<p> The age at which a juvenile female reaches sexual maturity can be modulated by a variety of environmental and social factors. Experiments described in this thesis were designed to enhance the current understanding of the relationships among three variables that influence the onset of sexual maturation in female mice (Mus musculus), including: [1] exposure to dietary phytoestrogens during development, [2] variations in prenatal androgens, and [3] the presence or absence of genetically-unrelated males after weaning. For the first time, age at onset of male-induced female puberty was investigated using non-invasive behavioural and fertility measures. Through enzyme immunoassay procedures, daily output of urinary creatinine, 17P-estradiol, and progesterone was profiled in developing females that were either isolated or exposed to adult males. Uterine and ovarian tissue was also measured in such females, and male exposure was observed to increase reproductive tissue mass and was influenced by prior androgen exposure in interaction with diet and male presence. Male-exposed females fed a diet containing phytoestrogens immediately became sexually receptive when housed directly with males, and they conceived earlier than females in other conditions. Females with longer anogenital distance, which reflects higher in utero androgen exposure, displayed more escape attempts and aggressive posturing in the direct presence of males, especially when they had been housed near males and fed the phytoestrogen-containing diet. Urinary 17P-estradiol was substantially reduced in females raised on the phytoestrogenfree diet. Urinary output of progesterone was not strongly influenced by diet. Maleexposed females ' output of progesterone and 17P-estradiol was more dynamic in comparison to that of isolated females. The size of this effect depended on diet, prior androgen exposure, and whether urinary steroid measures were adjusted by urinary creatinine. Urinary creatinine was elevated by the low phytoestrogen diet and reduced by male exposure. These data suggest that dietary phytoestrogens and in utero androgen exposure interact with presence or absence of males in determining the age at onset of sexual maturity in developing females. </p> <p> A final experiment was designed to examine two components of adult male urine, preputial gland emissions and unconjugated estrogens, that have been posited to act on females to advance reproductive maturation. Intact and preputialectomized males were compared in their output of urinary creatinine, 17~-estradiol, and testosterone, and in their influence on reproductive tissue in juvenile females. Lack of preputial glands did not hinder the capacity of males to induce uterine and ovarian growth in females. Male urinary creatinine was reduced by exposure to juvenile females. Creatinine-adjusted 17~estradiol and testosterone were greater in female-exposed males, regardless of whether the preputial glands were present. Based on these findings and those reported elsewhere, it is probable that male excreted urinary steroids are important in regulating reproductive changes in developing females exposed to males. </p> / Thesis / Doctor of Philosophy (PhD)

Introdução: A introdução da agricultura e pecuária foram muito recentes para que o genoma humano se adaptasse e a tecnologia avançada pós revolução Industrial foi ainda mais. Segundo Cordain, a substituição de alimentos minimamente processados por alimentos pós-agrícolas e pós-industriais influenciaram os indicadores nutricionais: carga glicêmica, composição de ácidos graxos e macronutrientes, densidade de micronutrientes, equilíbrio ácido-base, relação sódio/potássio e teor de fibras, levando a um desequilíbrio que é causa de várias doenças atuais da civilização. A Protein Leverage Hypothesis (PLH) propõe que a queda na ingestão de proteínas possa levar a obesidade e doenças cardiometabólicas associadas. Objetivos: Estudar o efeito do consumo de alimentos ultraprocessados nos indicadores nutricionais na população dos EUA, incluindo a composição de macronutrientes, densidade de fibras e micronutrientes e fitoestrógenos urinários; avaliar se a contribuição calórica de alimentos ultraprocessados é determinante para a qualidade nutricional das dietas contemporâneas; e finalmente estudar se a associação entre o consumo de alimentos ultraprocessados, proteína e energia correspondem às previsões do modelo PLH. Métodos: Foram avaliados os participantes do National Health and Nutrition Examination Survey 2009-2010, com pelo menos um recordatório alimentar de 24 horas. Os itens foram classificados em: alimentos in natura ou minimamente processados, processados, ultraprocessados e ingredientes de uso culinário. O manuscrito 1 examina a relação entre a contribuição calórica de alimentos ultraprocessados e qualidade nutricional da dieta, avaliando individual e globalmente a