Submitted by PATRÍCIA VERSUTI ARANTES ALVARENGA null (patyversuti@gmail.com) on 2016-01-19T12:39:30Z No. of bitstreams: 1 Patricia_Versuti_Arantes_Alvarenga.pdf: 1619217 bytes, checksum: 77807866047847ff35850a530f25e9a5 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-01-20T11:31:29Z (GMT) No. of bitstreams: 1 alvarenga_pva_me_jabo.pdf: 1588864 bytes, checksum: 5045ef30af54c138b961dab4b885ba3f (MD5) / Made available in DSpace on 2016-01-20T11:31:29Z (GMT). No. of bitstreams: 1 alvarenga_pva_me_jabo.pdf: 1588864 bytes, checksum: 5045ef30af54c138b961dab4b885ba3f (MD5) Previous issue date: 2016-01-08 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A utilização de prebióticos na nutrição animal vem sendo amplamente estudada, uma vez que a utilização de antimicrobianos está restrita e/ou banida de vários países. Dentre estes, destacam-se os oligossacarídeos de parece celular de levedura, mananas e glucanas, os quais têm seus efeitos ainda não totalmente esclarecidos sobre a nutrição de leitões desmamados. O objetivo do estudo foi avaliar o efeito da inclusão de mananas e glucanas na dieta de leitões recém-desmamados, dos 24 aos 66 dias de idade, sobre o desempenho, incidência de diarreia, tempo de trânsito gastrintestinal, e características morfofisiológicas e microbiológicas do trato gastrintestinal [pH, concentração de ácidos graxos de cadeia curta (AGCC), ácido lático, análise microbiológica e estrutura do intestino delgado]. Foram utilizados 96 leitões desmamados aos 24 dias de idade (7,7 ± 1,76kg), distribuídos em um delineamento em blocos casualizados de acordo com os tratamentos: Controle positivo: dieta com antimicrobiano (40ppm de sulfato de colistina, na 1ª fase); Controle negativo: dieta sem adição de aditivos; N+CA: Controle negativo com adição de 0,10% de MOS e 0,18% de β-glucano; N+CB: Controle negativo com adição de 0,036% de MOS e 0,040% de β-glucano; com 8 repetições por tratamento e 3 animais por unidade experimental. A suplementação com MOS e β-glucano não influenciou (P>0,05) as variáveis de desempenho, microbiologia, AGCC, ácido lático e o tempo de trânsito gastrintestinal. Os animais do tratamento CN apresentaram as menores (P<0,05) alturas de vilosidades no duodeno e jejuno. Os leitões do tratamento N+CA apresentaram maiores (P<0,05) alturas de vilosidades no duodeno, comparados aos do CP. Para a variável profundidade de cripta, os leitões do tratamento CN apresentaram os maiores valores (P<0,05) no duodeno, e para o jejuno, aqueles que receberam N+CA e N+CB apresentaram as menores (P<0,05) profundidades, comparados aos do CP e CN, e os do CP foram menores (P<0,05) que os do CN. A relação AV:PC foi maior no duodeno, nos animais que receberam N+CA e N+CB, comparados ao CP e CN, e os do CP apresentaram melhores (P<0,05) resultados comparados aos do CN. No jejuno, os leitões do tratamento CN apresentaram piora (P<0,05) na relação AV:PC comparados aos dos demais tratamentos. Os tratamentos não influenciaram (P<0,05) a contagem de células caliciformes no duodeno, coloração Alcian Blue, porém, no jejuno, houve aumento na expressão destas células para os animais suplementados com os prebióticos e antibiótico. Para a contagem de células caliciformes, coloração PAS, os animais do tratamento CN apresentaram as menores (P<0,05) contagens no duodeno e jejuno. A incidência de diarreia foi maior para os animais do tratamento CN comparados aos dos demais tratamentos. Houve diminuição (P<0,05) do pH estomacal para os animais dos tratamentos N+CB e CP, e diminuição no pH do duodeno para os dos tratamentos N+CA, comparados ao CP. Os prebióticos estudados levaram os animais a apresentarem resultados semelhantes ou melhores que os que receberam antibióticos para algumas variáveis, o que demonstra que a utilização destes prebióticos foi capaz de modular o trato gastrintestinal, mantendo o mesmo padrão de desempenho e, assim, podem ser utilizados em substituição ao antimicrobiano. / The use of prebiotics in animal nutrition has been extensively studied, since the use of antimicrobials is restricted and/or banned in many countries. Among these, we highlight the oligosaccharides from yeast cell wall, mannans and glucans, which have uncleared effects on nutrition of weaned piglets. The aim of the study was to evaluate the effect of inclusion of mannans and glucans in the diet of weanling pigs, from 24 to 66 days of age on performance, diarrhea incidence, rate passage of digesta, and morphophysiological and microbiological characteristics of the gastrointestinal tract [pH, concentration of short-chain fatty acids (SCFA), lactic acid, microbiological analysis and structure of the small intestine]. It was used 96 newly weaned piglets at 24 days old (7.7 ± 1.76kg), distributed in a randomized block design according to the treatments: Positive control: diet with antimicrobial (40ppm of colistin sulfate, in the first phase); Negative control: diet free of antimicrobial and prebiotics; N + CA: negative control with the addition of 0.10% MOS and 