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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

In-vitro- und in-vivo-Untersuchungen zur Bedeutung des intestinalen Arzneimittelmetabolismus und -transportes beim Menschen

Gläser, Hartmut, January 2003 (has links)
Stuttgart, Univ., Diss., 2003.
2

Mutational analysis of the rifampicin glycosyl-transferase (rgt) inactivation protein from Nocardia brasiliensis and its relationship to the vancomycin resistance of this organism

Baker, Alison Saxe 16 November 2006 (has links)
Student Number : 0418251N - MSc research report - School of Molecular and Cell Biology - Faculty of Science / Rifampicin is a chemotherapeutic agent used to combat mycobacterial and nocardial infections. Four enzymatic inactivation mechanisms have been identified which are partially responsible for the increasing number of rifampicin resistant strains. These are ADP-ribosylation, phosphorylation, decomposition and glucosylation. The gene encoding the latter, rgt, has been cloned and characterized from the opportunistic pathogen Nocardia brasiliensis. However, as of yet nothing is known of these inactivation enzymes. Thus in order to study the properties of the mechanism it is necessary to observe structure-function relationships through the characterization of mutants. Furthermore, the rgt gene confers a small yet reproducible increase to the vancomycin MIC. This has indicated that there may be other enzymatic mechanisms which are involved in the inactivation of vancomycin. Vancomycin is an important antibiotic as it is used to treat gram-positive infections by multi-drug resistant strains. Hitherto, no mechanisms of enzymatic inactivation have been identified for vancomycin. Thus in order to identify regions of DNA which may play a role in the high level resistance to vancomycin as observed in N. brasiliensis it was necessary to screen a genomic library of this organism. This was performed in a gram-positive background. No clones were identified in this study that had an increased resistance to vancomycin, indicating that the DNA involved in the phenotype is greater than that of the average insert size of the library, 1.9 kb. Future work will thus involve the generation of a genomic library with larger fragments and the subsequent screening of this. Additionally, performing a mutational analysis on the rgt gene may provide further insight into the specifics of the inactivation enzymes and thus will contribute to combating infection by opportunistic and other pathogens.
3

A metabolomics approach for characterising tuberculosis / Ilse Olivier

Olivier, Ilse January 2012 (has links)
In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012
4

A metabolomics approach for characterising tuberculosis / Ilse Olivier

Olivier, Ilse January 2012 (has links)
In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012
5

Deciphering the impact of rpoB mutations on the gene expression profile of Mycobacterium tuberculosis