contribuição de cada ingrediente crítico, usando a análise de componentes principais (ACP). O manuscrito 2 estuda a associação entre a contribuição calórica dos alimentos ultraprocessados e consumo de açúcares de adição. O manuscrito 3 avalia como o consumo de alimentos ultraprocessados influencia o conteúdo proteico relativo da dieta e as ingestões absolutas de energia e proteína, e se essas relações se encaixam nas previsões da PLH. O manuscrito 4 avalia a relação entre a contribuição calórica de alimentos ultraprocessados e níveis de fitoestrógenos urinários. Resultados: O teor médio de proteínas, fibras, vitaminas A, C, D e E, zinco, potássio, fósforo, magnésio e cálcio na dieta diminuiu ao longo dos quintis de contribuição calórica de alimentos ultraprocessados, enquanto o de carboidratos, açúcares de adição e gordura saturada aumentou. Uma associação inversa de dose-resposta foi encontrada entre o consumo de alimentos ultraprocessados e qualidade nutricional total, medida através de um escore de padrão balanceado de nutrientes derivado usando ACP. Consistente com a PLH, a contribuição calórica de alimentos ultraprocessados foi inversamente associada à densidade proteica e diretamente ao consumo energético total, enquanto a ingestão absoluta de proteínas permaneceu constante com aumento do consumo de alimentos ultraprocessados. Os níveis médios de enterolignanos urinários diminuíram ao longo dos quintis de consumo de alimentos ultraprocessados, enquanto os níveis de isoflavonas permaneceram inalterados. Conclusões: Este estudo mostra que a diminuição da contribuição calórica de alimentos ultraprocessados é um meio racional e eficaz de melhorar a qualidade nutricional das dietas dos EUA / Background: The introduction of agricultural and animal husbandry has not provided the human genome time enough to adapt, much less the advancing technology after Industrial Revolution. According to Cordain et al., displacement of minimally processed foods by post-agricultural and post-industrial food items adversely affected the following dietary indicators: glycemic load, fatty acid and macronutrient compositions, micronutrient density, acid-base balance, sodium-potassium ratio and fiber content. Many current diseases of civilization, in turn may be ascribable to those unbalanced dietary indicators. Indeed, Raubenheimer and Simpson have proposed the Protein Leverage Hypothesis (PLH) to explain how a drop in dietary protein content might lead to obesity and associated cardiometabolic disease. Objective: This thesis aims to study the effect of an increased consumption of ultra-processed foods on dietary indicators in the US population, including macronutrient composition, micronutrient and fiber densities, and urinary phytoestrogens. It also explores whether the dietary share of ultra-processed foods, expressed as a percentage of total energy intake, is a meaningful determinant of overall nutritional quality of contemporary diets. Lastly, it also looks into whether the association between ultra-processed food, protein and energy consumptions fit predictions of the PLH model. Methods: Participants from cross-sectional 2009-2010 National Health and Nutrition Examination Survey with at least one 24-hour dietary recall were evaluated. Food items were classified according to extent and purpose of industrial food processing as: unprocessed or minimally processed foods, processed culinary ingredients, processed foods and ultra-processed foods. Manuscript 1, examines the relationship between dietary contribution of ultra-processed foods and nutritional quality of US diet through the evaluation of dietary contents of critical nutrients individually and also overall, using Principal Component Analysis (PCA). Manuscript 2 studies the association between dietary contribution of ultra-processed foods and energy intake from added sugars. Manuscript 3 examines how consumption of ultra-processed food influences relative dietary protein content and, absolute energy and protein intakes; it furthermore, tests whether the relationships fit PLH predictions. Manuscript 4 assesses the relationship between dietary contribution of ultra-processed foods and urinary levels of phytoestrogens. Results: The average content of protein, fiber, vitamins A, C, D and E, zinc, potassium, phosphorus, magnesium and calcium in US diet decreased significantly across quintiles of energy contribution of ultra-processed foods, while carbohydrate, added sugars and saturated fat contents increased. An inverse dose-response association was found between ultra-processed food consumption and overall dietary quality measured through a Nutrient balanced pattern PCA derived factor score. Consistent with PLH, dietary contribution of ultra-processed foods was inversely associated with protein density and directly associated with total energy intake, while absolute protein intake remained relatively constant with increases in ultra-processed food consumption. Average urinary mammal lignan levels decreased across quintiles of ultra-processed food consumption, while isoflavone levels remained unchanged. Conclusions: This study suggests that decreasing the dietary share of ultra-processed foods is a rational and effective way to improve the nutritional quality of US diets

