em 24, 12 e 08h, o tratamento prévio em solução salina hipertônica e posteriormente com antibióticos resultou em um aumento de até 40, 70 e 80% da sobrevida de G. mellonella, respectivamente. / ESKAPE pathogens (ie, vancomycin-resistant (VRE) Enterococcus faecium; methicillin-resistant (MRSA) Staphylococcus aureus; extended spectrum β-lactamase-producing Klebsiella spp., and Escherichia coli; Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Resistant to carbapenems), represents an important group of multidrug-resistant (MDR) bacteria related to healthcare-associated infections (HAIs), whereas endemicity have been associated with selection and predominance of clones. Although the MR phenotype derives from the expression of mechanisms mediated by intrinsic and/or acquired genes, bacterial growth in the biofilm form contributes to an important physiological phenomenon of resistance, which is non-specific to the antimicrobial substrate. The present study aimed to evaluate the efficacy of physical and chemical agents against biofilms produced by clones of MDR bacteria of clinical and epidemiological importance, in Brazil. Polyamide bristles were used as adhesion surface model for the growth of biofilms, which were monitored by scanning electron microscopy (SEM). In vivo, the biofilm model was evaluated by the insertion of the bristles into the proleg of larvae of Galleria mellonella, while different treatments and physicochemical conditions were applied to inhibit biofilm formation. Additionally, the biofilm model produced by the P. aeruginosa PAO1/lecA::lux strain was evaluated in the presence of hypertonic solutions of sodium chloride (NaCl). In vitro, hypertonic solutions of sodium chloride presented a bacteriostatic effect (MIC90 = 1.7 M) against biofilm formation of all the isolates analyzed. Moreover, through the prophylactic use of hypertonic solutions of NaCl, it was possible to observe the inhibition of the motility of the isolates. On the other hand, the ammonium quaternary compounds (QACs) benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) had a bactericidal effect (CBM90 = 256 µg / mL) against previously formed biofilms in 24h. The activity of both QACs was potentiated in the presence of hypertonic saline solutions, as evaluated by the checkerboard methodology, having a synergistic effect against E. coli (ST10, ST101) MCR-1 (∑FIC = 0.5); (ST340) (∑FIC = 0.75), E. faecium VRE (ST478) and K. pneumoniae KPC-2 (ST340), E. cloacae CTX-M-8 (ST131); and indifferent effect against strains of P. aeruginosa SPM-1 (ST277) and S. aureus MRSA (ST5). Furthermore, the MIC of carbapenems, fluoroquinolones and aminoglycosides against biofilms of MDR Gram-negative bacteria was potentiated in the presence of hypertonic saline solution resulting in a decrease in MIC >=2-fold. Finally, for all MDR species studied, biofilms formed at 08, 12 and 24h resulted in 100% death of G. mellonella larvae within 96h post-infection. In fact, the same behavior was observed for the strain PAO1/lecA::lux, and it is possible to detect intense bioluminescence signals in the larvae infected with biofilms. However, for biofilms previously treated with hypertonic saline solution, bioluminescence signs decreased by up to 60%. As for biofilms formed at 24, 12 and 8h, pretreatment in hypertonic saline solution and later with antibiotics resulted in an increase of up to 40, 70 and 80% of the survival of G. mellonella, respectively.

