Project I 2-Deoxystreptamine (2-DOS), the most conserved central scaffold of aminoglycosides, is known to specifically recognize the 5'-GU-'3 sequence step through highly conserved hydrogen bonds and electrostatic interactions within and without the context of aminoglycosides (Figure 1a). We proposed that a novel monomeric unnatural amino acid building block using 2-DOS as a template would allow us to develop RNA binding molecules with higher affinity and selectivity than those currently available. Conjugating two or more of the monomeric building blocks by an amide bond would introduce extra hydrogen bonding donors and acceptors that are absent in natural aminoglycosides and increase specificity of binding to a target RNA through a network of hydrogen bonds. In addition, the amide conjugation between the monomeric building blocks places two GU-base recognizing amines at 5 Å… distance, which is equal to the distance of neighboring base stacks in dsRNAs We hypothesized that targeting dsRNAs containing multiple consecutive 5'-GU-'3 sequence steps would become possible by connecting two or more of the monomeric building blocks by amide bonds. According to the proposed hypothesis, we designed three dimeric 2-DOS compounds connected by an amide bond. These three targets include the dimeric 2-DOS substrate connected by an amide bond, the dimeric 2-DOS containing the sugar moiety from Neamine, and a dimeric 2-DOS connected by a urea linker. These compounds were then tested for sequence specific binding against 8 different RNA strands, and for antibacterial activity against E. coli, actinobacter baumannii and klebsiella. Project II A dual optimization approach was used for to enhance the catalytic activity and chemoselectivity for nitro reduction. In this approach the composition of the nanoparticles and electronics effects of the polymer were studied towards nitro reduction. Bimetallic Ruthenium-Cobalt nanoparticles showed exceptional catalytic activity and chemoselectivity compared to monometallic Ruthenium nanoparticles. The electronic effects of the polymer also had a significant effect on the catalytic activity of the bimetallic nanoparticles. The electron-deficient poly(4-trifluoromethylstyrene) supported bimetallic nanoparticles undergo nitro reduction in 20 minutes at room temperature, whereas electron-rich poly(4-methylstyrene) and poly(4-methoxystyrene) supported bimetallic nanoparticles to longer reaction times to go to completion. Electronics of the polymers also effects the change in mechanism of nitroreduction. Polystyrene bimetallic Ruthenium-Cobalt nanoparticles showed excellent yields and chemoselectivity towards nitro functional group in the presence of easily reducible functional groups like alkenes, alkynes, allyl ethers, propargyl ethers. Monometallic ruthenium nanoparticles also showed excellent reactivity and chemoselectivity towards azide reduction in the presence of easily reducible functional groups. Interestingly monometallic ruthenium nanoparticles showed regioselective reduction of primary azides in the presence of secondary and benzylic azides, also aromatic azides can be selectively reduced in the presence of secondary azides. These polystyrene supported nanoparticles are heterogeneous and are easily separated from the reaction mixture and reused multiple times without significant of catalytic activity.

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.

