After menopause, a woman's production of 17-estradiol, the predominant female sex hormone, declines. This change is associated with increased risk of osteoporosis/osteopenia and atraumatic bone fracture, cardiovascular disease, and breast and ovarian cancers. Phytoestrogens are non-steroidal compounds isolated from plants that have antagonistic, weak agonistic, or super-agonistic estrogenic effects in mammalian tissues; they have emerged as a potential therapeutic to alleviate post-menopausal symptoms. While some epidemiological evidence indicates that dietary consumption of phytoestrogens can alleviate post-menopausal health risks, other research suggests that phytoestrogens may not be completely safe. The research presented in this thesis indicates that a high concentration and sustained dose of phytoestrogens may be necessary to achieve antiestrogenic effects. MCF-7 cells, an estrogen-sensitive breast adenocarcinoma cell line, were used as a model system, and proliferating cell nuclear antigen (PCNA) was used as a marker of cell proliferation. Immunoblotting shows that genistein, a commercially purified phytoestrogen, promotes cell proliferation when administered for 24 hours, but may reduce proliferation when cells were treated for 48 hours. Genistein and estrogen have an additive effect on cells that were treated simultaneously with both hormones for 24 hours. In contrast, Promensilâ„¢, an over-the-counter phytoestrogen dietary supplement, was able to abolish expression of PCNA after 48 hours, and at high concentrations prevented estrogen-induced upregulation of PCNA after 48 hours. The clinical significance of these findings is that phytoestrogens may reduce the risk of breast cancer, but only after sustained high doses, which may be difficult if patient non-compliance is at issue. Additionally, because cell proliferation and not cell survival was investigated, we cannot say whether phytoestrogens are cytotoxic to breast cancer cells, only that they reduce proliferation.

O presente estudo visou avaliar os efeitos da administração de isoflavonas da soja (ISOs) sobre parâmetros bioquímicos e atividade imunológica em ratas ovariectomizadas. As ISOs foram administradas na apresentação não isolada nas doses de 100, 300 e 900 mg/kg, por gavage, durante 28 dias. Ao longo do tratamento foram avaliados o peso e o consumo de ração que evidenciaram efeito moderador na ingesta de alimentos e perda de peso com as maiores doses das ISOs. Foram avaliadas também a bioquímica sérica e urinária, que revelaram alteração do perfil lipídico (⇑ HDL) de forma dose-dependente, aumento de glicemia e diminuição de glicosúria, embora esta última não diferente estatisticamente do controle. No grupo de 900 mg/kg, o estresse oxidativo foi maior e caracterizado pelo aumento de glutationa oxidada (GSSG). No sistema imune foram avaliados diversos parâmetros, a saber: peso relativo de baço e timo e suas celularidades; celularidade de medula óssea; atividades de neutrófilos circulantes e macrófagos peritoneais; resposta imune do tipo tardia (DTH); fenotipagem de linfócitos T e B no sangue e baço e proliferação de linfócitos esplênicos em resposta a ConA e LPS. As maiores doses de ISOs causaram diminuição do peso absoluto do timo e aumento do peso relativo do baço, além de aumento da intensidade de fagocitose de macrófagos na dose de 900 mg/kg. Com a dose de 300 mg/kg de ISOs foi observado menor intensidade na fagocitose de neutrófilos. Assim, pode-se concluir que a utilização de ISOs, na apresentação não isolada, não induziu melhora no perfil lipídico, na proteção ao estresse oxidativo e nem alterou a capacidade de resposta do sistema imune de ratas ovariectomizadas, e, ainda, que a exposição a doses mais elevadas de ISOs apresenta potente efeito sobre a ingesta de alimentos, piora do perfil lipídico, aumento de glicemia e alteração na sinalização redox das células, portanto, futuros experimentos são necessários para melhor caracterizar os efeitos destas agliconas sobre o metabolismo dos lipídeos