Cheung Hoi Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 151-166). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / CONTENTS --- p.X / Chapter CHAPTER ONE: --- GENERAL INTRODUCTION / Chapter 1.1 --- Non-coding RNAs in transcriptional output --- p.2 / Chapter 1.2 --- Diverse functions of non-coding RNAs --- p.5 / Chapter 1.3 --- HI9: imprinted non-coding RNA --- p.6 / Chapter 1.4 --- Objective --- p.7 / Chapter CHAPTER TWO: --- The ROLE OF H19 RNA IN MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- H19-Igf2 locus as a model for genomic imprinting --- p.10 / Chapter 2.1.2 --- HI9 as a non-protein coding regulatory RNA --- p.12 / Chapter 2.1.3 --- Controversial roles of H19 RNA --- p.13 / Chapter 2.1.4 --- Novel role of H19 RNA in drug resistance --- p.15 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.17 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell culture --- p.19 / Chapter 2.2.2.2 --- Plasmid construction and stable cell transfection --- p.19 / Chapter 2.2.2.3 --- Transient gene transfection --- p.20 / Chapter 2.2.2.4 --- RNA isolation and RT-PCR --- p.21 / Chapter 2.2.2.5 --- MTT drug sensitivity assay --- p.22 / Chapter 2.2.2.6 --- Western blot analysis --- p.22 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Differential expression of H19 RNA in different human cancer cell lines --- p.24 / Chapter 2.3.2 --- R-HepG2 cells over-expressed P-glycoprotein and H19 RNA --- p.24 / Chapter 2.3.3 --- Development of H19-silenced cell lines in HepG2 cells by RNA interference --- p.26 / Chapter 2.3.4 --- Altered drug sensitivity in H19-silenced cells --- p.28 / Chapter 2.3.5 --- Expression of P-glycoprotein in H19-silenced cells --- p.31 / Chapter 2.3.6 --- Overexpression of H19 RNA in HepG2 cells --- p.34 / Chapter 2.3.7 --- Induction of H19 RNA and MDR1 in HepG2 cells --- p.34 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- H19 regulation of MDR1 associated drug resistance --- p.38 / Chapter 2.4.2 --- The puzzle of riboregulation in drug resistance --- p.40 / Chapter CHAPTER THREE: --- The ROLES OF PTB AND IMP1 IN H19-RELATED MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- H19 RNA binding proteins --- p.43 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.46 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.48 / Chapter 3.2.2.2 --- Plasmid construction and stable cell transfection --- p.48 / Chapter 3.2.2.3 --- RNA extraction and RT-PCR --- p.48 / Chapter 3.2.2.4 --- MTT drug sensitivity assay --- p.48 / Chapter 3.2.2.5 --- Western blot analysis --- p.48 / Chapter 3.2.2.6 --- Real-time PCR analysis of gene expression --- p.49 / Chapter 3.2.2.7 --- DOX efflux assay --- p.49 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PTB knockdown increased P-glycoprotein expression --- p.51 / Chapter 3.3.2 --- IMP1 knockdown decreased MDR1 /P-glycoprotein expression --- p.54 / Chapter 3.3.3 --- Altered drug sensitivity in IMP 1 -knockdown cells --- p.60 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Antagonistic effect of PTB and IMP1 on H19/MDR1 expressions --- p.64 / Chapter 3.4.2 --- Complexity of riboregulation --- p.65 / Chapter CHAPTER FOUR: --- IDENTIFICATION OF H19 RNA BINDING PROTEINS FROM HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Overview of RNA-protein interactions --- p.69 / Chapter 4.1.2 --- Methodology in the study of RNA-protein interactions --- p.71 / Chapter 4.1.3 --- Identification of RNA-binding proteins --- p.72 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.75 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Screening of H19 cDNA from human placenta cDNA library --- p.78 / Chapter 4.2.2.2 --- Preparation of nuclear and cytoplasmic extracts from HepG2 cells / Chapter 4.2.2.3 --- In vitro RNA transcription and RNA labeling --- p.80 / Chapter 4.2.2.4 --- RNA electrophoretic mobility shift assay --- p.81 / Chapter 4.2.2.5 --- In vitro UV-crosslinking assay --- p.82 / Chapter 4.2.2.6 --- Preparation of RNA-affinity column and isolation of RNA binding proteins --- p.83 / Chapter 4.2.2.7 --- In-gel digestion and MALDI-TOF mass spectrometry --- p.84 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Screening of H19 cDNA and preparation ofH19 RNA --- p.86 / Chapter 4.3.2 --- Electrophoretic mobility shift analysis of H19 RNA with HepG2 cytoplasmic extract --- p.87 / Chapter 4.3.3 --- UV-crosslinking of H19 RNA with HepG2 nuclear and cytoplasmic extract --- p.90 / Chapter 4.3.4 --- Isolation of H19 RNA binding proteins by RNA-affmity chromatography --- p.94 / Chapter 4.3.5 --- Confirmation of PTB and IMP1 as H19 RNA binding protein --- p.96 / Chapter 4.3.6 --- MALDI-TOF mass spectrometric analysis of isolated H19 RNA binding proteins --- p.96 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- RNA-protein interactions: an initial step for mechanistic study --- p.99 / Chapter 4.4.2 --- In vitro and in vivo methods for isolation of RNA binding proteins --- p.101 / Chapter 4.4.3 --- Novel