A presença de compostos de uso veterinário em produtos de origem animal é uma grande preocupação em termos de saúde pública. A utilização descontrolada de antibióticos como medicamentos veterinários pode levar ao desenvolvimento de resistência bacteriana, podendo prejudicar a eficácia destas drogas no uso em humanos. Os aminoglicosídeos são antibióticos extensivamente utilizados na criação de animais para o tratamento de infecções bacterianas ou para a promoção do crescimento. A União Europeia estabeleceu níveis máximos de resíduos permitidos (LMRs) para os aminoglicosídeos em vários alimentos de origem animal. O governo brasileiro implementou um plano de controle que avalia os resíduos de medicamentos veterinários em produtos de origem animal. Neste trabalho, foram desenvolvidos método quantitativo por cromatografia a líquido acoplada a espectrometria de massas triplo quadrupolo em modo tandem (LC-MS/MS) e método de screening utilizando cromatografia a líquido acoplada a espectrometria de massas com analisador por tempo-de-voo (LC-qTOF-MS) para a determinação simultânea dos aminoglicosídeos espectinomicina, tobramicina, gentamicina, kanamicina, higromicina, apramicina, estreptomicina, diidroestreptomicina, amicacina e neomicina em leite e em músculo bovino, de aves e suíno. Como método de extração utilizou-se procedimento com ácido tricloroacético e clean up por precipitação a baixa temperatura e C18 a granel. Os métodos por LC-MS/MS e LC-qTOF-MS foram validados de acordo com a Diretiva da Comissão Europeia 2002/657/CE. Os resultados obtidos no método quantitativo para recuperação, precisão, linearidade, especificidade, limites de decisão (CCα) e capacidades de detecção (CCβ) nestas matrizes foram considerados satisfatórios. Os dados de limite de detecção (LOD) e limite de quantificação (LOQ) foram estabelecidos a partir dos LMRs, variando de 5 a 100 ng g-1 para LOD e 12,5 a 250 ng g-1 para LOQ. As recuperações variaram de 36,8 a 98%, e os coeficientes de variação ficaram entre 0,9 e 20,2%, observando-se que todas as curvas foram feitas com nas próprias matrizes a fim de minimizar os efeitos de matriz que são intrínsecos para os casos estudados neste trabalho. Os valores de CCβ obtidos no método qualitativo foram entre 25 e 250 ng g-1, considerados satisfatórios para os analitos nessas matrizes. O método proposto mostrou-se simples, fácil e adequado para a análise de um grande número de amostras por dia a um baixo custo. / The presence of several substances in animal products has been a major public concern. The use of antibiotics drugs as veterinary medicines might lead to the development of bacterial resistance, which might undermine the efficacy of these drugs in human use. The aminoglycosides are antibiotics that have been extensively employed in animal husbandry for the treatment of bacterial infections, but also as growth promotion. The European Union has issued strict maximum residue levels (MRLs) for aminoglycosides in several animal origin products. The Brazilian government implemented a control plan that assesses the residues of veterinary medicines in animal products. A quantitative method based on liquid chromatography tandem mass spectrometry (LC–MS/MS) and a screening method by liquid chromatography mass spectrometry with time-of-flight system (LC-qTOF-MS) has been developed for simultaneous determination of the aminoglycosides spectonomycin, tobramycin, gentamicin, kanamycin, hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin and neomycin in milk and in bovine, poultry and swine muscles. A simple extraction method was developed using trichloroacetic acid and clean up with low temperature precipitation and C18 bulk. The LC–MS/MS and LC-qTOF-MS methods were validated according to the European Union Commission Directive 2002/657/EC. Adequate performance characteristics were obtained for recovery, precision, calibration curve, specificity, decision limits (CCα) and detection capabilities (CCβ) in all matrices tested. The detection limit (LOD) and limits of quantification (LOQ) was established from MRL ranging from 5 to 100 ng g-1 for LOD and 12.5 to 250 ng g-1 to LOQ. Recoveries ranged from 36.8 to 98.0% and the coefficient of variation from 0.9 to 20.2%, noting that all curves have been made into their own matrices in order to minimize the matrix effects which are intrinsic to the cases studied in this work. The CCβ values obtained in qualitative method were between 25 and 250 ng g-1, considered satisfactory for the analytes in those matrices. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.

