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SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL DRUG CANDIDATES TO ADDRESS DRUG RESISTANCE IN TUBERCULOSIS AND FUNGAL DISEASESNgo, Huy 01 January 2018 (has links)
Tuberculosis (TB) and fungal infections are two of the most lethal infectious diseases worldwide due to the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) and fungal strains that can resist the most potent antimicrobial drugs currently employed. Due to the rise of these drug resistant strains, effective treatment options for these two infections are limited. This dissertation aims at exploring novel drug scaffolds to help combat drug resistance in TB and fungal infections.
TB caused by the pathogenic Mtb is, alongside with human immunodeficiency virus acquired immunodeficiency virus (HIV), the deadliest infectious disease worldwide with approximately 2-3 billion people infected yearly. The situation has become increasingly intensified due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb strains.Aminoglycoside (AG) antibiotics such as amikacin and kanamycin A (KAN) are heavily relied upon for the treatment of MDR- and XDR-Mtb strains. However, the success rate for the treatment of these MDR- and XDR-TB cases is decreasing as a result of increased KAN resistance. It was reported by the Centers for Disease Control and Prevention (CDC) that upregulation of the enhanced intracellular survival (eis) gene was the cause of resistance to KAN in a large portion of Mtb clinical isolates. Our lab previously demonstrated that Eis is an AG acetyltransferase that can inactivate AGs via chemoenzymatic modification of the AG scaffolds. As Eis has been shown to acetylate a wide variety of AG scaffolds, the development of novel AGs that can completely escape the action of Eis remains highly challenging. Therefore, we suggested an alternative therapeutic approach involving inhibiting Eis enzyme and still employing the current FDA-approved KAN. As exemplified by the clinically successful combination of penicillin and b-lactamase inhibitors, we hypothesized that an Eis inhibitor may be used as adjuvant therapy in combination with KAN to treat MDR- and XDR-tuberculosis. Using high-throughput screening, we were able to identify several small-molecule scaffolds capable of inhibiting Eis. We performed structure activity relationship (SAR) studies using purified Eis enzyme and optimized lead compounds. Additionally, we also showed that co-administration of Eis lead inhibitors with KAN led to recovery of KAN activity against a KAN-resistant Mtb cell line that overexpressed Ei
Invasive fungal infections are on the rise due to an increased population of critically ill patients as a result of HIV infections, chemotherapies, and organ transplantations. Unlike antibiotics that are greatly diverse in categories and mechanisms of action, our current antifungal drug repertoire is greatly limited and insufficient in addressing the problem of drug-resistant fungal infections. Thus, there is a growing need for novel antimycotics that are safe and effective. We report a number of lead compounds with potent antifungal activitiy. The MIC values of these compounds were as low as 0.02 mg/mL against the fungal strains tested. Our compounds are derived from the ebselen core structure, which has been shown to be safe in multicenter clinical trials. Notably, fungal cells treated with our compounds showed the accumulation of ROS, which may further contribute to the growth inhibitory effect against fungi. This study provides new lead compounds for the development of antimycotic agents.
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Multiple Antibiotic Resistance Of Surface Mucus Dwelling Bacterial Populations In Freshwater FishOzaktas, Tugba 01 December 2007 (has links) (PDF)
Surface mucus of a freshwater fish, Alburnus alburnus (bleak), caught from Lake Mogan, situated in south of Ankara, was collected in different seasons. The total cultivable bacteria were enumerated by spread plate method on nine different media. Bacteria were isolated based on colony morphologies and pigmentation. A total of sixty bacterial isolates obtained. The mucus-dwelling bacteria were first tested for resistance against ampicillin and kanamycin / then streptomycin and chloramphenicol were added to the experimental set up. The resistance levels of isolates were determined in terms of four antibiotics by tube dilution method. About 90% of the isolates were resistant to chloramphenicol, about 84% to kanamycin, about 88% to streptomycin and about 98% to ampicillin. These high levels of antibiotic resistance are rather interesting from a standpoint that the lake has no record of antibiotics exposure of any sort. The plasmid isolations were carried out to determine if the multiple antibiotic resistance could be attributed to plasmids for starting assumption. But we found no direct relationship between the presence of plasmids and multiple antibiotic resistance. Our study indicated that multiple antibiotic resistance at high levels is among the current phenotypes of the fish mucus-dwelling bacterial populations in Lake Mogan.