role of hnRNP M protein in H19 RNA binding --- p.103 / Chapter CHAPTER FIVE: --- THE ROLE OF PTB IN APOPTOSIS / Chapter 5.1 --- Introduction / Chapter 5.1.1 --- Overview of polypyrimidine tract-binding protein in RNA processing and post-transcriptional gene regulation --- p.106 / Chapter 5.1.2 --- Evidences of polyrimidine-tract binding protein in the regulation of apoptosis --- p.108 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- Materials --- p.111 / Chapter 5.2.2 --- Methods / Chapter 5.2.2.1 --- Cell culture --- p.114 / Chapter 5.2.2.2 --- Stable cell transfection in A431 cells --- p.114 / Chapter 5.2.2.3 --- Western Blot analysis --- p.114 / Chapter 5.2.2.4 --- MTT drug sensitivity assay --- p.114 / Chapter 5.2.2.5 --- DNA fragmentation assay --- p.115 / Chapter 5.2.2.6 --- Flow cytometry analysis of apoptosis --- p.115 / Chapter 5.2.2.7 --- Caspase activity assay --- p.116 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Taxol as an apoptosis inducer in HepG2 cells --- p.117 / Chapter 5.3.2 --- PTB was cleaved during Taxol-induced apoptosis --- p.118 / Chapter 5.3.3 --- PTB knockdown increased Taxol cytotoxicity and apoptosis --- p.118 / Chapter 5.3.4 --- Effect of PTB knockdown on drug sensitivity of cells --- p.121 / Chapter 5.3.5 --- Effect of PTB knockdown on other drug-induced apoptosis --- p.121 / Chapter 5.3.6 --- Effect of PTB knockdown on the basal expressions of genes in apoptosis pathway --- p.126 / Chapter 5.3.7 --- The role of caspase-9 activation in PTB-regulated apoptosis --- p.129 / Chapter 5.3.8 --- The effect of PTB knockdown on pro-caspase-9 expression and Taxol-induced apoptosis in A431 cells --- p.133 / Chapter 5.3.9 --- The role of PTB in the regulation of intrinsic apoptosis pathway --- p.136 / Chapter 5.4 --- Discussion / Chapter 5.4.1 --- The role of PTB in intrinsic apoptosis pathway --- p.138 / Chapter 5.4.2 --- PTB in regulation of pro-caspase-9 expression --- p.139 / Chapter CHAPTER SIX: --- GENERAL DISCUSSION AND CONCLUSION / Chapter 6.1 --- H19 as a potential target in anti-cancer gene therapy --- p.143 / Chapter 6.2 --- Conclusion --- p.144 / Chapter 6.3 --- Unanswered questions and future work --- p.145 / Chapter 6.4 --- A proposed model for H19 pathway --- p.148 / REFERENCES --- p.151

Made available in DSpace on 2016-01-26T12:51:29Z (GMT). No. of bitstreams: 1 vivianedeciceracolombooliveira_dissert.pdf: 908582 bytes, checksum: 3c95ae4a3fb33fb357397cfbcc4f9bdf (MD5) Previous issue date: 2011-05-02 / Hospital bacterial resistance to multiple antibiotics is a great concern worldwide. This study objective was to know the multidrug-resistantance (MDR) agents, clinical materials, origin, trends, and to correlate them with the bacterial sensitivity and antimicrobial consumption. A total of 53,316 nosocomial bacteria were assessed in a tertiary hospital during the period from 1999 to 2008. MDR was defined for gram negative bacteria (GNB) when it presented resistance to two or more classes/groups of antibiotics. GNB were predominant (66.1%). GNB MDR had a global increase of 3.7 times in the end of the period. Acinetobacter baumannii was the most prevalent (36.2%). The most frequent materials were urinary and respiratory. A significant increase occurred during the period of 4.8 and 14.6 times of the A. baumannii and K. pneumoniae, respectively. Sixty-seven percent of GNB MDR was from the Intensive Care Units. A. baumannii resistance to carbapenemics increased from 7.4% to 57.5% during the period, concomitant to the consumption increase. The resistance of K. pneumoniae to the cephalosporins had a high increase during the decade. P. aeruginosa decreased in these last two years with a recovery of the sensitivity. / A resistência bacteriana hospitalar a múltiplos antibióticos é uma grande preocupação mundial. O objetivo deste estudo foi conhecer os agentes multidroga-resistentes (MDR), materiais clínicos, origem, evolução e correlacionar com a sensibilidade bacteriana e consumo de antimicrobianos. Foram avaliadas 53.316 bactérias de origem nosocomial, num hospital terciário, durante o período de 1999 a 2008. Foi definida a MDR para as bactérias gram negativas (BGN) quando apresentava resistência a duas ou mais classes/grupos de antibióticos. As BGN foram predominantes (66,1%). As BGN MDR tiveram um aumento global de 3,7 vezes no final do período. O Acinetobacter baumannii foi o mais prevalente (36,2%). Os materiais mais freqüentes foram urinário e respiratório. Ocorreu um aumento significativo durante o período de 4,8 e 14,6 vezes do A. baumannii e K. pneumoniae, respectivamente. Sessenta e sete por cento das BGN MDR foram das UTIs. A resistência do A. baumannii aos carbapenêmicos aumentou de 7,4% para 57,5% durante o período, concomitante ao aumento do consumo. A resistência da K. pneumoniae às cefalosporinas teve um grande aumento durante a década. Houve diminuição da P. aeruginosa nos últimos dois anos com uma recuperação da sensibilidade.

