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published_or_final_version / Botany / Master / Master of Science

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The work in this thesis consisted of initial experiments designed to elucidate the role of hormones in a hormonal dependent tumor. Various aspects of nucleic acid synthesis in a hormone dependent tumor in the presence (growing) and absence (regressing) of the hormone were studied. The rates of nucleic acid synthesis were studied in whole animals by injecting radioactive formate and allowing the animal to incorporate radioactivity for various periods of time. Nucleic acids were extracted by PAS, phenol procedure and separated on a MAK column. Labelling of all species of nucleic acid was decreased in regressing tumors. In order to determine whether estrogen is acting directly on cells or at some indirect physiological level; the ability of cells from growing and regressing tumor to synthesize nucleic acids in vitro was determined. Results of experiments with these cell suspensions demonstrate that cells from the regressing tumor had a decreased ability to synthesize nucleic acids relative to growing tumor. The rate of DNA synthesis was decreased somewhat more than RNA. In preliminary experiments the activity of DNA dependent DNA polymerase and RNA polymerase from regressing tumor was compared with the same enzyme in growing tumor. The specific activity of both RNA and DNA polymerase was decreased in the regressing tumor. In target tissue like uterus stimulation with estradiol results in an increased rate of synthesis of several species of RNA. In the tumor system used in these preliminary experiments, stimulation with estrogens has a greater effect on the synthesis of DNA than RNA. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The in vivo synthesis of deoxyribonucleic acid from labeled precursors was studied in the rat intestinal mucosa in an attempt to elucidate the complex process of DNA replication. In one set of experiments, the rats were injected with ³H-thymidine and then starved for 24 hours, in which time the stable DNA became labelled with tritium. (14)C-thymidine was then administered and the animals were sacrificed 5 minutes later. By this procedure the newly synthesized DNA was labelled with (14)C. The DNA, was fractionated by chromatography on a methylated-albumin kieselguhr column. Only one main peak of DNA was eluted with a sodium chloride solution ranging in concentration from 0.5-0.6 M. The thermal denaturation temperature for the DNA in each.fraction from this peak was determined and the G + C content was calculated:, Within the DNA peak obtained from MAK chromatography, the G + C content of the DNA decreased with increasing fraction number. In addition to these differences in base composition, there were differences in metabolic activity between the fractions, which were indicated by their ³H/ (14)C ratios. The ³H/ (14)C ratio of the DNA fractions from MAK chromatography increased with fraction number to a maximum at fraction 4 or 5 and then decreased. It was found that the ³H/O.D. ratio of the fractions was not constant, thus suggesting that the tritium might be unevenly distributed throughout the fractions. If the time interval between the ³H and (14)C-thymidine injections was reduced to 3 ½ hours, the ³H/O.D. ratio became constant while the pattern of the ³H/14C ratios remained unchanged. If (14)C-thymidine was administered 20 minutes before the animals were sacrificed, the ³H/(14)C ratio of the DNA fractions from MAK chromatography increased with increasing fraction number. From these results it was concluded that small molecular weight, newly synthesized DNA, which was highly labelled with (14)C, was being incorporated with time into the high molecular weight, stable DNA fraction, which is labelled with ³H. During these experiments it was observed that the pattern of ³H/(14)C ratio versus fraction number varied according to the treatment given to the DNA sample prior to the preparation for radioactive counting. If the sample was denatured by heating to obtain its T(M) value, and then dialyzed against distilled water, small molecular weight nucleotides passed into the dialysate. The denatured DNA sample also gave different results from the native DNA sample on digestion with snake venom phosphodiesterase. On the denatured sample, the pattern of release of ³H and (14)C labelled material into the acid soluble material, indicated that both these labels were uniformly distributed along the DNA chain. On the other hand, with the native 5 min. DNA samples, the release of (14)C labelled material into the acid soluble fraction was that expected for DNA which had incorporated (14)C-preferentially into the 3’ terminal positions. The separation of the pyrimidine clusters of DNA indicated that those were not uniformly labelled with (14)C and ³H. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Peroxinitrito (ONOO- + ONOOH), o produto da rápida reação do óxido nítrico com o ânion radical superóxido, tem recebido muita atenção como possível mediador dos efeitos deletérios associados a uma superprodução de •NO. O peroxinitrito é um potente oxidante que é capaz de oxidar e nitrar várias biomoléculas por mecanismos que contribuímos para esclarecer no decorrer desta tese. Especificamente, estudamos a oxidação de urato e tirosina por peroxinitrito. Demonstramos que o urato é oxidado por peroxinitrito a alantoina, aloxana e ao radical aminocarbonila. Como a reação direta entre urato e peroxinitrito tem uma constante de velocidade relativamente baixa (k= 4,8 x 102 M -1.s-1) em comparação com àquelas de outras biomoléculas, sugerimos que o urato é um potente sequestrador dos radicais derivados do peroxinitrito (•NO2 e CO3•-, na maioria dos ambientes biológicos; a pH ácido, o radical •OH também pode se tornar relevante). No caso da tirosina, confirmamos que ela não reage