Die niedrige Inzidenz chronisch-entzündlicher Darmerkrankungen (CED) in ostasiatischen Ländern im Vergleich zu Westeuropa und den USA könnte auf unterschiedliche Lebensstile und Ernährungsgewohnheiten zurückzuführen sein. Asiaten nehmen mit der Nahrung viel höhere Mengen an Isoflavonen zu sich als Europäer und US-Amerikaner. Diese sind in der Lage, wie natürliche Östrogene an Östrogenrezeptoren (ER) zu binden. Für das Östrogen 17β-Östradiol (E2) sowie selektive Liganden des ERβ sind antiinflammatorische Wirkungen im Darm bereits nachgewiesen worden. Diese Arbeit untersuchte in Modellsystemen für CED die antiinflammatorischen Eigenschaften von Isoflavonen, speziell von Genistein, und stellte einen Vergleich mit synthetischen ER-selektiven Liganden sowie E2 her, um die Involvierung der beiden ER-Subtypen zu evaluieren. In tierexperimentellen Studien wurde der Einfluss der Testsubstanzen auf Ausprägung und Verlauf einer Kolitis in zwei Nagermodellen (HLA-B27 transgene Ratte und TNBS-induzierte Kolitis) analysiert. Ein Ernährungsexperiment, in dem eine Gruppe der Tiere bereits in utero sowie postnatal über Muttermilch und Futter hohen Phytoöstrogenspiegeln ausgesetzt war, zeigte wider Erwarten keine antiinflammatorischen Effekte auf die akute Ausprägung der induzierten Kolitis. Stattdessen waren die untersuchten Parameter bei dieser Ernährungsform gegenüber prä- und postnatal normal ernährten Tieren verstärkt. Dagegen bewirkte oral verabreichtes Genistein in der chronischen Phase der TNBS-induzierten Kolitis eine Unterdrückung der Entzündungsparameter im Darm. Die subkutane Verabreichung von Genistein, eines steroidalen ERβ-selektiven Agonisten, oder von E2 führte hingegen zu keiner signifikanten Einflussnahme auf die untersuchten Parameter in der akuten Phase der Inflammation. Zur Charakterisierung der molekularen Grundlagen einer antiinflammatorischen Wirkung von E2, synthetischen ER-selektiven Agonisten und Genistein wurden in vitro Studien mit Kolonkarzinomzelllinien (HT-29 und Caco-2) durchgeführt. Hierzu wurden die Zellen mit Interleukin-1β (IL-1β) stimuliert, was eine Induktion der inflammationsassoziierten Gene Cyclooxygenase-2 und Interleukin-6 auf mRNA Ebene bewirkte. Bis auf Genistein konnten für die getesteten Substanzen keine antiinflammatorischen Effekte auf die mRNA-Expression der induzierten Markergene beobachtet werden. Genistein bewirkte in Caco-2 Zellen eine Hemmung der untersuchten Gene. Weitere Analysen ergaben, dass die beiden Zelllinien ER nur schwach bzw. gar nicht exprimieren. Eine Transfektion von HT-29 Zellen mit ERα führte zu einer deutlichen Hemmung der Expression der Markergene durch E2, während eine Transfektion mit ERβ lediglich einen schwach hemmenden Effekt bewirkte. Die Ergebnisse der vorliegenden Arbeit legen nahe, dass die niedrigen CED-Inzidenzraten in Ostasien wohl nicht allein auf dem dortigen hohen Isoflavonkonsum beruhen, sondern auch anderen Komponenten des Lebensstils zuzuschreiben sind. Dennoch deutet sich an, dass das Genistein, bei oraler Administration, die Regeneration des geschädigten Darmgewebes im chronischen Erkrankungsverlauf unterstützen und damit auch zur Prävention von Kolonkarzinomen beitragen könnte. Bei antiinflammatorischen Effekten von ER-Liganden spielt die Transaktivierung von ER eine entscheidende Rolle. Die Wirkung von Genistein in untransfizierten Caco-2 Zellen legt jedoch auch die Teilnahme weiterer Mechanismen nahe, die noch zu untersuchen sind. Vor diesem Hintergrund erscheinen weiterführende Untersuchungen zum Einsatz von steroidalen ER-Agonisten und Genistein bei CED und den zugrunde liegenden Mechanismen als sinnvoll.

Introdução: A introdução da agricultura e pecuária foram muito recentes para que o genoma humano se adaptasse e a tecnologia avançada pós revolução Industrial foi ainda mais. Segundo Cordain, a substituição de alimentos minimamente processados por alimentos pós-agrícolas e pós-industriais influenciaram os indicadores nutricionais: carga glicêmica, composição de ácidos graxos e macronutrientes, densidade de micronutrientes, equilíbrio ácido-base, relação sódio/potássio e teor de fibras, levando a um desequilíbrio que é causa de várias doenças atuais da civilização. A Protein Leverage Hypothesis (PLH) propõe que a queda na ingestão de proteínas possa levar a obesidade e doenças cardiometabólicas associadas. Objetivos: Estudar o efeito do consumo de alimentos ultraprocessados nos indicadores nutricionais na população dos EUA, incluindo a composição de macronutrientes, densidade