A reemergência de algumas bactérias Gram-positivas, em particular, do gênero Staphylococcus, como principal foco causador de infecções hospitalares, tem se intensificado nas últimas décadas, e, apesar da existência de potentes fármacos voltados para o tratamento de infecções causadas por este gênero de bactéria, as taxas de morbidade e mortalidade prevalecem com perfil crescente. Além disso, um grande problema associado a cepas de MRSA (Methicillin-Resistant Staphylococcus aureus) é o fenótipo de multi-resistência, característica que confere a este microrganismo resistência não apenas à meticilina como também a uma série de outros fármacos, exceto frente à vancomicina e à teicoplanina. Muito tem se feito, mas ainda são poucos os resultados efetivamente aplicáveis no tratamento de infecções com caráter de multi-resistência, justificando, desta forma, a necessidade de desenvolvimento de sucedâneos que sejam consideravelmente mais efetivos para a solução deste problema. Baseado nestes fatos, a proposta deste estudo envolveu o planejamento, síntese, identificação e estudos de QSAR (Quantitative Structure-Activity Relationships) em duas e três dimensões de derivados 5-nitro-2-tiofilidênicos com atividade antimicrobiana frente a cepas padrão e multi-resistente de Staphylococcus aureus. A escolha dos grupos substituintes foi realizada em duas etapas. Na primeira delas seguiu-se metodologia de substituição em anéis aromáticos proposta por Topliss para a otimização da bioatividade de compostos. Em uma segunda etapa, predominantemente quantitativa, foram selecionados mais alguns derivados baseando-se em faixa de hidrofobicidade ótima pré-determinada experimentalmente e na variação de efeito estérico dos grupos substituintes. Quatorze derivados 5-nitro-2-tiofilidênicos foram sintetizados, estruturalmente identificados e avaliados quanto à atividade antimicrobiana frente às cepas padrão (ATCC 25923) e multi-resistente (3SP/R33) de Staphylococcus aureus por determinação da concentração inibitória mínima empregando-se método de macrodiluição sucessiva em tubos. Salienta-se que a cepa 3SP/R33 se mostra resistente a dezenove antibióticos empregados na prática médica e apresenta suscetibilidade apenas à vancomicina. As concentrações inibitória e bactericida mínimas apresentadas pelos compostos sintetizados mostraram sofrer influência significativa da hidrofobicidade sobre as referidas atividades de acordo com os estudos de QSAR-2D e QSAR-3D, sendo os resultados obtidos para a cepa multi-resistente absolutamente compatíveis com os anteriormente determinados para a cepa padrão. Os estudos de QSAR-2D indicaram que a atividade antimicrobiana das 5nitro-2-tiofilideno benzidrazidas substituídas sofre influência significativa de duas propriedades físico-químicas que são a hidrofobicidade e a distribuição eletrônica. A relevância dos descritores estruturais σ e efe na determinação da atividade antimicrobiana, sinalizam que a distribuição eletrônica influencia fortemente o aumento da potência antimicrobiana dos compostos em estudo tanto pela influência dos efeitos indutivo e de ressonância na estrutura química do ligante, como também pelos campos moleculares gerados ao redor de grupos substituintes, sugerindo uma possível interação dos mesmos com uma área específica do sítio receptor. Nos estudos de QSAR-3D, foi evidenciado, em concordância com o estudo clássico anteriormente realizado, que a hidrofobicidade prevalece como propriedade de fundamental importância no estabelecimento da atividade antimicrobiana. Foi observada a importância da presença de regiões hidrofílicas pontuais nos compostos de forma a propiciar processos de solvatação e dessolvatação que são críticos na difusão através de membranas biológicas. Pode-se afirmar que a análise de QSAR, considerando os aspectos tridimensionais ligantes, ressaltou a necessidade de um balanço lipofílico-hidrofílico para um bom desempenho das 5-nitro-2-tiofilideno benzidrazidas ρ-substituídas como agentes antimicrobianos. A partir dos resultados obtidos evidenciou-se, neste estudo, o forte potencial de derivados 5-nitro-2-tiofilidênicos como possível alternativa para o desenvolvimento racional, em nível molecular, de fármacos voltados para o tratamento de infecções causadas por cepas multi-resistentes de Staphylococcus aureus. / In the last decade, there has been a reemergence of Gram-positive bacteria, in particular Staphylococcus, which isconsidered one of the. most causing of nosocomial infections. Although potent antistaphylococcal drugs are available, this infection continues presenting increasing morbidity and mortality rates. Besides, a serious problem associated with MRSA (Methicillin-Resistant Staphylococcus aureus) is the phenotype of multidrug resistance, which is, resistance not only to methicillin but also to many