diretamente com o peroxinitrito mas com os radicais dele derivados. Como antecipado, o rendimento relativo dos produtos (3-nitrotirosina, 3,3-bitirosina e 3-hidroxitirosina (DOPA)) variou com o pH e a presença de CO2. Esses estudos nos levaram a propor a co-localização de proteínas nitradas e hidroxiladas como um possível biomarcador de peroxinitrito. Para testar essa hipótese, um anticorpo monoclonal anti-DOPA foi desenvolvido e utilizado em modelos de infecção por Leishmania amazonenses (macrófagos (J774), e camundongos resistentes (C56Bl/6) e suscetíveis (BALB/c). A co-localização de proteínas hidroxiladas e nitradas ficou evidênciada em todos os modelos testados e ocorreu concomitantemente a máxima produção de •NO. Infelizmente, o anticorpo obtido perdeu a atividade e ainda não pudemos confirmar esses dados. / Peroxynitrite (ONOO- + ONOOH), which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of the deleterious effects associated with an overproduction of •NO. The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that this work has contributed to establish. Specifically, we studied the oxidation of urate and tyrosine by peroxynitrite. Urate oxidation produced allantoin, alloxan and the amiocarbonyl radical. Since the rate constant of the direct reaction between urate and peroxynitrite (k= 4,8 x 102 M-1.s-1) is low in comparison with those of other biotargets, we proposed that urate is an efficient scavenger of peroxynitrite-derived radicals (•NO2 and CO3•- in most biological environments; at acid pH, the •OH radical may also become relevant). ln the case of tyrosine, we confirmed that it does not react directly with peroxynitrite but, instead, with the radicals derived form it. As anticipated, the relative yield of the products (3-nitrotyrosine, 3,3-bityrosine and 3-hydroxytyrosine (DOPA)) varied with the pH and CO2 presence. These results led us to propose that co-localization of nitrated and hydroxylated proteins could be a peroxynitrite biomarker. To test this hypothesis, a monoclonal anti-DOPA antibody was developed and tested in Leishamnia amazonensis infection models (macrophages (J774), and resistant (C56Bl/6) and susceptible mice (BALB/c). It was possible to evidence co-localization of hydroxylated and nitrated proteins in all tested models in a time when •NO synthesis was maximum. Unfortunetly, we were unable to confirm these results due to antibody inactvation; new antibody baches are being obtained.

Peroxinitrito (ONOO- + ONOOH), o produto da rápida reação do óxido nítrico com o ânion radical superóxido, tem recebido muita atenção como possível mediador dos efeitos deletérios associados a uma superprodução de •NO. O peroxinitrito é um potente oxidante que é capaz de oxidar e nitrar várias biomoléculas por mecanismos que contribuímos para esclarecer no decorrer desta tese. Especificamente, estudamos a oxidação de urato e tirosina por peroxinitrito. Demonstramos que o urato é oxidado por peroxinitrito a alantoina, aloxana e ao radical aminocarbonila. Como a reação direta entre urato e peroxinitrito tem uma constante de velocidade relativamente baixa (k= 4,8 x 102 M -1.s-1) em comparação com àquelas de outras biomoléculas, sugerimos que o urato é um potente sequestrador dos radicais derivados do peroxinitrito (•NO2 e CO3•-, na maioria dos ambientes biológicos; a pH ácido, o radical •OH também pode se tornar relevante). No caso da tirosina, confirmamos que ela não reage diretamente com o peroxinitrito mas com os radicais dele derivados. Como antecipado, o rendimento relativo dos produtos (3-nitrotirosina, 3,3-bitirosina e 3-hidroxitirosina (DOPA)) variou com o pH e a presença de CO2. Esses estudos nos levaram a propor a co-localização de proteínas nitradas e hidroxiladas como um possível biomarcador de peroxinitrito. Para testar essa hipótese, um anticorpo monoclonal anti-DOPA foi desenvolvido e utilizado em modelos de infecção por Leishmania amazonenses (macrófagos (J774), e camundongos resistentes (C56Bl/6) e suscetíveis (BALB/c). A co-localização de proteínas hidroxiladas e nitradas ficou evidênciada em todos os modelos testados e ocorreu concomitantemente a máxima produção de •NO. Infelizmente, o anticorpo obtido perdeu a atividade e ainda não pudemos confirmar esses dados. / Peroxynitrite (ONOO- + ONOOH), which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of the deleterious effects associated with an overproduction of •NO. The compound is a strong oxidant that is able to oxidize and nitrate a variety of biotargets by mechanisms that this work has contributed to establish. Specifically, we studied the oxidation of urate and tyrosine by peroxynitrite. Urate oxidation produced allantoin, alloxan and the amiocarbonyl radical. Since the rate constant of the direct reaction between urate and peroxynitrite (k= 4,8 x 102 M-1.s-1) is low in comparison with those of other biotargets, we proposed that urate is an efficient scavenger of peroxynitrite-derived radicals (•NO2 and CO3•- in most biological environments; at acid pH, the •OH radical may also become relevant). ln the case of tyrosine, we confirmed that it does not react directly with peroxynitrite but, instead, with the radicals derived form it. As anticipated, the relative yield of the products (3-nitrotyrosine, 3,3-bityrosine and 3-hydroxytyrosine (DOPA)) varied with the pH and CO2 presence. These results led us to propose that co-localization of nitrated and hydroxylated proteins could be a peroxynitrite biomarker. To test this hypothesis, a monoclonal anti-DOPA antibody was developed and tested in Leishamnia amazonensis infection models (macrophages (J774), and resistant (C56Bl/6) and susceptible mice (BALB/c). It was possible to evidence co-localization of hydroxylated and nitrated proteins in all tested models in a time when •NO synthesis was maximum. Unfortunetly, we were unable to confirm these results due to antibody inactvation; new antibody baches are being obtained.