de fibras e micronutrientes e fitoestrógenos urinários; avaliar se a contribuição calórica de alimentos ultraprocessados é determinante para a qualidade nutricional das dietas contemporâneas; e finalmente estudar se a associação entre o consumo de alimentos ultraprocessados, proteína e energia correspondem às previsões do modelo PLH. Métodos: Foram avaliados os participantes do National Health and Nutrition Examination Survey 2009-2010, com pelo menos um recordatório alimentar de 24 horas. Os itens foram classificados em: alimentos in natura ou minimamente processados, processados, ultraprocessados e ingredientes de uso culinário. O manuscrito 1 examina a relação entre a contribuição calórica de alimentos ultraprocessados e qualidade nutricional da dieta, avaliando individual e globalmente a contribuição de cada ingrediente crítico, usando a análise de componentes principais (ACP). O manuscrito 2 estuda a associação entre a contribuição calórica dos alimentos ultraprocessados e consumo de açúcares de adição. O manuscrito 3 avalia como o consumo de alimentos ultraprocessados influencia o conteúdo proteico relativo da dieta e as ingestões absolutas de energia e proteína, e se essas relações se encaixam nas previsões da PLH. O manuscrito 4 avalia a relação entre a contribuição calórica de alimentos ultraprocessados e níveis de fitoestrógenos urinários. Resultados: O teor médio de proteínas, fibras, vitaminas A, C, D e E, zinco, potássio, fósforo, magnésio e cálcio na dieta diminuiu ao longo dos quintis de contribuição calórica de alimentos ultraprocessados, enquanto o de carboidratos, açúcares de adição e gordura saturada aumentou. Uma associação inversa de dose-resposta foi encontrada entre o consumo de alimentos ultraprocessados e qualidade nutricional total, medida através de um escore de padrão balanceado de nutrientes derivado usando ACP. Consistente com a PLH, a contribuição calórica de alimentos ultraprocessados foi inversamente associada à densidade proteica e diretamente ao consumo energético total, enquanto a ingestão absoluta de proteínas permaneceu constante com aumento do consumo de alimentos ultraprocessados. Os níveis médios de enterolignanos urinários diminuíram ao longo dos quintis de consumo de alimentos ultraprocessados, enquanto os níveis de isoflavonas permaneceram inalterados. Conclusões: Este estudo mostra que a diminuição da contribuição calórica de alimentos ultraprocessados é um meio racional e eficaz de melhorar a qualidade nutricional das dietas dos EUA / Background: The introduction of agricultural and animal husbandry has not provided the human genome time enough to adapt, much less the advancing technology after Industrial Revolution. According to Cordain et al., displacement of minimally processed foods by post-agricultural and post-industrial food items adversely affected the following dietary indicators: glycemic load, fatty acid and macronutrient compositions, micronutrient density, acid-base balance, sodium-potassium ratio and fiber content. Many current diseases of civilization, in turn may be ascribable to those unbalanced dietary indicators. Indeed, Raubenheimer and Simpson have proposed the Protein Leverage Hypothesis (PLH) to explain how a drop in dietary protein content might lead to obesity and associated cardiometabolic disease. Objective: This thesis aims to study the effect of an increased consumption of ultra-processed foods on dietary indicators in the US population, including macronutrient composition, micronutrient and fiber densities, and urinary phytoestrogens. It also explores whether the dietary share of ultra-processed foods, expressed as a percentage of total energy intake, is a meaningful determinant of overall nutritional quality of contemporary diets. Lastly, it also looks into whether the association between ultra-processed food, protein and energy consumptions fit predictions of the PLH model. Methods: Participants from cross-sectional 2009-2010 National Health and Nutrition Examination Survey with at least one 24-hour dietary recall were evaluated. Food items were classified according to extent and purpose of industrial food processing as: unprocessed or minimally processed foods, processed culinary ingredients, processed foods and ultra-processed foods. Manuscript 1, examines the relationship between dietary contribution of ultra-processed foods and nutritional quality of US diet through the evaluation of dietary contents of critical nutrients individually and also overall, using Principal Component Analysis (PCA). Manuscript 2 studies the association between dietary contribution of ultra-processed foods and energy intake from added sugars. Manuscript 3 examines how consumption of ultra-processed food influences relative dietary protein content and, absolute energy and protein intakes; it furthermore, tests whether the relationships fit PLH predictions. Manuscript 