O objetivo deste estudo foi caracterizar bactérias multirresistentes (MDR) isoladas de cavalos saudáveis (fezes) e doentes no Brasil e na França. De março de 2012 a dezembro de 2014, amostras clínicas coletadas de cavalos saudáveis e doentes no Brasil foram selecionadas para pesquisa da presença de bactérias MDR. A investigação sobre as amostras franceses foi restria a isolados de Escherichia coli (EC) recuperados a partir de amostras clínicas coletadas entre 2014 e 2015. Nos cavalos brasileiros, a análise de amostras de fezes de animais saudáveis revelou a presença de clones de EC não relacionados pertencentes aos filogrupos A, D ou B2 que carreavam genes como: blaCTX-M-1, blaCMY-2, qnr- e genes add-tipo (amino-transferases); enquanto que nos cavalos doentes foram encontradas EC, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa e Serratia marcescens carreando genes blaCTX-M-15, blaCTX-M-1, rmtD 16S rRNA metilase, qnr-tipo, aac(6´)-Ib-cr e aad-tipo. Nos cavalos doentes franceses de EC MDR foram positivas para CTX-M-1, seguido de M-2- e M-9. Estes resultados destacam a importância de cavalos como um novo reservatório de bactérias MDR. / The aim of this study was to characterize multidrug-resistant (MDR) bacteria isolated from healthy and infected horses in Brazil and France. From March 2012 to December 2014, clinical samples collected from healthy and infected horses, in Brazil, were screened for the presence of MDR bacteria. Investigation on French isolates was restricted to E. coli strains recovered from clinical samples collected between 2014 and 2015. In Brazilian horses, the analysis of fecal samples from healthy animals revealed the presence of clonally unrelated A, D or B2 phylogroups of E. coli strains carrying blaCTX-M-1, blaCMY-2, qnr- and aminoglycoside adenyl transferase (aad)-type genes, whereas in infected horses, E. coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens isolates carrying blaCTX-M-15, blaCTX-M-1, rmtD 16S rRNA methylase, qnr-type, aac(6´)-Ib-cr and aad-type genes. In French infected horses, most MDR E. coli isolates were positive for CTX-M-1-, followed by CTX-M-2- and CTX-M-9-type extended-spectrum beta-lactamases. These results highlight the importance of horses as a new reservoir of MDR bacteria.

A presença de compostos de uso veterinário em produtos de origem animal é uma grande preocupação em termos de saúde pública. A utilização descontrolada de antibióticos como medicamentos veterinários pode levar ao desenvolvimento de resistência bacteriana, podendo prejudicar a eficácia destas drogas no uso em humanos. Os aminoglicosídeos são antibióticos extensivamente utilizados na criação de animais para o tratamento de infecções bacterianas ou para a promoção do crescimento. A União Europeia estabeleceu níveis máximos de resíduos permitidos (LMRs) para os aminoglicosídeos em vários alimentos de origem animal. O governo brasileiro implementou um plano de controle que avalia os resíduos de medicamentos veterinários em produtos de origem animal. Neste trabalho, foram desenvolvidos método quantitativo por cromatografia a líquido acoplada a espectrometria de massas triplo quadrupolo em modo tandem (LC-MS/MS) e método de screening utilizando cromatografia a líquido acoplada a espectrometria de massas com analisador por tempo-de-voo (LC-qTOF-MS) para a determinação simultânea dos aminoglicosídeos espectinomicina, tobramicina, gentamicina, kanamicina, higromicina, apramicina, estreptomicina, diidroestreptomicina, amicacina e neomicina em leite e em músculo bovino, de aves e suíno. Como método de extração utilizou-se procedimento com ácido tricloroacético e clean up por precipitação a baixa temperatura e C18 a granel. Os métodos por LC-MS/MS e LC-qTOF-MS foram validados de acordo com a Diretiva da Comissão Europeia 2002/657/CE. Os resultados obtidos no método quantitativo para recuperação, precisão, linearidade, especificidade, limites de decisão (CCα) e capacidades de detecção (CCβ) nestas matrizes foram considerados satisfatórios. Os dados de limite de detecção (LOD) e limite de quantificação (LOQ) foram estabelecidos a partir dos LMRs, variando de 5 a 100 ng g-1 para LOD e 12,5 a 250 ng g-1 para LOQ. As recuperações variaram de 36,8 a 98%, e os coeficientes de variação ficaram entre 0,9 e 