Thesis (doctoral)--München, 1983.

No description available.

Myotonic dystrophy (DM) is an inherited disease characterized by myotonia, insulin resistance, cardiomyopathy, and cognitive deficiencies. DM is a triplet repeat disorder, meaning that affected individuals carry anywhere between 50 and thousands of CTG/CCTG repeats in their genetic makeup. When transcribed into RNA, these repeats become “toxic” in the sense that they serve to bind and sequester important RNA binding proteins. One such family of proteins, the Muscleblind-like (MBNL) family, is important in the regulation of alternative mRNA splicing, and thus the sequestration of MBNL proteins leads to a number of mis-splicing events. Many of these events are directly correlated to DM symptoms. While there is no known cure for DM, the use of small molecules to treat symptoms is a well-characterized therapeutic tactic with immense promise. Pentamidine is a small molecule that was found to reverse mis-splicing in both DM cell and mouse models. Mechanistically, this molecule is particularly unique because unlike many small molecules, which physically displace MBNL from the toxic CUG RNA, pentamidine reduces CUG RNA levels, possibly through inhibition of CTG transcription. Chapter I summarizes alternative splicing mechanisms and regulation, defines MBNL protein structure and function, describes DM pathophysiology and molecular mechanism, and finally provides an overview of pentamidine characterization as a small molecule therapeutic. Chapter II reports the development of an in vitro T7 transcription assay, which allowed us to compare the relative efficacy by which pentamidine is able to inhibit the transcription of various repeat and non-repeat DNA sequences. This chapter further reports the characterization of a series of methylene linker analogues of pentamidine, which were also characterized through the T7 transcription assay. Chapter III details our thorough structure-activity relationship investigation of bisbenzamidine analogues of pentamidine, both in in vivo and in vitro models. Chapter IV describes our characterization of actinomycin D, a known transcription inhibitor and chemotherapeutic, within the DM disease framework. Chapter V summarizes these data, which ultimately serve as a proof of concept for the potential of CTG transcription inhibition in therapeutic contexts and broadly describe their application in other repeat diseases. This dissertation contains previously published and unpublished co-authored material. / 10000-01-01

MDMX has emerged as a negative regulator of p53 transcriptional activity following DNA damage, loss of ribosomal integrity, and aberrant mitogenic signaling. Disruption of rRNA biogenesis by ribosomal stress activates p53 by releasing ribosomal proteins from nucleoli which bind MDM2 and inhibit p53 degradation. We found that p53 activation by ribosomal stress requires degradation of MDMX by MDM2. This occurs by L11 binding to the acidic domain of MDM2 which promotes its E3 ligase function preferentially towards MDMX. Further, unlike DNA damage which regulates MDMX stability through ATM-dependent phosphorylation events, ribosomal stress does not require MDMX phosphorylation suggesting p53 may be more sensitive to suppression by MDMX under these conditions. Indeed, we find that tumor cells overexpressing MDMX are less sensitive to ribosomal stress-induced growth arrest by the addition of actinomycin D due to formation of inactive p53-MDMX complexes that fail to transcriptionally activate downstream targets such as p21. Knockdown of MDMX increases sensitivity to actinomycin D, whereas MDMX overexpression abrogates p53 activation. Furthermore, MDMX expression promotes resistance to the chemotherapeutic agent 5-fluorouracil (5-FU), which at low concentrations activates p53 by inducing ribosomal stress without significant DNA damage signaling. Knockdown of MDMX abrogates HCT116 tumor xenograft formation in nude mice. MDMX overexpression does not accelerate tumor growth but increases resistance to 5-FU treatment in vivo. In addition to MDMX regulation at the protein level, we found that regulation of cellular MDMX levels, like MDM2, can occur at the transcriptional level by inducing the Ras/Raf/MEK/ERK pathway. We found MDMX levels in tumor cell lines closely correlate with promoter activity and mRNA level. Activated K-Ras and growth factor IGF-1 induce MDMX expression at the transcriptional level through mechanisms that involve the MAPK kinase and c-Ets-1 transcription factors. Pharmacological inhibition of MEK results in down-regulation of MDMX in tumor cell lines. MDMX overexpression is detected in ~50% of human colon tumors and showed strong correlation with increased Erk phosphorylation. Taken together, the data show that MDMX has multiple modes of regulation, which ultimately determine the overall extent of p53 activation.

No description available.

