Thesis (M.S.)--Michigan State University. Dept. of Microbiology & Molecular Genetics, 2008. / Title from PDF t.p. (viewed on July 29, 2009) Includes bibliographical references (p. 162-166). Also issued in print.

Thesis (Ph. D)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-137).

Resumo: Objetivos: Dois estudos controlados cegos aleatórios foram realizados para avaliar os resultados clínicos e microbiológicos iniciais e a longo prazo, da tetraciclina aplicada com o microbrush no tratamento de sítios com bolsa periodontal persistente 5mm e sangramento à sondagem, tratados previamente por raspagem e alisamento radicular (RAR). Material e método: Quarenta e dois pacientes com pelo menos 4 dentes com bolsas periodontais 5 mm e sangramento à sondagem foram divididos em 2 grandes grupos: tetraciclina e controle. Nos grupos tetraciclina, dois dentes receberam aplicação de solução de cloridrato de tetraciclina (100mg/mL) com microbrush uma vez por semana por 4 semanas (grupo T) e os outros dois dentes receberam o mesmo tratamento associado a uma sessão de RAR na primeira consulta (grupo RAR+T). Nos grupos controle, dois dentes receberam uma sessão de RAR (grupo RAR) e outros dois dentes receberam aplicação de solução salina com microbrush uma vez por semana por 4 semanas associada a uma sessão de RAR na primeira consulta (grupo RAR+S). Medidas clínicas de índice de placa visível (IP), índice de sangramento gengival (IG), sangramento à sondagem (SS), profundidade de sondagem (PS), nível de inserção clínica (NIC), recessão gengival (RG), e coletas microbiológicas para identificação das bactérias Aggregatibacter actinomycetemcomitans (Actinobacillus actinomycetemcomitans), Porphyromonas gingivais, Prevotella intermédia, Prevotella nigrescens, Tannerella forsythia, e Treponema denticola foram tomadas no baseline, 30, 90 e 180 dias após o tratamento. Amostras de biofilme subgengival foram analisadas por reação de polimerase em cadeia. Resultados: Todas as terapias resultaram em melhora na PS, NIC e SS, embora as diferenças clínicas entre os grupos não tenham alcançado significância estatística. (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Objectives: Two randomized controlled single-blind studies were carried out to evaluate the initial and the long term clinical and microbiological results of tetracycline applied with a microbrush in treatment of sites with persistent periodontal pockets 5mm and bleeding on probing previous treated by scaling and root planning (SRP). Methods: For each patient in the test groups, two teeth received 4 applications of tetracycline solution (100mg/ml) with a microbrush once a week (T group) while the other two teeth received the same treatment plus one session of SRP at the first appointment (SRP+T group). For each patient in the control groups, two teeth received one session of SRP (SRP group) and the other two received 4 applications of saline with a microbrush once a week plus one session of SRP (SRP+S group). Clinical measurements were taken at baseline, 30, 90 and 180 days after last treatment. Subgingival biofilme samples were analyzed by polymerase chain reaction for Aggregatibacter actinomycetemcomitans (Actinobacillus actinomycetemcomitans), Porphyromonas gingivais, Prevotella intermédia, Prevotella nigrescens, Tannerella forsythia, e Treponema denticola identification. Results: All therapies provided significant improvement in probing depth, clinical attachment level and bleeding on probing, although the clinical difference did not reach statistically significance. At 1 month post-therapy the prevalence of P. gingivalis was significantly reduced in groups that received SRP plus additional therapy and T. forsythia was significantly reduced in only test groups. Until 6 months P. gingivalis, T. forsythensis,and A. actinomycetemcomitans significantly decrease in prevalence only for tetracycline groups. Conclusion: The results of the present study suggest that tetracycline solution applied with a microbrush may be an alternative in management of persistent periodontal pockets with further microbiologic benefits. / Orientador: Rosemary Adriana Chiérici Marcantonio / Coorientador: Denise Madalena Palomari Spolidorio / Banca: Adriana Campos Passanezi Sant'Ana / Banca: José Roberto Cortelli / Banca: Sérgio Luiz de Souza Salvador / Banca: Silvana Regina Perez Orrico / Doutor

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.

Class of 2015 Abstract / Objectives: To assess the solubility and stability of tetracycline in compounded miracle mouthwash solutions over time, and at different temperatures (room temperature versus refrigerated) and pH (unaltered versus pH 7). Methods: Miracle mouthwash (MMW) solutions were compounded using tetracycline HCl capsules and 1:1 pseudo-dexamethasone elixir and diphenhydramine. High-performance liquid chromatography (HPLC) was used to measure the tetracycline concentrations in the MMW samples tested. Data on tetracycline crystal composition over time were also collected using powder x-ray diffraction, differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA). Results: For the tetracycline MMW solutions stored at room temperature, only 16% of the original tetracycline remained in solution after 24 hours, stabilizing at 65-81 mcg/mL on day 5 then decreasing further down to 45 mcg/mL by day 15. Similar results were obtained for the refrigerated tetracycline MMW solution (11% of original concentration after 5 days, with a decrease from 31-54 mcg/mL on day 5 to 22 mcg/mL on day 15). Tetracycline concentrations appeared to undergo a steeper decline in MMW solutions of pH 7 than in unadjusted MMW solutions (pH 4.68). All MMW samples exhibited a conversion from tetracycline HCl to tetracycline hexahydrate. Conclusions: Tetracycline solubility decreases rapidity in MMW within 24 hours of compounding regardless of temperature. MMW solutions at pH 7 may have further reduced solubility. Stability decreases at a stable rate from tetracycline HCl to tetracycline hexahydrate.

