No description available.

No description available.

This investigation involved the production of specific antisera against known International Streptomyces project strains of Streptomyces.

Submitted by Pedro Barros (pedro.silvabarros@ufpe.br) on 2018-09-13T21:08:34Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Sandrine Maria de Arruda Lima.pdf: 3262152 bytes, checksum: a8690471279e77c24ff02a21a444cecc (MD5) / Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-09-18T14:46:05Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Sandrine Maria de Arruda Lima.pdf: 3262152 bytes, checksum: a8690471279e77c24ff02a21a444cecc (MD5) / Made available in DSpace on 2018-09-18T14:46:05Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Sandrine Maria de Arruda Lima.pdf: 3262152 bytes, checksum: a8690471279e77c24ff02a21a444cecc (MD5) Previous issue date: 2017-01-18 / CAPES / Os micro-organismos, em particular bactérias do gênero Streptomyces, são conhecidos por produzir um vasto número de metabólitos secundários bioativos de interesse farmacêutico. O objetivo deste estudo foi investigar as atividades antioxidante, antimicrobiana e anticâncer de metabólitos bioativos produzidos por Streptomyces spp., bem como sua toxicidade. A identificação taxonômica das linhagens foi realizada utilizando as técnicas de caracterização fenotípica e molecular. A produção dos metabólitos bioativos foi avaliada através de fermentação submersa e semi-sólida. Paralelamente, a atividade antioxidante foi analisada por meio dos ensaios DPPH, ABTS e fosfomolibdênio e a atividade antimicrobiana investigada pela concentração mínima inibitória. Os testes de atividade anticâncer foram realizados através do ensaio antiproliferativo do MTT, seguido dos testes de mecanismos de ação por citometria de fluxo para analisar a capacidade do composto em despolarizar a membrana mitocondrial, promover parada de ciclo celular e causar fragmentação no DNA. Para determinar a DL50 (dose letal mediana responsável por matar 50% de uma população em teste) foi realizada uma toxidade aguda in vivo utilizando camundongos albinos Swiss. Streptomyces sp UFPEDA 3370 foi identificado como S. hygroscopicus. Os extratos apresentaram baixa atividade antioxidante, sendo o extrato metanólico da fermentação submersa o melhor para atividade antimicrobiana e citotóxica comparado aos demais. A elaiofilina foi isolada a partir desse extrato, sendo identificada por métodos espectroscópicos, apresentando menor concentração mínima inibitória frente Enterococcus faecalis. Na atividade anticâncer, a elaiofilina apresentou potencial inibitório em todas as linhagens neoplásicas testadas, com melhor atividade frente às células leucêmicas. Os testes por citometria de fluxo revelaram que a substância causa despolarização mitocondrial, parada de ciclo na fase G1 e fragmentação do DNA, o que indica uma provável atuação na via apoptótica de morte celular. A toxidade aguda revelou que a fração EB1, rica em elaiofilina, apresenta DL50 de 1000 mg/kg. Estes resultados demonstram que a substância em estudo tem potencial antimicrobiano e antiproliferativo, tornando-se um candidato para estudos clínicos. / Microorganisms, particularly bacteria of the Streptomyces genus, are known for producing several active secondary metabolites of pharmaceutical interest. The main goal of this study was to evaluate the antioxidant, antimicrobial and anticancer activities of the bioactive secondary metabolites produced by Streptomyces spp., and their toxicity. The taxonomic identification of the strains lineages was accessed by utilizing techniques of molecular and phenotypic characterization. The production of the bioactive metabolites was evaluated through submerged and semi-solid fermentation. In parallel, the antioxidant activity was analyzed through DPPH, ABTS and phosphomolybdenum and the antimicrobial activity was investigated using the microdilution method. The anticancer activity test was accessed by the MTT test followed by mechanisms of action tests using a flow cytometer, which could analyze the ability of the compound to depolarize the mitochondrial membrane, promote cell cycle arrest, and cause DNA fragmentation. To determine the LD 50 (median lethal dose responsible for killing 50% of a population under test) an acute toxicity was performed in vivo using Swiss albino mice. Ours results shows that Streptomyces sp UFPEDA 3370 was identified as S. hygroscopicus. The extracts present low antioxidant activity, being the methanolic extract of the fermentation in submerged culture the best for antimicrobial and cytotoxic activity compared to the others. The elaiophyline isolated starting from this extract, identified by spectroscopic methods, showed the smallest minimum inhibitory concentration against Enterococcus faecalis. In the anticancer activity, elaiophiline showed inhibitory potential in all tested cancer cell lines, with the best results against leukemic cells. The flow cytometer tests showed that the substance causes D mitochondrial depolarization, cycle stop in G1 phase and DNA fragmentation, which indicates that the compound might act through apoptosis. The acute toxicity test revealed that an EB1 fraction, rich in elaiophylin, had a LD50 of 1000 mg/kg. These results demonstrate that our substance has antimicrobial and antiproliferative properties, which makes it a promissory candidate for clinical studies.

