Neste trabalho desenvolveu-se um sensor eletroquímico a partir da modificação de superfície de um eletrodo de carbono vítreo com nanopartículas metálicas de Pt e intermetálicas de NiSb suportadas em carbono Vulcan® XC72. Tal modificação visou à obtenção de um sensor de baixo custo, simples e rápido para determinação do cloridrato de tetraciclina (TTC). A tetraciclina é um fármaco com propriedades antibióticas amplamente utilizado na saúde humana e animal devido sua baixa toxicidade e fácil obtenção. A modificação de superfície do eletrodo de trabalho foi realizada a partir da adição de uma suspensão das nanopartículas em dimetilformamida (DMF) (1 mg/1 mL) sobre a superfície do eletrodo previamente limpo e os estudos voltamétricos conduzidos utilizando as técnicas de voltametria cíclica, voltametria de pulso diferencial e cronoamperometria. Como consequência dos resultados obtidos, prosseguiu-se apenas com os estudos da nanopartícula de Pt na construção de um sensor para determinação da TTC. As nanopartículas apresentam propriedades diferentes daquelas exibidas pelo mesmo material quando estes se encontram em partículas de maior tamanho como bulks, e dentre suas principais características, o aumento da área superficial provocado pela modificação do eletrodo mostrou resultados promissores na detecção e quantificação do analito. Após diversos estudos sobre o eletrólito de suporte e o pH, obteve-se a melhor resposta voltamétrica em solução de tampão fosfato 0,10 mol L-1 pH 3,0 através da voltametria cíclica tanto na determinação do analito em água purificada quanto na determinação em urina sintética. O eletrodo modificado com nanopartículas de Pt apresentou resposta linear na faixa de 9,99 e 44,01 µmol L-1, limite de detecção 4,28 µmol L-1 e limite de quantificação 14,26 µmol L-1. A aplicação do eletrodo em uma amostra de urina sintética ocorreu com sucesso, alcançando um valor de concentração com diferença de 3% para o valor real. / In this work it was developed an electrochemical sensor by the surface modification of a glassy carbon electrode with metallic Pt nanoparticles and intermetallic NiSb nanoparticles in Vulcan® XC72 carbon as the support. Such surface modification sought to obtain a low cost sensor, easy to work and quick working to determine tetracycline hydrochloride. The tetracycline is a drug with antibiotics properties widely used in human and animal treatment due to its low toxicity and easy obtainability. The surface modification of the work electrode was accomplished through the addition of a suspension of the nanoparticles in dimethylformamide (1 mg/1 mL) in the electrode surface previously cleaned and the voltammetric studies conducted with the use of techniques such as cyclic voltammetry, differential pulse voltammetry and chronoamperometry. As a consequence of the obtained results, only the studies with the sensor developed with Pt nanoparticles were continued for the tetracycline determination. The nanoparticles presents different properties of those shown by the same material when these are in bigger forms, such as bulks and among the main characteristic, the superficial area increase caused by the electrode modification shown promising results for the detection and quantification of the analyte. After several studies about the supporting electrolyte and the pH, it was obtained a better voltammetric response in phosphate buffer solution 0.1 mol L-1 and pH 3.0 for the determination of the analyte in purified water and synthetic urine. The modified electrode with metallic Pt nanoparticles had a linear response in the range 9.99 to 44.01 µmol L-1 with a detection limit of 4.28 µmol L-1 and quantification limit of 14.26 µmol L-1. The electrode application in a synthetic urine sample was a success, reaching concentration values with a difference within 3% of the real value.