other drugs, except to vancomycin and teicoplanin. Many efforts have been made in a tentative to reduce this problem, nevertheless there is only a few number of alternatives to combat Staphylococcus aureus multidrug-resistant strains, justifying the necessity of development of more effective compounds to the treatment of these infections. Based in these facts, the purpose of this study was the design, synthesis, structural identification and 2D-QSAR and 3D-QSAR (Quantitative Structure-Activity Relationships) studies of 5-nitro-2-thiophylidene derivatives with antimicrobial activity against multidrug-resistant strains of Staphylococcus aureus. The choice of substituent groups was made in two stages. The first stage comprises on application of Topliss operational scheme for aromatic substitution. In a second quantitative stage, more derivatives were selected according by hydrophobicity range previously determined. Other standard considered at the selection of substituent groups was the variation of steric effect. Fourteen 5-nitro-2-thiophylidene derivatives were synthesized, structural identified and tested against standard (A TCC 25923) and multidrug-resistant (3SP/R33) strains of Staphylococcus aureus. The Minimal Inhibitory Concentration, MIC, was determined using the serial dilution tests in two sequential stages. The 3SP/R33 strain is resistant to nineteen antimicrobial agents in use, except to vancomycin. The minimal inhibitory and bactericidal concentrations of synthesized compounds showed, according by 2D-QSAR and 3D-QSAR studies, a significant influence of hydrophobic properties on antimicrobial activity determination and the results obtained for multidrug-resistant strain were consistent with those determined for A TCC 25923 strain. 2D-QSAR studies showed that antimicrobial activity are mainly influenced by two physico-chemical properties: hydrophobicity and electronic distribution. The relevance of σ e ephe parameters on antimicrobial activity determination, denotes the contribution of inductive and resonance effects for the polar performed by the substituent groups, probably suggesting an interaction between them and specific receptor site. 3D-QSAR studies showed that hydrophobicity is a essential property to antimicrobial activity determination, sustained the same conclusions previously obtained by Hansch Analysis. It was observed a great concern of small hydrophilic regions distributed on derivatives in order to promote solvation and desolvation process, that have critical importance on diffusion process through the biological membranes. QSAR studies considering three-dimensional properties of ligands indicated the necessity of accurate hydrophilic-hydrophobic balance on nitrothiophene derivatives for their good performance as antimicrobial agents. The results obtained in this preliminary study have shown the potential of synthesized compounds as alternatives to the treatment of infections caused by multidrug-resistant strains of Staphylococcus aureus.

INTRODUÇÃO: A incidência de tuberculose (TB) em Santos (SP) situa-se em 73/100.000 habitantes-ano. A prevalência média de coinfecção TB/HIV é de 16%, taxas de cura e abandono de tratamento, entre casos novos são, respectivamente, 71% e 12%. Tais indicadores sugerem elevado risco para TB multidroga resistente (TBMR) no município, com incidência estimada em 1,9/100.000 habitantes-ano. OBJETIVO: Descrever e analisar o perfil de sensibilidade às drogas (TS) de primeira e segunda linha de tratamento entre pacientes com TB pulmonar (TBP), estimar a incidência de TBMR e a proporção de TB extensivamente resistente (TBXR); analisar aspectos moleculares, epidemiológicos e institucionais dos casos de TBP resistentes em Santos (SP). MÉTODOS: Estudo descritivo de uma coorte de pacientes de TBP, com início de tratamento ou retratamento entre 01 de janeiro de 2011 a 31 de dezembro de 2012. Definiu-se como caso de TBP, indivíduos com 15 anos ou mais, de ambos os sexos, residentes no município de Santos, com manifestações clínicas compatíveis com TBP e confirmação por cultura com isolamento de Mycobacterium tuberculosis. As variáveis de interesse para o estudo foram as características sociodemográficas, história atual e pregressa de TB, aspectos relativos ao tratamento, co-morbidades, ao diagnóstico e resistência a drogas. Para as análises comparativas entre proporções foram usados os testes qui-quadrado de Pearson e o Exato de Fisher e para variáveis contínuas o teste T de Student ou o de Kruskal - Wallis. Os perfis genéticos dos isoladas resistentes a ao menos uma droga foram obtidos pela técnica RLFP e analisados pelo programa Bionumerics versão 5.0 (Applied Maths - Bélgica). A descrição da distribuição espacial da TB resistentes e clusters foram feitas mediante a inserção dos casos no mapa de