Du Plessis, Juanelle 04 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Mycobacterium tuberculosis is the etiological agent for tuberculosis, an infectious disease which is one of the leading causes of morbidity and mortality in developing countries. The emergence of drug resistant tuberculosis has negatively impacted the efficacy of current treatment regimens and threatens to undermine tuberculosis control programs worldwide. Rifampicin forms the backbone of the World Health Organization’s recommended treatment regimen for the treatment of drug susceptible tuberculosis. Resistance to rifampicin is caused by mutations in the 81 bp core region of the rpoB gene which encodes the β subunit of RNA polymerase. Numerous studies have shown that mutations at codons 531 and 526 are the most frequent in clinical isolates, yet little is known concerning the mechanistic effect of these mutations on the fidelity of RNA polymerase. In the present study, we aimed to determine the influence of rpoB mutations on the gene expression profile of M. tuberculosis cultured in vitro. To accomplish this, rifampicin resistant clinical isolates and spontaneous mutants (selected in vitro from H37Rv and a drug-sensitive clinical strain) harbouring rpoB H526Y and S531L mutations were subjected to whole genome sequencing and genome-wide transcriptional profiling. When comparing the transcription profile of H37Rv to the in vitro rpoB mutants, a large proportion of the differentially expressed genes were found to encode for proteins involved in intermediary metabolism and respiration; and cell wall and cell processes. The majority of these differentially expressed genes were downregulated. Prominent differential expression in the same functional categories was also evident when comparing the clinical isolates with these mutations; however, a greater number of genes were differentially expressed in this case. Furthermore, expression of genes that are part of the WhiB7 regulon were found to be upregulated in the rpoB526 mutants, and downregulated in the rpoB531 mutants. These findings indicate that both the position of the rpoB mutation, as well as the genetic background of the strain, play an important role in the gene expression profile of rpoB mutants. Surprisingly, transcriptional profiling of cultures that were exposed to the critical concentration of rifampicin for 24 hours did not exhibit significant differential gene expression. Whole genome sequencing, followed by bioinformatic analysis, revealed that the in vitro mutants harbour synonymous and non-synonymous single nucleotide polymorphisms in addition to the respective rpoB mutations. This suggests that the mycobacterial genome is constantly evolving, challenging previous assumptions of relatively static mycobacterial genomes. The findings from this research have provided novel insight into understanding the influence of resistance-conferring mutations on the biology of M. tuberculosis and have shown that further studies are urgently needed to better understand the complex physiology of this pathogen. This knowledge will be critical for the success of future drug development endeavours. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is die etiologiese agent vir tuberkulose, een van die grootste oorsake van morbiditeit en sterftes in ontwikkelende lande. Die verskyning van middelweerstandige tuberkulose het 'n negatiewe impak op die effektiwiteit van die huidige behandeling van tuberkulose en dreig om tuberkulose beheerprogramme wêreldwyd te ondermyn. Weerstandigheid teen rifampisien, een van die eerste-lyn anti-tuberkulose middels, word veroorsaak deur mutasies in die 81 bp kerngedeelte van die rpoB geen. Die mees algemene mutasies in kliniese isolate word gevind in kodons 531 en 526; alhoewel daar min inligting beskikbaar is oor die effek van hierdie mutasies op die funksie van RNS polimerase. Die doel van hierdie studie was om die effek van verskillende rpoB mutasies op die geen-uitdrukkingsprofiel van M. tuberculosis te bepaal. Rifampisien-weerstandige kliniese isolate en in vitro mutante (geselekteer vanaf H37Rv en 'n rifampisien-sensitiewe kliniese isolaat) met rpoB H526Y en S531L mutasies was vir hierdie doel geselekteer en gebruik om die heel genoom volgorde te bepaal en geenuitdrukking te kwantifiseer. Vergelyking van die H37Rv transkripsieprofiel met in vitro geselekteerde rpoB mutante het getoon dat 'n groot aantal gene wat differensieel uitgedruk is, vir proteïene kodeer wat betrokke is in selwand-prosesse en intermediêre metabolisme en respirasie. Die meerderheid van hierdie differensieel uitgedrukte gene was afgereguleer. ’n Soortgelyke verskynsel is ook waargeneem in kliniese isolate, met die verskil dat 'n groter aantal gene in hierdie geval differensieel-uitgedruk was. Gene wat deel vorm van die WhiB7 regulon is opgereguleer in die rpoB526 mutante, terwyl dit afgereguleer was in die rpoB531 mutante. Hierdie resultate is ’n baie sterk aanduiding dat beide die posisie van die rpoB mutasie, asook die genetiese agtergrond van die organisme, ’n belangrike rol speel in die uitdrukking van gene in rifampisin weerstandige M. tuberculosis. Kulture wat aan die kritiese konsentrasie van rifampisien vir 24 uur blootgestel is, het teenverwagting geen differensiële geen-uitdrukking getoon nie. Verder het die heel genoom volgorde bepaling van die in vitro mutante getoon dat sinonieme en nie-sinonieme enkel-nukleotied polimorfismes teenwoordig is in die onderskeie rpoB mutante. Dit dui daarop dat die mikobakteriële genoom voortdurend verander, moontlik nie so stadig as wat voorheen vermoed is nie. Die bevindinge van hierdie navorsing bied nuwe insigte om die invloed van mutasies wat middel-weerstandigheid veroorsaak op die biologie van M. tuberculosis te verstaan. Dit het ook getoon dat verdere studies dringend nodig is om die komplekse fisiologie van hierdie patogeen te verstaan. Hierdie kennis sal van kardinale belang wees vir die sukses van toekomstige ondernemings om nuwe anti-tuberkulose middels te ontwikkel.
6

Adrenal function in hospitalised patients with pulmonary tuberculosis treated with rifampicin