Pesquisas prévias evidenciaram que a desmineralização óssea com ácido cítrico melhora a consolidação de enxertos autógenos em bloco e favorece o espalhamento e a morfologia de pré-osteoblastos em cultura. Os resultados promissores encontrados com o ácido cítrico levantaram a suspeita de que a tetraciclina ácida pudesse suscitar efeitos semelhantes. Assim, este estudo se propôs a avaliar comparativamente o comportamento de células pré-osteoblásticas MC3T3-E1 cultivadas sobre superfícies ósseas de calvária de ratos desmineralizadas com tetraciclina ácida (50mg/mL) e com ácido cítrico (10%, pH1) em diferentes tempos de aplicação. Foram removidas 126 amostras ósseas bicorticais da calvária de 63 ratos Wistar adultos machos empregando broca trefina de 5 milímetros de diâmetro. As amostras foram distribuídas aleatoriamente em um dos seguintes grupos de estudo (n=18): AC 15 no qual as amostras foram desmineralizadas por ácido cítrico durante 15 segundos; AC 30, no qual as amostras foram desmineralizadas por ácido cítrico durante 30 segundos; AC 60, no qual as amostras foram desmineralizadas por ácido cítrico durante 60 segundos; TCN 15 no qual as amostras foram desmineralizadas por tetraciclina durante 15 segundos; TCN 30, no qual as amostras foram desmineralizadas por tetraciclina durante 30 segundos; TCN 60, no qual as amostras foram desmineralizadas por tetraciclina durante 60 segundos e C, grupo controle formado por amostras não desmineralizadas. Os pré-osteoblastos foram cultivados sobre as amostras por 24, 48 e 72 horas (n= 6) para serem examinadas à microscopia eletrônica de varredura. Vinte e uma amostras coletadas de outros 11 animais foram distribuídas entre os mesmos grupos e analisadas com espectroscopia de energia dispersiva (EDS) para análise da composição química superficial (n=3). A área de recobrimento superficial por células foi significantemente maior após 24 e 48 horas de cultura nos grupos AC15 (60,38% e 100% respectivamente), AC30 (99,32% e 100% respectivamente), AC60 (99,22% e 100% respectivamente), TCN15 (96,73% e 70,24% respectivamente) e TCN30 (64,72% e 57,40% respectivamente) do que nos grupos TCN60 (9,67% e 51,45% respectivamente) e C (5,99% e 31,83% respectivamente). Às 72 horas os grupos apresentaram recobrimento praticamente completo das superfícies ósseas por células, com exceção do grupo TCN60 (56,15%). As células apresentaram-se com morfologia compatível com em estágios mais avançados de diferenciação nos grupos que sofreram desmineralização do que no controle. As variações nas porcentagens anatômicas (A%) dos elementos C, O, Na, Mg, P e Ca foram insuficientes para justificar mudanças no comportamento celular. Concluiu-se que a desmineralização quer por ácido cítrico ou tetraciclina de superfícies ósseas são favoráveis para o crescimento e diferenciação de células pré-osteblásticas especialmente quando empregada conforme os grupos AC30 e TCN15. Os mecanismos por trás desses resultados ainda carecem de elucidação. / Previous researches have demonstrated that bone demineralisation by citric acid improves the consolidation of bone autografts and promotes spreading and morphology of pre-osteoblasts in culture. The promising results with citric acid raised the suspicion that tetracycline could elicit similar effects. Thus, the aim of this study was to comparatively evaluate the behavior of pre-osteoblastic MC3T3-E1 cultured on bone surfaces of rat calvaria demineralized with tetracycline (50mg / ml) and citric acid (10%, pH1) at different times of application. 126 bicortical samples were removed from the calvarial bone of 63 adult male Wistar rats using trephine drill of 5 mm in diameter. Samples were randomly assigned to one of the following study groups (n = 18) AC 15 in which the samples were demineralized by citric acid for 15 seconds; AC 30, in which the samples were demineralized by citric acid for 30 seconds; AC 60 wherein the samples were demineralized by citric acid for 60 seconds; TCN 15 in which the samples were demineralized tetracycline for 15 seconds; TCN 30, in which the samples were demineralized tetracycline for 30 seconds; TCN 60 wherein the samples were demineralized tetracycline for 60 seconds, and C, control group of samples not demineralized. The pre-osteoblasts were cultured on the samples for 24, 48 and 72 hours (n = 6) to be examined in the scanning electron microscope. Twenty-one samples were collected from other 11 animals and were distributed among the same groups for analysis of the surface chemical composition by energy dispersive spectroscopy (EDS) (n = 3). The average percentage of the bone surfaces covered by cells was significantly higher after 24 and 48 hours of culture in groups AC15 (60.38% and 100% respectively), AC30 (99.32% and 100% respectively), AC60 (99.22% and 100% respectively) TCN15 (96.73% and 70.24% respectively) and TCN30 (64.72% and 57.40% respectively) than in groups TCN60 (9.67% and 51.45% respectively), and C (5.99% and 31.83% respectively). At 72 hours, all the groups presented almost complete covering of the surfaces by cells, with the exception of TCN60 group (56.15%). Cells presented with morphology compatible with more advanced stages of differentiation in groups undergone to demineralization than control. Variations in the anatomical percentages (A%) of the elements C, O, Na, Mg, Ca and P were insufficient to justify changes in cell behavior. The conclusion was that both demineralization of citric acid or tetracycline are favorable for the growth and differentiation of pre-osteoblasts especially when used according to AC30 and TCN15 groups. The mechanisms behind these results still need elucidation.