0.18% β-glucan; N + CB: negative control with the addition of 0.036% MOS and 0.040% β-glucan; with 8 replicates per treatment and 3 animals each. Supplementation with MOS and β-glucan did not influence (P>0.05) the performance variables, microbiology, SCFA, lactic acid and gastrointestinal transit time. The animals of CN treatment showed lower (P<0.05) villus height in the duodenum and jejunum. The piglets fed N+CA treatment showed higher (P<0.05) villus heights in the duodenum, compared to those of CP. For the variable crypt depth, the piglets of CN treatment showed the highest values (P<0.05) in the duodenum, and in the jejunum, those fed N+CA and N+CB had lower (P<0.05) crypt depths, compared to those fed CP and CN, and animals fed CP had lower (P<0.05) than those of CN. The AV:PC ratio was higher in the duodenum, for the animals fed N+CA and N+CB, compared to CP and CN, and those of CP had higher (P<0.05) results compared to those of the CN. In the jejunum, the pigs of CN treatment showed worse (P<0.05) relation AV:PC compared to the animals of the other treatments. The treatments did not affect (P<0.05) the count of goblet cells in the duodenum, Alcian Blue staining. For counto f globet cells, PAS staining, the animals of the CN treatment had the lowest (P<0.05) scores in the duodenum and jejunum. The incidence of diarrhea was higher for animals of CN treatment compared to those of the other treatments. There was a decreased (P<0.05) in the stomach pH of animals fed N+CB and CP treatments, and decreased pH in the duodenum for animals of N+CA treatment compared to animals fed CP. The prebiotics studied led the animals to exhibit similar or better results than those who received antibiotics for some variables, what shows that the use of these prebiotics has been able to modulate the gastrointestinal tract, keeping the same standard of performance, and thus, they can be used to replace the antimicrobial. / CNPq: 131970/2014-3

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-26Bitstream added on 2014-06-13T19:53:38Z : No. of bitstreams: 1 000737512.pdf: 448616 bytes, checksum: ca9cfa4680cedc19ad31f8680f15521b (MD5) / The florfenicol is an antibiotic used for the treatment of respiratory disease in cattle. Study and understand its pharmacokinetics are important tools for effective control of these diseases and appropriate, thereby minimizing the appearance of resistant bacterial strains. Therefore, this study aimed to determine the pharmacokinetic profile of florfenicol in bovine plasma after intramuscular injection of two doses and evaluate their effectiveness against susceptible bacteria. In the study, we used eight cattle that received two applications of florfenicol and intramuscularly at a dose of 20 mg/kg at intervals of 48 hours. The plasma concentration was determined by high performance liquid chromatography coupled to a mass spectrometer. The minimum inhibitory concentration was performed in a veterinary laboratory following the international standards of the Clinical and Laboratory Standards Institute. Pharmacokinetic parameters were calculated: Cmax = 1.21 ± 0.25 µg/mL, Tmax = 3.43 ± 2.23 h, AUC0-t = 34.16 ± 4.50 h.µg/mL, T½ = 63.46 ± 23.76 h, and Cmax = 1.17 ± 0.20 µg/mL, Tmax = 6.00 ± 0.00 h, AUC0-t = 52.37 ± 5.50 h.µg/mL, T½ = 77.74 ± 43.65 h, respectively for the first and second application. The florfenicol levels in plasma remained above the minimum inhibitory concentration for bacteria: Mannheimia haemolytica (0.75 µg/mL), Pasteurella multocida capsular type A (0.75 µg/mL) and Histophilus sommus (0.30 µg/mL). These results indicate the effectiveness of florfenicol against bacteria that cause respiratory disease of cattle

As endometrites representam um entrave à produção de bovinos leiteiros, à medida que a enfermidade diminui a fertilidade dos rebanhos e causa muitos prejuízos ligados ao custo de tratamento e perda produtiva. Este trabalho avaliou a validade e a eficiência do tratamento com Cefapirina, Ceftiofur e Oxitetraciclina. Para isso 120 animais diagnosticados com endometrite graus II e III pela técnica de histeroscopia fluida foram divididos em 4 grupos de 30 animais, sendo grupo controle (não tratado), um grupo tratado com 500 mg i.u. de Cefapirina, um grupo tratado com 6,6 mg/Kg de Ceftiofur e um grupo recebeu uma infusão de 4g de Oxitetraciclina i.u.. Os animais foram avaliados 4 vezes ou até alcançarem a cura. Avaliou-se o escore de condição corporal, a involução uterina por meio da mensuração dos diâmetros de cérvix e cornos uterinos, a presença e a característica do conteúdo uterino, o padrão hemodinâmico do útero, a resposta de citologia endometrial e a saúde endometrial por meio da histeroscopia. Os animais tratados com Cefapirina e Ceftiofur apresentaram diminuição da proporção de polimorfonucleares nas células recuperadas para citologia endometrial (P = 0,028). Todos os tratamentos apresentaram taxas de cura superior a do grupo controle, os tratamentos apresentaram taxa de cura pelo menos 23% superior a do controle (P = 0,042), porém não houve diferença entre os grupos. Não houve diferença na velocidade de cura entre os tratamentos. Em conclusão o emprego de tratamentos para endometrite é uma conduta aconselhável. Todos os tratamentos testados neste estudo obtiveram eficiência semelhante / Endometritis is a great barrier to the dairy production, as it diminishes fertility and causes economical losses with treatment costs and lowering production. This work has evaluated the value and the efficacy of the use of Cephapirin, Ceftiofur, and Oxitetracyclin for endometritis treatment. 