e também na proteção ao estresse oxidativo. / The present study aimed to evaluate the effects of administration of soybean isoflavones (ISOs) on biochemical and immunological parameters in ovariectomized rats. For this, the ISOs were administered in non-isolated presentation at doses of 100; 300 and 900 mg/kg by gavage for 28 days. Throughout the treatment, the body weight gain and feed intake were evaluated and higher doses of ISOs showed a moderating effect on food intake and weight loss. It were also evaluated the serum and urine biochemistry, which showed altered lipid profile (⇑ HDL), increase in blood glucose and decreased glycosuria in a dose-dependent fashion, although the latter is not statistically different from the control. In the group of 900 mg/kg, the oxidative stress was higher, characterized by increased in oxidized glutathione (GSSG) levels. In the immune system several parameters were evaluated, relative weight of thymus and spleen and their cellularity, bone marrow cellularity, neutrophils and peritoneal macrophages activity, delayed type immune response (DTH), T and B lymphocytes phenotyping in the blood and spleen and splenic lymphocyte proliferation in response to ConA and LPS. Higher doses of ISOs caused decreased in the absolute thymus weight and increase in relative spleen weight. In addition, the dose of 900 mg/kg increased the intensity of phagocytosis of macrophages. On the other hand, it was observed lower phagocytic activity of the neutrophils in the group of 300 mg/kg. Thus, it can be concluded that the use of ISOs in a non-isolated presentation did not induce an improvement in the lipid profile, in the cellular protection to oxidative stress and did not alter the immune response of ovariectomized rats; furthermore, the exposure to higher doses of ISOs has potent effect on food intake, worsening lipid profile, increased blood glucose and changes in redox signaling cells, so further experiments are needed to better characterize the effects of these aglycones on the metabolism of lipids and also in protecting the oxidative stress.

by Szeto Yuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 257-283). / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.vi / Abstract --- p.ix / Contents --- p.xi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoies --- p.is / Chapter 1.1.1 --- Ontogeny of the hematopoietic system --- p.1 / Chapter 1.1.2 --- Hierarchy of hematopoietic cells --- p.3 / Chapter 1.1.3 --- Characteristics of a functional blood system and the need for regulation --- p.11 / Chapter 1.1.4 --- Interrupted hematopoiesis -- Leukemia --- p.13 / Chapter 1.2 --- Regulation of myeloid cell differentiation / Chapter 1.2.1 --- Regulation of hematopoiesis --- p.16 / Chapter 1.2.2 --- Models of hematopoiesis --- p.18 / Chapter 1.2.3 --- Genes regulation of myeloid cell differentiation and its study --- p.21 / Chapter 1.2.4 --- Genes differentially expressed and involved in myeloid cell differentiation --- p.24 / Chapter 1.3 --- Induced myeloid cell differentiation / Chapter 1.3.1 --- Induced myeloid cell differentiation --- p.46 / Chapter 1.3.2 --- WEHI-3B JCS cells --- p.48 / Chapter 1.3.3 --- Chemical inducers -- Flavonoids and benzodiazepines --- p.51 / Chapter 1.4 --- The aim of study --- p.59 / Chapter Chapter Two --- Cytokine Expression in Biochanin A- and Midazolam-treated JCS cells / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cytokine and myeloid differentiation --- p.62 / Chapter 2.1.2 --- Phenotypic studies biochanin A- and midazolam-treated JCS cells --- p.65 / Chapter 2.1.3 --- Cytokine regulation at transcriptional level --- p.68 / Chapter 2.1.4 --- Cytokine mRNA phenotyping by a semi-quantitative approach --- p.69 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Cell line --- p.72 / Chapter 2.2.2 --- Chemicals and buffers --- p.72 / Chapter 2.2.3 --- DIG system --- p.73 / Chapter 2.2.4 --- Enzymes and nucleic acids --- p.73 / Chapter 2.2.5 --- Solutions --- p.74 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of total RNA by guanidinium thiocyanate/cesium chloride isopycnic gradient --- p.75 / Chapter 2.3.