Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: J. R. Nodwell. Includes bibliographical references (leaves 121-147) Also available online.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2000. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-04-10T19:33:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação.pdf: 2311263 bytes, checksum: 2cd90cdab2e2c81506db007951c48578 (MD5) / Made available in DSpace on 2017-04-10T19:33:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação.pdf: 2311263 bytes, checksum: 2cd90cdab2e2c81506db007951c48578 (MD5) Previous issue date: 2015-02-24 / Actinobactérias são bactérias Gram-positivas que se destacam pela sua grande potencialidade de produzir diversos metabólitos secundários bioativos de interesse científico e industrial. Mais de 50% dos metabólitos microbianos bioativos descobertos são produzidos, tais como antimicrobianos e antitumorais, pelas actinobactérias principalmente pelo gênero Streptomyces.O câncer é considerado um importante problema de saúde pública em países desenvolvidos e em desenvolvimento, sendo a segunda causa de morte no Brasil e no mundo. Diante da diversidade de tecnologias e pesquisas aplicadas no campo das neoplasias, ainda não há uma terapia eficaz para o tratamento de todos os tipos de câncer e os quimioterápicos utilizados atualmente apresentam elevada toxicidade. Neste contexto, o presente projeto objetivou avaliar o potencial antimicrobiano e anticâncer dos extratos brutos produzidos pela por Streptomyces UFPEDA 3407. Após fermentação e extração dos metabólitos com diferentes solventes, foram realizados ensaios para avaliar a atividade antimicrobiana e citotóxica nas diferentes linhagens de células cancerígenas humanas (HL- 60, HT-29 e MCF- 7) e em eritrócitos murinos. Os resultados obtidos demonstraram que os extratos da biomassa extraídos com acetona, etanol e metanol apresentaram um bom espectro de ação contra bactérias Gram-positivas e leveduras. Nos ensaios de citotoxicidade, os mesmos extratos reduziram de maneira significativa a viabilidade das linhagens tumorais, mostrando resultados bastante promissores. / Actinomycetes are Gram-positive bacteria with high potential to produce various bioactive secondary metabolites of scientific and industrial interest. More than 50% of bioactive microbial metabolites discovered to date are produced by actinomycetes mainly by gender Streptomyces. Cancer is considered a major public health problem in developed and developing countries, and the second cause of death in Brazil and in the world. Given the diversity of technologies and applied research in the field of cancer, there is still no effective therapy for the treatment of all types of cancer and the chemotherapy drugs used today have high toxicity. In this context, this project aimed to evaluate the antimicrobial potential anticancer and crude extracts produced by Streptomyces actinobacteria UFPEDA 3407. After fermentation and extraction of metabolites with different solvents, tests were conducted to evaluate the antimicrobial and cytotoxic activity in different strains of human cancer cells (HL- 60, HT-29 and MCF-7) and murine erythrocytes. Partial results showed that the extracts of biomass extracted with acetone, ethanol and methanol had a good spectrum of action against Gram-positive bacteria and yeasts. In the cytotoxicity assays, the same extracts significantly reduced the viability of tumor cell lines, showing promising results.

To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.

𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘮𝘺𝘤𝘦𝘴 initiate a complex developmental program during their 5-day life cycle, consisting of aerial mycelium formation and antibiotic production. Several developmental genes are involved in regulating these events, one of which is 𝘣𝘭𝘥𝘉. I have complemented a 𝘣𝘭𝘥𝘉 null mutant, which demonstrated the loss of aerial mycelium formation and antibiotic production, restoring both characteristics. This demonstrated that 𝘣𝘭𝘥𝘉 is essential for 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 morphogenesis but not viability. Using a bacterial two hybrid system devised, I screened the 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 genome using BldB as "bait" for binding partners of BldB. The two most compelling candidates were bbpl, a homologue of UspA in 𝘌. 𝘤𝘰𝘭𝘪, and bbp2, a homologue of SrmR in 𝘚. 𝘢𝘮𝘣𝘰𝘧𝘢𝘤𝘪𝘦𝘯𝘴. Furthermore, I have investigated these interactions biochemically by affinity chromatography to further elucidate the details involved in these interactions. Preliminary results showed three protein bands obtained at approximately 68kDa, 55kDa and 35kDa, respectively. / Thesis / Master of Science (MS)