20,2%, observando-se que todas as curvas foram feitas com nas próprias matrizes a fim de minimizar os efeitos de matriz que são intrínsecos para os casos estudados neste trabalho. Os valores de CCβ obtidos no método qualitativo foram entre 25 e 250 ng g-1, considerados satisfatórios para os analitos nessas matrizes. O método proposto mostrou-se simples, fácil e adequado para a análise de um grande número de amostras por dia a um baixo custo. / The presence of several substances in animal products has been a major public concern. The use of antibiotics drugs as veterinary medicines might lead to the development of bacterial resistance, which might undermine the efficacy of these drugs in human use. The aminoglycosides are antibiotics that have been extensively employed in animal husbandry for the treatment of bacterial infections, but also as growth promotion. The European Union has issued strict maximum residue levels (MRLs) for aminoglycosides in several animal origin products. The Brazilian government implemented a control plan that assesses the residues of veterinary medicines in animal products. A quantitative method based on liquid chromatography tandem mass spectrometry (LC–MS/MS) and a screening method by liquid chromatography mass spectrometry with time-of-flight system (LC-qTOF-MS) has been developed for simultaneous determination of the aminoglycosides spectonomycin, tobramycin, gentamicin, kanamycin, hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin and neomycin in milk and in bovine, poultry and swine muscles. A simple extraction method was developed using trichloroacetic acid and clean up with low temperature precipitation and C18 bulk. The LC–MS/MS and LC-qTOF-MS methods were validated according to the European Union Commission Directive 2002/657/EC. Adequate performance characteristics were obtained for recovery, precision, calibration curve, specificity, decision limits (CCα) and detection capabilities (CCβ) in all matrices tested. The detection limit (LOD) and limits of quantification (LOQ) was established from MRL ranging from 5 to 100 ng g-1 for LOD and 12.5 to 250 ng g-1 to LOQ. Recoveries ranged from 36.8 to 98.0% and the coefficient of variation from 0.9 to 20.2%, noting that all curves have been made into their own matrices in order to minimize the matrix effects which are intrinsic to the cases studied in this work. The CCβ values obtained in qualitative method were between 25 and 250 ng g-1, considered satisfactory for the analytes in those matrices. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.

A presença de compostos de uso veterinário em produtos de origem animal é uma grande preocupação em termos de saúde pública. A utilização descontrolada de antibióticos como medicamentos veterinários pode levar ao desenvolvimento de resistência bacteriana, podendo prejudicar a eficácia destas drogas no uso em humanos. Os aminoglicosídeos são antibióticos extensivamente utilizados na criação de animais para o tratamento de infecções bacterianas ou para a promoção do crescimento. A União Europeia estabeleceu níveis máximos de resíduos permitidos (LMRs) para os aminoglicosídeos em vários alimentos de origem animal. O governo brasileiro implementou um plano de controle que avalia os resíduos de medicamentos veterinários em produtos de origem animal. Neste trabalho, foram desenvolvidos método quantitativo por cromatografia a líquido acoplada a espectrometria de massas triplo quadrupolo em modo tandem (LC-MS/MS) e método de screening utilizando cromatografia a líquido acoplada a espectrometria de massas com analisador por tempo-de-voo (LC-qTOF-MS) para a determinação simultânea dos aminoglicosídeos espectinomicina, tobramicina, gentamicina, kanamicina, higromicina, apramicina, estreptomicina, diidroestreptomicina, amicacina e neomicina em leite e em músculo bovino, de aves e suíno. Como método de extração utilizou-se procedimento com ácido tricloroacético e clean up por precipitação a baixa temperatura e C18 a granel. Os métodos por LC-MS/MS e LC-qTOF-MS foram validados de acordo com a Diretiva da Comissão Europeia 2002/657/CE. Os resultados obtidos no método quantitativo para recuperação, precisão, linearidade, especificidade, limites de decisão (CCα) e capacidades de detecção (CCβ) nestas matrizes