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Molekulární analýza transgenních rostlin rododendronů, získaných po transformaci vektorem 35SGUSint. / Molecular analysis of transgenic rhododendron plants obtained by transformation with 35SGUSint. constructSKOTNICOVÁ, Petra January 2011 (has links)
The goal of this thesis was molecular analysis of rhododendron plants obtained after transformation by Agrobacterium tumefaciens and subsequent selection on kanamycin. Presence of incorporated genes gusA and nptII was determined by polymerase chain reaction (PCR) and Southern analysis. GUS activity was determined by fluorimetric and histochemical assay too.
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[pt] DESENVOLVIMENTO DE MÉTODOS ESPECTROFLURIOMÉTRICOS PARA A DETERMINAÇÃO DE ERITROMICINA E CANAMICINA E APLICABILIDADE NA VACINA CONTRA A FEBRE AMARELA / [en] DEVELOPMENT OF SPECTROFLUORIMETRIC METHODS FOR DETERMINATION OF ERYTHROMYCIN AND KANAMYCIN AND THEIR APPLICATION IN THE YELLOW FEVER VACCINEVIRGINIA DE LOURDES MENDES FINETE 27 December 2005 (has links)
[pt] Os antibióticos eritromicina e canamicina, pertencentes às
classes dos
macrolídeos e aminoglicosídeos respectivamente, têm sua
importância como
agentes preservativos no processo de preparação e durante
o processo de
utilização da vacina contra a febre amarela - apresentação
de cinco doses -
produzida no Instituto de Tecnologia em Imunobiológicos,
Bio-Manguinhos,
FIOCRUZ, RJ. Este trabalho propõe apresentar duas novas
abordagens para a
determinação espectrofluorimétrica desses antibióticos,
considerando o
desempenho, inclusive com relação à seletividade, na
análise dessa matriz
específica (vacina). Um estudo preliminar do comportamento
luminescente
(fosforescência e fluorescência) dessas substâncias foi
realizado em diferentes
condições experimentais a fim de encontrar as que
permitissem o
desenvolvimento dos métodos analíticos. Para a
eritromicina, que não apresentou
luminescência nativa, foi utilizado um procedimento de
derivação fotoquímica de
soluções do analito preparadas em meio contendo ácido
sulfúrico. Como resultado
obteve-se um foto-produto que apresentou fluorescência em
412/465 nm. As
condições experimentais foram otimizadas visando a
maximização desse sinal
fluorescente. Nesse caso, foram estudados o tempo de
irradiação com UV, o tipo e
a concentração do ácido utilizado, o tempo e a temperatura
de aquecimento. O
método foi parcialmente validado apresentando limites de
detecção e de
quantificação iguais a 0,25 ug mL-1 e 0,85 ug mL-1
respectivamente. A faixa
linear do método estendeu-se até 200 ug mL-1 e os
parâmetros de precisão e
robustez foram bastante satisfatórios. O método foi
aplicado na análise de uma
simulação de vacina contra a febre amarela e na análise de
medicamentos
comerciais com um percentual de recuperação entre 98,2 e
105,2 %. A
metodologia desenvolvida para a canamicina, que também não
apresentou fluorescência natural, baseou-se na reação de
oxirredução deste composto com o
Ce (IV) produzindo o Ce (III), que é uma espécie
fortemente fluorescente
(255/360 nm). As intensidades dos sinais fluorescentes
medidos no par
excitação/emissão do Ce (III) foram diretamente
proporcionais às concentrações
de canamicina. As condições para a realização da reação
foram estudadas,
avaliando a quantidade de Ce (IV), a concentração e tipo
de ácido usado no meio
reacional e o tempo e temperatura de aquecimento. A ampla
faixa linear (até 1000
ug mL-1) foi obtida, com limites de detecção e
quantificação de 1,22 ug mL-1 e
4,08 ug mL-1, respectivamente. O percentual de recuperação
obtido na análise da
vacina diluída foi de 109,8 % e em amostra diluída de
urina enriquecida com o
analito foi de 103,4 %. / [en] The antibiotics erythromycin and kanamycin, members of the
macrolide and
aminoglycoside classes respectively, have their importance
as preservative agents
for the preparation process and during the using of the
yellow fever vaccine, five
doses presentation, produced in Technology on
Immunobiologicals Institute, Bio-
Manguinhos, FIOCRUZ, RJ. In this work two new approaches
for the
spectrofluorimetric determination of these antibiotics are
presented, taking into
consideration their selectivity performance for this
specific matrix (vaccine). A
preliminary study was performed to evaluate the