Venter, Willem Daniel Francois 13 February 2009 (has links)
Abstract Introduction: Tuberculosis carries a high mortality in the days immediately after treatment. It is also the commonest cause of adrenal insufficiency in the developing world. Rifampicin is a potent hepatic enzyme inducer, and may contribute to adrenal insufficiency by accelerating cortisol breakdown. The aim of the study was to determine whether rifampicin induced accelerated catabolism of corticosteroids. Methods: A prospective, randomised study comparing adrenal function in 20 patients with pulmonary tuberculosis in the first five days treated with two different antituberculosis regimens, one containing rifampicin, and the other ciprofloxacin. Results: Demographic, clinical and laboratory results were similar in both groups. Both groups showed a statistically significant and similar decrease in morning cortisol, with similar responses to ACTH stimulation at both 30 and 60 minutes before and after four days of treatment. In the entire cohort, 40% demonstrated an incremental cortisol rise of <250nmol/l after ACTH stimulation on day 1. Mean basal cortisol concentrations were substantially elevated and DHEA-S levels were consistently subnormal, resulting in a high cortisol:DHEA-S ratio. No patient demonstrated overt adrenal insufficiency. There were no significant differences between the two groups before or during therapy for any electrolytes, hormones or calculated serum osmolality. Conclusions: Rifampicin did not additionally impair adrenocortical function during the initial period of therapy.
7

Development and characterization of biodegradable microspheres containing selected antimycobacterials

Bain, David F. January 1998 (has links)
Prolonged therapy required to effectively treat mycobacterial infection frequently results in severe dose-limiting side-effects and drug resistance due to patient non-compliance with protracted dosage regimens. Biodegradable poly-a-hydroxy acid microspheres and microcapsules containing rifampicin (RIF) and isoniazid (INH) respectively have been prepared with the intention of providing high sustained site-specific concentrations to overcome some of the shortcomings of existing oral treatments. Due to the high dose, hydrophilicity and instability of both drugs, formulation strategies to attain high drug loading and methodologies to characterize in vitro drug release during ongoing decomposition were required. Stability indicating HPLC assays to quantify drug release have been developed, validated and applied to monitor drug release based on cumulative quantification of drug and degradates. A mathematical correction for serial decompositions associated with RIF was made based on the terminal pseudo equilibrium observed during stability studies. An isocratic HPLC assay was prospectively developed for the quantification of both drugs and their major metabolites in biological samples. Further preformulation studies confirmed the absence of significant polymorphs for both drugs when recrystallized from solvents later used in formulation development. Furthermore, thermal analysis revealed only modest interaction between the drugs and Resomer®. The high and moderate water solubilities of INH (145 mgmL-1) and RIF (1 mgmL-1) determined the selection of spray-drying (SD) and emulsion solvent evaporation (ESE) for RIF, whereas preparation of INH microcapsules relied solely on the former technique. Examination of the effects of varying RIF: polymer ratio, phase volumes and continuum presaturation with selected poly(L-lactide) and poly(D, L-lactide-co-glycolide) (PDLGA) Resomer® identified optimum conditions to maximize drug loading during a comparison of aqueous ESE with spray-drying with a range of nine further amorphous Resomer® polymers. Although yields were generally higher with ESE (85-90 %), SD (45- 75 %) was considered a superior preparation technique on the basis of the rapid production of microspheres of high and predictable drug loading (100 % of that attempted), with monodisperse granulometry and superior morphology. Release profiles were typically asymptotic characterized by a rapid `burst' of release followed by a slow release of residual entrapped RIF, irrespective of the preparative technique or polymer used. Poor yields (7.2 %) when SD low molecular weight (MW) PDLGA (8 kD) were greatly enhanced (74.8 %) by reduction in drying temperature and substitution of chloroform: dichloromethane (CFM: DCM) (1: 1) cosolvent with DCM. These conditions were adopted as the optimum parameters for further studies of blends of low (2 kD, R104) and moderate (11 kD, R202H) MW poly(D, L-lactide) (PDLLA); materials which demonstrated excellent sprayability and dramatically modulated the release of drug when combined compared to their use alone. Drug release showed a remarkable dependence on blend, dramatic acceleration being observed between 44 and 48 %w/w R104. Release over this range showed a marked dependence on medium temperature and led to the proposal of an autohydration mechanism linked to the hydrophilicity and glass transition (T9) of the blend which accounted for the sigmoidal profiles observed. First order dependence of release allowed calculation of Arrhenius derived activation energies of drug release in glassy anhydrous and rubbery plastic matrices of 630 and 320 J mol-1. Hydration and thermal studies supported the postulated diffusion mechanism, whereas granulometric and morphological examinations demonstrated that erosion did not contribute significantly. The criticality of matrix composition was further highlighted when interchange with nominally identical polymer, R202H, shifted the critical composition to 30 %w/w R104. Moreover, this observation contested the batch-to-batch reproducibility of commercial polymer. Substitution of DCM with halothane (HAL) and acetone (ACT) had a profound influence on the properties of compositionally identical ('R104: R202'H, 30: 70) microspheres, particularly release kinetics. This was attributed to the more rapid drying kinetics with the poor solvent, ACT, and the generation of a porous matrix. Consequently, drug was largely released during the 'burst' phase. Superior solvents, HAL and DCM resulted in enhanced matrix coherence at the expense of considerable residual solvent burdens (6 - 12.5 %), which allowed extensive matrix relaxation as solvent was lost with first order kinetics. This ageing process was followed by the development of an endotherm associated with the Tg as the matrix stabilized with a resultant increase in the induction period and a general retardation of drug release. Extension of the concept of blending R104 as release 'initiator' to a range of MW PDLGA of 50: 50 and 75: 25 comonomer ratio as release 'modulator' was of limited success generating release profiles reminiscent of each polymer when used alone. The magnitude of the 'burst' correlated to the precipitation kinetics of the predominant complementary PDLGA polymer as determined by cloud-point titration. Due to the more hydrophilic nature of the copolymers, at the critical concentration uncontrolled hydration resulted in a single rapid release phase. Spray-dried biodegradable INH microcapsules were prepared by a two stage process whereby SD cores of drug or in combination with biodegradable albumin or casein were subsequently coated with PDLGA by SD. The highly crystalline, aggregated and irregular morphology of SD drug resulted in poor coating efficiency and a rapid release of encapsulated drug. Protein microspheres of superior sphericity allowed more effective coating and hence slower INH release. It is concluded that SD has excellent industrial potential for the preparation of biodegradable poly-a-hydroxy acid microspheres for high dose drugs to be delivered directly to their site of action, e. g., intra-pulmonary. Indeed, the granulometry of these particles and, in particular, the hydrophilic character of blends of PDLLA described have considerable potential for the sustained delivery of drugs in the low volumes of fluid that prevails in the lung. These formulations might offset some of the limitations of current oral antimycobacterial therapy.
8