Santos, por endereço de residência, segundo o índice de vulnerabilidade social. RESULTADOS: Dos 263 casos de TBP selecionados, 68,4% (180/263) eram do sexo masculino, a mediana da idade foi de 38 anos, 8,7% (23/263) eram diabéticos; 20,4% (42/206) dos casos novos apresentavam ao menos um fator de risco para TBMR; destacando-se entre estes casos 10,7% (22/206) de confecção HIV/TB; 47,3% (123/260) tiveram tratamento supervisionado, 14,7% (91/617) dos contatos foram examinados, 18,6% (49/263) foram hospitalizados durante o tratamento, perfazendo uma média de 145,4 dias por paciente. Entre os casos resistentes a ao menos uma droga, a resistência à isoniazida foi 8,4% (22/263) e à rifampicina 3,8% (10/263) dos casos. A TBMR primária foi encontrada em 1,9% (4/206) dos casos e destes 25,0% (1/4) eram TBXR. A incidência média anual de TBMR foi de 0,57/100,000 habitantes. Dos 25 isolados resistentes ao menos uma droga, submetidos à RFLP, 12 (48,0%) foram agrupados em seis grupos genéticos, com dois pacientes em cada grupo. CONCLUSÕES: A elevada proporção TBMR primária, com um caso de TBXR enfatizam a necessidade de universalizar a cultura e TS, ampliar a cobertura do tratamento supervisionado, a investigação rotineira dos contatos e o monitoramento da resistência a drogas. O fortalecimento da vigilância da resistência às drogas é indispensável para o contínuo aperfeiçoamento do Programa de Controle da TB, especialmente em regiões de elevada carga da doença / INTRODUCTION: The incidence of tuberculosis (TB) in Santos (SP) is located around 73 / 100,000-year, approximately double that found on average in the country. The average prevalence of TB / HIV is 16% cure rates and treatment dropout among new cases are, respectively, 71% and 12%. Such indicators suggest high risk for multidrug-resistant TB (MR-TB) in the city, with the incidence estimated at 1.9 / 100,000-year. OBJECTIVE: To describe and analyze the sensitivity to drugs of first and second line treatment of patients with pulmonary TB (PTB) to estimate the incidence of MR-TB and extensively drugresistant TB (TBXR), describe molecular and institutional aspects, spatial distribution, epidemiological PTB resistant cases in the city of Santos (SP). METHODS: A descriptive study of a cohort of patients with PTB residing in the city who started treatment or retreatment in the period January 2011 to December 31, 2012. The case definition PTB individuals 15 years or more, both sexes, living in the city of Santos (SP), who present clinical manifestations compatible with PTB and whose confirmation was made by culture with isolation of M. tuberculosis. The variables of interest for the study were: bacteriological / laboratory socio-demographic characteristics, current and previous history of TB, aspects related to treatment, and comorbidities. For comparative analyzes of proportions the chi-squared tests and Fisher\'s exact were used for continuous variables and the Student t test or the Kruskal - Wallis. The genetic profiles of isolates resistant to at least one drug were obtained by RFLP (length polymorphism restriction fragment) and analyzed using version BioNumerics 5.0 (Applied Maths - Belgium) software. The description of the spatial distribution of resistant TB and the clusters was made by inserting the cases in Santos map, by address of residence, which was according to the index of social vulnerability. RESULTS: Of the 263 cases of PTB selected, 68.4% (180/263) were male, th median age was 38 years, 8.7% (23/263) were diabetes; 20.4% (42/206) of new cases had at least one risk factor for MR-TB, especially 10.7% (22/206) of making HIV / TB; 47.3% (123/260) underwent supervised treatment, 14.7% (91/617) of the contacts were examined, 18.6% (49/263) were hospitalized during treatment, totaling 7127 days of hospitalization with a mean 145.4 days per patient. Among the cases resistant to at least one drug resistance to isoniazid 8.4% (22/263) and rifampin 3.8% (10/263) of the cases was found. The primary MR-TB was found in 1.9% (4/206) of MR-TB cases and of these 25.0% (1/4) were TBXR. The average annual incidence of MDR-TB was 0.57/100,000 inhabitants. Of the 25 isolates resistant least one drug, subjected to molecular characterization of IS6110, 12 (48.0%) were grouped in six clusters, with each group including two isolates. CONCLUSIONS: A high proportion of primary MR-TB, including a case of TBXR emphasizes the need to universalize culture and TS, expand the coverage of supervised treatment, routine investigation of contacts and monitoring of drug resistance. The strengthening of the surveillance of drug resistance is essential for continuous improvement of the TB Control Program, especially in regions of high disease burden