foram considerados satisfatórios. Os dados de limite de detecção (LOD) e limite de quantificação (LOQ) foram estabelecidos a partir dos LMRs, variando de 5 a 100 ng g-1 para LOD e 12,5 a 250 ng g-1 para LOQ. As recuperações variaram de 36,8 a 98%, e os coeficientes de variação ficaram entre 0,9 e 20,2%, observando-se que todas as curvas foram feitas com nas próprias matrizes a fim de minimizar os efeitos de matriz que são intrínsecos para os casos estudados neste trabalho. Os valores de CCβ obtidos no método qualitativo foram entre 25 e 250 ng g-1, considerados satisfatórios para os analitos nessas matrizes. O método proposto mostrou-se simples, fácil e adequado para a análise de um grande número de amostras por dia a um baixo custo. / The presence of several substances in animal products has been a major public concern. The use of antibiotics drugs as veterinary medicines might lead to the development of bacterial resistance, which might undermine the efficacy of these drugs in human use. The aminoglycosides are antibiotics that have been extensively employed in animal husbandry for the treatment of bacterial infections, but also as growth promotion. The European Union has issued strict maximum residue levels (MRLs) for aminoglycosides in several animal origin products. The Brazilian government implemented a control plan that assesses the residues of veterinary medicines in animal products. A quantitative method based on liquid chromatography tandem mass spectrometry (LC–MS/MS) and a screening method by liquid chromatography mass spectrometry with time-of-flight system (LC-qTOF-MS) has been developed for simultaneous determination of the aminoglycosides spectonomycin, tobramycin, gentamicin, kanamycin, hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin and neomycin in milk and in bovine, poultry and swine muscles. A simple extraction method was developed using trichloroacetic acid and clean up with low temperature precipitation and C18 bulk. The LC–MS/MS and LC-qTOF-MS methods were validated according to the European Union Commission Directive 2002/657/EC. Adequate performance characteristics were obtained for recovery, precision, calibration curve, specificity, decision limits (CCα) and detection capabilities (CCβ) in all matrices tested. The detection limit (LOD) and limits of quantification (LOQ) was established from MRL ranging from 5 to 100 ng g-1 for LOD and 12.5 to 250 ng g-1 to LOQ. Recoveries ranged from 36.8 to 98.0% and the coefficient of variation from 0.9 to 20.2%, noting that all curves have been made into their own matrices in order to minimize the matrix effects which are intrinsic to the cases studied in this work. The CCβ values obtained in qualitative method were between 25 and 250 ng g-1, considered satisfactory for the analytes in those matrices. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.

Os aminoglicosídeos são antibióticos utilizados para o tratamento de muitas infecções bacterianas graves. A maioria é produzida por microorganismos (gêneros Streptomyces e Actinomyces), mas a semi-síntese resultam na descoberta de notáveis aminoglicosídeos. Apesar de seu mecanismo de ação seletivo, os aminoglicosídeos são extremamente tóxicos. A nefrotoxicidade e ototoxicidade são mais freqüentemente observadas. Sabe-se que a Doença de Ménière pode ser tratada através da destruição seletiva das células vestibulares, preservando-se as células cocleares (tecidos da orelha interna). Antibióticos aminoglicosídeos são usados para esta finalidade, mas podem paralelamente causar danos cocleares (surdez). O estudo de relação estrutura-toxicidade dos resíduos de fragmentação de antibióticos aminoglicosídeos pode originar produtos simplificados, com atividade vestibular seletiva, dissociada da atividade coclear, mais seguros para o tratamento da Doença de Ménière. Em trabalhos anteriores, os ensaios envolvendo 2-desoxi-estreptamina e estreptidina demonstraram que não são tóxicos ao tecido coclear, quando comparados com os compostos originais. Neamina, outro fragmento de neomicina, se mostrou mais tóxica ao vestíbulo que a própria neomicina, mas aprensentou também grande toxicidade coclear. A substituição da unidade diamino-glicosídica de neamina, contendo o grupo 2-desoxi-estreptamina, por outras unidades