Student number:0312412A Faculty of Health Sciences School of Public Health / Management of multidrug-resistant tuberculosis (MDR-TB) is more expensive, lengthy and is associated with less favourable outcomes and more adverse reactions than management of susceptible tuberculosis. The aim of this study was to review the management and treatment outcomes of registered MDR-TB patients hospitalized at Sizwe hospital during a five-year period. A cross-sectional study with both descriptive and analytic features was done on 237 MDR-TB patients hospitalized from the beginning of June 1998 to the end of May 2003. Data were analysed using SPSS version 12 Software. Main outcome measures were interim treatment outcomes at the end of hospitalization period. These outcomes comprised culture conversion rates, time to culture conversion, transfer out, interruption, and death rates. Multiple logistic regression analysis was performed to determine risk factors for poor treatment outcomes. These poor outcomes were defined as treatment interruption, failure and mortality rates. The burden of institutional care for MDR-TB patients in this setting was found to involve high numbers of MDR-TB patients for whom the allocated hospital beds were insufficient. Patients with primary MDR-TB, who had no history of nonadherence to treatment, were paradoxically more likely to be hospitalized shortly after diagnosis. Acquired MDR-TB patients were mostly managed as outpatients immediately after diagnosis only to be hospitalized later due to persistent nonadherence or disease severity. Overall, acquired MDR-TB patients were hospitalized in larger numbers than those with primary disease. This reflects the higher prevalence of acquired MDR-TB compared to primary MDR-TB. Page v Abstract Culture turnaround time was on average 19 days. The overall culture conversion rate of the hospitalized patients was low at 41.9 percent. This low culture conversion rate resulted in protracted hospitalization periods and high interim mortality rates. The mean duration of hospitalization, 3.52 months, correlated favourably with the time interval to the first culture conversion of 2.96 months. Hospitalization did not guarantee the expected adherence to treatment. Surgical interventions were done belatedly with resultant high mortality outcomes. The main reasons given by patients for refusing hospital treatment were visiting traditional healers, solving socioeconomic problems and attending to family matters. A large percentage of hospitalized patients were co-infected with HIV. HIV care and support was incomplete as antiretroviral drugs were not available at the hospital. Among the main findings of the study was the powerful influence HIV status had on poor hospitalization outcomes. Recommendations arising from the study include the need to provide ARVs at the Sizwe hospital. Admission and discharge guidelines aimed at ensuring adequate beds are reserved for deserving patients should be formulated. Continuing education for service providers must be encouraged and rewarded. Infection control procedures at both community and health institution level ought to be vigorously promoted. Patients known to be hopelessly non-adherent should at least be partially hospitalized in the interest of public health.

Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (φSPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (φSPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.

Orientador: Antonio Carlos Paes / Banca: Hélio Langoni / Banca: Rogério Giuffrida / Resumo: A passagem de bactérias resistentes dos animais ao homem é possível. As amostras foram coletadas de cães, machos e fêmeas, de diferentes raças e idade, com infecções bacterianas variadas. Foram realizados cultura e antibiograma das bactérias isoladas (n=100), sendo avaliadas como sensíveis ou resistentes. Grupo das bactérias Gram-negativas: tetraciclina 83,02%, azitromicina 81,48%, doxiciclina 77,78%, ampicilina 62,96%, ceftiofur e florfenicol 50%, cefalexina 46,3%, enrofloxacino 44,44%, norfloxacino 18,52%, gentamicina 20,37%, levofloxacino 27,78%, amoxicilina + ácido clavulânico 31,48%, ciprofloxacino 31,48%, amicacina e ceftriaxona 33,33%, cloranfenicol e sulfa + trimetoprin 35,19%. Grupo dos Streptococcus: tetraciclina 80%, eritromicina 72%, enrofloxacino e levofloxacino 52%, ampicilina, azitromicina, ciprofloxacino, norfloxacino, penicilina G e sulfa + trimetoprin 48%, amoxicilina + ácido clavulânico 4%, cefalexina 12%, florfenicol 24%, ceftiofur, ceftriaxona e oxacilina 28%, cloranfenicol 32%. Grupo dos Staphylococcus spp: ampicilina 57,14%, sulfa + trimetoprin e tetraciclina 52,38%, amicacina, amoxicilina + ácido clavulânico, gentamicina, levofloxacino, 4,76%; cefalexina, ceftiofur, ceftriaxona e cloranfenicol, 9,52%; vancomicina 13,33%, norfloxacino 19,05%; ciprofloxacino, enrofloxacino