The RamC protein is required for the production of spore-forming cells called aerial hyphae in colonies of 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘮𝘺𝘤𝘦𝘴 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳. RamC can be detected during the period between 24 and 48 hours following spore germination however there is a dramatic drop in RamC levels thereafter. This could be explained either by the existence of an active means of RamC removal or by the fact that at later time points in the 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 lifecycle non-RamC producing cells vastly outnumber RamC-producing cells. characterized a large number of 𝘣𝘭𝘥 mutants and found that most of them do not produce RamC. In the majority of the 𝘣𝘭𝘥 mutants that do produce RamC, we observed the same pattern of accumulation and loss during colony growth as in wildtype colonies. Furthermore, we identified a small number of mutants that produced RamC such that it persisted at detectable levels for a longer duration or only appeared after a substantial delay relative to the wildtype. None of these RamC-producing 𝘣𝘭𝘥 mutants was complemented by plasmids containing a cloned 𝘣𝘭𝘥𝘔, 𝘣𝘭𝘥𝘕 or 𝘳𝘢𝘮𝘙 gene, mutations in which also cause persistent or delayed RamC production. These results suggest either that there is more than one differentiated cell type within the substrate mycelium or that 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 colonies actively rid themselves of RamC once the protein's biological function has passed. / Thesis / Master of Science (MS)

Resistance to aminoglycosides in the organisms that produce them is often ascribed to the well characterised and clinically important antibiotic modifying enzymes. However, at least three aminoglycoside producing actinomycetes, namely Micromonospora purpurea, Streptomyces tenjimariensis, and Streptomyces tenebrarius possess ribosomes that are refractory to some members of this class of drugs. In these cases, resistance is due to methylation of rRNA of the small ribosomal subunit. This study supports the possibility that this mechanism might be more widespread than hitherto suspected. Two of the methylase genes have been analysed at the nucleotide level and their transcripts mapped. The gentamicin resistance methylase gene (kgmA) from M. purpurea codes for a 36 kDa protein consisting of 249 amino acids. Like most actinomycete genes, kgmA is not expressed in E. coli from its own promoter, although the determinant was expressed in this Gram-negative host as a result of DNA rearrangement. Sequence analysis of the mutated plasmid suggested that the methylase was expressed as a translational fusion with the lacZ' gene of pUC18, a view that was later confirmed. Transcript mapping revealed that kgmA is probably read from a single promoter but that it might be part of a polycistron. The second gene examined confers resistance to kanamycin and apramycin, and originated in S. tenjimariensis. This determinant (kamA) was shown to encode a predicted protein of 155 amino acids with a molecular weight of 19 kDa. Unlike kgmA, this gene could not be expressed as either a transcriptional or translational fusion in E. coli. Transcription of kamA is directed by tandem promoters and is a monocistron since the the transcript terminates only 160 bp downstream of the stop codon.

The coupled transcription-translation system previously developed for Streptomyces lividans was modified such that it functioned using purified ribosomal subunits, a crude initiation factor preparation and a high speed supernatant fraction. This system was used to investigate antibiotic resistance mechanisms in two Streptomyces which synthesise inhibitors of translation. Resistance to either pactamycin in Streptomyces pactum or celesticetin in Streptomyces caelestis was due to ribosome modification. In each case, high level resistance was attributed solely to one ribosomal subunit, the 30S subunit of the S. pactum ribosome and the 50S subunit of the S. caelestis ribosome. Shotgun cloning experiments have enabled a pactamycin resistance determinant from S. pactum to be isolated in S. lividans. However, in the original pactamycin resistant clone the plasmid was unstable and in the absence of pactamycin selection pressure, only a deleted form could be recovered. When ribosomes from resistant subclones were analysed, it appeared that a ribosome modification system from S. pactum had been cloned. Ribosome reconstitution studies indicated that a property of 16S rRNA was responsible for resistance. Since the cloned resistance determinant was not homologous to 16S rRNA (as judged by Southern analysis), pactamycin resistance in S. pactum is probably due to post-transcriptional modification of 16S rRNA.

No description available.

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