Fosfoetanolamina sintética (Pho-s) é um monoéster análogo à fosfoetanolamina da membrana celular fosforilada artificialmente, com propriedades antiinflamatórias e apoptóticas para vários tipos de células tumorais humanas e murinas. Neste projeto foram avaliados os efeitos antitumorais in vitro da Pho-s e da formulação lipossomal DODAC/Pho-s na linhagem tumoral de leucemia mielóide crônica humana (K-562), em comparação ao modelo resistente a múltiplas drogas K-562 Lucena (MDR+). Os efeitos de citotoxicidade da Pho-s na linhagem tumoral K-562 e K-562 Lucena (MDR+) foram avaliados pela viabilidade celular utilizando o teste da Sulforodamina B, e os valores da IC50% obtidos foram de 43.1 mM e 145.9 mM, respectivamente após 24 horas de tratamento. O tratamento com o carreador DODAC vazio nas células K-562 e K-562 Lucena (MDR+) a IC50% foi de 0,0003mM e 0,008mM respectivamente, e com o tratamento com a formulação lipossomal DODAC/Pho-s a IC50% obtida respectivamente de, 0,56 mM e 0,31 mM. A viabilidade celular foi determinada a exclusão pelo azul de tripan 0,2%, em sistema automatizado Vi-Cell e foram significativas as diminuições da viabilidade celular em comparação ao grupo controle não tratado nas diversas concentrações e em diferentes períodos de tempo de tratamento. As alterações nas distribuições nas populações celulares nas fases do ciclo celular determinadas por citometria de fluxo mostraram aumento do DNA fragmentado (Sub/G1) em 12 e 24 horas de tratamento. Atividade apoptótica das células tumorais pela expressão da Anexina V/PI, no estágio de apoptose inicial, apoptose tardia e necrose foram quantificadas em citometria de fluxo, o tratamento com Pho-s, quando comparado ao grupo controle K-562 (40 e 80 mM) e K-562 Lucena (MDR+) (146 e 292 mM) ocorreu aumento significativo do número de células apoptóticas e em menor percentual o número de células necróticas. O tratamento com a Pho-s em célula leucêmica K-562 e K-562 Lucena (MDR+) tratadas, respectivamente com 40 e 80 mM e 146 e 292 mM mostraram que independente da expressão do fenótipo de resistência há uma redução significativa no potencial elétrico mitocondrial, analisado pelo o ensaio da rodamina-123. Os marcadores de controle e progressão do ciclo celular e da apoptose, mostraram efeitos moduladores da Pho-s dependentes da p53 na expressão da moléculas pró-apoptóticas. Esse conjunto de informações demonstrou os efeitos apoptóticos da Pho-s e da formulação lipossomal nas células tumorais independentemente do perfil molecular de resistência (MDR+), o que possibilita dizer que esse composto possui significativo potencial terapêutico nesse grupo de leucemias / Synthetic phosphoethanolamine (Pho-s) is a monoester analogous to the phosphoethanolamine which composes the membrane of an artificially phosphorylated cell, with anti-inflammatory and apoptotic properties for various types of human and murine tumor cells. In this project, we evaluated in vitro antitumor effects of Pho-s and the DODAC/Pho-s liposomal formulation in the human chronic myeloid leukemia (K-562) tumor line in comparison with the K-562 Lucena (MDR+). The effects of cytotoxicity of Pho-s on K-562 and K-562 Lucena (MDR +) tumor cells lines were evaluated by cell viability using the Sulforhodamine B test, and the IC50% values obtained were 43.1 mM and 145.9 mM after 24 hours of treatment, respectively. Treatment with the empty DODAC carrier on the K-562 and K-562 Lucena (MDR+) at IC50% cells was 0.0003 mM and 0.008 mM, and the treatment with the DODAC/Pho-s liposomal formulation obtained results of 0.56 mM and 0.31 mM, respectively. Cell viability was determined by 0.2% trypan blue exclusion in automated Vi-Cell system and the decreases in cell viability were significant in comparison to the untreated control group at various concentrations and different treatment time periods. Alterations in the distributions of cell populations in the cell cycle phases determined by flow cytometry showed increment of fragmented DNA (Sub/G1) in 12 and 24 hours of treatment. Apoptotic activity of tumor cells by the expression of Annexin V/PI, in the early apoptosis, late apoptosis phase and necrosis stage were quantified in flow cytometry, the treatment with Pho-s, when compared to the control group K-562 (40 and 80 mM) and K-562 Lucena (MDR +) (146 and 292 mM) demonstrated that there was a significant increase in the number of apoptotic cells and, in a lower percentage, the number of necrotic cells. Treatments with Pho-s in leukemic cell K-56 with 40 and 80 mM and in K-562 Lucena (MDR+) with 146 and 292 mM showed that independent of the expression of the resistance phenotype there is a significant reduction in the electrical potential of the mitochondrial membrane, analyzed with the rhodamine-123 assay. Control and progression markers of cell cycle and apoptosis showed p53-dependent modulating effects on the expression of pro-apoptotic molecules. This set of information demonstrated the apoptotic effects of Pho-s and liposomal formulation on tumor cells independently of the molecular resistance profile (MDR+), which makes it possible to say that this compound has significant therapeutic potential in this group of leukemias