Metabólitos secundários produzidos por actinobactérias são uma inesgotável fonte de compostos com potentes atividades biológicas e estruturas intrínsecas. O desenvolvimento em instrumentação analítica tem contribuído significantemente para acelerar o processo de identificação e caracterização desses metabólitos bioativos. Sem dúvida alguma, a espectrometria de massas (MS) e o seu acoplamento com técnicas de separação, especialmente a cromatografia líquida (UHPLC-MS), tem sido reconhecida como a técnica mais eficiente em análises de produtos naturais. Nesta dissertação foi explorado o potencial da espectrometria de massas como ferramenta analítica para a identificação e caracterização estrutural de fitotoxinas produzidas por actinobactérias isoladas da rizosfera de plantas da caatinga. Foram produzidos noventa extratos de actinobactérias, dos quais quinze apresentaram alguma atividade para o bioensaio da Lemna minor e seis apresentaram atividade para o bioensaio da Chlorella vulgaris. Os extratos brutos ativos das actinobactérias Caat 7-38, Caat 8-6 e Caat 5-29 foram selecionados para caracterização dos compostos ativos, os quais foram isolados empregando o fracionamento guiado por bioensaios. No extrato bruto Caat 7-38, a actinomicina D foi identificada como fitotoxina, ao passo que para o extrato bruto Caat 8-6, foi possível inferir a atividade fitotóxica à presença do griseorhodin A. Já para o extrato bruto Caat 5-29, o composto identificado com atividade fitotóxica apresenta uma estrutura inédita, provavelmente pertencente à classe das anguciclinonas. Foi realizado ainda um estudo para avaliar o efeito da adição de terras raras ao meio de cultivo da actinobacteria Caat 7-38. Para os meios de cultivos contendo neodímio e, principalmente, lantânio ocorreu uma superprodução da actinomicina D, indicando assim, o grande potencial da aplicação das terras raras nos estudos de micro-organismos. / Secondary metabolites produced by actinomycetes are an inexhaustible source of compounds with potent biological activities and intrinsic structures. The development analytical instrumentation has contributed significantly to accelerate the identification and characterization of these bioactive metabolites. Undoubtedly, mass spectrometry (MS) and its coupling with separation techniques, especially liquid chromatography (UHPLC-MS) has been recognized as the most \"efficient\" technique in natural product analysis. In this work was explored the potential of mass spectrometry as an analytical tool for identification and structural characterization of phytotoxins produced by actinomycetes isolated from the rhizosphere of plants from the Caatinga biome. Ninety actinomycetes extracts were produced, of which fifteen showed some activity for the bioassay with Lemna minor and six showed activity for the bioassay with Chlorella vulgaris. The crude active extract of actinomycetes Caat 7-38, Caat 8-6 and Caat 5-29 were selected to characterize the active compounds, which were isolated using bioassay-guided fractionation. In the crude extract Caat 7-38, actinomycin D was identified as phytotoxin, while for crude extract Caat 8-6, it was possible to infer phytotoxic activity to the presence of griseorhodin A. For the crude extract Caat 5-29, the compound identified with phytotoxic activity presents a new structure, probably belonging to the class of anguciclinones. A study to evaluate the effect of addition of rare earths to the culture medium of actinobacteria Caat 7-38 was also carried out. To the culture medium containing neodymium and especially lanthanum occurred overproduction of actinomycin D, thus indicating the great potential of application of rare earths in the studies of microorganisms.

Metabólitos secundários produzidos por actinobactérias são uma inesgotável fonte de compostos com potentes atividades biológicas e estruturas intrínsecas. O desenvolvimento em instrumentação analítica tem contribuído significantemente para acelerar o processo de identificação e caracterização desses metabólitos bioativos. Sem dúvida alguma, a espectrometria de massas (MS) e o seu acoplamento com técnicas de separação, especialmente a cromatografia líquida (UHPLC-MS), tem sido reconhecida como a técnica mais eficiente em análises de produtos naturais. Nesta dissertação foi explorado o potencial da espectrometria de massas como ferramenta analítica para a identificação e caracterização estrutural de fitotoxinas produzidas por actinobactérias isoladas da rizosfera de plantas da caatinga. Foram produzidos noventa extratos de actinobactérias, dos quais quinze apresentaram alguma atividade para o bioensaio da Lemna minor e seis apresentaram atividade para o bioensaio da Chlorella vulgaris. Os extratos brutos ativos das actinobactérias Caat 7-38, Caat 8-6 e Caat 5-29 foram selecionados para caracterização dos compostos ativos, os quais foram isolados empregando o fracionamento guiado por bioensaios. No extrato bruto Caat 7-38, a actinomicina D foi identificada como fitotoxina, ao passo que para o extrato bruto Caat 8-6, foi possível inferir a atividade fitotóxica à presença do griseorhodin A. Já para o extrato bruto Caat 5-29, o composto identificado com atividade fitotóxica apresenta uma estrutura inédita, provavelmente pertencente à classe das anguciclinonas. Foi realizado ainda um estudo para avaliar o efeito da adição de terras raras ao meio de cultivo da actinobacteria Caat 7-38. Para os meios de cultivos contendo neodímio e, principalmente, lantânio ocorreu uma superprodução da actinomicina D, indicando assim, o grande potencial da aplicação das terras raras nos estudos de micro-organismos. / Secondary metabolites produced by actinomycetes are an inexhaustible source of compounds with potent biological activities and intrinsic structures. The development analytical instrumentation has contributed significantly to accelerate the identification and characterization of these bioactive metabolites. Undoubtedly, mass spectrometry (MS) and its coupling with separation techniques, especially liquid chromatography (UHPLC-MS) has been recognized as the most \"efficient\" technique in natural product analysis. In this work was explored the potential of mass spectrometry as an analytical tool for identification and structural characterization of phytotoxins produced by actinomycetes isolated from the rhizosphere of plants from the Caatinga biome. Ninety actinomycetes extracts were produced, of which fifteen showed some activity for the bioassay with Lemna minor and six showed activity for the bioassay with Chlorella vulgaris. The crude active extract of actinomycetes Caat 7-38, Caat 8-6 and Caat 5-29 were selected to characterize the active compounds, which were isolated using bioassay-guided fractionation. In the crude extract Caat 7-38, actinomycin D was identified as phytotoxin, while for crude extract Caat 8-6, it was possible to infer phytotoxic activity to the presence of griseorhodin A. For the crude extract Caat 5-29, the compound identified with phytotoxic activity presents a new structure, probably belonging to the class of anguciclinones. A study to evaluate the effect of addition of rare earths to the culture medium of actinobacteria Caat 7-38 was also carried out. To the culture medium containing neodymium and especially lanthanum occurred overproduction of actinomycin D, thus indicating the great potential of application of rare earths in the studies of microorganisms.