glicosídicas (glicose, galactose, glicosamina) representa uma tentativa de eliminar a atividade cocleotóxica e manter a atividade vestibulotóxica original (100%). A mesma idéia pode ser também aplicada ao resíduo de estreptidina. Desta forma, foram sintetizados, dois pseudos-dissacarídeos, 2-desoxi-estreptamina ligado a galactose (48) e 2-desoxi-estreptamina ligado a glicose (49), ambas as ligações em posição referente ao carbono glicosídico anomérico. Apenas o pseudo-dissacarídeo 2-desoxi-estreptamina ligado a galactose (48) foi obtido com massa suficiente para analise ototóxica, o qual apresentou atividade vestibular seletiva como desejado, no tratamento da doença de Ménière. Ensaios de atividade antimicrobiana foram realizados empregando ambos pseudos-dissacarídeos sintetizados, 2-desoxi-estreptamina ligada a galactose (48) e 2-desoxi-estreptamina ligada a glicose (49), porém não apresentaram uma concentração inibitória mínima (MIC) significativa para as cepas testadas. / Aminoglycosides are antibiotics used for the treatment of many serious bacterial infections. Most are produced by microorganisms (genera Streptomyces and Actinomyces), but products obtained by semi-synthesis resulted in the discovery of remarkable aminoglycosides. Despite their selective mechanism of action, the aminoglycosides are highly toxic. The nephrotoxicity and ototoxicity are more frequently observed. It is known that Ménière\'s disease can be treated by selective destruction of the vestibular cells, preserving the cells cochlear (inner ear tissues). Aminoglycoside antibiotics are used for this purpose but may cause cochlear damage (deafness). The study of structure-toxicity of residues fragmentation of aminoglycoside antibiotics may lead to simplified products, with selective vestibular activity, dissociated from the cochlear activity, safer for the treatment of Ménière\'s disease. In previous work, the experiments involving 2-deoxy-streptamine and streptidine demonstrated that they are not toxic to the cochlear tissue, when compared with the original compound. Neamina, another fragment of neomycin, was more toxic to the vestibular tissue than neomycin, but also presented great cochlear toxicity. The replacement of the diamino-glycoside unit of neamina containing the 2-deoxy-streptamine by other glycosidic units (glucose, galactose, glucosamine) is an attempt to eliminate the cochlear toxicity and maintain the original vestibular toxicity (100%). The same idea can also be applied to the streptidine residue. Thus, two pseudo-disaccharides, 2-deoxy-streptamine linked to galactose (48) and 2-deoxy-streptamine linked to glucose (49), both linked to the position on the glycoside anomeric carbon. Only the pseudo-disaccharide 2-deoxy-streptamine linked to galactose (48) was obtained in sufficient quantity to perform the ototoxic assay, which presented selective vestibular activity as desired in the treatment of Ménière\'s disease. Antimicrobial activity assays were performed with both pseudo-disaccharides synthesized 2-deoxy-streptamine linked to galactose (48) and 2-deoxy- streptamine linked to glucose (49), but did not show a minimum inhibitory concentration (MIC) significant against the strains tested.

Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance. Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of an antibiotic. This makes ribonucleases a potential target for novel antibiotics. It was shown that Escherichia coli mutants that were deficient for RNase III, RNase E, RNase R, RNase G, or RNase PH had an increase in ribosomal subunit assembly defects. These mutant bacterial cells also displayed an increased sensitivity to neomycin and paromomycin antibiotics. My research has also shown that an inhibitor of RNases, vanadyl ribonucleoside complex, potentiated the effects of an aminoglycoside and a macrolide antibiotic in wild type Escherichia coli, methicillin sensitive Staphylococcus aureus, and methicillin resistant Staphylococcus aureus. RNases are essential enzymes in both rRNA maturation and degradation. Based on this and previous work, the inhibition of specific RNases leads to an increased sensitivity to antibiotics. This work demonstrates that the inhibition of RNases might be a new target to combat antibiotic resistance.