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.76 / Chapter 2.3.3 --- Southern blotting --- p.79 / Chapter 2.3.4 --- Cycle titration and dot blotting --- p.79 / Chapter 2.3.5 --- DIG 3' end labeling of probes --- p.81 / Chapter 2.3.6 --- Hybridization and stringency wash --- p.81 / Chapter 2.3.7 --- Chemiluminescent detection --- p.82 / Chapter 2.3.8 --- Quantitation by densitometry --- p.82 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Analysis of total RNA --- p.83 / Chapter 2.4.2 --- mRNA phenotyping --- p.85 / Chapter 2.4.3 --- Summary of mRNA phenotyping results --- p.98 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- mRNA phenotyping --- p.100 / Chapter 2.5.2 --- Cytokine gene regulation --- p.106 / Chapter 2.5.3 --- mRNA quantitation using the current method --- p.108 / Chapter Chapter Three --- Identification and Isolation of Genes that are Differentially Expressed during Midazolam-induced JCS Cell Differentiation / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Methods for studying differentially expressed genes --- p.110 / Chapter 3.1.2 --- RNA fingerprinting by arbitrarily-primed PCR (RAP-PCR) and differential display (DDRT-PCR) --- p.113 / Chapter 3.1.3 --- Re-amplification of PCR products by touchdown PCR --- p.118 / Chapter 3.1.4 --- Strategies to avoid false positives --- p.119 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Cell line and bacterial culture --- p.121 / Chapter 3.2.2 --- Chemicals --- p.121 / Chapter 3.2.3 --- Enzymes and nucleic acids --- p.122 / Chapter 3.2.4 --- Kits --- p.122 / Chapter 3.2.5 --- Solutions --- p.122 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Isolation of total RNA --- p.124 / Chapter 3.3.2 --- First strand cDNA synthesis --- p.124 / Chapter 3.3.3 --- RNA fingerprinting by arbitrarily-primed PCR --- p.124 / Chapter 3.3.4 --- First round cDNA probe screening --- p.126 / Chapter 3.3.5 --- Subcloning of differentially amplified fragments --- p.129 / Chapter 3.3.6 --- Second round cDNA probe screening --- p.133 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Spectrophotometric analysis of total RNA --- p.134 / Chapter 3.4.2 --- Normalization of samples --- p.135 / Chapter 3.4.3 --- RNA fingerprinting of arbitrarily-primed PCR --- p.136 / Chapter 3.4.4 --- Re-amplification of PCR products --- p.138 / Chapter 3.4.5 --- First round cDNA probe screening --- p.139 / Chapter 3.4.6 --- Subcloning of the differentially amplified fragments --- p.143 / Chapter 3.4.7 --- Second round cDNA probe screening --- p.145 / Chapter 3.4.8 --- A comparison of the first and second screening --- p.149 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Towards the steps to isolate differentially expressed genes --- p.151 / Chapter 3.5.2 --- Expression profiles predicted at different stage of the procedures --- p.156 / Chapter 3.5.3 --- Representation of the total mRNA in the cell --- p.158 / Chapter 3.3.4 --- Comparison of the original and modified protocol of RAP-PCR --- p.159 / Chapter 3.3.5 --- Advantages of the modified protocol and further refinements --- p.163 / Chapter Chapter Four --- Characterization of the Putative Differentially Expressed Genesin Midazolam-induced JCS cells / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- DNA sequencing --- p.165 / Chapter 4.1.2 --- Automated DNA sequencing and analysis --- p.168 / Chapter 4.1.3 --- Genbank and BLAST homology search --- p.171 / Chapter 4.1.4 --- Internal primer design for RT-PCR --- p.174 / Chapter 4.1.5 --- Genes involved in both myeloid cell differentiation and embryonic development --- p.177 / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Selected recombinant plasmids --- p.180 / Chapter 4.4.2 --- Total RNAs --- p.180 / Chapter 4.2.3 --- Chemicals --- p.180 / Chapter 4.2.4 --- Enzymes and nucleic acids --- p.181 / Chapter 4.2.5 --- Kits --- p.181 / Chapter 4.2.6 --- Solutions --- p.181 / Chapter 4.3 --- Methods / Chapter 