Orientadores: Lucia Helena Innocentini Mei, Sergio Persio Ravagnani / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Quimica / Made available in DSpace on 2018-08-04T01:52:20Z (GMT). No. of bitstreams: 1 Cruz_MariaClaraPinto_D.pdf: 5943553 bytes, checksum: b12150e79224fd5dadd1465609f322d1 (MD5) Previous issue date: 2004 / Resumo: Neste trabalho as análises de difusão de oxitetracilina (OTC) como espécie ativa, foram realizadas a 37 +/- 0,5 °C; em soluções tampão pH=7,4 e 1,2, condições estas similar ao trato gastro-intestinal, com microesferas de alginato de cálcio; microcápsulas de alginato de cálcio coacervado com quitosana (baixa, média e alta viscosidade); microcápsulas de alginato de cálcio coacervada com a quitosana baixa viscosidade, e revestidas com o polietileno glicol - PEG (2000, 4600 e 10000); microcápsulas de alginato de cálcio revestidas com o PEG e, finalmente, microcápsulas de alginato de cálcio revestidas com EUDRAGIT®. Na primeira parte do trabalho, priorizou-se o estudo da estrutura, sequência e composição das unidades repetitivas dos biopolímeros alginato e quitosana, as quais certamente exercem profunda influência na liberação controlada do fármaco. Na segunda parte, o coeficiente de difusão (D) de OTC foi calculado através de equações estabelecidas por Crank, para difusão em esferas, que seguem a segunda lei de Fick. Considerou-se a difusão do interior para o exterior das micropartículas, utilizando o método dos mínimos quadrados e método iterativo de Newton Raphson para ajuste dos dados. Foi verificado que o modelo matemático para a difusão de OTC tem uma representatividade muito boa em meio básico, mesmo com o efeito de disparo de OTC em forma de cristais alojados na superfície das micropartículas, conforme observado por Microscopia Eletrônica de Varredura (MEV). No entanto, em meio ácido, o ajuste não foi o esperado, pois fatores como a alta solubilidade do fármaco no meio; além da presença de fissuras nas microesferas, devido aos cristais na superfície, contribuíram para que o perfil desviasse do obtido em outros meios, resultando em um comportamento não-Fickiano. Finalmente, observou-se a possibilidade de modulação da velocidade de liberação nos diversos tipos de microesferas / Abstract: Diffusion studies of oxytetracycline (OTC) entrapped in microbeads of calcium alginate, calcium alginate coacerved with chitosan (high, medium and low viscosity) and calcium alginate coacerved with chitosan low viscosity, covered with Poly (ethylene glycol) - PEG (2 000, 4 600 and 10 000) and alginate covered with EUORAGITI®, were carried out at 37 +/- 0.5 °C, in buffer solutions at pH 7.4 and pH 1.2, similar to the conditions of the gastric-intestinal system. The diffusion coefficient, or diffusivity (D), of OTC was calculated by equations provided by Crank for diffusion, which follows Fick's second law, considering the diffusion from the inner parts to the surface of the microbeads. The least square and the Newton Raphson methods were used to obtain the diffusion coefficients. The microbeads swelling in pH 7.4 and OTC diffusion is classically Fickian, suggesting that the OTC transport, in this case, is controlled by the exchange rates of free water and relaxation of calcium-alginate chains. In case of acid media, it was observed that the phenomenon did not follow Fick's law, due probably to the high solubility of the OTC in this environment and the presence of cracks formed during the drying process of the microbeads. It was possible to model the release rate of OTC in several types of microbeads / Doutorado / Ciencia e Tecnologia de Materiais / Doutor em Engenharia Química