e oxacilina 23,81% e azitromicina 33,33%. Os cães são reservatórios de bactérias multidrogas resistentes que podem transmitir por meio de plasmídios os genes de resistência, explicando a resistência de bactérias isoladas de cães à antimicrobianos de uso humano como a vancomicina. / Abstract: The transmission of resistant bacteria from animals to humans is possible. Samples were collected from different breeds of dogs in different ages including males and females, with a variety of bacterial infections. The culture and antibiograma of isolated bacteria were analysed (n = 100), being evaluated as sensitive or resistant. Group of Gram-negative bacteria tetracycline 83.02%, azithromycin 81.48%, doxycycline 77.78%, ampicillin 62.96%, ceftiofur and florfenicol 50%, cephalexin 46,3%, enrofloxacin 44.44%, norfloxacin 18.52%, gentamicin 20.37%, levofloxacin 27.78%, amoxicillin + clavulanic acid 31.48%, ciprofloxacin 31.48%, amikacin and ceftriaxone 33.33%, chloramphenicol and trimethoprim + sulfa (35.19%). Streptococcus' group: tetracycline 80%, erythromycin 72%, enrofloxacin and levofloxacin 52%, ampicillin, azithromycin, ciprofloxacin, norfloxacin, penicillin G and sulfamethoxazole + trimethoprim 48%, amoxicillin + clavulanic acid 4%, cephalexin 12%, florfenicol 24%, ceftiofur, ceftriaxone, and oxacillin 28%, chloramphenicol 32%. Group of Staphylococcus spp: ampicillin 57.14%, sulfamethoxazole + trimethoprim and tetracycline 52.38%, amikacin, amoxicillin + clavulanic acid, gentamicin, levofloxacin, 4.76%, cephalexin, ceftiofur, ceftriaxone, and chloramphenicol 9.52%, vancomycin 13.33%, norfloxacin 19.05% ciprofloxacin, enrofloxacin and oxacillin 23.81% and azithromycin 33.33%. Dogs have resistant-multidrug bacteria that might pass through the plasmid resistant genes, explaining the resistance of isolated bacteria from dogs to human use of antimicrobials such as vancomycin. / Mestre

Chai, Stella. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 108-126). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.v / PATENT AND PUBLICAION --- p.vii / CONFERENCE ABSTRACTS AND PRESENTATIONS --- p.viii / AWARDS --- p.ix / ABBREVIATIONS --- p.x / LIST OF TABLES --- p.xii / LIST OF FIGURES --- p.xiv / TABLE OF CONTENT --- p.xviii / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Multidrug resistance (MDR) --- p.1 / Chapter 1.1.1 --- Cancer --- p.1 / Chapter 1.1.2 --- Mechanisms of MDR in cancer --- p.1 / Chapter 1.1.2.1 --- Drug entry --- p.3 / Chapter 1.1.2.2 --- Drug metabolism --- p.3 / Chapter 1.1.2.3 --- Drug sequestration --- p.4 / Chapter 1.1.2.4 --- Mechanisms activated after nuclear entry --- p.5 / Chapter 1.1.2.5 --- Evasion of drug-induced apoptosis --- p.5 / Chapter 1.1.3 --- Approaches in treating MDR --- p.5 / Chapter 1.1.3.1 --- Overcoming MDR by inhibiting transporters --- p.6 / Chapter 1.1.3.2 --- Overcoming MDR by altering signaling pathway --- p.6 / Chapter 1.1.4 --- ATP Binding Cassette (ABC) Transporters --- p.7 / Chapter 1.1.4.1 --- P-glycoprotein (P-gp) --- p.7 / Chapter 1.1.4.2 --- Multidrug resistance-associated protein 1 (MRP 1) --- p.9 / Chapter 1.1.4.3 --- Breast cancer resistant protein (BCRP) --- p.10 / Chapter 1.1.4.4 --- ABC drug transporters and drug absorption --- p.11 / Chapter 1.2 --- The Use of Traditional Chinese Medicine (TCM) in circumventing P-gp-mediated MDR --- p.12 / Chapter 1.2.1 --- Active ingredients in TCM - Alkaloid --- p.12 / Chapter 1.2.2 --- Active ingredients in TCM - Saponin --- p.14 / Chapter 1.2.3 --- Active ingredients in TCM - Flavonoid --- p.15 / Chapter 1.2.4 --- Active ingredients in TCM - Others --- p.17 / Chapter 1.3 --- Polyoxypregnane compounds (POPs) --- p.17 / Chapter 1.3.1 --- Characterization --- p.17 / Chapter 1.3.2 --- POPs isolated from M. tenacissima --- p.18 / Chapter 1.4 --- Objectives of Current Study --- p.22 / Chapter CHAPTER 2. --- EFFECTS OF POLYOXYPREGNANE COMPOUNDS ON VIABILITY AND PROLIFERATION OF HUMAN RESISTANT CANCER CELLS --- p.24 / Chapter 2.1 --- Materials and Methods --- p.25 / Chapter 2.1.1 --- "Chemicals, Materials and Reagents" --- p.25 / Chapter 2.1.2 --- Methods --- p.26 / Chapter 2.1.2.1 --- Cell Lines and Cell Culture --- p.26 / Chapter 2.1.2.2 --- Preparation of POPs --- p.27 / Chapter 2.1.2.3 --- Sulforhodamine B assay --- p.27 / Chapter 2.1.2.4 --- Statistical analysis --- p.29 / Chapter 2.2 --- Results --- p.29 / Chapter 2.2.1 --- Effects of POPs on the viability of parental SW620 and P-gp-overexpressing resistant SW620/Ad300 cells --- p.29 / Chapter 2.2.2 --- Effects of POPs on the viability of parental MCF-7 and MRP1-overexpressing resistant MCF-7/VP cells --- p.33 / Chapter 2.2.3 --- Effects of POPs on the viability of parental MCF-7 and ABCG2-overexpressing resistant MCF-7/FLV1000 cells --- p.37 / Chapter 2.3 --- Discussion --- p.41 / Chapter 2.3.1 --- Structure