4.3.1 --- Preparation of selected recombinant plasmid DNA --- p.182 / Chapter 4.3.2 --- Sequencing --- p.182 / Chapter 4.3.3 --- Data analysis and assessment by ALF manager and DNAsis --- p.184 / Chapter 4.3.4 --- Sequence search by BLASTN program --- p.185 / Chapter 4.3.5 --- Primer design by Oligo´ёØ ver. 34 --- p.186 / Chapter 4.3.6 --- Differential expression confirmed by RT-PCR --- p.186 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Analysis of selected recombinant plasmid DNA --- p.187 / Chapter 4.4.2 --- Sequencing results --- p.191 / Chapter 4.4.3 --- BLASTN search results --- p.212 / Chapter 4.4.4 --- Primer design of the sequenced fragments --- p.222 / Chapter 4.4.5 --- "Expression profile of the isolated genes in midazolam-, biochanin A- induced JCS cells and mouse embryos" --- p.223 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Sequence analysis of the isolated gene fragments --- p.233 / Chapter 4.5.2 --- Expression profiles of the isolated genes --- p.236 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Studies on leukemic cell differentiation / Chapter 5.1.1 --- Differentiation pathways revealed by different inducers --- p.241 / Chapter 5.1.2 --- Lineage preference during differentiation --- p.243 / Chapter 5.2 --- Differentiation program triggered by midazolam / Chapter 5.2.1 --- Signaling pathways initiated by biochanin A and midazolam --- p.245 / Chapter 5.2.2 --- Differentially expressed genes during midazolam-induced differentiation --- p.247 / Chapter 5.2.3 --- Expression patterns of the isolated differentially expressed genesin midazolam and biochanin A-induced JCS cells --- p.248 / Chapter 5.2.4 --- Myeloid genes in embryonic development --- p.250 / Chapter 5.3 --- Future studies of the isolated fragments --- p.252 / Chapter 5.4 --- Conclusion --- p.256 / Reference --- p.257 / Append --- p.ix / Chapter A1. --- Ambiguity codes for sequencing --- p.i / Chapter A2. --- Myeloid cell lines --- p.ii / Chapter A3. --- Details of manufacturer's products --- p.iii / Chapter A4. --- List of machine and equipment --- p.v

O presente estudo visou avaliar os efeitos da administração de isoflavonas da soja (ISOs) sobre parâmetros bioquímicos e atividade imunológica em ratas ovariectomizadas. As ISOs foram administradas na apresentação não isolada nas doses de 100, 300 e 900 mg/kg, por gavage, durante 28 dias. Ao longo do tratamento foram avaliados o peso e o consumo de ração que evidenciaram efeito moderador na ingesta de alimentos e perda de peso com as maiores doses das ISOs. Foram avaliadas também a bioquímica sérica e urinária, que revelaram alteração do perfil lipídico (⇑ HDL) de forma dose-dependente, aumento de glicemia e diminuição de glicosúria, embora esta última não diferente estatisticamente do controle. No grupo de 900 mg/kg, o estresse oxidativo foi maior e caracterizado pelo aumento de glutationa oxidada (GSSG). No sistema imune foram avaliados diversos parâmetros, a saber: peso relativo de baço e timo e suas celularidades; celularidade de medula óssea; atividades de neutrófilos circulantes e macrófagos peritoneais; resposta imune do tipo tardia (DTH); fenotipagem de linfócitos T e B no sangue e baço e proliferação de linfócitos esplênicos em resposta a ConA e LPS. As maiores doses de ISOs causaram diminuição do peso absoluto do timo e aumento do peso relativo do baço, além de aumento da intensidade de fagocitose de macrófagos na dose de 900 mg/kg. Com a dose de 300 mg/kg de ISOs foi observado menor intensidade na fagocitose de neutrófilos. Assim, pode-se concluir que a utilização de ISOs, na apresentação não isolada, não induziu melhora no perfil lipídico, na proteção ao estresse oxidativo e nem alterou a capacidade de resposta do sistema imune de ratas ovariectomizadas, e, ainda, que a exposição a doses mais elevadas de ISOs apresenta potente efeito sobre a ingesta de alimentos, piora do perfil lipídico, aumento de glicemia e alteração na sinalização redox das células, portanto, futuros experimentos são necessários para melhor