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Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised. / Doctor of Philosophy / Antibiotic-resistant bacteria are increasingly recognized as a threat to global health, and new antibacterial drugs are urgently needed. Before a chemical compound can advance far in the journey to becoming a new drug it must be tested for toxicity against mammalian cells. A portion of this dissertation research involved testing the toxicity of several organometallic compounds (OMCs) previously shown to have antibacterial potential. Mouse-derived mammalian cells were treated with several of the OMCs, and initial results indicated that one of the OMCs is non-toxic and is likely a safe option for additional analysis. This OMC was further tested to see if it could inhibit mycobacterial growth inside of the mammalian cells. It did not effectively clear bacteria from inside of the mammalian cells, likely because of poor penetration of the cell membrane. Further research with this compound should focus on ways to effectively transport the OMC inside infected mammalian cells so that it can reach the bacteria it is meant to target. A second portion of this research involved using a peptide nucleic acid (PNA) to try and reverse tetracycline antibiotic resistance in the bacterial strain Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Peptide nucleic acids are short linear molecules that can bind strongly to complementary DNA and RNA sequences and thus be used to interfere with expression of specific genes. A PNA was designed to inhibit expression of the bacterial tetA gene that codes for a protein called the TetA tetracycline efflux pump, which imparts resistances to tetracycline. Treating the bacteria with the PNA resulted in a lower dose of tetracycline needed to inhibit bacterial growth, indicating a successful increase in tetracycline susceptibility. By using a molecular analysis technique called reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), it was possible to measure the amount of tetA messenger RNA (mRNA) in cultures of DT104 treated only with tetracycline or with a combination of tetracycline and the PNA. As expected, bacteria treated with both the antibiotic and the PNA had less tetA mRNA than the cultures treated only with tetracycline, supporting the hypothesis that the PNA prevents the bacteria from effectively expressing the tetA gene. The PNA was next used in conjunction with tetracycline as an experimental treatment for mice infected with DT104. The PNA did not provide the expected protective effect under these circumstances. The overall conclusion for this part of the research is that PNAs offer an exciting potential avenue for counteracting antibiotic resistance, but additional experimentation is needed. Future research should focus on investigating more effective ways to get the PNAs inside the bacteria and on understanding more about how the PNAs behave in live animals. Several other PNAs targeting different genes involved in antibiotic resistance or essential bacterial functions were also tested against DT104 with variable success.

Considerando a crescente contaminação da água e os vários problemas ao meio ambiente e a saúde humana decorrentes dessa contaminação, os produtos farmacêuticos e de higiene pessoal, como antibióticos e outros, constituem uma grande preocupação, pois não são completamente removidos nos sistemas de tratamento de esgoto, além de serem resistentes à biodegradação. Antibióticos como a tetraciclina (TeC), por exemplo, são excretados em grande parte eliminados pela urina e/ou fezes, sendo cada vez mais detectados em uma grande variedade de matrizes ambientais, causando inúmeros efeitos tais como, alergias e aumento da resistência de bactérias. Assim, esse estudo visou a degradação da TeC em meio de urina artificial por método eletroquímico utilizando um ânodo dimensionalmente estável (ADE), o qual foi selecionado pela alta concentração de cloreto no meio. Foram realizados estudos de densidade de corrente e pH inicial, avaliando a concentração remanescente de TeC, creatinina, uréia e COT e, comparando os resultados com os obtidos em meio aquoso contendo NaCl 0,1 mol L-1. A TeC sofreu degradação eletroquímica devido à eletrogeração de espécies oxidantes de cloro ativo a partir do cloreto presente no meio. O decaimento da concentração da TeC ajustou-se ao modelo de cinética de pseudo-primeira ordem e aumentou como o aumento da densidade de corrente aplicada. O tratamento eletroquímico pode ser realizado em valores de pH próximos ao neutro, em que a TeC na forma aniônica e a espécie HOCl, favorecem a degradação, mesmo na presença de EDTA. A creatinina e a uréia interferem no processo eletroquímico, fazendo com que a degradação da TeC apresente velocidades de reação mais baixas e o consumo energético do processo seja mais elevado do que em meio aquoso contendo NaCl. / Considering the increase contamination of water and various environmental and human health problems resulting from this contamination, the pharmaceuticals and personal care products, such as antibiotics, etc., constitute a major concern because they are not removed completely from the sewage treatment systems, and they are resistant to biodegradation. Antibiotics such as tetracycline (TeC), for example, are largely excreted in the urine and/or feces increasingly being detected in a wide variety of environmental matrices and causing numerous effects such as allergies and increasing resistance to bacteria. Thus, this study aimed in degradation TeC amid in artificial urine medium by electrochemical method using a dimensionally stable anode (DSA), which has been selected from a highly concentrated chloride in the medium. The studies were resulted from current density and initial pH by assessing the remaining concentration of TeC, creatinine, urea and TOC and comparing the results with those obtained in an aqueous medium containing NaCl 0,1 mol L-1. It was observed that, TeC undergoes electrochemical degradation due to the electrogeneration of oxidizing species of active chlorine from the chloride present in the medium. The decaing of TeC concentration was adjusted to a pseudo-first order kinetic model and increased as current density was increased. It was also observed that the electrochemical treatment may be performed on pH close to the neutral, wherein the anionic form TEC species and HOCl favor degradation, even in the presence of EDTA. The presence of creatinine and urea interfere in the electrochemical process, causing the degradation of TeC to present lowers reaction rates and highers energy consumption in the process than in aqueous medium containing NaCl.