120 animals were diagnosed with grade II and III endometrites trough hyteroscopic examination, and were allocated in four groups of 30 animals, control group (no treatment), Cephapin, that received i.u. Infusion of 500 mg of Cephapirin, Ceftiofur, received s.c. 6.6 mg/kg of Ceftiofur, and Oxitetracyclin, that got treated with i.u. Infusion of 4g of Oxitetracyclin. The animals were evaluated for four times or until they reached cure. Body condition score, Uterine involution assessed by ultrasound measurement of cervix and uterine horns, presence and the characteristic of uterine content, uterine hemodynamic patterns, cytology response, and uterine health response to treatment assessed by hysteroscopy were evaluated at each examination. Cows treated with Cephapirin and Ceftiofur presented a drop on the proportion of PMN retrieved at endometrial cytology examination (P = 0,028). All the treatments presented a bigger rate of cure than control group, at least 23% higher (P = 0,042); however, there was no difference among treatments. There was no difference on cure time among treatments. In conclusion, treating endometritis is an advisable conduct. All the treatments tested in this trial were equally efficient

O objetivo do presente trabalho foi estudar o efeito de diferentes limitações nutricionais sobre a produção de retamicina por Streptomyces olindensis ICB20. Realizaram-se cultivos contínuos empregando meios limitados em carbono, nitrogênio ou fosfato, variando-se a vazão específica de alimentação entre 0,025 e 0,075 h -1 . A análise dos dados dos cultivos mostrou que a produção de retamicina foi favorecida sob limitação por fosfato, resultando em velocidades específicas de produção (qRTM) da ordem de 9,2 mg/g.h em D=0,075 h -1 ; adicionalmente, qRTM variou linearmente com D, isto é, com a velocidade específica de crescimento, tal relação não foi observada sob limitação por carbono ou nitrogênio. O emprego de meio limitado em nitrogênio resultou nas menores velocidades específicas de produção, com valor máximo de 4,2 mg/g.h em D=0,043 h -1 . Cultivos empregando meio limitado em carbono levaram a valores intermediários de qRTM, variando entre 3,0 e 6,6 mg/g.h. Os maiores valores de fator de conversão glicose a célula (YX/GLC) foram obtidos em cultivos empregando meio limitado em carbono, aproximadamente, 0,40, enquanto que sob limitação por nitrogênio e fosfato, YX/GLC variou ao redor de 0,30. Para estudar o efeito de diferentes concentrações de glicose na alimentação, realizaram-se cultivos contínuos empregando meio limitado em fosfato com concentração de glicose variando entre 10 e 25 g/L. Os resultados mostraram que o emprego de concentrações de glicose superiores a 10 g/L levou a menor produção de retamicina, possivelmente devido à ocorrência de repressão catabólica. Os dados relativos à análise de imagens não indicaram nenhuma relação clara entre as diferentes limitações nutricionais e a morfologia nem entre as dimensões dos objetos e a produção. Entretanto, parece haver relação entre a porcentagem em área de diferentes classes morfológicas e a produção de retamicina, sendo que aparentemente, a produção é inversamente proporcional à porcentagem de clumps. / The aim of the present work was to assay the influence of different nutrient limitation on the production of retamycin by Streptomyces olindensis ICB20. A series of continuous cultures was performed using carbon-limited, phosphate-limited or nitrogen-limited media, varying the dilution rate (D) between 0.025 and 0.075 h -1 . The analysis of the cultures data showed that the production of retamycin was favored under phosphate limitation, resulting in values of specific production rate (qRTM) as high as 9.2 mg/g.h at D=0.075 h -1 , additionally, qRTM varied linearly with D, hence, with the specific growth rate; however such relationship was not observed in carbon-limited neither nitrogen-limited cultures. The use of nitrogen-limited medium led to the lowest production rates, with a maximum value of 4.2 mg/g.h at D=0.043 h -1 . Cultures using carbon-limited medium resulted in intermediary values of qRTM, varying between 3.0 and 6.6 mg/g.h. The highest values of biomass yield (YX/GLC) were obtained in cultures using carbon-limited medium, approximately 0.40, while under nitrogen and phosphate limitation, YX/GLC varied around 0.30. To study the effect of different glucose concentration in the feed medium, continuous cultures using phosphate-limited medium with glucose concentration varying between 10 g/L and 25 g/L were performed. The culture results showed that the use of glucose concentration higher than 10 g/L in the feeding medium led to lower production of retamycin, possibly due to catabolic repression. The data of image analysis showed no clear relation between nutrient limitation and morphology neither between objects dimensions and retamycin production. However, there seems to be a relation between the percentage in area of different morphological classes and retamycin production, apparently the production is inversely proportional to the percentage of clumps.