4 assesses the relationship between dietary contribution of ultra-processed foods and urinary levels of phytoestrogens. Results: The average content of protein, fiber, vitamins A, C, D and E, zinc, potassium, phosphorus, magnesium and calcium in US diet decreased significantly across quintiles of energy contribution of ultra-processed foods, while carbohydrate, added sugars and saturated fat contents increased. An inverse dose-response association was found between ultra-processed food consumption and overall dietary quality measured through a Nutrient balanced pattern PCA derived factor score. Consistent with PLH, dietary contribution of ultra-processed foods was inversely associated with protein density and directly associated with total energy intake, while absolute protein intake remained relatively constant with increases in ultra-processed food consumption. Average urinary mammal lignan levels decreased across quintiles of ultra-processed food consumption, while isoflavone levels remained unchanged. Conclusions: This study suggests that decreasing the dietary share of ultra-processed foods is a rational and effective way to improve the nutritional quality of US diets

Eine ständig steigende Nachfrage nach einer auf pflanzlichen Stoffen basierenden Hormonersatztherapie für menopausale Beschwerden, macht eine verstärkte Untersuchung sogenannter Phytoöstrogene erforderlich. Bei den hier untersuchten Substanzen handelt es sich um die chemisch synthetisierten Flavanone 6-(1,1-Dimethylallyl)Naringenin (6DMAN) und 8-Prenylnaringenin (8PN), die ursprünglich in Pflanzen identifiziert wurden. 7-Oxyprenylnaringenin-4'-Azetat (7OPN) ist ein weiteres Mitglied dieser Stoffgruppe, zu dem jedoch bisher noch praktisch keine Daten vorlagen. Ebenfalls in die Untersuchungen einbezogen wurde ein isopropanolischer Extrakt aus dem Rhizom von Cimicifuga racemosa (L.) Nutt. (iCR), der schon seit langer Zeit erfolgreich zur Bekämpfung menopausaler Beschwerden eingesetzt wird, dessen Wirkmechanismus jedoch unbekannt ist und zu dem bisher noch wenige molekulare Daten bezüglich der Wirkung auf einzelne Zielorgane, insbesondere nach einer Behandlung über einen längeren Zeitraum vorliegen. Die durchgeführten Untersuchungen betrafen zum Einen den Uterus als eines der Hauptzielorgane weiblicher Sexualhormone und zum Anderen die Vena cava, repräsentativ für das Gefäßsystem. Als Modellorganismus wurden Ratten gewählt. Dabei wurde die Genexpression Östrogen (E2)-regulierter Gene auf mRNA-Ebene mittels Real-Time-PCR bestimmt. Die Wirkung von 7OPN wurde in einem in vitro-System (MVLN-Zellen) und im Uterus getestet. Zusätzlich wurden humane Nabelschnurvenen-Endothelzellen als in vitro-Modell für das Gefäßsystem eingesetzt. Mit ihnen wurde die Wirkung der Testsubstanzen auf Angiogenese und Differenzierung getestet. Die durch die E2 hervorgerufenen Effekte entsprachen weitestgehend den Erwartungen. Es konnte sowohl eine Zunahme des Uterusgewichts der Ratten als auch eine Regulation der Expression von Proliferationsmarkern und E2-abhängig regulierten Genen im Uterus auf mRNA-Ebene festgestellt werden. Auch im Gefäßsystem konnte sowohl eine Regulation E2-abhängig exprimierter Gene beobachtet werden, als auch ein proliferationsfördernde und differentiationsfördernde Wirkung auf venöse Endothelzellen in vitro. Die untersuchten Flavanone zeigten einen von den vorhandenen funktionalen Gruppen abhängigen Effekt auf die E2-regulierten Prozesse und Gene. Während es sich bei 8PN um eine rein östrogen wirkende Substanz zu handeln scheint, wirkt 6DMAN offenbar als selektiver Östrogenrezeptor-Modulator (SERM) und zeigt für einen Einsatz in der HRT sehr interessante Eigenschaften, da weder ein uterotrophe Wirkung in vivo, noch eine angiogene Wirkung in vitro beobachtet werden konnte, während gleichzeitig eine östrogene, potentiell kardioprotektive Wirkung auf die Genexpression im Gefäßsystem gezeigt werden konnte. Auch 7OPN zeigt ein differenziertes Wirkmuster. Ein in vitro-Reportergentest in einer stabil transfizierten Brustkrebszellinie deutete auf eine in Abhängigkeit von der Konzentration E2-antagonistische oder agonistische Wirkung hin. Die Genexpressionsstudien im Uterus der Versuchstiere nach dreitägiger Behandlung zeigten, daß die Wirkung von 7OPN jedoch auch abhängig vom untersuchten Gen ist. Da auch dieses Flavanon keine uterotrophe Wirkung zu besitzen scheint, ist 7OPN ebenfalls eine interessante Zielsubstanz weiterer Untersuchungen im Hinblick auf die Anwendung in der HRT. Für den in einem Arzneimittel enthaltenen Extrakt iCR konnte in einem 17-tägigen Tierversuch keine östrogene Wirkung auf Uterus und Gefäßsystem gezeigt werden. Während eine Anwendung dieses Extrakts zur Behandlung menopausaler Beschwerden im Lichte dieser Ergebnisse und langjähriger praktischer Erfahrungen zwar sicher erscheint, bleibt der Wirkmechanismus von iCR weiterhin unbekannt.

