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Environmental pollution and its effect on the living organisms has attracted lots of attention recently. There is a growing body of evidence that we are exposed to environmental pollutants at low concentrations in everyday life. The cells and organisms have tools to identify, neutralize and excrete the majority of the toxic compounds. The most dangerous are those that can escape this process or act at low trace concentrations. Endocrine disruptors (EDs) belong to the latter group. Endocrine disruptors can be of natural and anthropogenic origin. EDs target corresponding hormonal receptors and can act at low concentrations. A wide family of nuclear receptors recognize steroid hormones. The majority of EDs can pass through the cytoplasmic membrane, use the hydrophobic nature of the receptor-ligand binding, trigger hormone response and change the expression of the sensitive genes. By interfering with estrogen and androgen signaling, EDs can have effect on the whole organism, but the reproductive system is influenced most. In the present work, our aim was to develop the methods for ED detection and monitoring, analyze the estrogenic potency of EDs, and evaluate the effects of natural estrogens and EDs on male reproductive functions, including sperm and testicular physiology and endocrine functions. First, we...

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β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs. There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Na agricultura moderna, apesar de serem empregadas as mais modernas tecnologias de cultivo de plantas e apesar dos progressos que têm sido feitos em programas de melhoramento, o máximo potencial das culturas de interesse agronômico está ainda longe de ser plenamente explorado. Assim, os bioestimulantes – uma categoria de produtos relativamente novos de diversas formulações, os quais afetam positivamente processos vitais de uma planta e apresentam grande potencial para uso na agricultura brasileira – podem ser uma alternativa promissora para os orizicultores. O objetivo deste trabalho foi conceituar e discutir o uso de agroquímicos de regulação hormonal e avaliar a influência de duas fontes bioestimulantes de aplicação foliar com Tecnologia AZAL5 (extrato de Ascophyllum nodosum) contendo silicato de potássio e micronutrientes, em aspectos nutricionais e vegetativos, na cultura do arroz de sequeiro. O primeiro capítulo teve a finalidade de, a partir de uma revisão bibliográfica, discutir o uso, os conceitos, os percalços e os benefícios de bioestimulantes, biorreguladores, bioativadores, ácidos húmicos e fúlvicos, bem como extrato de algas na agricultura brasileira. Os capítulos seguintes consistiram de dois experimentos (testes biológicos) em que foram avaliadas as duas fontes bioestimulantes – uma contendo silicato de potássio + molibdênio (Capítulo 2) e outra contendo silicato de potássio + zinco (Capítulo 3) – aplicadas via foliar na cultura do arroz. Os experimentos foram realizados em casa de vegetação, em vasos de 5 kg, com solo classificado como Neossolo Quartzarênico, utilizando-se a cultivar BRS Primavera. O delineamento experimental foi inteiramente casualizado, com quatro repetições, em arranjo 5 x 2. Os tratamentos consistiram de cinco doses (0; 1,50; 3,00; 4,50; 6,00 L ha-1), em dois modos de aplicação (parcelado e não parcelado). Foram avaliados: teores de clorofilas A, B e Total; teores foliares de silício e macro e micronutrientes; massa de matéria seca de raiz e parte aérea. Foram testadas as pressuposições estatísticas dos dados obtidos com os testes de normalidade de Kolmogorov-Smirnov e de homogeneidade das variâncias de Levene, ambos a 0,01 de significância e submetidos à análise de variância pelo teste de Tukey a 0,05 de significância. Os dados significativos para o fator quantitativo (doses) foram submetidos à análise de regressão. A aplicação da fonte bioestimulante contendo silicato de potássio + molibdênio alterou os teores foliares de K, Ca, S, Zn, Cu e a produção de massa de matéria seca de raiz; porém, não houve alteração nos teores foliares de N, P, Mg, Fe, Mn e Si, nos teores das clorofilas A, B e Total aos 55 DAE e na produção de massa de matéria seca da parte aérea em função das diferentes doses da fonte bioestimulante e dos diferentes modos de aplicação. A aplicação da fonte bioestimulante contendo silicato de potássio + zinco alterou os teores foliares de N, Ca, Mg, S, Zn e Cu e a produção de massa de matéria seca de raiz; contudo, não houve alteração nos teores foliares de Si, P, K, Fe e Mn, na produção de massa de matéria seca de parte aérea e nos teores das clorofilas A, B e Total aos 55 DAE em função das diferentes doses da fonte bioestimulante e dos diferentes modos de aplicação. / Despite the use of the most modern technologies of plant cultivation and the progress in breeding programs, the full potential of crops of agronomic interest is still far from being fully exploited. Biostimulants, a relatively new product category of various formulations, positively affect vital processes of plants and have shown great potential for use in the Brazilian agriculture, especially in the rice agriculture. The aim of this study is to discuss the use of hormonal regulation agrochemicals and assess the agronomic and nutritional efficiency of the foliar application of two biostimulants based on AZAL5 Technology (extract of Ascophyllum nodosum) containing potassium silicate and micronutrients. Chapter 1 draws on a review of the literature to discuss uses, concepts, benefits of and obstacles to biostimulants, bioregulators, bio-activators, humic and fulvic acids, as well as seaweed extract in the Brazilian agriculture. The following chapters report on two experiments (biological testing) that assessed both biostimulants – one containing potassium silicate + molybdenum (Chapter 2) and the other containing potassium silicate + zinc (Chapter 3) – applied to upland rice foliage. The experiments were carried out in a greenhouse, using 5-kg vases with Quartzipsamment soil and BRS Primavera cultivar. The experimental design was completely randomized, with four repetitions in a 5 x 2 structure. The treatments consisted of five doses (0; 1.50; 3.00; 4.50; 6.00 L ha-1) used in two modes of application (single application or in portions). The following parameters were evaluated: concentrations of chlorophyll A, B and Total; leaf content of macro and micronutrients and Si; and dry matter of root and aerial part. Statistical assumptions were assessed for the obtained data using Kolmogorov-Smirnov normality test and Levene’s test for homogeneity of variances, both set at 1%. Tukey’s test was used for analysis of variance and set at 5% significance. The significant data for the quantitative factor (doses) were included in a regression analysis. The application of biostimulant containing potassium silicate + molybdenum significantly changed the leaf content of K, Ca, S, Zn and Cu, as well as the production of dry matter of root. However, the leaf content of N, P, Mg, Fe, Mn and Si, the concentrations of chlorophyll A, B and Total, and the production of dry matter of aerial part were not significantly influenced by the different doses of said biostimulant and modes of application. The application of biostimulant containing potassium silicate + zinc significantly impacted on the leaf content of N, Ca, Mg, S, Zn and Cu, and on the production of dry matter of root. However, the leaf content of Si, P, K, Fe, and Mn, the production of dry matter of aerial part and the concentrations of chlorophyll A, B and Total were not significantly influenced by the different doses of said biostimulant applied in different modes. / Dissertação (Mestrado)