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this work, a summary is provided of our current understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria (Chapter 1). The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities. Although this thesis primarily explores structure and function, it also elucidates the hidden regulatory mechanism (post-translational) behind the association of a small protein called AcrZ with the tripartite complex AcrAB/TolC, in connection with the lipid environment, and the resulting changes in the latter's functionality (Chapter 2). A regulatory role of the native membrane lipid environment as well as of small proteins for efflux pump activity have previously been hypothesised. I present the first example of a function-regulating role of the lipid cardiolipin in combination with a small protein binding partner (AcrZ) for the substrate selectivity and transport activity of an efflux pump protein (AcrB). This regulation happens through induced structural changes which have remained unseen so far. Alongside with these results, a nanodisc reconstitution method was experimentally adapted for a structure-function investigation of an efflux pump (complex) using cryo-EM (Chapter 2). Beyond some fundamental regulatory insights, hidden intrinsic transport mechanisms for some transporters have also remained to be explored and studied. The discovery of a mechanism for active influx by a prominent efflux pump model system (Chapter 3) provides hope that this phenomenon is more common amongst multidrug transporters and that it could be utilised for drug discovery purposes. This novel feature explains the contradictory findings on this transporter in the past and raises new questions about the little-known physiological role and evolution of efflux pumps. The development and evolution of antimicrobial resistance has frequently shown to be a multifactorial and fast-moving process. One of these factors is the evolution of pumps itself towards an altered functionality (e.g. towards a broader or altered substrate spectrum or higher efflux rates). Against this background, the role of key carboxylate residues for efflux-energising proton trafficking was investigated for a prominent study model of a secondary-active transporter (Chapter 4). The re-allocation and/or addition of acidic residues was demonstrated to result in the preservation of wild type activity or the generation of hyper-efflux activity, respectively. These findings suggest that rapid emergence of antimicrobial resistance could be enhanced by the 'plasticity' in the location of key carboxylate residues with a role in proton coupling. It also demonstrates the necessity of antimicrobial drug design programmes to anticipate possible trajectories of an adaptive evolution of efflux pump. The 'cryo-EM revolution' has boosted the pace at which new structural and functional insights into multidrug efflux pumps are gained. Nevertheless, in order to derive the structure of individual pump components or of a full assembly, it is sometimes necessary to identify and characterise homologues and mutants, which would allow the application of cryo-EM for obtaining near-atomic maps. Functional analyses presented in this work helped to characterise a homologue and mutants of the MacAB/TolC tripartite complex to justify the obtained protein structures and strategies for further functional characterisation (Chapter 5). Given (1) the unusual stoichiometry of a MacB dimer in complex with a hexameric membrane-fusion protein (MacA), which leads to a seeming leakiness of the assembly, and (2) the fact that substrate has to pass through a narrow aperture in the membrane-fusion protein for extrusion, it is rather surprising that MacB was previously shown to transport an entire toxin. An experimental approach was developed that could enable the structure determination of a toxin-bound full assembly of MacAB/TolC (Chapter 5). Finally, the role of multidrug efflux pumps for the evolution of multidrug resistance is yet to be studied and better explored. For instance, evolutionary trajectories of pump overexpression, as compared to those of regular expression or no expression, are unknown yet could have the potential to reveal useful insights for spread prevention and drug design. The outline of an experimental design with some preliminary validating data is presented in Chapter 6.

The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.

The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.