Developing treatments for antibiotic resistant bacterial infections is among the most urgent public health challenges worldwide. Tetracyclines are one of the most important classes of antibiotics, but like other antibiotics classes, have fallen prey to antibiotic resistance. Key small changes in the tetracycline structure can lead to major and distinct pharmaceutically essential improvements. Thus, the development of new synthetic capabilities has repeatedly been the enabling tool for powerful new tetracyclines that combatted tetracycline-resistance. Traditionally, tetracycline antibiotics were accessed through bacterial natural products or semisynthetic analogs derived from these products or their intermediates. More recently, total synthesis provided an additional route as well. Importantly however, key promising antibiotic candidates remained inaccessible through existing synthetic approaches. Heterologous biosynthesis is tackling the production of medicinally important and structurally intriguing natural products and their unnatural analogs in tractable hosts such as Saccharomyces cerevisiae. Recently, the heterologous biosynthesis of several tetracyclines was achieved in Streptomyces lividans through the expression of their respective biosynthetic pathways. In addition, the heterologous biosynthesis of fungal anhydrotetracyclines was shown in S. cerevisiae. This dissertation describes the use of Saccharomyces cerevisiae towards the biosynthesis of target tetracyclines that have promising prospects as antibiotics based on the established structure-activity relationship of tetracyclines but have been previously synthetically inaccessible. Chapter 1 provides an introduction to the pursuit of tetracycline antibiotics using S. cerevisiae. Following an overview of tetracycline drugs, the chapter describes the methods for making tetracyclines and their limitations in accessing the tetracycline analogs targeted in this study. The desirability of making these target analogs as well as key desired properties are then exemplified by natural products, totally synthetic and semisynthetic derivatives. The target tetracycline analogs pursued in this study are then outlined and the considerations in choosing their desired properties are discussed, as well as the reasons for employing S. cerevisiae in their synthesis. Chapter 2 describes the use of Saccharomyces cerevisiae for the final steps of tetracycline biosynthesis, setting the stage for total biosynthesis of tetracyclines in Saccharomyces cerevisiae. Chapter 3 describes the work towards biosynthesis of the target tetracycline analogs using Saccharomyces cerevisiae, utilizing successful expression optimization and gene biomining approaches. Chapter 4 describes the work towards the target tetracycline analogs from fungal anhydrotetracyclines in Saccharomyces cerevisiae. The challenge of enzyme evolution towards unnatural substrates and the complex environment of cells require metabolic engineering efforts to be performed in libraries, as it is currently impossible to predetermine which modifications will prove beneficial. Traditional methods in DNA mutagenesis and increasingly, advances in DNA synthesis, DNA assembly and genome engineering are enabling high throughput strain construction. Thus, there is a need for a general, high-throughput, versatile and readily implemented assay for the detection of target molecule biosynthesis. The development of such an assay is described in Chapter 5. The assay is demonstrated to detect tetracycline derivatives, and differentiate a producer and a nonproducer strain of the fungal anhydrotetracycline TAN-1612. The yeast three hybrid assay for metabolic engineering of tetracycline derivatives described in this chapter could be used in the next steps towards the heterologous biosynthesis of the target tetracycline analogs in S. cerevisiae and beyond.

Thesis (Master of veterinary science)--Washington State University, December 2007. / Includes bibliographical references (p. 13-17).