activity relationship (SAR) --- p.43 / Chapter 2.3.2 --- Nine compounds relating to P-gp-mediated MDR --- p.46 / Chapter CHAPTER 3. --- MECHANISM OF NINE SELECTED POPS IN MODULATING P-GP-MEDIATED MDR --- p.49 / Chapter 3.1 --- Materials and Methods --- p.49 / Chapter 3.1.1 --- "Chemicals, Materials and Reagents" --- p.49 / Chapter 3.1.2 --- Methods --- p.53 / Chapter 3.1.2.1 --- Cell Lines and Cell Culture --- p.53 / Chapter 3.1.2.2 --- Extraction of nine POPs from M. tenacissima --- p.54 / Chapter 3.1.2.3 --- Sulforhodamine B (SRB) assay --- p.55 / Chapter 3.1.2.4 --- Flow cytometry assay --- p.55 / Chapter 3.1.2.5 --- P-gp ATPase assay --- p.56 / Chapter 3.1.2.6 --- Immuno-blot/Western blot analysis --- p.58 / Chapter 3.1.2.7 --- Reverse transcription and quantitative real-time PCR --- p.59 / Chapter 3.1.2.8 --- Statistical analysis --- p.60 / Chapter 3.2 --- Results --- p.60 / Chapter 3.2.1 --- Effects of nine selected POPs on the viability of sensitive human breast cancer MCF-7 cells --- p.60 / Chapter 3.2.2 --- Effects of nine selected POPs on the viability of MDR 1 -transfected HEK1 MDR1 cell line and its control vector transfected cell line HEK293 pcDNA3 --- p.61 / Chapter 3.2.3 --- Effects of nine selected POPs in inhibiting efflux of P-gp substrate --- p.64 / Chapter 3.2.4 --- Effects of nine selected POPs in modulating P-gp ATPase activity --- p.68 / Chapter 3.2.5 --- Effects of nine selected POPs in regulating P-gp protein expression --- p.69 / Chapter 3.2.6 --- MDR1 mRNA expression in various cell lines --- p.72 / Chapter 3.3 --- Discussion --- p.72 / Chapter 3.3.1 --- Effective POPs are targeting specifically P-gp overexpression --- p.73 / Chapter 3.3.2 --- Mechanistic understanding the circumvention of MDR by the effective POPs --- p.74 / Chapter 3.3.2.1 --- Relative potency for the reversal of P-gp-mediated MDR --- p.75 / Chapter 3.3.2.2 --- Inhibition of P-gp-mediated drug efflux across cell membrane by the effective POPs --- p.75 / Chapter 3.3.2.3 --- Stimulation of ATPase by the effective POPs --- p.76 / Chapter 3.3.2.4 --- No effect of POPs on the alteration of P-gp expression --- p.77 / Chapter 3.3.2.5 --- An overall summary of the mechanism of MDR reversal by the effective POPs --- p.78 / Chapter 3.3.3 --- Implication in drug disposition and drug-drug interactions --- p.79 / Chapter 3.3.4 --- Additional information for the structure activity relationship (SAR) --- p.80 / Chapter CHAPTER 4. --- EFFECTS OF CRUDE EXTRACT AND THREE MAJOR POLYOXYPREGNANES (POPS) OF MARS DEN I A TENACISSIMA --- p.81 / Chapter 4.1 --- Materials and Methods --- p.82 / Chapter 4.1.1 --- "Chemicals, Materials and Reagents" --- p.82 / Chapter 4.1.2 --- Methods --- p.82 / Chapter 4.1.2.1 --- "Preparation of M. tenacissima extract, artificial mixture and three fractions" --- p.82 / Chapter 4.1.2.2 --- Sulforhodamine B assay --- p.85 / Chapter 4.1.2.3 --- "Biotransformation study of POP68, POP69 and POP70" --- p.85 / Chapter 4.1.2.4 --- HPLC-MS analysis --- p.86 / Chapter 4.1.2.5 --- Animal care and housing conditions --- p.87 / Chapter 4.1.2.6 --- Toxicity studies of fraction 2 in mice --- p.88 / Chapter 4.1.2.7 --- Statistical analysis --- p.89 / Chapter 4.2 --- Results --- p.89 / Chapter 4.2.1 --- "Effects of crude extract, artificial mixture on the viability of sensitive human breast cancer MCF-7 cells" --- p.89 / Chapter 4.2.2 --- "Effects of crude extract, artificial mixture on the viability of sensitive SW620 and P-gp-overexpressing resistant SW620/Ad300 cells" --- p.90 / Chapter 4.2.3 --- "Metabolites of POP68, POP69 and POP70 after incubation with human intestinal microbiota" --- p.91 / Chapter 4.2.4 --- Toxicity of fraction 2 in mice --- p.94 / Chapter 4.3 --- Discussion --- p.98 / Chapter CHAPTER 5. --- FINAL DISCUSSION AND CONCLUSIONS --- p.105 / REFERENCES --- p.108

ABSTRACT Extended-spectrum beta-lactamase (ESBL)-producing Salmonella enterica serotype Isangi has emerged as a common Salmonella serotype affecting mainly children in hospitals throughout South Africa. Between 2000 and 2002, 279 S. Isangi isolates from single infection episodes were referred from 21 hospitals in 5 provinces to the Enteric Diseases Reference Unit of the National Institute for Communicable Diseases of South Africa. All isolates were subjected to antibiotic susceptibility testing and three disk-diffusion methods confirmed ESBL-production in 273 isolates. PCR and nucleotide sequencing of 101 isolates identified TEM-1 (2%), TEM-63 (91%), a novel TEM-131 (7%), and SHV-5 (2%), but CTX-M was not found. Plasmid profiling produced types with 1 to 6 plasmids, 7.4kb to 166kb in size, which were neither serotype nor ESBL-type specific. Pulsed-field gel electrophoresis revealed four major clusters while sub-clusters with identical, or near identical banding patterns suggested extensive intra-hospital transmission and clonal spread between hospitals and provinces in South Africa.