Due to the recent rise in antibiotic resistant pathogens, and the difficulties surrounding the quest for new antibiotics, many researchers have started revisiting antibiotic interactions in hopes of finding new treatment options. The primary outcome of this project was to examine the efficacy of concomitant antibiotic use under varying nutrient conditions, to identify variations in antibiotic interactions. Antibiotic interactions were studied, utilizing E. coli as a model bacterial system, grown in four different media types. E. coli cultures were treated with streptomycin, tobramycin, erythromycin, and amikacin individually and in a pairwise fashion at varying doses. We found that at least some antibiotic efficacies were dependent on the environmental nutrient conditions E. coli was grown in, as the antibiotics were not equally effective in all media types. E. coli grown in potato dextrose broth, in particular, showed extremely high tolerance to antibiotic inhibition. In addition, we observed several variations in antibiotic interactions, depending on the combination of antibiotics and environmental conditions utilized. It is predicted that differences in available nutrients is the primary cause of the observed discrepancies in antibiotic properties between media. The observation of changes in antibiotic efficacy under different environmental and nutrient conditions has serious implications for use of antibiotic combinations as drug treatments. Not all microenvironments within the human body have identical nutrient make-up. If the interactions antibiotics are reported to have in one environmental condition change under another, reckless prescription of combinations could lead to a serious adverse reaction. Thus, this is an important area for future in vitro and in vivo research.

Bacterial infections are a major cause of morbidity and mortality in both the community and nosocomial settings, particularly among the elderly and chronically ill. Sepsis is the body’s response to antigens and toxins released by the invasive pathogenic organisms that cause infection. When infection is not effectively controlled, sepsis may develop and progress to severe sepsis and septic shock. Early diagnosis and treatment is pivotal for survival in severe sepsis and particularly, septic shock. Our research focuses on developing a novel treatment strategy for septic shock by using single, ultra-high doses of aminoglycosides. In this project, the effect of a single, ultra-high dose of gentamicin in clearing bacteria from the blood and reducing the bacterial burden in vital organs was evaluated in a rat model of E. coli (Bort strain) induced peritonitis with severe sepsis/septic shock. Serum cytokine levels and serum lactate levels were serially measured. Further, the potential adverse effects of ultra-high dosing of aminoglycoside antibiotics in a short-term (9 h) invasive study and long term (180 days) non-invasive study were assessed. Neuromuscular paralyses due to ultra-high doses of aminoglycosides were assessed. In addition, renal injury markers such as serum creatinine and urinary Neutrophil Gelatinase Associated Lipocalin (NGAL) were assayed. The auditory and vestibular function was also assessed after ultra-high dosing of aminoglycoside in the long-term study. We conclude that animals can tolerate ultra-high doses of aminoglycosides with appropriate support. Animals were under neuromuscular paralysis for 28 – 50 minutes and were on ventilator support after single ultra-high doses (80 and 160 mg/kg) of aminoglycoside antibiotics (gentamicin and tobramycin). There was no significant acute or delayed renal or ototoxicity associated with the single, ultra-high dose aminoglycoside therapy. Histology studies of the kidneys and the cochlea of single, ultra-high aminoglycoside dosed animals and untreated control animals were performed after 180 days (6 months). Results indicated that there were no morphological differences between the treated and untreated control animals. Terminal deoxy-nucleotidyl transferase dUTP nick end labeling (TUNEL) assay of kidney tissue indicated that there was no apoptosis of endothelial cells in the tubular and glomerular regions with single, ultra- high dose of aminoglycosides consistent with an absence of ultrahigh dose induced nephrotoxicity. In the septic shock model, the E. coli Bort was below the limit of detection from the blood of the animals within minutes after single, ultra-high dose aminoglycoside administration. After necropsy, bacterial load was determined from all the vital organs and peritoneal fluid (site of infection). The bacterial levels were below the detection limit from the kidneys and there was a significant reduction in bacterial counts from all the remaining organs compared to the infected control animals. A decrease in serum cytokine and serum lactate levels compared to baseline was observed after ultra-high doses of aminoglycosides in the septic shock animals. Our studies have indicated that the ultra-high dose gentamicin is well tolerated by rats. It is highly effective in clearing E. coli Bort from the blood and reducing the bacterial burden in the organs in an experimental model of bacterial peritonitis/septic shock. Further studies in larger animals such as rabbits, sheep, pigs or dogs are required to confirm these results. If these findings are replicated in larger animals, this therapy may be developed further from ‘lab to bedside’ to treat septic shock patients in intensive care units (ICUs). / October 2015