Submitted by Alisson Mota (alisson.davidbeckam@gmail.com) on 2015-07-06T20:07:08Z No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-06T20:34:18Z (GMT) No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-06T20:36:21Z (GMT) No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-06T20:40:04Z (GMT) No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Made available in DSpace on 2015-07-06T20:40:04Z (GMT). No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) Previous issue date: 2012-02-24 / Não Informada / The increasing number of antibiotic-resistant bacteria encourages the search for new antibacterial substances. Therefore, the selection of microorganisms with potential for production of new antimicrobial compounds have been extensively studied. Among these organisms a special attention is given to the actinomycetes that have the capacity to produce a variety of bioactive compounds such as antibiotics, antifungal, antitumor and other compounds that can be applied in various industry segments. The genus Streptomyces is considered of great industrial importance due to its ability to produce many secondary metabolites, accounting for 80% of currently used antibiotics. Considering the importance of actinomycetes and existing biodiversity in the Amazon, this study aims to isolate and select actinomycetes producing antibiotics and optimize their production. In this sense, he was made an initial screening to detect the antimicrobial activity of 371 actinomycetes isolated from soil from different localities in the Amazon region. Antibiosis trials were conducted to evaluate the antimicrobial activity against the indicator microorganisms isolated Gram-positive and Gram-negative. From these preliminary results, three isolates were considered promising because it showed inhibitory activity against Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATCC 292123. These were selected for the study of production, using the response surface model to assess the best physical and chemical conditions that might interfere with production of the antibiotic of interest. The results presented here demonstrate that the isolate No. 01 is a potential producer of new bioactive metabolites. Morphological characterization and partial sequence analysis of 16S rDNA, demonstrate the great diversity of this group of microorganisms, and can thus identify the genus level. It has been shown that environmental conditions and the substrate are critical in the production of secondary metabolites, especially antibiotics. / O aumento crescente de bactérias resistentes a antibióticos incentiva à pesquisa por novas substâncias antibacterianas. Diante disso, a seleção de microrganismos com potencial para a produção de novos compostos antimicrobianos tem sido amplamente estudada. Dentre estes microrganismos uma especial atenção é dada aos actinomicetos que apresentam capacidade de produzir uma variedade de compostos bioativos como antibióticos, antifúngicos, antitumorais entre outros compostos que podem ser aplicados nos mais diversos segmentos da indústria. O gênero Streptomyces é considerado de grande importância industrial devido à sua capacidade de produzir muitos metabólitos secundários, respondendo por 80% dos antibióticos utilizados atualmente. Considerando a importância dos actinomicetos e a biodiversidade existente na Amazônia, este trabalho tem como objetivo isolar e selecionar actinomicetos produtores de antibióticos e otimizar a produção dos mesmos. Neste sentido, foi feito uma triagem inicial para detectar a atividade antimicrobiana dos 371 actinomicetos isolados de solo de diferentes localidades da região Amazônica. Foram realizados ensaios de antibiose para avaliar a atividade antimicrobiana dos isolados frente aos microrganismos indicadores Gram-positivos e Gram-negativos. A partir desses resultados preliminares, 3 isolados foram considerados promissores, pois apresentaram atividade inibitória frente a Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 e Enterococcus faecalis ATCC 292123. Estes,foram selecionados para o estudos de produção, utilizando o modelo de superfície de resposta, para avaliar as melhores condições físicas e químicas que possam interferir na produção do antibiótico de interesse. Os resultados apresentados neste trabalho demonstraram que o isolado n° 01 é um potencial produtor de novos metabólitos bioativos. A caracterização morfológica e a análise da seqüência parcial da região 16S do rDNA, demonstram a grande diversidade deste grupo de microrganismos, sendo possível assim, a identificação a nível de gênero. Foi demonstrado que as condições ambientais e do substrato são fundamentais na produção de metabólitos secundários, principalmente antimicrobianos.