The number of fungal infections is rising in Germany and worldwide. These infections are mainly caused by the opportunistic fungal pathogen C. albicans, which especially harms immunocompromised people. With increasing numbers of fungal infections, more frequent and longer lasting treatments are necessary and lead to an increase of drug resistances, for example against the clinically applied therapeutic fluconazole. Drug resistance in C. albicans can be mediated by the Multidrug resistance pump 1 (Mdr1), a membrane transporter belonging to the major facilitator family. However, Mdr1-mediated fluconazole drug resistance is caused by the pump’s regulator, the transcription factor Mrr1 (Multidrug resistance regulator 1). It was shown that Mrr1 is hyperactive without stimulation or further activation in resistant strains which is due to so called gain of function mutations in the MRR1 gene. To understand the mechanism that lays behind this constitutive activity of Mrr1, the transcription factor should be structurally and functionally (in vitro) characterized which could provide a basis for successful drug development to target Mdr1-mediated drug resistance caused by Mrr1. Therefore, the entire 1108 amino acid protein was successfully expressed in Escherichia coli. However, further purification was compromised as the protein tended to form aggregates, unsuitable for crystallization trials or further characterization experiments. Expression trials in the eukaryote Pichia pastoris neither yielded full length nor truncated Mrr1 protein. In order to overcome the aggregation problem, a shortened variant, missing the N-terminal 249 amino acids named Mrr1 ‘250’, was successfully expressed in E. coli and could be purified without aggregation. Similar to the wild type Mrr1 ‘250’, selected gain of function variants were successfully cloned, expressed and purified with varying yields and with varying purity. The Mrr1 `250’ construct contains most of the described regulatory domains of Mrr1. It was used for crystallization and an initial comparative analysis between the wild type protein and the variants. The proposed dimeric form of the transcription factor, necessary for DNA binding, could be verified for both, the wild type and the mutant proteins. Secondary structure analysis by circular dichroism measurements revealed no significant differences in the overall fold of the wild type and variant proteins. In vitro, the gain of function variants seem to be less stable compared to the wild type protein, as they were more prone to degradation. Whether this observation holds true for the full length protein’s stability in vitro and in vivo remains to be determined. The crystallization experiments, performed with the Mrr1 ‘250’ constructs, led to few small needle shaped or cubic crystals, which did not diffract very well and were hardly reproducible. Therefore no structural information of the transcription factor could be gained so far. Infections with M. tuberculosis, the causative agent of tuberculosis, are the leading cause of mortality among bacterial diseases. Especially long treatment times, an increasing number of resistant strains and the prevalence of for decades persisting bacteria create the necessity for new drugs against this disease. The cholesterol import and metabolism pathways were discovered as promising new targets and interestingly they seem to play an important role for the chronic stage of the tuberculosis infection and for persisting bacteria. In this thesis, the 3-ketoacyl-CoA thiolase FadA5 from M. tuberculosis was characterized and the potential for specifically targeting this enzyme was investigated. FadA5 catalyzes the last step of the β-oxidation reaction in the side-chain degradation pathway of cholesterol. We solved the three dimensional structure of this enzyme by X-ray crystallography and obtained two different apo structures and three structures in complex with acetyl-CoA, CoA and a hydrolyzed steroid-CoA, which is the natural product of FadA5. Analysis of the FadA5 apo structures revealed a typical thiolase fold as it is common for biosynthetic and degradative enzymes of this class for one of the structures. The second apo structure showed deviations from the typical thiolase fold. All obtained structures show the enzyme as a dimer, which is consistent with the observed dimer formation in solution. Thus the dimer is likely to be the catalytically active form of the enzyme. Besides the characteristic structural fold, the catalytic triad, comprising two cysteines and one histidine, as well as the typical coenzyme A binding site of enzymes belonging to the thiolase class could be identified. The two obtained apo structures differed significantly from each other. One apo structure