Angelman syndrome (AS) is a rare genetic disorder affecting 1/10,000 to 1/20,000 births. This disorder arises through the genetic disruption of the maternal UBE3A allele, which when coupled with epigenetic silencing of the paternal allele UBE3A allele, gives rise to an absence of UBE3A protein in the central nervous system. Clinical manifestations of the syndrome vary in severity and include poor motor function, deficits in language and severe intellectual impairments. Previous research in the Angelman syndrome mouse model revealed abnormalities in dendritic spine density and morphology of hippocampal pyramidal cells. As seen in humans with AS, mice show abnormal behavioral characteristics that include deficits in motor coordination and ability as well as hippocampal dependent associative fear conditioning. Physiologically, these animals exhibit severe deficits in long-term potentiation (LTP) when compared to wildtype littermates. In an attempt to reduce the time from laboratory study to human translation, we began to search a small molecule library for established compounds with the ability to improve the behavioral and physiological defects normally associated with the AS mouse. One compound, minocycline, was found to normalize the density of dendritic spines in the hippocampus as well as recover the associative memory of AS mice. Moreover, a significant increase in LTP after theta-burst stimulation was also observed in area CA1 hippocampal pyramidal neurons of AS mice treated with minocycline when compared to saline vehicle control mice. These results suggest treatment with minocycline improves synaptic function and learning and memory of AS mice and may provide similar improvements to patients with Angelman syndrome. Twenty-five participants ages 4-12 were enrolled in a clinical trial examining the safety and tolerability of minocycline in children with Angelman syndrome. Patients were evaluated at 3 time points, baseline (T1), after 8 weeks of treatment (T2) and again 8 weeks after the drug was discontinued (T3). Each evaluation was identical and included laboratory testing, EEG recording and neuropsychological examination. Results of the study showed minocycline was safe and well tolerated with only minor adverse effects reported. While no change was observed in EEG recordings, significant increases in the mean clinical global impressions severity scale score were observed after treatment with minocycline. Moreover, participants showed significant improvement in the raw scores of the communication and self-care domains of the Bayley Scale of Infant and Toddler Development, 3rd Edition. These results show for the first time, a therapeutic with the ability to improve the characteristic behaviors of Angelman syndrome. Unfortunately, currently available neuropsychological measures were found to be insensitive to the behavioral phenotype of AS. The primary outcome measure, the Bayley Scale of Infant and Toddler Development, 3rd Edition relies on verbal communication and for the examinee to perform specific tasks on command. These testing methods are not compatible with this patient population and resulted in raw scores outside of 2 standard deviations from the mean. The inability of the participants to perform on these exams led us to develop a novel outcome measure; one that relies on observation rather than verbal communication. 9 children with AS and 7 healthy children were enrolled in an observational study in which 30 minutes of free play activity was video recorded. Using behavioral coding software, 3 independent raters quantified stereotypical AS behaviors as well as communication methods. Speech attempts were categorized into five difference types of vocalizations and revealed children with AS used less advance forms of vocalization consisting mostly of phonation control. Phonetic inventories show mostly front or back vowel usage suggesting little tongue movement occurs during speech production. These results suggest deficits in verbal ability may be related to a childhood apraxia of speech. Impairments in balance and motor coordination have been associated with AS. In an attempt to measure gross motor function, spatiotemporal gait parameters were collected using an electronic walkway and gait analysis software. Results show the gait of children with AS most resembles that of patients with ataxia but without cognitive impairment. In an attempt to develop a single quantitative measure able to describe the severity of gait-related disability, statistical methods were used to create a gait index for patients with AS. The results of this study provides AS researchers with the tools necessary to accurately measure changes in behavior and gait during the clinical evaluation of potential therapeutics

Tetraciklinų nustatymas piene atliktas efektyviąja skysčių chromatografija ir įteisintas remiantis ES išleista direktyva 2002/657/EB. Metodas atitinka visus kriterijus aprašytus šioje direktyvoje. Antibakterinių medžiagų likučiai iš pieno ekstrahuojami Mcllvaine-EDTA buferiniu tirpalu, o po to valymas atliekamas naudojant OASIS HLB kietafazės ekstrakcijos kolonėles. Chromatografinėje kolonėlėje atskyrimas atliekamas naudojant 10 mM oksalo rūgšties vandeninio tirpalo ir ACN gradientinį režimą, tėkmės greitis 0,9 ml/min, 100 μl injekcija, bangos ilgis 355 nm ir optimali kolonėlės temperatūra (LiChroCART Superspher 60 RP C8 (250 x 4,6 mm, dalelių dydis 5 m)) – 30 oC. Visi rezultatai apskaičiuoti naudojant kompiuterinę duomenų apdorojimo programą “Interval”. Tetraciklinų išgavos nuo 86,20 % iki 107,19 %, o nustatymo ribos yra intervale nuo 26,7 μg/l iki 49,3 μg/l. / Analysis of tetracycline compounds was performed using high performance liquid chromatography and validated according to EU commition decision 2002/657/EC. Method is compliant at all criteria described in the decision. Antibacterial material residues were extracted from milk with Mcllvaine-EDTA buffer followed by clean-up on OASIS HLB cartridges. Separation was performed using 10 mM oxalic acid/ACN gradient with flow rate 0.9 ml/min, 100 μl injection, detection at 355 nm and the optimal temperature of column (LiChroCART Superspher 60 RP C8 (250 x 4.6 mm, size of particles 5 m)) – 30 oC. All the results were evaluated statistically with “Interval” data processing program. Recoveries are at the range from 86.20 % to 107.19 %. The determination limits for tetracyclines were carried out from 26.7 μg/L to 49.3 μg/L in this study.