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-20T13:21:58Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-20T13:22:46Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-20T13:23:00Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) / Made available in DSpace on 2017-04-20T13:23:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) Previous issue date: 2016-12-16 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / The increase of antibiotic-resistant bacteria encourages the search for new antibacterial substances. Therefore, the selection of microorganisms with potential for the production of new antimicrobial compounds has been extensively studied. Among these microorganisms, special attention is given to actinomycetes that are capable of producing a wide variety of bioactive compounds, such as antibiotics, antifungals, antitumorals and other compounds that can be applied in various segments of the industry. The genus Streptomyces is considered of great industrial importance due to its capacity to produce many secondary metabolites, accounting for 80% of the antibiotics currently used. In previous studies, it was possible to report the antimicrobial activity of a strain of Streptomyces spp. Which showed inhibitory activity against Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATCC 292123. Subsequently, its tuberculicidal activity was also inhibited by the growth of M. tuberculosis H37Rv (ATCC 27294). From these preliminary results, the present work had the objective of analyzing extracts and fractions of metabolites of Streptomyces sp. Aiming to isolate, purify and chemically identify the compound (s) with antimicrobial and tuberculicidal activity. The fractions in hexane (fr-Hex), ethyl acetate (fr-AcoEt) and chloroform (fr-Clo) were obtained from the metabolites obtained in liquid AC medium and by liquid-liquid partition using solvents of increasing polarity. The fr-Clo was chosen to continue the tests because its yield was higher, presenting high antibacterial activity in the Minimum Inhibitory Concentration (MIC) tests against the bacteria Staphylococcus aureus and Mycobacterium smegmatis. Thus, the fr-Clo was subjected to thin layer chromatography (CCD) using several eluent systems and then the Bioautography technique was performed to identify the compounds with activity. From this purification two bioactive sub-fractions were obtained, which were subjected to the isolation of the compounds by preparative CCD and HPLC-DAD-MS technique. The isolated substances were named Clarin A and Clarin B, which presented a Minimum Inhibitory Concentration (MIC) of 1.0 μg / mL for Clarin A and 0.5 μg / mL for Clarin B against Staphylococcus aureus and 16 μg / Ml against Mycobacterium smegmatis. They did not present cytotoxicity to 3T3-L1 non-tumor cells. The Clarin A and Clarin B substances were analyzed using Nuclear Magnetic Resonance (NMR) and low resolution and high resolution Mass Spectrometry techniques for chemical identification. The isolated substances were identified as Actinomycin D and Actinomycin X2 with m / z of 1277 [M + Na] and 1291 [M + Na]. The strong antibacterial activity of the isolated substances makes this product an important source of natural antibacterial compounds. / O aumento de bactérias resistentes a antibióticos incentiva a pesquisa por novas substâncias antibacterianas. Diante disso, a seleção de microrganismos com potencial para a produção de novos compostos antimicrobianos tem sido amplamente estudada. Dentre estes microrganismos uma especial atenção é dada aos actinomicetos que apresentam capacidade de produzir uma ampla variedade de compostos bioativos como antibióticos, antifúngicos, antitumorais entre outros compostos que podem ser aplicados nos mais diversos segmentos da indústria. O gênero Streptomyces é considerado de grande importância industrial devido à sua capacidade de produzir muitos metabólitos secundários, respondendo por 80% dos antibióticos utilizados atualmente. Em trabalhos anteriores, foi possível reportar a atividade antimicrobiana de uma linhagem de Streptomyces spp. que apresentou atividade inibitória frente a Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 e Enterococcus faecalis ATCC 292123. Posteriormente, verificou-se ainda a sua atividade tuberculicida inibindo o crescimento da M. tuberculosis H37Rv (ATCC 27294). A partir desses resultados preliminares, o presente trabalho teve por objetivo analisar os extratos e frações de metabólitos de Streptomyces sp. visando isolar, purificar e identificar quimicamente o(s) composto(s) com atividade antimicrobiana e tuberculicida. A