is in agreement with the characteristic thiolase fold and the well-known dimer interface could be identified in our structure. The same characteristics were observed in all complex structures. In contrast, the second apo structure followed the thiolase fold only partially. One subdomain, spanning 30 amino acids, was in a different orientation. This reorientation was caused by the formation of two disulfide bonds, including the active site cysteines, which rendered the enzyme inactive. The disulfide bonds together with the resulting domain swap still permitted dimer formation, yet with a significantly shifted dimer interface. The comparison of the apo structures together with the preliminary activity analysis performed by our collaborator suggest, that FadA5 can be inactivated by oxidation and reactivated by reduction. If this redox switch is of biological importance requires further evaluation, however, this would be the first reported example of a bacterial thiolase employing redox regulation. Our obtained complex structures represent different stages of the thiolase reaction cycle. In some complex structures, FadA5 was found to be acetylated at the catalytic cysteine and it was in complex with acetyl-CoA or CoA. These structures, together with the FadA5 structure in complex with a hydrolyzed steroid-CoA, revealed important insights into enzyme dynamics upon ligand binding and release. The steroid-bound structure is as yet a unique example of a thiolase enzyme interacting with a complex ligand. The characterized enzyme was used as platform for modeling studies and for comparison with human thiolases. These studies permitted initial conclusions regarding the specific targetability of FadA5 as a drug target against M. tuberculosis infection, taking the closely related human enzymes into account. Additional analyses led to the proposal of a specific lead compound based on the steroid and ligand interactions within the active site of FadA5. / Die Zahl der Pilzinfektionen, welche hauptsächlich durch den opportunistisch-pathogenen Pilz C. albicans verursacht werden, ist nicht nur in Deutschland, sondern weltweit steigend. Die auftretenden Infektionen betreffen vor allem immunsupprimierte Personen. Dieser Anstieg an Pilzinfektionen verursacht häufigere und immer länger andauernde Behandlungen und resultiert auch im vermehrten Auftreten von Resistenzen gegen Antimykotika, unter anderem gegen das klinisch eingesetzte Fluconazol. Eine Möglichkeit der Resistenzbildung in C. albicans ist die Expression der ‚Multidrug resistance pump 1‘ (Mdr1), einer Membranpumpe, die zur Major-Facilitator-Superfamilie zählt. Diese durch Mdr1-vermittelte Fluconazolresistenz wird durch den Mdr1 regulierenden Transkriptionsfaktor Mrr1 (‚Multidrug resistance regulator 1‘) gesteuert. In resistenten C. albicans Stämmen befindet sich Mrr1 ohne weitere Stimulation oder externe Aktivierung bereits in einem hyperaktiven Zustand, der durch Mutationen mit Funktionsgewinn im MRR1 Gen verursacht wird. Um die Mechanismen, die sich hinter der konstitutiven Aktivität von Mrr1 verbergen, zu entschlüsseln, sollte dieser Transkriptionsfaktor in vitro strukturell und funktionell charakterisiert werden. Diese Charakterisierung könnte im Anschluss genutzt werden, um Wirkstoffe gegen die von Mrr1 gesteuerte und von Mdr1-vermittelte Resistenz zu entwickeln. Zu diesem Zweck, wurde das gesamte, 1108 Aminosäuren umfassende, Protein in Escherichia coli exprimiert. Die anschließende Proteinreinigung war allerdings durch Aggregatbildung beeinträchtigt, welche Kristallisationsansätze oder eine weitere Charakterisierung dieses Proteinkonstruktes verhinderten. Im Eukaryot Pichia pastoris durchgeführte Expressionsanalysen, waren leider erfolglos und weder die Expression des Volllängen-Mrr1 noch seiner verkürzten Proteinvarianten konnte nachgewiesen werden. Um Proteinaggregation zu umgehen, wurde deshalb ein N-terminal, um 249 Aminosäuren, verkürztes Proteinkonstrukt, Mrr1 ‚250‘, in E. coli exprimiert und erfolgreich, ohne Aggregation, gereinigt. Zusätzlich zum wildtypischen Mrr1 ‚250‘ Protein wurden auch ausgewählte Varianten kloniert, exprimiert und gereinigt, allerdings mit unterschiedlicher Ausbeute und Reinheit. Da das verkürzte Mrr1 ‚250‘ Protein noch immer fast alle in der Literatur beschriebenen Regulierungsdomänen besitzt, wurde es zur Kristallisation und für einen initialen Vergleich zwischen Wildtyp und Varianten genutzt. So konnte zum Beispiel die vermutete Dimerisierung des Transkriptionsfaktors sowohl für das Wildtypprotein als auch für die Varianten gezeigt werden. Eine weiterführende Untersuchung der Sekundärstruktur mittels zirkular Dichroismus Messungen zeigte keine signifikanten Unterschiede zwischen den Mutanten und dem Wildtypprotein. Allerdings erscheinen die Funktionsgewinn Varianten von Mrr1 in vitro instabiler