caracterizar os efeitos destas agliconas sobre o metabolismo dos lipídeos e também na proteção ao estresse oxidativo. / The present study aimed to evaluate the effects of administration of soybean isoflavones (ISOs) on biochemical and immunological parameters in ovariectomized rats. For this, the ISOs were administered in non-isolated presentation at doses of 100; 300 and 900 mg/kg by gavage for 28 days. Throughout the treatment, the body weight gain and feed intake were evaluated and higher doses of ISOs showed a moderating effect on food intake and weight loss. It were also evaluated the serum and urine biochemistry, which showed altered lipid profile (⇑ HDL), increase in blood glucose and decreased glycosuria in a dose-dependent fashion, although the latter is not statistically different from the control. In the group of 900 mg/kg, the oxidative stress was higher, characterized by increased in oxidized glutathione (GSSG) levels. In the immune system several parameters were evaluated, relative weight of thymus and spleen and their cellularity, bone marrow cellularity, neutrophils and peritoneal macrophages activity, delayed type immune response (DTH), T and B lymphocytes phenotyping in the blood and spleen and splenic lymphocyte proliferation in response to ConA and LPS. Higher doses of ISOs caused decreased in the absolute thymus weight and increase in relative spleen weight. In addition, the dose of 900 mg/kg increased the intensity of phagocytosis of macrophages. On the other hand, it was observed lower phagocytic activity of the neutrophils in the group of 300 mg/kg. Thus, it can be concluded that the use of ISOs in a non-isolated presentation did not induce an improvement in the lipid profile, in the cellular protection to oxidative stress and did not alter the immune response of ovariectomized rats; furthermore, the exposure to higher doses of ISOs has potent effect on food intake, worsening lipid profile, increased blood glucose and changes in redox signaling cells, so further experiments are needed to better characterize the effects of these aglycones on the metabolism of lipids and also in protecting the oxidative stress.

Estudos recentes têm demonstrado o efeito das isoflavonas da soja, especialmente da genisteína (GEN), administrada topicamente, na prevenção do fotoenvelhecimento e fotocarcinogênesis. Esse efeito tem sido relacionado com a sua atividade inibidora de tirosina quinase e antioxidante. Recentemente, demonstramos um lento perfil de permeação da GEN in vitro (usando células de difusão de Franz) a partir de nanoemulsões através de pele de orelha suína (SILVA et al., Pharmazie, v. 64, p. 32- 35, 2009). Entretanto, estudos relativos à distribuição da GEN através das camadas da pele ainda não foram realizados. Neste contexto, o principal objetivo deste estudo foi avaliar a distribuição da GEN através da pele suína a partir de nanoemulsões, antes e após incorporação em hidrogéis de ácido acrílico, a fim de ajustar a viscosidade das mesmas para aplicação tópica. Nanoemulsões contendo GEN apresentaram valores de potencial  negativos e baixa viscosidade. Os hidrogéis derivados contendo as nanoemulsões de GEN demonstraram um comportamento não-Newtoniano pseudoplástico. Um método isocrático de cromatografia líquida para determinar a GEN nas camadas da pele de orelha suína foi desenvolvido e validado. O método mostrou ser específico, linear, preciso e exato para a determinação da GEN nas camadas da pele de orelha suína (i.e. stratum corneum, epiderme e derme). Nas condições validadas, o perfil de permeação/retenção da GEN foi claramente influenciado pela natureza do núcleo oleoso empregado. Nanoemulsões constituídas de um núcleo oleoso de triglicerídeos de cadeia média apresentaram uma permeação mais lenta e uma maior retenção da GEN na pele quando comparado com nanoemulsões contendo octildodecanol. A incorporação das nanoemulsões de GEN em hidrogéis aumenta a retenção da GEN, especialmente na epiderme. Tal resultado pode estar relacionado com o aumento da