Antibiotic resistance has become a major problem in the United States. Approximately 2 million people are affected by hospital-acquired infections. Each year about 90,000 people are killed from them. Of the infections 70% of them are resistant to at least one drug. In order to trigger antibiotic resistance in bacteria, the antibiotics need to be detected by sensors in the bacteria. Riboswitches may act as toxin sensors in bacteria. Riboswitches are RNA aptamers that regulate gene expression via allosteric structural changes triggered by binding of a small molecule. Most identified riboswitches specifically recognize the metabolic product of the gene to be regulated. When the concentration of the metabolite reaches its threshold it binds to the riboswitch causing a structural change that in most cases turns off transcription or translation of the metabolite-producing gene. The ykkCD riboswitch appears to recognize the antibiotic, tetracycline to up-regulate expression of an efflux pump (also called ykkCD) that exports toxic drugs from the bacterial cell. In this work we present initial characterization of the previously uncharacterized ykkCD riboswitch. With the help of tetracycline derivatives and mutagenesis studies on the riboswitch we will (1) determine the substrate specificity of this riboswitch; (2) assess the importance of aromatic character and/or functional groups in antibiotic recognition. To achieve this goal we have developed a fluorescent binding assays. The binding assays will measure the binding affinity (Kd) of the riboswitch-antibiotic complex. Since substrates of the efflux pump are toxic to the bacterial cell, we posit that the ykkCD riboswitch might work as a toxin sensor and could serve as a target in the fight against bacterial pathogens. Afterwards we will evaluate how the ykkCD riboswitch sensor works in vivo. In order to do this we will have to quantify the amount of protein produced in the presence of tetracycline derivatives and mutant sensors. However quantifying the level of a particular protein in the cell is difficult so instead we replace the sequence of the efflux pump with the B-gal gene in B subtilis cell and quantify B-gal enzymatic activity using a colorimetric assay. This is a widely used technique in which the fluorescence corresponds to how much protein is produced. / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

Riboswitches are naturally occurring RNA aptamers that form a precise three-­dimensional structure and selectively bind to cellular target molecules. Binding of the target molecule initiates an allosteric structural change in the riboswitch that in turn regulates expression of a relevant target gene. Most riboswitches specifically recognize the metabolic product of the gene that is being regulated. Expression may be regulated at either transcription or translation stage of gene expression. Most riboswitches are off switches meaning they turn off expression of metabolite producing gene when metabolite concentration is high enough. The ykkCD putative riboswitch appears to increases production of an efflux pump that expels toxic drugs from the cell by binding to the antibiotic tetracycline. Based on previous data collected the ykkCD putative riboswitch seems to regulate the efflux pump at the transcriptional level. To confirm this hypothesis we want to map the structural change that takes place upon binding of the antibiotic tetracycline to the mRNA. Nucleic acid footprinting studies will be used to map the binding site of tetracycline and the allosteric change that takes place upon tetracycline binding. / Department of Chemistry

Thesis (M.Sc. (Biochemistry))--Mahidol University, 1986.