als das Wildtypprotein, was sich durch stärkeren Abbau der Variantenproteine zeigt. Ob diese Beobachtungen allerdings vom verkürzten Protein auf das Gesamtprotein und dessen in vitro und in vivo Stabilität übertragbar sind, ist derzeit noch unklar. Kristallisationsansätze, die mit den verschiedenen Varianten des Mrr1 ‚250‘ Konstrukts durchgeführt wurden, führten zu sehr wenigen, nadelförmigen oder kubischen Kristallen, die kaum reproduzierbar waren und schlecht diffraktierten. Bisher konnten deshalb keine strukturellen Daten für den untersuchten Transkriptionsfaktor erhalten werden. Noch immer sind Infektionen, die durch M. tuberculosis, dem Erreger der Tuberkulose, verursacht werden die Haupttodesursache im Bereich der bakteriellen Infektionen. In diesem Zusammenhang stellen vor allem lange Behandlungszeiten, das vermehrte Auftreten resistenter Stämme und das Vorkommen persistierender Bakterien, die Jahrzehnte in ihrem Wirt überdauern können, nach wie vor große Herausforderungen dar und die Entwicklung neuer Tuberkulosemedikamente ist dringend erforderlich. Sowohl der Cholesterinimport als auch dessen Stoffwechselweg wurden als vielversprechende Wirkstoffziele identifiziert. Nicht zuletzt, da beide Mechanismen eine wichtige Rolle während der chronischen Phase der Tuberkuloseinfektion und für persistierende Bakterien zu spielen scheinen. Im Laufe dieser Arbeit wurde die 3-ketoacyl-CoA Thiolase FadA5 aus M. tuberculosis strukturell charakterisiert und auf ihre Tauglichkeit als spezifisches Wirkstoffziel hin untersucht. FadA5 katalysiert den letzten Schritt der β-Oxidation im Zuge des Seitenkettenabbaus von Cholesterin. Wir konnten die Proteinstruktur des FadA5 Proteins mittels Röntgenkristallographie ermitteln und erhielten zwei unterschiedliche apo-Strukturen sowie drei Komplexstrukturen. In den Komplexstrukturen waren entweder Acetyl-CoA, CoA oder ein hydrolisiertes Steroid-CoA, welches das natürliche Produkt von FadA5 darstellt, an das Enzym gebunden. Die Strukturanalyse der apo-Strukturen lies für eine der beiden Modelle die typische Thiolasefaltung erkennen, welche für biosynthetische und degradative Enzyme dieser Klasse üblich ist. In der zweiten apo-Struktur konnte diese Faltung nur teilweise identifiziert werden. Das Protein liegt in allen erhaltenen Strukturen als Dimer vor, was auch in Lösung beobachtet werden konnte und darauf hinweist, dass das Dimer die katalytisch aktive Form des Proteins darstellt. Neben der charakteristischen Faltung, wurde das aktive Zentrum, bestehend aus zwei Cysteinen und einem Histidin, sowie die für Thiolasen übliche Coenzym A Bindetasche identifiziert. Die erhaltenen apo-Strukturen unterschieden sich deutlich voneinander. Die zuvor beschriebene typische Dimer-Interaktionsfläche wird auch in den Komplexstrukturen beobachtet. Dahingegen war die Thiolasefaltung in der zweiten Apo-Struktur nur teilweise vorhanden, da beispielsweise eine Domäne, die 30 Aminosäuren umfasst, umorientiert vorlag. Die Bildung zweier Disulfidbrücken, welche beide katalytischen Cysteine involviert, verursachte die beschriebene Umorientierung und damit gepaart eine wahrscheinliche Inaktivität des Enzyms. Trotz der beschriebenen Umorientierung und Disulfidbrückenbildung liegt das Protein noch immer als Dimer vor, allerdings mit einer deutlich verschobenen Interaktionsfläche. Der Vergleich der beiden apo-Strukturen in Kombination mit einer vorläufigen Aktivitätsanalyse, die von unseren Kollaborationspartnern durchgeführt wurde, lassen vermuten, dass FadA5 durch Oxidation inaktiviert und durch Reduktion reaktiviert werden kann. Ob diese Redoxregulierung biologisch relevant ist, muss noch geklärt werden, allerdings wäre dies der erste beschriebene Fall einer redoxregulierten bakteriellen Thiolase. Die Komplexstrukturen stellen verschiedene Stufen der Thiolasereaktion dar. In einigen dieser Strukturen lag FadA5 am katalytischen Cystein acetyliert vor und befand sich im Komplex mit acetyl-CoA oder CoA. Durch eine weitere Struktur, in der FadA5 im Komplex mit einem hydrolisierten Steroid-CoA vorlag, konnten wichtige Einblicke in die Enzymdynamik während der Ligandenbindung und Freisetzung gewonnen werden. Die Steroid gebundene Struktur stellt derzeit ein einzigartiges Beispiel einer Thiolase im Komplex mit einem großen, mehrere Ringsysteme umfassenden Liganden dar. Das charakterisierte Enzym diente als Ausgangspunkt für Modellierungsversuche und Vergleiche mit humanen Thiolasen. Diese Analysen erlaubten initiale Schlussfolgerungen bezüglich einer Verwendung von FadA5 als spezifisches Wirkstoffziel gegen Tuberkuloseinfektionen, im Kontext verwandter humaner Enzyme. Zusätzliche Untersuchungen ermöglichten die Ausarbeitung einer spezifischen Leitsubstanz, die auf den analysierten Interaktionen zwischen dem aktiven Zentrum von FadA5 und den gebundenen Liganden basiert.