DNA microarray based gene expression profiling in human hepatocyte cells to serve as a basis for dynamic modelling of the human liver a systems biology approach /

Reichart, Thomas, January 2008 (has links)
Stuttgart, Univ., Diss., 2008.
9

Evolution of Antibiotic Resistance

Pietsch, Franziska January 2015 (has links)
The emergence of antimicrobial resistance is a major global threat to modern medicine. The rapid dissemination of resistant pathogens and the associated loss of efficacy of many important drugs needs to be met with the development of new antibiotics and alternative treatment options. A better understanding of the evolution of resistance could help in developing strategies to slow down the spread of antimicrobial drug resistance. In this thesis we investigated the evolution of resistance to two important antibiotics, rifampicin and ciprofloxacin, paying special attention to the resistance patterns occurring with high frequency in clinical isolates. Rifampicin is a first-line drug in tuberculosis treatment and resistance to this valuable drug limits treatment options. Our work on rifampicin resistance helps to explain the extreme bias seen in the frequency of specific resistance mutations in resistant clinical isolates of M. tuberculosis. We identified an important interplay between the level of resistance, relative fitness and selection of fitness-compensatory mutations among the most common resistant isolates. Fluoroquinlones are widely used to treat infections with Gram-negatives and the frequency of resistance to these important drugs is increasing. Resistance to fluoroquinolones is the result of a multi-step evolutionary process. Our studies on the development of resistance to the fluoroquinolone drug ciprofloxacin provide insights into the evolutionary trajectories and reveal the order in which susceptible wild-type E. coli acquire multiple mutations leading to high level of resistance. We found that the evolution of ciprofloxacin resistance is strongly influenced by the mutation supply rate and by the relative fitness of competing strains at each successive step in the evolution. Our data show that different classes of resistance mutations arise in a particular, predictable order during drug selection. We also uncovered strong evidence for the existence of a novel class of mutations affecting transcription and translation, which contribute to the evolution of resistance to ciprofloxacin.
10