hidratação da epiderme como sugerido nas observações histológicas. Em conclusão, o conjunto dos resultados demonstra o efeito das formulações sobre a distribuição da GEN através das camadas da pele de orelha suína. / Recent studies have shown the effect of soy isoflavones, especially genistein (GEN), topically administrated, in preventing skin photoaging and photocarcinogenesis. This effect has been related to tyrosine kinase inhibition and antioxidant activities. Recently, we have demonstrated a slow permeation profile of GEN in vitro (using Franz type diffusion cells) from nanoemulsions through porcine ear skin (SILVA et al., Pharmazie, v. 64, p. 32-35, 2009). However, studies concerning the distribution of GEN in the skin layers have yet to be performed. In this context, the main purpose of this study was to evaluate the GEN distribution through the porcine skin from topical nanoemulsions, before and after their incorporation into acrylic acid-hydrogels, in order to have their viscosity adjusted to topical applications. Nanoemulsions containing GEN exhibit negative -potential and low viscosity. The derived hydrogels containing GEN-loaded nanoemulsions presents non-Newtonian pseudoplastic behavior. An isocratic liquid chromatographic method to determine GEN in porcine ear skin layers was developed and validated. The method proved to be specific, linear, precise and accurate to determine GEN in the porcine skin layers (i.e. stratum corneum, epidermis and dermis). In the validated conditions, the GEN permeation/ retention profile was clearly influenced by the nature of the oil core used. Nanoemulsions composed by a medium chain triglycerides oil core exhibit both a slower permeation and a higher retention profile of GEN as compared to octyldodecanol nanoemulsions. The incorporation of GEN-loaded nanoemulsion into hydrogels improved the GEN retention, especially in the epidermis. Such a result might be related to the increased hydration of epidermis as suggested in histological observations. In conclusion, the overall results show the effect of the formulation on distribution of GEN through the porcine skin layers.

Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans, including urothelial carcinoma. Genistein have been reported to regulate genes that are involved in several cellular events. However, the molecular mechanism of genistein -induced upregulation of epithelial membrane protein 2 (EMP2), candidate urothelial tumor suppressor, is not entirely understood. At first, we found that the mRNA and protein expression levels of EMP2 were significantly greater in the normal urothelial tissues and human urothelial cells than those in urothelial bladder carcinoma tissues and urothelial cell carcinoma-derived cell lines. Second, EMP2 knockdown via RNA interference markedly enhanced cell proliferation, colony formation, migration and invasiveness. By contrast, EMP2 overexpression suppressed these malignant behaviors. Third, we showed that genistein-induced inhibition in cell proliferation is associated with an increase in EMP2 expression. Using various deleted EMP2 promoter constructs, we defined that the EMP2 core promoter is enough to observe the genistein-induced upregulation of EMP2 transcriptional activity. Using site direct mutagenesis and chromatin immunoprecipitation assays demonstrated that cyclic-AMP response element binding protein 1 (CREB1) acts as a positive regulator of EMP2 transcription by directly binding to its promoter. These results showed EMP2 suppressed urothelial cell carcinoma-derived cell growth, motility and invasion and for the first time that genistein promoted EMP2 expression in urothelial cell carcinoma-derived cells by inducing EMP2 transcriptional activity via CREB1 binding.

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed Jan. 29, 2010). Includes bibliographical references (p. 55-60).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 117-132).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 103-131).

No description available.