Ziel der vorliegenden Arbeit waren pharmakologische Untersuchungen zum antiproliferativen Effekt der beiden Laktatdehydrogenase (LDH)-Inhibitoren Natriumoxamat und Galloflavin sowie des MRP1-Inhibitors Reversan einzeln und in Kombination bei verschiedenen Sauerstoffkonzentrationen in vitro zu untersuchen. Zusätzlich wurde der antiproliferative Effekt der drei Inhibitoren mit dem antiproliferativen Effekt von 5-FU verglichen. Das Konzept zu dieser Arbeit basiert auf Gemeinsamkeiten zwischen LDH und MRP1 in malignen Zellen. Eine ist, dass beide Moleküle von zahlreichen Tumoren überexprimiert werden. Weiter sind beide an der Ausbildung von Chemoresistenz beteiligt und beide werden auch in Hypoxie exprimiert. Zudem wird das für die Funktion von MRP1 notwendige ATP in malignen Zellen hauptsächlich mit der hyperaktiven Glykoloyse gebildet, deren Stoffumsatz auch von der LDH-Aktivität abhängig ist. Eine kombinierte Inhibition beider Zielstrukturen scheint somit geeignet zu sein, um die Proliferation maligner Zellen gezielt zu hemmen. Da in großen Teilen solider Tumoren hypoxische bzw. anoxische Bedingungen vorherrschen, wurde die Wirksamkeit der drei Inhibitoren auch bei 5 % und 1 % Sauerstoff, die als tumorphysiologisch gelten, untersucht. Die wichtigsten Ergebnisse aus dieser Arbeit sind, dass die beiden LDH-Inhibitoren Natriumoxamat und Galloflavin und der MRP1-Inhibitor Reversan einen antiproliferativen Effekt bei kolorektalen Karzinomzellen auslösen, der auch für tumorphysiologische Sauerstoffkonzentrationen nachzuweisen war. So verringerte sich durch Natriumoxamat bzw. Galloflavin der Anteil vitaler Zellen um bis zu 45 % und durch Reversan um bis zu 60 % bei 5 % und 1 % Sauerstoff im Vergleich zur unbehandelten Kontrolle. Auch unterschiedliche Kombination aus Natriumoxamat, Galloflavin und Reversan führten zu einer Steigerung des antiproliferativen Effektes, der auch immer bei tumorphysiologischen Konzentrationen nachzuweisen war. Den stärksten antiproli-ferativen Effekt wies die Dreifachkombination aus Galloflavin, Natriumoxamat und Reversan auf. So verringerte sich der Anteil vitaler Zellen bei 1 % Sauerstoff durch diese Kombination auf bis zu 28 % bei vier der fünf kolorektalen Karzinomzelllinien. Die Dreifachkombination wies einen gleichstarken bzw. stärkeren antiproliferativen Effekt auf als das Chemotherapeutikum 5-FU und zwar ebenfalls bei 5 % und 1 % Sauerstoff. Die Ergebnisse der vorliegenden Arbeit zum antiproliferativen Effekt von Natriumoxamat, Galloflavin (beides LDH-Inhibitoren) und Reversan (MRP1-Inhibitor) in vitro lassen den Schluss zu, dass das Konzept der Arbeit, einen antiproliferativen Effekt auch bei tumorphysiologischen Sauerstoffkonzentrationen zu induzieren, grundsätzlich bestätigt wurde. Auch löste die gemeinsame Hemmung von LDH und MRP1 einen teilweise stärkeren antiproliferativen Effekt aus als 5-FU. Weitere Untersuchungen sind aber ohne Frage nötig, um die molekularen Interaktion zwischen LDH und MRP1 sowie ihrer Inhibition im Detail zu verstehen. / The aim of the present study was to investigate the antiproliferative effect of the two lactate dehydrogenase (LDH) inhibitors sodium oxamate and galloflavin and the MRP1 inhibitor reversan at different oxygen concentrations in vitro. The inhibitors were used individually and in combination. In addition, the antiproliferative effect of the three inhibitors was compared with the antiproliferative effect of 5-FU. The concept of this study is based on similarities between LDH and MRP1 in malignant cells: their overexpression by numerous tumors; their contribution to chemoresistance and their expression in hypoxia. In addition, the ATP necessary for the function of MRP1 is mainly formed in malignant cells by an increased turnover of the hyperactive glycolysis, which also depends on the LDH activity. Thus, a combined inhibition of both targets appears to inhibit tumor cell proliferation effectively. Since hypoxic or anoxic conditions prevail in large parts of solid tumors, the efficacy of the three inhibitors was also investigated at 5% and 1% oxygen, which are considered to be physiological for solid tumors. The most important results of the study are that both sodium oxamate and galloflavin, as well as reversan trigger an antiproliferative effect in colorectal carcinoma cells, even in the presence of tumor physiological oxygen concentrations. For example, the pro-portion of viable cells decreased up to 45% with sodium oxamate or galloflavin and up to 60% with reversan, even at 5% and 1% oxygen compared to untreated control cells. Different combinations of sodium oxamate, galloflavin and reversan resulted in en-hanced antiproliferative effects, which were also demonstrated at tumor physiological oxygen concentrations. The