Determinação do perfil pré-clínico da atividade anti-tuberculose in vitro e in vivo de complexos heterolépticos de Rutênio(II) com fosfinas. diiminas e picolinato como ligantes

Pavan, Fernando Rogério [UNESP] 11 August 2011 (has links) (PDF)
Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-11Bitstream added on 2014-06-13T19:22:41Z : No. of bitstreams: 1 pavan_fr_dr_arafcf.pdf: 966119 bytes, checksum: c9d048c219b0b202a52cb7f70f5d5846 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / Tuberculose (TB) é uma doença infecciosa e curável transmitida pelo ar. A taxa de mortalidade reduziu globalmente em 2007, entretanto, TB-MDR, XDR e a coinfecção TB/HIV têm sufocado as tentativas de controle da TB, causando sofrimento e morte em todo o mundo. Adicionalmente, um terço da população mundial está infectada com o Mycobacterium tuberculosis (MTB) em estado de latência, o qual serve como reservatório para TB ativa. A rifampicina (RMP), descoberta há mais de 40 anos, representa a última classe de antibióticos introduzida como fármaco de primeira-linha no tratamento da TB. No esforço de sanar esta falha, é crescente a importância da Química Medicinal Inorgânica como um aliado na pesquisa de novos fármacos contra TB. É bem conhecido que alguns elementos metálicos desempenham papel fundamental nos seres vivos. Tem sido relatado desde 1970 que compostos de rutênio (Ru) exibem atividade anti-tumoral. Inclusive, alguns destes compostos se encontram na fase clínica de avaliação. Compostos de Ru foram avaliados também em nosso laboratório demonstrando atividade contra MTB. Os complexos com Ru(II) demonstraram ser mais ativos contra o MTB em relação a forma do ligante livre, em até 150 vezes, com baixa citotoxicidade e alta seletividade. Os resultados promissores inspiraram a procura de uma melhor abordagem para explorar o perfil pré-clínico in vitro e in vivo desses compostos. Assim, o objetivo deste trabalho foi desenvolver uma linha de pesquisa (pipeline) com ensaios fenotípicos rápidos, sensíveis, específicos e de baixo custo na pesquisa de um novo fármaco contra TB. Paralelamente, avaliou-se os melhores complexos heterolépticos de Ru(II) com fosfinas, diiminas e picolinato como ligantes, pré-selecionados, frente a esse pipeline. Os resultados obtidos com os complexos... (Resumo completo, clicar acesso eletrônico abaixo / Tuberculosis (TB) is a preventable and curable infectious disease transmitted through the air. The mortality rates decreased globally in 2007, however, MDR and XDR TB, and co-infection TB/HIV have stifled attempts to control TB causing suffering and death worldwide. Additionally, a third of the world population is infected with Mycobacterium tuberculosis (MTB) in state of latency, which serves as a reservoir for active TB. Rifampicin (RMP), discovered more than 40 years ago, represents the last class of antibiotics introduced as first-line drug to treat TB. In an effort to correct this failure is an increasingly importance of Inorganic Medicinal Chemistry as an ally in the search for new drugs against TB. It is well known that some metallic elements play a fundamental role in living organisms. It has been reported since 1970 that compounds of ruthenium (Ru) exhibit anti-tumor activity. Even some of these compounds are in clinical phase of evaluation. Ru compounds were evaluated in our laboratory showing activity against MTB. Complexes with Ru(II) showed activity against MTB in relation to the free ligand up to 150 higher, with low cytotoxicity and high selectivity. The promising results inspired the search for a better approach to explore the pre-clinical in vitro and in vivo profile of these compounds. So, the objective of this study was to develop a research line (pipeline) fast, sensitive, specific and low-cost phenotypic testing in the search for a new drug against TB. At the same time the best complexes of heteroleptic Ru(II) phosphine/diimines/picolinate, pre-selected, against several biological in vitro and in vivo assays. Those evaluated assays were inserted into the pipeline developed at our laboratory. The in vitro results obtained with the Ru(II) (compounds SCAR) complexes are comparable and/or better than the first choice... (Complete abstract click electronic access below)

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