luminescent behavior
(phosphorescence and fluorescence) of these substances in
different experimental
conditions aiming to find the best ones that would lead to
the development of the
analytical methods. For erythromycin, which does not
present native
luminescence, a photochemical derivatization was employed
using analite
solutions prepared in sulfuric acid. As a result, a photo-
product was obtained,
which presented fluorescence at 412/465 nm. The
experimental conditions were
optimized aiming the maximization of the fluorescent
signal. In this case, the
studied parameters were the UV irradiation time, the type
and concentration of the
acid utilized, and the time and temperature used for the
heating step. The method
was partially validated, indicating limits of detection
and quantification of
0.25 ug mL-1 and 0.85 ug mL-1, respectively. The linear
dynamic range of the
method extended up to 200 ug mL-1 and the parameters
related to precision and
robustness were very satisfactory. The method was applied
in the analysis of a
simulated yellow fever vaccine and in commercial
pharmaceutical formulations.
Recovery percent between 98.2 and 105.2 % were achieved.
The methodology
developed for kanamycin, which also do not presented
natural fluorescence, was
based on the oxirreduction reaction of this compound with
Ce (IV), producing Ce
(III), a strongly fluorescent species (255/360 nm). The
fluorescence intensities, measured in the
excitation/emission pair of Ce (III), was directly
proportional to
the kanamycin concentration. The reaction conditions were
studied by the
evaluation of the amount of Ce (IV), the type and
concentration of the acid
utilized in reactional medium and heating time and
temperature. Large linear
range (up to 1000 ug mL-1) was obtained, with detection
and quantification limits
of 1.22 ug mL-1 and 4.08 ug mL-1, respectively. The
recovery percent obtained in
the analysis of a diluted vaccine was 109.8 % while the
recovery achieved for
diluted spiked urine was 103.4 %.
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[en] SPECTROANALYTICAL METHODS USING GRAPHENE QUANTUM DOTS AS PHOTOLUMINESCENT PROBES FOR THE DETERMINATION OF ANALYTES OF BIOLOGICAL AND PHARMACOLOGICAL INTEREST / [pt] MÉTODOS ESPECTROANALÍTICOS UTILIZANDO PONTOS QUÂNTICOS DE GRAFENO COMO SONDAS FOTOLUMINESCENTES PARA A DETERMINAÇÃO DE ANALITOS DE INTERESSE BIOLÓGICO E FARMACOLÓGICOCARLOS ALBERTO TOLOZA TOLOZA 20 December 2018 (has links)
[pt] O presente trabalho teve como objetivo o desenvolvimento de métodos espectroanalíticos capazes de determinar indiretamente analitos de interesse biológico e farmacológico que possuem fraca atividade óptica no UV-vis (no caso, captopril, histamina e sulfato de canamicina). Embora muitos métodos para quantificar esses analitos estejam reportados, muitos dependem da derivatização química do analito, procedimento considerado complexo e trabalhoso para fazer tais analitos absorverem e emitirem no UV-vis. Por isso, a proposta de uso de pontos quânticos fotoluminescentes é interessante, pois permitem, em condições ajustadas, respostas analíticas que proporcionam a determinação indireta dos analitos de interesse em concentrações da ordem de até 10-8 mol L-1. A determinação do captopril foi proposta utilizando pontos quânticos de grafeno aminofuncionalizados com uso de glutationa (GQDs-amino). O captopril induziu a supressão e o deslocamento espectral (para o vermelho) da fotoluminescência da dispersão aquosa dos GQDs-amino. Por outro lado, quando Fe3+ foi usado como um mediador que gera uma supressão da fotoluminescência da dispersão de GQDs-amino, a adição de captopril restabelece a fotoluminescência original dos pontos quânticos. Em condições experimentais ajustadas, a magnitude da supressão ou de deslocamento espectral da fotoluminescência dos GQDs-amino pode ser relacionada com a concentração de captopril. Em ambos os casos, a resposta linearizada abrangeu três ordens de grandeza (10-6 a 10-4 mol L-1). Em contrapartida, a abordagem de restauração do sinal da sonda, previamente suprimida com Fe3+, também se mostrou útil do ponto de vista analítico. As abordagens propostas foram testadas com a determinação de captopril em amostras simuladas e em formulações farmacêuticas comerciais. O deslocamento