Due to the current rise of exposure to natural and synthetic compounds in our daily life, the debate concerning the safety of many substances is becoming increasingly relevant. The estrogenic activity of various compounds, described as xenoestrogens, is the major part of this debate. Humans beings are exposed to these substances from different environmental contaminations ranging from conscious intake of estrogenic substances, as in contraception or in hormone replace therapy (HRT), to unconscious exposure, from food, the use of synthetic material in daily life and air and water pollution. At this point the need for methods to investigate the activity and the safety of these substances is becoming increasingly important. Classical methods for the analysis of the estrogenic activity of substances, like batteries of in vivo test systems on the rat uterotrophic assay are not able to describe the different pathways of action of recently discovered estrogenic substances. This evidence was already shown by the Organization for Economic Cooperation and Development (OECD), introducing new test guidelines for the investigation of effects of endocrine disruptors (according to enhanced Test Guideline 407). As reviewed by Nilsson (Nilsson et al., 2001), after the interaction of the estrogens with the Estrogen Receptor (ER) in the cells, the mechanism of activation possible is not only via direct binding of the ER to the Estrogen Responsive Elements (EREs) present in the promoter region of the target gene, very well described for many target genes, but that also other mechanisms are used: the interaction of the ER with the AP 1, Sp 1 and NFkB modes, that are discovered but not yet comprehensively described. The aim of my work is to produce a microarray DNA chip for the investigation of the estrogenic activity of different compounds present in the environment. The chip will consist of a selection of 100 genes that are estrogen responsive and it will cover the spectrum of activities of estrogenic compounds in various organs of the body. In the gene selection, genes were chosen that are estrogen responsive in the classical target tissues of estrogens, linked to reproduction, like uterus and mammary gland, and also in tissues not related to reproduction like liver, bones and capillars. In addition, other genes are included to monitor different pathways that are related to disease states; control of cell proliferation, apoptosis or cancer related genes. Currently these kinds of investigations are already in process, but by other methods which are more time consuming and with a lower throughput e.g. the gene expression profiling using the real time RT-PCR. The use of microarray’s satisfies the need for a less time consuming, high throughput method, to obtain a fast characterization of the gene expression finger print of the candidate substances and their mechanism of action in the organism. In my work I investigated the estrogenic potency of different Xenoestrogens that commonly occur in our daily life, in rat cells and tissue using well known estrogen sensitive genes like C3, Clu, IGFBP1 and CaBP9k. I focused on their effect on cell proliferation, studying PCNA expression. For the first time sensitivity of the gene CA2 was proofed in liver and uterus. A new identified mRNA sequence, r52, was characterized for its sensitivity to estrogenic exposure. This sequence was investigated at the molecular level expanding the known nucleic sequence. I produce a microarray chip with 16 genes to investigate the estrogenic potency of different compounds. As proof of principle of the microarray method completely produced in house I compared the result of gene expression obtained by the chip to that obtained by real time RT PCR finding a similarity of results. This new established method is less sensitive than the real-time RT PCR but allows a high throughput of gene expression analysis producing at the end a more complete picture of the expression signature of a compound.

