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.

<p>An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis.</p><p>In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. </p><p>The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by <i>Streptococcus thermophilus</i> and the other a homopolysaccharide produced by <i>Propionibacterium freudenreichii</i>. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from <i>Escherichia coli</i> strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the <i>Salmonella enteritidis</i> O-antigen and a bacteriophage are described which were performed with NMR experiments.</p>

An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis. In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by Streptococcus thermophilus and the other a homopolysaccharide produced by Propionibacterium freudenreichii. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from Escherichia coli strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the Salmonella enteritidis O-antigen and a bacteriophage are described which were performed with NMR experiments.

Today, recombinant expression of proteins is used for a variety of purposes. One of these is the production of allergens, which are vital components in allergy diagnostics. However, traditional expression systems such as ​Escherichia coli​ and ​Pichia pastoris​ might not have the capacity to express all proteins of interest. Thermo Fisher, which is a leading producer of allergy tests, has requested an evaluation of different microorganisms and their capacity for heterologous protein expression in order to expand their existing toolbox of expression systems. This summary was made through a literature study, where twelve organisms were evaluated. Six eukaryotic and six prokaryotic expression systems are compared based on their ability to properly glycosylate protein, need for specific culture conditions, safety, protease activity, duration, protein yield and protein solubility. The prokaryotic systems – Corynebacterium glutamicum​ , ​Lactococcus lactis​ , ​Pseudomonas fluorescens​ , Pseudoalteromonas haloplanktis​ , ​Ralstonia eutropha​ and ​Streptomyces lividans​ – are characterized by being easy to cultivate, operating in different temperature ranges and providing relatively high yields of recombinant protein. The eukaryotic systems – ​Aspergillus fungi, the green algae ​Chlamydomonas reinhardtii​ , the yeast ​Hansenula polymorpha​ , the parasite ​Leishmania tarentolae​ , the moss ​Physcomitrella patens​ and suspension-based plant cells – all have very different morphology and properties. In comparison with the prokaryotic systems, it can be concluded that they are generally better at folding and providing the correct glycosylation patterns for mammalian and plant proteins. However, they require more time and effort to establish a competent cell line. Furthermore, the resulting protein yield is usually less than for the prokaryotic systems. The conclusion can be drawn that no expression system is perfect. The solution is a toolbox, containing various expression systems and vector systems, providing the basis for successful expression of all kinds of complex proteins. Based on the evaluation of expression systems in this review, such toolbox can be obtained.