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espectral a partir da sonda GQDs-amino e ativação/desativação da fotoluminescência utilizando GQDs-amino-Fe3+ resultou em recuperações satisfatórias, mostrando o potencial de detecção quantitativo do método. No estudo com a histamina, avaliou-se o comportamento fotoluminescente da dispersão aquosa de GQDs-amino na presença de histamina com interação mediada por diferentes íons metálicos. Os resultados revelaram que uma interação mais forte e seletiva existia na presença de Eu3+, Fe3+ e Cu2+. A sensibilidade das curvas de supressão de fotoluminescência normalizada (Ks) indicou uma interação dez vezes mais forte da histamina com a superfície dos GQDs na presença de Fe3+. A resposta linear observada nos GQDs-amino-Fe3+ (luminescência medida a 345/435 nm) abrangeu a concentração de histamina de 4,3 × 10-7 mol L-1 (limite de quantificação) até 3,2 × 10-5 mol L-1. A dispersão de GQDs-amino-Fe3+ foi usada como sonda na análise de amostras de atum após extração do analito em cartucho fase sólida catiônica. Os resultados analíticos foram estatisticamente semelhantes aos obtidos com um método baseado na cromatografia líquida com detecção fluorimétrica (após derivatização química da histamina). A determinação do sulfato de canamicina foi feita medindoo efeito que ela exerce sobre a fotoluminescência dos GQDs-amino associados às nanopartículas de ouro (AuNPs), que foram produzidas pela redução de AuCl4 com NaBH4 em uma dispersão aquosa de GQDs-amino (obtido pela pirólise de ácido cítrico e glutationa) contendo também o agente tensoativo catiônico CTAB. O sistema AuNPs-GQDs-amino-CTAB apresentou fotoluminescência suprimida, que foi amplificada na presença de canamicina. Sob condições experimentais ajustadas, a ampliação da fotoluminescência do nanomaterial em função da concentração de analito se mostrou linear e abrangeu três ordens de grandeza (10-7 a 10-5 mol L-1). O uso de extração em fase sólida com um cartucho empacotado com um polímero molecularmente impresso (seletivo para aminoglicosídeos) assegurou a seletividade nas determinações de sulfato de canamicina feitas em vacina da febre amarela e em formula� / [en] The objective of the present work was the development of spectroanalytical methods capable of indirectly determining analytes of biological and pharmacological interest that present inherent weak optical activity in UV-vis (in this case, captopril, histamine and kanamycin sulfate). Although many methods to quantify these analytes are reported, many of these depend on chemical derivatization, a procedure considered complex and laborious to promote UV-vis absorption and luminescence. Therefore, the proposed use of photoluminescent quantum dots is interesting since they allow, under adjusted conditions, analytical responses that allow the indirect determination of the analytes of interest in concentrations of the order of down to 10-8 mol L-1. The determination of captopril was proposed using graphene quantum dots aminofunctionalized using glutathione as a precursor (GQDs-amino). Captopril induced photoluminescence suppression and spectral red-shift from the aqueous dispersion of GQDs-amino. In contrast, when Fe3+ is used as a mediator, it generates a suppression of the photoluminescence of the GQD-amino dispersion and the addition of captopril restored the original photoluminescence of the quantum dots. In adjusted experimental conditions, photoluminescence suppression of the GQDs-amino, as a function of the captopril concentration, can be related both to the magnitude of the suppression and to the spectral shift. In both cases, the linearized response covered three orders of magnitude (10-6 to 10-4 mol L-1). In contrast, the probe signal restoration of the previously Fe3+ suppressed photoluminescent GQDs, also proved to be analytically useful. The proposed approaches were tested by the determination of captopril in simulated samples and in commercial pharmaceutical formulations. Spectral shift from the GQDs-amino probe and the photoluminescence on/off approach (using GQDs-amino-Fe3+ probe) resulted in satisfactory recoveries, showing the quantitative capability of the method. In the work concerning histamine, the photoluminescent behavior of the aqueous dispersion of GQDs-amino in the presence of this amino acid was studied in function of different interaction mediators (metal ions). The results revealed that strong and selective interaction existed in the presence of Eu3+, Fe3+ and Cu2+. The sensitivity of normalized photoluminescence (Ks) suppression curves indicated a ten-fold stronger interaction of histamine with the surface of GQDs in the presence of Fe3+. The linear response observed in the GQDs-amino-Fe3+ (luminescence measured at 345/435 nm) covered the histamine concentration of 4.3 × 10-7 mol L-1 (quantification limit) to 3.2 × 10-5 mol L-1. The GQDs-amino-Fe3+ was applied as a probe in the analysis of tuna fish samples after solid phase extraction (SPE) of the analyte using a cationic solid phase. The analytical results were statistically similar to those obtained with a method based on liquid chromatography with fluorimetric detection (after chemical derivatization of histamine). The determination of kanamycin sulfate was made by measuring the effect it exerts on the photoluminescence of gold nanoparticles (AuNPs) associated GQDs, that were produced by the reduction of AuCl4 with NaBH4 in an aqueous dispersion of GQDs-amino (obtained by pyrolysis of citric acid and glutathione) also containing the cationic surfactant CTAB. The AuNPs-GQDs-amino-CTAB system showed a suppressed photoluminescence, which was amplified in the presence of kanamycin. Under adjusted experimental conditions, the magnification of the photoluminescence of the nanomaterial as a function of the analyte concentration was linear and covered three orders of magnitude (10-7 to 10-5 mol L-1). The use of solid phase extraction with a cartridge packed with a molecularly imprinted polymer (selective for aminoglycosides) ensured selectivity in the determinations made in yellow fever vaccine and in veterinary pharmaceutical formulations. The analytical results were statistically similar to those obtained with an HPLC based method with fluorimetri
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Molekulární charakteristika transformantů \kur{L. esculentum} po vnesení genu pro manóza-6-fosfátizomerázu / The molecular analysis of transgenic \kur{Lycopersicon esculentum} plants harbouring incorporated pmi gene for phosphomannose isomerasePŘIKRYLOVÁ, Pavla January 2007 (has links)
The aim of this study was to analyze number of incorporated T-DNA harbouring pmi and nptII transgenes and expression of the pmi gene in tomato transformants using Southern blot and Northern blot methods. The presence of a functional PMI protein was assesed using PMI-assay with Chlorophenol red dye. Mannose selection of tomato seeds was established and segregation patterns in T1 progeny were studied.
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[en] DETERMINATION OF QUERCETIN, KANAMYCIN AND PYRACLOSTROBIN USING METHODS PHOTOLUMINESCENT AND MOLECULARLY IMPRINTED POLYMERS / [pt] DETERMINAÇÃO DE QUERCETINA, KANAMICINA E PIRACLOSTROBINA USANDO MÉTODOS FOTOLUMINESCENTES E POLÍMEROS DE IMPRESSÃO MOLECULAR08 November 2021 (has links)
[pt] Neste trabalho, polímeros de impressão molecular (MIP) foram desenvolvidos para aplicação em procedimentos de extração em fase sólida (SPE) visando separar e concentrar quercetina (flavonóide), piraclostrobina (pesticida) e kanamicina (antibiótico) em amostras contendo substâncias interferentes na determinação dos analitos-alvo. A seletividade em relação aos analitos de interesse foi conseguida pela interação específica dessas espécies químicas com os sítios de reconhecimento dos polímeros. A produção desses MIPs foi baseada na polimerização em bulk e, de modo a se comprovar a efetividade dos mesmos, o desempenho destes foram comparados com polímeros não-impressos (NIP) correspondentes, que foram produzidos sem o uso da molécula-molde. Os procedimentos para síntese são simples e o material produzido é quimicamente resistente nas condições de uso. A caracterização do material produzido foi feita por meio de microscopia de varredura eletrônica (MEV) e espectrometria de absorção na região do infravermelho (IV). O MIP preparado com quercetina foi empregado na extração seletiva deste flavonóide em amostras de urina e de suplemento alimentar e permitiu a obtenção de limite de detecção de 4,58 x 10-8 mol L-1 usando espectrofotometria de absorção no UV-vis com recuperações superiores a 90 porcento e separação efetiva de outros flavonóides como a flavona e