partir dos metabólitos obtidos em meio AC líquido e por partição líquido-líquido utilizando solventes de polaridade crescente, obteve-se as frações em hexano (fr-Hex), acetato de etila (fr-AcoEt) e clorofórmio (fr-Clo). A fr-Clo foi escolhida para dar continuidade aos ensaios pois o seu rendimento foi maior, apresentando alta atividade antibacteriana nos testes de Concentração Inibitória Mínima (CIM), contra as bactérias Staphylococcus aureus e Mycobacterium smegmatis. Dessa forma, a fr-Clo foi submetida à Cromatografia em Camada Delgada (CCD) utilizando vários sistemas de eluentes e, em seguida, a técnica de Bioautografia foi realizada para identificar os compostos com atividade. Dessa purificação foram obtidas duas subfrações bioativas, as quais foram submetidas ao isolamento dos compostos pela técnica de CCD preparativa e HPLC – DAD-MS. As substâncias isoladas foram denominados Clarina A e Clarina B, as mesmas apresentaram uma Concentração Inibitória Mínima (CIM) de 1,0 μg/mL para a Clarina A e 0,5 μg/mL para Clarina B frente a bactéria Staphylococcus aureus e 16 μg/mL frente a Mycobacterium smegmatis. As mesmas não apresentaram citotoxidade para céluas não tumorais 3T3-L1. As substâncias Clarina A e Clarina B foram analisadas por meio das técnicas de Ressonância Magnética Nuclear (RMN) e Espectrometria de Massas de baixa e alta resolução para identificação química. As substâncias isoladas foram identificadas como Actinomicina D e Actinomicina X2 com m/z de 1277 [M + Na] e 1291 [M+Na]. A forte atividade antibacteriana apresentada pelas substâncias isoladas torna este produto uma importante fonte de compostos antibacterianos naturais

Metabólitos secundários produzidos por Streptomyces com atividade antibiótica apresentam relevante importância biotecnológica para as indústrias farmacêuticas e agroquímicas. Dentre estes metabólitos, podemos destacar a nigericina, um antibiótico poliéter usado como aditivo em ração animal atuando como promotor de crescimento e no tratamento de algumas doenças, como a malária, em carcinoma nasofaríngeo, a vaccínia, entre outras. Neste trabalho foram avaliadas duas cepas de actinobactérias potenciais produtoras de nigericina, a EUCAL 26 e a EUCAL 74. As duas actinobatérias foram fermentadas em cinco meios de cultivo diferentes (BD, Czapek, ISP2, M29 e TSB). A cepa EUCAL 26 foi a mais promissora na produção de nigericina em meio Czapeck. A partir da EUCAL 26, foi feito um estudo da máxima produção de nigericina em meio Czapek variando o pH do meio, temperatura de fermentação, e período de fermentação. As melhores condições encontradas foram em pH 7,0 a 25 °C por 27 dias. Foi realizado também um estudo de otimização de aumento de escala de fermentação, de um volume de meio Czapeck de 50 mL, para um volume de 4 L. Também foram avaliados dois resíduos agroindustriais (Farmal e Melaço de Soja) para a produção de nigericina. O meio de Melaço de Soja aumento a produção em aproximadamente 300x quando comparado com o meio Czapeck padrão. Os efeitos dos nutrientes do meio Czapeck também foram avaliados. A retida do K2HPO4 do meio produziu um aumento de 50x na produção de nigericina, quando comparado com o meio Czapeck controle. Também foi avaliado o efeito da adição de -butirolactonas sintéticas, moléculas de sinalização hormonal, para a produção de nigericina. Das 15 -butirolactonas testadas, a DP21A foi a mais eficiente, pois além de aumentar a produção de nigericina em 23x, também diminui o período máximo de sua produção. Todas as analises realizadas neste trabalho para o monitoramento da produção de nigericina, foram feitas empregando a espectrometria de massas sequencial acoplada à cromatografia liquida de ultra eficiência. / Secondary metabolites produced by Streptomyces with antibiotic activity have significant biotechnological importance for the pharmaceutical and agrochemical industries. Among them, nigericin stands out as an antibiotic polyether used as growth promoter in animal feed and for treatment of some diseases such as malaria, nasopharyngeal carcinoma, and vaccinia. In this study, two actinobacteria strains considered potential producers of nigericin named EUCAL 26 and 74 were tested. The two actinobacteria were fermented in five different culture media (BD, Czapek, ISP2, M29 and TSB). EUCAL 26 strain was the most promising in producing nigericin in amid Czapeck media. For EUCAL 26, a study of maximum production of nigericin in Czapek medium at varying the pH, fermentation temperature and fermentation period have been performed. As a result, the best conditions were pH 7.0, at 25 °C for 27 days. In addition, an optimization study for scale-up fermentation have been done, where a volume of 50 mL Czapeck medium have been expanded to 4 L, in order to obtain the highest production of nigericin. Two agroindustrial residues (FARMAL and Honey Soy) have also been evaluated for nigericin production. The honey soy medium increased nigericin production in the rate of 300 when compared with standard Czapeck medium. The effects of nutrients from Czapeck medium have also been evaluated. Removal of K2HPO4 from culture medium resulted in an increase of 50 times when compared with the control Czapeck medium. The effect of adding synthetics -butyrolactones (hormone signaling molecules) for the production of nigericin have also been evaluated. From 15 tested -butyrolactones, DP21A was the most efficient. In addition to increase nigericin yield in 23x it also reduced the period for its maximum production. All analyzes performed in this study to monitor the nigericin production were performed using tandem mass spectrometry coupled to ultra high performance liquid chromatography.