Six initial phage isolates with high titer lysates were obtained using Streptomyces toxytricini B-5426 as the host bacterium. These isolates were named Goby, Toma, Yosif, Yara, Deema, and Hsoos. However, upon completion of the sequencing, it was found that the Yara and Hsoos isolates were identical, as were Goby and Deema. As a result, final analysis was completed on only the four unique isolates. All of the phages mentioned above were isolated from soil samples from different locations. Also, they had different sizes of plaques, ranging from 0.3 – 0.9mm. Yosif had the largest plaque size. Yara's head diameter was 79nm with tail diameter of 94nm.

Sortase enzymes are cell wall-associated transpeptidases that facilitate the attachment of proteins to the peptidoglycan. Exclusive to Gram positive bacteria, sortase enzymes contribute to many processes, including virulence and pilus attachment, but their role in Streptomyces coelicolor biology remained elusive. Previous work suggested that the sortases anchored a subset of a group of hydrophobic proteins known as the long chaplins. The chaplins are important in aerial hyphae development, where they are secreted from the cells and coat the emerging aerial hyphae to reduce the surface tension at the air-aqueous interface. Two sortases (SrtE1 and SrtE2) were predicted to anchor these long chaplins to the cell wall of S. coelicolor. Deletion of both sortases or long chaplins revealed that although the long chaplins were dispensable for wild type-like aerial hyphae formation, the sortase mutant had a severe defect in growth. These two sortases were found to be nearly redundant, as deletion of individual enzymes led to only a modest change in phenotype. In vitro analysis of sortase cleavage activity showed that both sortases recognized the unique LAXTG pentapeptide sequence found in the long chaplins, and 11 other putative substrate proteins. Transcriptional analysis revealed that a number of genes typically expressed during aerial hyphae development were not expressed in a sortase deletion mutant. This suggests that the sortases have a role in transcriptional regulation, a phenomenon that has not been described previously. Current work is focused on addressing the mechanism(s) by which sortases affect transcriptional regulation, with a specific focus on the role of the proteins that they anchor to the cell wall (sortase substrates) in aerial growth. / Thesis / Master of Science (MSc)

No description available.

No description available.

No description available.

Amyloid proteins are found in all domains of life, and have a number of defining characteristics, including considerable β-sheet secondary structure and the ability to self-assemble into large, insoluble fibers. These insoluble aggregates are often deleterious to the cell, evidenced by amyloid proteins being involved in Alzheimer’s, Parkinson’s, and Huntington’s disease. Remarkably however, some organisms have found ways to circumvent the toxicity of amyloid proteins, and instead co-opt them as beneficial aspects of their development and survival. An example of this can be seen in the bacteria genus Streptomyces. Streptomyces bacteria have a complex, multicellular life cycle that involves progressing through a number of distinct developmental stages. The reproductive phase of the life cycle requires the activity of amyloid-forming proteins known as chaplins - hydrophobic proteins that polymerize on the cell surface, ultimately promoting reproductive development. Due to limitations in other model Streptomyces, key questions regarding the function of chaplins have not yet been addressed. The emerging model species Streptomyces venezuelae is unique given its rapid growth, its ability to develop in liquid, and its potential to adopt two distinct life cycle programs. This work sought to characterize how chaplins influence these processes. We created a number of chaplin mutants, and determined that chaplins contribute to these process in mostly redundant ways, but when deleted in bulk cause significant phenotypic changes. We have also shown that the requirement of chaplins in development goes beyond what was previously understood - as their loss affects development in all classical life cycle stages, and further impacts alternative life cycle programs. To understanding how chaplins are regulated in S. venezuelae, mutagenesis screens were conducted to identify mutants with altered chaplin regulation. These yielded promising candidates for further investigation. Collectively, this work has advanced our understanding of chaplin proteins, specifically how they are regulated, and how they affect various modes of Streptomyces growth and development. / Thesis / Master of Science (MSc)