Die niedrige Inzidenz chronisch-entzündlicher Darmerkrankungen (CED) in ostasiatischen Ländern im Vergleich zu Westeuropa und den USA könnte auf unterschiedliche Lebensstile und Ernährungsgewohnheiten zurückzuführen sein. Asiaten nehmen mit der Nahrung viel höhere Mengen an Isoflavonen zu sich als Europäer und US-Amerikaner. Diese sind in der Lage, wie natürliche Östrogene an Östrogenrezeptoren (ER) zu binden. Für das Östrogen 17β-Östradiol (E2) sowie selektive Liganden des ERβ sind antiinflammatorische Wirkungen im Darm bereits nachgewiesen worden. Diese Arbeit untersuchte in Modellsystemen für CED die antiinflammatorischen Eigenschaften von Isoflavonen, speziell von Genistein, und stellte einen Vergleich mit synthetischen ER-selektiven Liganden sowie E2 her, um die Involvierung der beiden ER-Subtypen zu evaluieren. In tierexperimentellen Studien wurde der Einfluss der Testsubstanzen auf Ausprägung und Verlauf einer Kolitis in zwei Nagermodellen (HLA-B27 transgene Ratte und TNBS-induzierte Kolitis) analysiert. Ein Ernährungsexperiment, in dem eine Gruppe der Tiere bereits in utero sowie postnatal über Muttermilch und Futter hohen Phytoöstrogenspiegeln ausgesetzt war, zeigte wider Erwarten keine antiinflammatorischen Effekte auf die akute Ausprägung der induzierten Kolitis. Stattdessen waren die untersuchten Parameter bei dieser Ernährungsform gegenüber prä- und postnatal normal ernährten Tieren verstärkt. Dagegen bewirkte oral verabreichtes Genistein in der chronischen Phase der TNBS-induzierten Kolitis eine Unterdrückung der Entzündungsparameter im Darm. Die subkutane Verabreichung von Genistein, eines steroidalen ERβ-selektiven Agonisten, oder von E2 führte hingegen zu keiner signifikanten Einflussnahme auf die untersuchten Parameter in der akuten Phase der Inflammation. Zur Charakterisierung der molekularen Grundlagen einer antiinflammatorischen Wirkung von E2, synthetischen ER-selektiven Agonisten und Genistein wurden in vitro Studien mit Kolonkarzinomzelllinien (HT-29 und Caco-2) durchgeführt. Hierzu wurden die Zellen mit Interleukin-1β (IL-1β) stimuliert, was eine Induktion der inflammationsassoziierten Gene Cyclooxygenase-2 und Interleukin-6 auf mRNA Ebene bewirkte. Bis auf Genistein konnten für die getesteten Substanzen keine antiinflammatorischen Effekte auf die mRNA-Expression der induzierten Markergene beobachtet werden. Genistein bewirkte in Caco-2 Zellen eine Hemmung der untersuchten Gene. Weitere Analysen ergaben, dass die beiden Zelllinien ER nur schwach bzw. gar nicht exprimieren. Eine Transfektion von HT-29 Zellen mit ERα führte zu einer deutlichen Hemmung der Expression der Markergene durch E2, während eine Transfektion mit ERβ lediglich einen schwach hemmenden Effekt bewirkte. Die Ergebnisse der vorliegenden Arbeit legen nahe, dass die niedrigen CED-Inzidenzraten in Ostasien wohl nicht allein auf dem dortigen hohen Isoflavonkonsum beruhen, sondern auch anderen Komponenten des Lebensstils zuzuschreiben sind. Dennoch deutet sich an, dass das Genistein, bei oraler Administration, die Regeneration des geschädigten Darmgewebes im chronischen Erkrankungsverlauf unterstützen und damit auch zur Prävention von Kolonkarzinomen beitragen könnte. Bei antiinflammatorischen Effekten von ER-Liganden spielt die Transaktivierung von ER eine entscheidende Rolle. Die Wirkung von Genistein in untransfizierten Caco-2 Zellen legt jedoch auch die Teilnahme weiterer Mechanismen nahe, die noch zu untersuchen sind. Vor diesem Hintergrund erscheinen weiterführende Untersuchungen zum Einsatz von steroidalen ER-Agonisten und Genistein bei CED und den zugrunde liegenden Mechanismen als sinnvoll.

<p>U ovoj doktorskoj disertaciji&nbsp; razvijen&nbsp; je fluorescentni test u kvascu za identifikaciju potencijalnih prirodnih ili sintetičkih liganada&nbsp; ER&alpha;, ER&beta; ili AR i kvantifikaciju&nbsp; njihovog&nbsp; afiniteta&nbsp; vezivanja sa mogućno&scaron;ću testiranja čitavih biblioteka modifikovanih steroida i ksenoestrogena. Takođe, opisana&nbsp; je primena optimizovanog biosenzora&nbsp; za&nbsp; procenu&nbsp; estrogenog&nbsp; potencijala sintetskih steroida i odabranih biljnih ekstrakata bogatih jedinjenjima fitoestrogenih osobina. U cilju potpunijeg sagledavanja mehanizma&nbsp; delovanja&nbsp; odabranih&nbsp; modifikovanih&nbsp; steroida&nbsp; ispitana&nbsp; je&nbsp; njihova antiproliferativna aktivnost prema ćelijskim&nbsp; linijama estrogen receptor pozitivnog kancera dojke&nbsp; (MCF-7) i kancera prostate (PC-3), dok su&nbsp; in silico metodom molekularnog&nbsp; dokinga&nbsp; predviđene&nbsp; energije&nbsp; i&nbsp; geometrije&nbsp; vezivanja&nbsp; ovih&nbsp; jedinjenja za ligand-vezujuće&nbsp; domene&nbsp; ER&alpha; i ER&beta;. Drugi deo ovog rada obuhvata razvoj testa za&nbsp; ispitivanje aktivnosti humanog enzima aromataze,&nbsp; heterologno eksprimiranog u ćelijama&nbsp; kvasca&nbsp; Saccharomyces cerevisiae&nbsp; i/ili&nbsp; bakterija Escherichia coli, u prisustvu&nbsp; ili&nbsp; odsustvu&nbsp; inhibitora.