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus cotrimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to cotrimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Cotrimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium. IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.

Master of Agribusiness / Department of Agricultural Economics / Vincent R. Amanor-Boadu / Mycoplasma; it is not a household name for many Americans or people around the world, but for those in the livestock industry, it has been a major concern. Mycoplasma, a member of the class Mollicutes, has had and continues to have a major impact on the cattle, swine and poultry industry, causing conditions such as arthritis, otitis media, reduced growth rate and reduced egg production (Journal of Veterinary Internal Medicine 2011) (Okwara 2016). This class of bacteria is unlike other classes, as defined by the lack of a cell wall, and is considered by many to be the smallest self-replicating prokaryote (Jack Maniloff 1992). Due to its small size, it can reside within cells and even pass through some of the currently used sterilizing filters in the biological/pharmaceutical industry today (Pall Corporation n.d.). This creates a risk for Mycoplasma contamination for those facilities/research centers that use materials of animal origin, as Mycoplasma organisms have historically been a common contaminate of cell lines and laboratory cultures, affecting roughly 15-35% of cell cultures (Cara N. Wilder 2015). An added concern is the difficulty in treatment of infected animals once an infection is established. The Mollicutes class has been considered innately resistant to the antibiotic penicillin and other cephalosporins due to the lack of the cell wall (Jack Maniloff 1992). Due to the clinical significance and risk factors surrounding the Mollicutes class, it is a current regulatory requirement to test materials of animal origin for the presence or absence of Mycoplasma. The specific criteria for the presence or absence of Mycoplasma test is dependent upon the country in which the product is intended to be sold. For the purposes of this study, the required method and products will be for those intended for sale domestically in the United States, or countries accepting US methodologies. To test a material or product for the presence or absence of Mycoplasma according to the current USDA code of federal regulations (CFR), the method is not a rapid procedure or a simple traditional broth inoculation. The domestic method is a minimum 24 day test that requires complex broth and agar media for Mycoplasma recovery. The complex media requirement is due to the fact that Mycoplasma organisms have stringent nutritional requirements due to their simplified cell structure/genome, which often require materials of animal origin, such as serums for lipid supply/metabolism (Jack Maniloff 1992). The 24 day Mycoplasma test requires an initial inoculation into the aforementioned broth and agar media and then 4 subsequent subcultures from the broth media onto the agar media at specified time intervals. All of the broth and agar media plates are incubated at specific atmospheric conditions and temperature for the duration of the test. The initial inoculation and subcultures are all examined by a trained Microbiologist at specific time intervals to search for evidence of viable Mycoplasma growth. The examination by a trained Microbiologist/technician is a vital step as Mycoplasmas do not produce turbidity in media, such as in traditional bacterial growth, nor are they visible by traditional light microscopy (Farzaneh 2011). If a Mycoplasma contamination is found, a biological/pharmaceutical company can pay huge sums of money to investigate the cause of the contamination, initiate corrective action, decontaminate the facility and destroy impacted batches. As evidenced by the above description, Mycoplasma testing places a large burden on a biological/pharmaceutical production facility or even research institutions. The complex media and labor cost for the 24 day test is extensive, which must be repeated for each batch of new material received or produced. The cost skyrockets if any contamination event occurs or even appears to occur, as investigation and decontamination add cost due to delay of release or possible destruction.