naringenina. O MIP preparado com piraclostrobina foi usado na análise de amostra de água de rio e urina e permitiu o alcance de limite de detecção de 4,6 x 10-9 mol L-1, usando detecção por espectrofluorimetria, e recuperações maiores que 90 porcento na presença de outros pesticidas da classe das estrobilurinas. Para o MIP preparado com kanamicina, o limite de detecção alcançado usando detecção com sonda de nanopartículas fluorescentes foi 9 ng mL-1 (1,5 x 10-8 mol L-1) com aplicação na análise de urina e vacina contra febre amarela com recuperações maiores a 90 porcento. / [en] In this work, molecularly imprinted polymers (MIPs) have been developed for application procedures for solid phase extraction (SPE) in order to separate and concentrate quercetin (flavonoid), pyraclostrobin (pesticide) and kanamycin (antibiotic) in samples containing interfering substances in the determination of target analytes. The selectivity in respect of analytes has been achieved by specific binding of these chemical species with the recognition sites of the polymers. The production of MIP polymerization was based on "bulk" and in order to prove the effectiveness thereof, the performance of these polymers were compared with non-printed (NIP) thereof, that were produced without the use of template molecule. The procedures for synthesis are simple and relatively low cost and is chemically resistant material produced under the conditions of use. The characterization of the material produced was done by scanning electron microscopy (MEV), absorption spectroscopy in the infrared region and CHN elemental analysis. The MIP prepared with quercetin was used for the selective extraction of flavonoids in urine samples and dietary supplement and allowed to obtain a detection limit of 4,58 x 10-8 mol L-1 using absorption spectrophotometry in the UV-vis with recoveries exceeding 90 percent and effective separation of other flavonoids such as naringenin and flavone. The MIP prepared with pyraclostrobin was used to analyze samples of urine and river water and allowed to reach the detection limit of 4,6 x 10-9 mol L-1, detection using spectrofluorimetry and recoveries greater than 90 percent in presence of other pesticides from the class of strobilurins. For the MIP prepared with kanamycin, the detection limit using detection with fluorescent nanoparticles probe was 9 ng mL-1 (1,5 x 10-8 mol L-1) application to the analysis of urine and yellow fever vacine with recoveries greater than 90 percent.
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Model Development and Investigation of Antibiotic Cross Resistance and Decay in <i>E. Coli</i>Boyette, Rachel A. 22 June 2022 (has links)
No description available.
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Development of a Model for Bacterial Antibiotic Resistance in <i>E. coli</i>DeWeese, Claire 22 June 2022 (has links)
No description available.
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Synthesis and Biological Studies of Amphiphilic Compounds Derived from Saccharides and AminoglycosidesAlfindee, Madher N. 01 August 2019 (has links)
Adjacent cells communicate through gap junctions (GJs). These GJs are formed by head to head docking of two hemichannels (HCs) from two adjacent cells. HCs are connexin hexamer proteins. Connexin mutation is the most frequent cause of childhood hearing loss. This hearing impairment affects 2 in every 2000 children. Inhibition of the HCs might be the key factor to treat such disorders. A library of amphiphilic kanamycins was synthesized to be tested as HC inhibitors. These compounds showed excellent inhibition activity in comparison with the parent compound (kanamycin A) with less toxicity.
A library of monosaccharide esters with varying carbon chain lengths (acetyl (C2) to hexadecyl (C16)) were synthesized, characterized, and tested for bioactivity. Carbohydrate esters showed low toxicity while remaining active against bacteria and fungi. The compound 6-O-tetradecanoyl-D-mannopyranose (MAN014), a mannose ester with a fourteen-carbon chain, showed the greatest antibacterial and antifungal properties. A mode of action study was tested against Staphylococcus aureus (bacteria) and Fusarium graminearum (fungus) and found the compound perturbed the cell membrum.
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