&nbsp; Interakcije modifikovanih&nbsp; steroida, odabranih na osnovu strukture,&nbsp; sa&nbsp; aromatazom&nbsp; ispitane&nbsp; su&nbsp; osetljivim spektroskopskim metodama, praćenjem promene spinskog stanja Fe iz hem grupe ili promene fluorescencije ostatka&nbsp; triptofana&nbsp; iz&nbsp; aktivnog&nbsp; centra usled konformacione&nbsp; promene&nbsp; proteina, izazvane interakcijom sa ligandom. Razvijeni in vitro testovi bez upotrebe radioaktivnih izotopa su, osim&nbsp; visoke efikasnosti&nbsp; i&nbsp; bezbednosti&nbsp; po&nbsp; korisnika&nbsp; i&nbsp; okolinu, pokazali&nbsp; specifičnost&nbsp; i&nbsp; ekonomičnost&nbsp; u preliminarnom&nbsp; skriningu&nbsp; liganada&nbsp; steroidnih receptora&nbsp; i inhibitora aromataze. Jedinjenja&nbsp; kod kojih je detektovana&nbsp; značajna biolo&scaron;ka aktivnost mogu potencijalno poslužiti kao osnova za razvoj terapeutika u lečenju hormon-zavisnih bolesti i stanja, koja danas predstavljaju globalni zdravstveni problem.</p> / <p>In&nbsp; this&nbsp; doctoral&nbsp; dissertation,&nbsp; a&nbsp; fluorescent&nbsp; assay&nbsp; in&nbsp; yeast&nbsp; was&nbsp; developed&nbsp; for&nbsp; identification&nbsp; of&nbsp; potential&nbsp; natural or synthetic ligands of ER&alpha;, ER&beta; or AR and<br />quantification&nbsp; of&nbsp; their&nbsp; binding&nbsp; affinity,&nbsp; as&nbsp; well&nbsp; asevaluation&nbsp; of&nbsp; the&nbsp; estrogenic&nbsp; potential&nbsp; of&nbsp; synthetic steroids&nbsp; and&nbsp; selected&nbsp; plant&nbsp; extracts&nbsp; rich&nbsp; in phytoestrogen&nbsp; content.&nbsp; The&nbsp; assay&nbsp; could&nbsp; be&nbsp; used&nbsp; to&nbsp; screen&nbsp; libraries&nbsp; of&nbsp; modified&nbsp; steroids&nbsp; and xenoestrogens.&nbsp; In&nbsp; order&nbsp; to&nbsp; better&nbsp; understand&nbsp; the biomedical&nbsp; potential&nbsp; of&nbsp; selected&nbsp; modified&nbsp; steroids, results&nbsp; were&nbsp; compared&nbsp; to&nbsp; antiproliferative&nbsp; activity against&nbsp; estrogen&nbsp; receptor&nbsp; positive&nbsp; breast&nbsp; cancer (MCF-7)&nbsp; and&nbsp; prostate&nbsp; cancer&nbsp; (PC-3)&nbsp; cell&nbsp; lines. Binding&nbsp; energies&nbsp; and&nbsp; the&nbsp; geometry&nbsp; of&nbsp; binding&nbsp; of these&nbsp; compounds&nbsp; for&nbsp; ER&alpha;&nbsp; and&nbsp; ER&beta;&nbsp; ligand&nbsp; binding domains&nbsp; were&nbsp; predicted&nbsp; in&nbsp; silico&nbsp; by&nbsp; molecular&nbsp; docking&nbsp; methods.&nbsp; The&nbsp; second&nbsp; part&nbsp; of&nbsp; this&nbsp; study includes development&nbsp; of&nbsp; an&nbsp; assay&nbsp; for&nbsp; study&nbsp; of&nbsp; aromatase&nbsp; activity&nbsp; in&nbsp; the&nbsp; presence&nbsp; or&nbsp; absence&nbsp; of inhibitors&nbsp; by&nbsp; heterologous&nbsp; expression&nbsp; of&nbsp; human aromatase&nbsp; in&nbsp; Saccharomyces&nbsp; cerevisiae&nbsp; and/or Escherichia&nbsp; coli&nbsp; cells,&nbsp; as&nbsp; model-organisms. Furthermore, interactions between modified steroids, selected&nbsp; according&nbsp; to&nbsp; their&nbsp; structure,&nbsp; and&nbsp; aromatase were&nbsp; tested&nbsp; using&nbsp; sensitive&nbsp; spectroscopic&nbsp; methods based on ligand-induced changes&nbsp; in&nbsp; the&nbsp; spin state of Fe&nbsp; from&nbsp; the&nbsp; heme&nbsp; group&nbsp; or&nbsp; changes&nbsp; in&nbsp; the fluorescence&nbsp; of&nbsp; a&nbsp; tryptophan&nbsp; residue&nbsp; in&nbsp; the&nbsp; active site.&nbsp; The&nbsp; non-radioactive&nbsp; in&nbsp; vitro&nbsp; assays&nbsp; developed&nbsp; here, besides high efficiency, user and environmental safety,&nbsp; also&nbsp; have&nbsp; greater&nbsp; specificity&nbsp; and&nbsp; are&nbsp; more cost-effective&nbsp; for&nbsp; preliminary&nbsp; screening&nbsp; of&nbsp; steroid receptor&nbsp; ligands&nbsp; and&nbsp; aromatase&nbsp; inhibitors. Additionally,&nbsp; compounds&nbsp; identified&nbsp; to&nbsp; express significant biological activity can serve as a basis for the&nbsp; development&nbsp; of&nbsp; potential&nbsp; therapeutics&nbsp; in&nbsp; the treatment&nbsp; of&nbsp; hormone-dependent&nbsp; diseases&nbsp; and conditions, a global health issue today.</p>