Secondary multidrug transporters use the energy stored in transmembrane ion gradients to bind and extrude a variety of weakly related chemical structures. These polyspecific antiporters challenge the notions of high-affinity conformation and strict ion-substrate coupling, inherent to the alternating-access model of transport. In order to investigate the mechanism of secondary multidrug transport at a molecular level, we study LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, which relies on the proton-motive force to achieve the transport of its diverse substrates. We carried out Double Electron Electron (DEER) distance measurements to elucidate the conformational dynamics underlying the transport cycle. We monitored the conformational response of a library of labeled double cysteine mutants to the presence of ligand(s) and proton(s). We investigated the role of the lipid environment by performing the measurements on mutants reconstituted in nanoscale soluble lipid bilayers (nanodiscs). During this work, we have demonstrated that the transporter oscillates between two main conformations, the outward-open and the inward-open. We have shown that the protonation of conserved acidic residues is the driving force of the conformational transition. The lipid bilayer modulates the equilibrium and allows the transition to occur at higher and more physiological pH values. By using specific lipid compositions, we observe that the lipid headgroup is crucial in the regulation of the conformational equilibrium. Based on our data, we propose a model of secondary multidrug transport wherein substrate binding initiates the transport cycle by catalysing proton entrance from the extracellular side. Subsequent protonation of membrane-embedded acidic residues triggers a cascade of conformational changes that results in substrate extrusion to the extracellular side and proton release in the cytosol. We suggest the opening and closing of the extracellular site is tightly regulated while the cytoplasmic side is more flexible. To our knowledge, this work provides the first direct structural evidence of the role of the lipids in the regulation of the conformational dynamics of a membrane transporter. / La surexpression de transporteurs capables d’expulser des molécules cytotoxiques est un mécanisme connu de résistance aux antibiotiques de la cellule bactérienne. Certains transporteurs ont développé la capacité de reconnaitre et d’expulser des substrats de structures diverses, donnant lieu à une résistance multidrogue de la part de leur hôte. Ces transporteurs multidrogues sont présents dans une variété de classes de protéines, distribués dans tous les règnes du vivant. Parmi celles - ci, la famille MFS (Major Facilitator Superfamily) comprend la majorité des transporteurs multidrogues activé par une source d’énergie secondaire, et jouent un rôle crucial dans la propagation de maladies nosocomiales d’origine bactérienne. Une meilleure compréhension des mécanismes fondamentaux du transport multidrogue secondaire est le prérequis indispensable à l’élaboration de thérapies adaptées. En particulier, une description détaillée des changements conformationnels impliqués dans le transport, et une identification des mécanismes moléculaires qui permettent de lier la source d’énergie au transport fait actuellement défaut. Afin de pallier ce manque, ce travail vise à étudier LmrP (Lactococcus lactis multidrug resistance Protein) un transporteur MFS qui confère à son hôte Lactococcus lactis la résistance à divers antibiotiques et agents cytotoxiques de structure et de charge variable. Cette extrusion active est alimentée par un cotransport énergétiquement favorable de protons. Nous avons étudié le mécanisme de transport de LmrP à l’échelle moléculaire en utilisant la technique spectroscopique Double Electron Electron Resonance (DEER), qui permet de mesurer des variations de distances à l’échelle nanométrique, idéale pour observer les mouvements intramoléculaires d’un transporteur MFS. Différents aspects moléculaires susceptibles de réguler le cycle de transport sont étudiés de façon indépendante et couplée :le rôle des protons, des différents substrats, et de l’environnement lipidique. Sur base de cette cartographie conformationnelle, un mécanisme de transport couplant tous les acteurs moléculaires est proposé :la liaison du proton à un motif d’acides aminés conservé constitue la base de la transition conformationnelle, les divers substrats ayant pour rôle de permettre aux protons d’accéder à ce motif. La compétition substrat-proton est la base du transport couplé. Notre travail a mis en évidence le rôle fondamental de l’environnement lipidique, qui module l’équilibre conformationnel du transporteur en interagissant avec un ou plusieurs motif(s) conservé(s). Par ailleurs, notre étude questionne le paradigme actuel de transport au sein de la famille MFS car elle démontre que les changements conformationnels globaux passent par des réarrangements locaux et coordonnés. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished