Thesis (Ph. D.)--University of Wisconsin--Madison, 1943. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [99]-[104]).

SOUSA, T. S.; PESSOA, O. D. L. Micro-organismos marinhos produtores de metabólitos secundários biologicamente ativos. 2013. 228 f. Tese (Doutorado em Química Orgânica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by José Jairo Viana de Sousa (jairo@ufc.br) on 2014-10-15T17:59:31Z No. of bitstreams: 1 2013_tese_tssousa.pdf: 22088189 bytes, checksum: f347a4a8a1de4b931bb08902d3d1e6f7 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2015-11-24T19:18:11Z (GMT) No. of bitstreams: 1 2013_tese_tssousa.pdf: 22088189 bytes, checksum: f347a4a8a1de4b931bb08902d3d1e6f7 (MD5) / Made available in DSpace on 2015-11-24T19:18:11Z (GMT). No. of bitstreams: 1 2013_tese_tssousa.pdf: 22088189 bytes, checksum: f347a4a8a1de4b931bb08902d3d1e6f7 (MD5) Previous issue date: 2013 / This work describe the chemical and biological investigation of the extracts from the marine bacterias Pseudoalteromonas sp., Micromonospora sp., Streptomyces sp. and Kocuria sp., aiming the isolation and structural elucidation of new bioactive constituents. The chemical investigation carried out with the bacteria Pseudoalteromonas sp. lead to the isolation a red pigment identified as prodigiosin and two bile acids derivatives known as deoxycholic acid and cholic acid. The prodigiosin was evaluated against four tumor cell lines showing IC50 values similar to the positive control doxorubicin. The chemical study of Micromonospora sp. Resulted in the isolation of four new anthracyclinones designed as 4,6,11-trihydroxy-9-propryltetracene-5,12-dione; 4-methoxy-9-propyltetracene-6,11-dione; 7,8,9,10 - tetrahydro-9-hydroxy-4-methoxy-9-propiltetra-cene-6,11-dione and 10β-Carbomethoxy-7,8,9,10-tetrahydro-4,6,7α,9α,11-pentahydroxy-9-propyltetracene-5,12-dione . The cytotoxic potential of these compounds were evaluated against HCT-8cell line, two of which showed moderate cytotoxicity with IC50 values of 12.74 and 6.18 M, respectively. From Streptomyces sp. strain was isolated a ditiolpyrrolidin, established as 5-oxo-6-(N-methylformamide) -4,5 - dihydro-1,2-dithiol [4,3-b] pyrrole. This secondary metabolite was tested against six tumor cell lines, shown IC50 values of 1.66, 1.05 and 1.52 mM for the metastatic prostate lines, ovarium carcinoma and glioblastoma, respectively. The study of Kocuria sp. lead to the isolation of a new peptide, which was designed as kocurin. This compound was subjected to the tested its antimicrobial assays against several pathogens bacteria and fungal including Staphylococcus aureus strains methicillin resistant (MRSA) and Staphylococcus aureus strains tiazomicin resistant. Kocurin was strongly active against MRSA MB5393 exhibiting a MIC of 0,25µg/mL, moreover showed antibacterial activity against Bacillus subtilis and Enterococcus faecium. The structures of all isolated compounds in this work were stabilized employing spectroscopic methods such as 1H and 13C NMR (1D and 2D), mass spectrometry and infrared. / Este trabalho descreve o estudo químico e biológico dos extratos das bactérias marinhas Pseudoalteromonas sp., Micromonospora sp., Streptomyces sp. e Kocuria sp., visando o isolamento e a elucidação estrutural de novos constituintes bioativos. A investigação química realizada com a bactéria Pseudoalteromonas sp. resultou no isolamento de um pigmento vermelho identificado como prodigiosina e de dois ácidos biliares conhecidos como ácido desoxicólico e ácido cólico. A prodigiosina foi testada frente a quatro linhagens de células tumorais e apresentou valores de IC50 semelhantes ao padrão positivo. O estudo químico de Micromonospora sp. resultou no isolamento de quatro novas antraciclinonas: 4,6,11-triidroxi-9-propriltetraceno-5,12-diona; 4-metoxi-9-propiltetraceno-6,11-diona; 7,8,9,10-tetraidro-9-hidroxi-4-metoxi-9-propiltetra-ceno-6,11-diona e 10β-metoxicarbonil-7,8,9,10-tetraidro-4,6,7α,9α,11–pentaidroxi–9–propil-tetraceno-5,12-diona. Esses compostos foram avaliados quanto a sua atividade anti-tumoral frente a linhagem celular HCT-8, dois dos quais mostraram citotoxidade moderada com valores de IC50 de 12,74 e 6,18 M. O estudo da bactéria Streptomyces sp. possibilitou o isolamento de uma ditiolpirrolidina cuja estrutura foi estabelecida como 5-oxo-6-(N-metilformamida)-4,5- diidro-1,2-ditiol[4,3-b]pirrol. Esse metabólito teve sua atividade citotóxica testada frente a seis linhagens celulares tumorais, mostrando forte atividade com IC50 de 1,66, 1,05 e 1,52 µM para as linhagens de próstata metastática, carcinoma de ovário e glioblastoma, respectivamente. O estudo de Kocuria sp. resultou no isolamento de um novo peptídeo denominado como kocurina. Esse composto teve sua atividade antimicrobiana testada frente a várias bactérias e fungos patogênicos, incluindo cepas de Staphylococcus aureus resistentes a meticilina (MRSA) e cepas de Staphylococcus aureus resistentes a tiazomicina. Kocurina inibiu fortemente o crescimento de MRSA MB5393 com valores de CIM de 0,25µg/mL, além de exibir atividade antibacteriana contra as bactérias Bacillus subtilis e Enterococcus faecium. As estruturas de todos os compostos isolados neste trabalho foram determinadas empregando métodos espectroscópicos tais como RMN 1H e 13C (1D e 2D), espectrometria de massas e infravermelho.

Philosophiae Doctor - PhD / Actinomycetes are ubiquitous in many environments such as soil, activated sludge and water.Besides the genus Streptomyces, which has been extensively exploited, members of other genera including Micromonospora have been shown to be a promising source of novel secondary metabolites and enzymes.The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps. The first step, catalysed by a hydantoinase, yields an N-carbamylamino acid intermediate, which is subsequently broken down by an Ncarbamoylase to the amino acid. This process has been successfully applied in industry for the production of optically pure amino acids which are used in the synthesis of pharmaceuticals,insecticides, hormones, and food additives. The need for novel hydantoinases to hydrolyse a wider variety of substrates is increasing. This thesis describes the search for a novel hydantoinase from environmental isolates obtained from two Zambian hot-springs. The aim of this study was to isolate, characterise and screen novel actinobacteria for industrially relevant enzymes including hydantoinases. Fifty one actinobacteria were isolated. Isolates were characterized by a polyphasic approach using standard methods, combining phylogenetic analysis of the 16S rRNA gene, chemotaxonomic and phenotypic characterization. Results revealed that these sites were dominated by actinobacteria belonging to the family Micromonosporaceae, and a potentially novel Verrucosispora species was identified. Screening the isolate identified a Streptomyces species which has hydantoinase, carbamoylase, amidase and nitrilase activities.The Streptomyces sp. hydantionase was cloned and functionally expressed in E.coli. The recombinant enzyme showed 49 % similarity to a crystallised hydantoinase from a Bacillus species. Homology modelling revealed that the enzyme had the TIM barrel topology which is characteristic of hydantoinases. Amino acid residues predicted to be involved in the catalytic activity as well as substrate orientation were identified. The partially purified hydantoinase was characterised and showed optimally activity at 45 °C and pH 8. This study revealed that hot springs may represent a previously unexplored source of novel actinobacterial diversity. However, it also revealed that novel secondary metabolites are not only limited to novel organisms but that some of the answers for the challenges we face today maybe found in organisms we have already encountered and characterised.

A bacia do rio Tietê, é a maior região hidrográfica do Estado de São Paulo e possui biodiversidade pouco explorada. Actinomicetos são produtores de moléculas bioativas e são descritas em ambientes aquáticos. O objetivo deste estudo foi caracterizar e identificar actinomicetos isolados da água e sedimento da bacia do rio Tietê. Nove actinomicetos foram isoladas: 6 do gênero Streptomyces e 3 do gênero Micromonospora. Três dos isolados pertencem a novas espécies. Os demais isolados foram identificados como S. bingchenggensis, S. lavendulae, S. humi e S. gancidicus; M. sediminicula e M. tulbaghiae. Todos as linhagens foram capazes de hidrolisar ao menos um dos substratos lignocelulósicos testados. Todos os isolados do gênero Streptomyces apresentaram atividade antifúngica. Com exceção de 2 isolados, todos os isolados apresentaram atividade antibacteriana, inclusive contra bactérias multiresistentes a antibióticos. Os genomas dos isolados foram anotados por RAST e antiSMASH, e apresentaram clusters de PKS do tipo I, II e III, sideróforos, terpenos, NRPS, ectoínas, fenazinas, lantipeptídeos, butirolactonas e bacteriocinas. Todos os isolados deste estudo apresentaram grande potencial biotecnológico, comprovado in silico e in vitro. / The Tietê river basin is the largest hydrographic region of São Paulo and its biodiversity is underexplored. Actinomycetes are known to bioactive molecules, and are described in aquatic environments. The aim of this study was to characterize and identify actinomycetes isolated from water and sediment from the Tietê river basin. Nine strains of actinomycetes were isolated: 6 Streptomyces strains and 3 Micromonospora strains. Three isolates are new species of actinomycetes. The remaining isolates were identified as S. bingchenggensis, S. lavendulae, S. humi and S. gancidicus; M. sediminicula and M. tulbaghiae. All strains could hydrolyse at least one of the lignocellulosic substrates tested. All Streptomyces showed antifungal activity. Except for two strains, all isolates showed antibacterial activity, including against multiresistant bacteria. The genomes of all isolates were annotated by RAST and antiSMASH and showed clusters of: type I, II and III PKS, siderophores, terpenes, NRPS, ectoins, phenazines, lantipeptides, butyrolactones and bacteriocins. All isolates in this study showed a high biotechnological potential, proved by in silico and in vitro methods.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho descreve o estudo quÃmico e biolÃgico dos extratos das bactÃrias marinhas Pseudoalteromonas sp., Micromonospora sp., Streptomyces sp. e Kocuria sp., visando o isolamento e a elucidaÃÃo estrutural de novos constituintes bioativos. A investigaÃÃo quÃmica realizada com a bactÃria Pseudoalteromonas sp. resultou no isolamento de um pigmento vermelho identificado como prodigiosina e de dois Ãcidos biliares conhecidos como Ãcido desoxicÃlico e Ãcido cÃlico. A prodigiosina foi testada frente a quatro linhagens de cÃlulas tumorais e apresentou valores de IC50 semelhantes ao padrÃo positivo. O estudo quÃmico de Micromonospora sp. resultou no isolamento de quatro novas antraciclinonas: 4,6,11-triidroxi-9-propriltetraceno-5,12-diona; 4-metoxi-9-propiltetraceno-6,11-diona; 7,8,9,10-tetraidro-9-hidroxi-4-metoxi-9-propiltetra-ceno-6,11-diona e 10β-metoxicarbonil-7,8,9,10-tetraidro-4,6,7α,9α,11âpentaidroxiâ9âpropil-tetraceno-5,12-diona. Esses compostos foram avaliados quanto a sua atividade anti-tumoral frente a linhagem celular HCT-8, dois dos quais mostraram citotoxidade moderada com valores de IC50 de 12,74 e 6,18 M. O estudo da bactÃria Streptomyces sp. possibilitou o isolamento de uma ditiolpirrolidina cuja estrutura foi estabelecida como 5-oxo-6-(N-metilformamida)-4,5- diidro-1,2-ditiol[4,3-b]pirrol. Esse metabÃlito teve sua atividade citotÃxica testada frente a seis linhagens celulares tumorais, mostrando forte atividade com IC50 de 1,66, 1,05 e 1,52 ÂM para as linhagens de prÃstata metastÃtica, carcinoma de ovÃrio e glioblastoma, respectivamente. O estudo de Kocuria sp. resultou no isolamento de um novo peptÃdeo denominado como kocurina. Esse composto teve sua atividade antimicrobiana testada frente a vÃrias bactÃrias e fungos patogÃnicos, incluindo cepas de Staphylococcus aureus resistentes a meticilina (MRSA) e cepas de Staphylococcus aureus resistentes a tiazomicina. Kocurina inibiu fortemente o crescimento de MRSA MB5393 com valores de CIM de 0,25Âg/mL, alÃm de exibir atividade antibacteriana contra as bactÃrias Bacillus subtilis e Enterococcus faecium. As estruturas de todos os compostos isolados neste trabalho foram determinadas empregando mÃtodos espectroscÃpicos tais como RMN 1H e 13C (1D e 2D), espectrometria de massas e infravermelho. / This work describe the chemical and biological investigation of the extracts from the marine bacterias Pseudoalteromonas sp., Micromonospora sp., Streptomyces sp. and Kocuria sp., aiming the isolation and structural elucidation of new bioactive constituents. The chemical investigation carried out with the bacteria Pseudoalteromonas sp. lead to the isolation a red pigment identified as prodigiosin and two bile acids derivatives known as deoxycholic acid and cholic acid. The prodigiosin was evaluated against four tumor cell lines showing IC50 values similar to the positive control doxorubicin. The chemical study of Micromonospora sp. Resulted in the isolation of four new anthracyclinones designed as 4,6,11-trihydroxy-9-propryltetracene-5,12-dione; 4-methoxy-9-propyltetracene-6,11-dione; 7,8,9,10 - tetrahydro-9-hydroxy-4-methoxy-9-propiltetra-cene-6,11-dione and 10β-Carbomethoxy-7,8,9,10-tetrahydro-4,6,7α,9α,11-pentahydroxy-9-propyltetracene-5,12-dione . The cytotoxic potential of these compounds were evaluated against HCT-8cell line, two of which showed moderate cytotoxicity with IC50 values of 12.74 and 6.18 M, respectively. From Streptomyces sp. strain was isolated a ditiolpyrrolidin, established as 5-oxo-6-(N-methylformamide) -4,5 - dihydro-1,2-dithiol [4,3-b] pyrrole. This secondary metabolite was tested against six tumor cell lines, shown IC50 values of 1.66, 1.05 and 1.52 mM for the metastatic prostate lines, ovarium carcinoma and glioblastoma, respectively. The study of Kocuria sp. lead to the isolation of a new peptide, which was designed as kocurin. This compound was subjected to the tested its antimicrobial assays against several pathogens bacteria and fungal including Staphylococcus aureus strains methicillin resistant (MRSA) and Staphylococcus aureus strains tiazomicin resistant. Kocurin was strongly active against MRSA MB5393 exhibiting a MIC of 0,25Âg/mL, moreover showed antibacterial activity against Bacillus subtilis and Enterococcus faecium. The structures of all isolated compounds in this work were stabilized employing spectroscopic methods such as 1H and 13C NMR (1D and 2D), mass spectrometry and infrared.