strongest antiproliferative effects were observed with the triple combination of galloflavin, sodium oxamate and reversan. In this combination, the proportion of viable cells decreased to 28% at 1% oxygenation in four of the five colorectal carcinoma cell lines. The triple combination caused an antiproliferative effect that was equal to or even more potent than the antiproliferative effect of the chemotherapeutic agent 5-FU also at 5% and 1% oxygen. The results of this study on the antiproliferative effect of sodium oxamate, galloflavin (both LDH inhibitors) and reversan (MRP1 inhibitor) in vitro seems to confirm the aim of the study, which was to induce an antiproliferative effect even in tumor physiologi-cal oxygen concentrations. In part, the combined inhibition of LDH and MRP1 caused a stronger antiproliferative effect than 5-FU. However, further investigations are neces-sary to comprehend the molecular interaction between LDH and MRP1 as well as its inhibition in detail.

The overall aim of this research was to explore why some hospitals are more successful than others at reducing the acquisition rates of multidrug-resistant organisms. Using a socio-ecological perspective on health systems adapted from works in ecological restoration, ecosystems management, and healthcare, a participatory comparative case study design was employed. The study was collaboratively conducted on a surgical unit at a Netherlands hospital with very low rates of multidrug-resistant organisms and a surgical unit in a Canadian hospital with higher rates of these pathogens. The cases were selected on the basis that they were both academic health sciences centres of similar size in publicly funded systems; yet, they reported differing rates of MDRO infections. Research methods included a total of six unit observations, nine practitioner-led photo walkabouts of the units (n=13), six photo elicitation focus groups with practitioners (n=26), and the review of relevant policies and procedures and related infection prevention and control data. Common findings across both cases include the perceived importance of engaged leadership, the presence of environmental design issues, a lack of antibiotic prescribing restrictions, and the frequent use of workarounds that may be problematic for infection prevention and control. Disparate findings between cases include differences in ratios of hospital beds per capita, bed occupancy rates, staffing practices, equipment cleaning processes, bed cleaning systems (centralized versus manual) and the presence, in one hospital, of an active grass roots Hygiene in Practice group engaging practitioners in several ongoing activities to promote infection prevention and control. There is a lack of comparable findings between the two cases on hand hygiene audit protocols, surveillance strategies, reporting of acquisition rates, and the nature and extent of high risk populations for community-acquired methicillin-resistant Staphylococcus aureus in the two hospitals catchment areas. The findings and methodological challenges identified in this study suggest that case selection in future comparative infection prevention and control case studies should be based on an expanded list of criteria. These criteria should include comparable audits, surveillance, and reporting practices and comparable demographic and other relevant data, such as data on the agricultural practices within and demographic attributes of vulnerable populations within the hospital catchment areas.

The multidrug transporter LmrP, member of the major facilitator superfamily (MFS), confers L. lactis and recombinant E. coli cells resistance to an array of cytotoxic compounds including antibiotics. LmrP mediates drug extrusion from the plasma membrane by an electrogenic proton/drug exchange reaction, whereby a positively charged substrate may move towards the external medium in exchange for two or more protons moving towards the cytoplasm. Recent studies have suggested that MFS transporters require phosphatidylethanolamine (PE) for function and proper topology. However, the specificity of the PE requirement, as well as the contribution of the electrochemical gradient (the driving force of the substrate transport) to this lipid requirement was not addressed. Here we report a new approach for addressing PE specific requirement for the function and the structure of membranes transporters. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by phosphatidylcholine (PC) ( including energy transfer between the protein tryptophan residues and the lipid headgroups). We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins. HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA. Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.