Plusieurs éléments de la pathogenèse de la scoliose idiopathique de l’adolescent indiquent que les estrogènes pourraient intervenir dans le développement et la progression de cette maladie. Ce projet avait donc pour but d’explorer l’expression et la fonctionnalité des récepteurs aux estrogènes ERα et ERβ ainsi que leurs isoformes dans les ostéoblastes de patients scoliotiques et sains. L’induction des gènes de facteurs influençant la minéralisation et la différenciation des ostéoblastes par les estrogènes a également été étudié. Par immunofluorescence, nous avons remarqué une augmentation de la présence protéique de ERβ dans les ostéoblastes de patients SIA comparé aux sujets contrôles. Les récepteurs aux estrogènes provenant des ostéoblastes des patients sont fonctionnels tout comme ceux des contrôles et aucune différence dans l'interaction ADN-protéine n’a été observée. Il y a également une augmentation de l’expression génique de l’ostéopontine, l’ostéocalcine, le collagène de type I, la phosphatase alkaline et BMP2 dans les ostéoblastes des patients SIA. Un début de minéralisation in vitro a été observé dans les ostéoblastes de patients SIA et contrôles. ERα et ERβ sont présents et fonctionnels dans les ostéoblastes des patients SIA et sains. Leur expression est variable, mais ces variations existent chez les patients SIA et les contrôles. L’implication des estrogènes dans la SIA ne serait donc pas au niveau des récepteurs aux estrogènes mais au niveau de l’interactions des estrogènes avec d’autres facteurs étiologiques tels que la mélatonine, la formation/résorption osseuse ou autres facteurs neuro-endocriniens. / Recent developments in the research on pathogenesis of Adolescent Idiopathic Scoliosis indicate that estrogens could intervene in the development and progression of this disease. Therefore, this project focussed on the expression and functionality of estrogen receptors ERα and ERβ and their isoforms in osteoblasts of scoliotic patients and controls. The induction by estrogens of the expression of factors influencing mineralization and osteoblast differentiation was also studied. We observed by immunofluorescence, an increase of the presence of ERβ in osteoblasts of AIS patients compared to controls. A higher level of expression of osteopontin, osteocalcin, type I collagen, alkaline phosphatase and BMP2 was found in osteoblasts of AIS patients compared to controls. Estrogen receptors in osteoblasts from AIS patients are functional, as they were in controls and no difference in DNA-binding activity was observed. ERα et ERβ are present and functional in osteoblasts from AIS patients and controls. Their expression is variable, but these variations exist in both populations. The implication of estrogens in AIS would therefore be in the interaction between these hormones and other factors influencing the aetiology of AIS like melatonin, bone formation and resorption and neuro-endocrin factors, rather than in a default in the estrogens receptors themselves.

La croissance de deux tiers des tumeurs mammaires dépend des œstrogènes. Le réseau de gènes responsable de propager les signaux prolifératifs des œstrogènes est encore mal connu. Des micropuces d’ADN de cellules de carcinome mammaire MCF7 traitées à l’œstradiol (E2) avec ou sans l’inhibiteur de synthèse protéique cycloheximide (CHX) ont permis d’identifier de nombreux gènes cibles primaires et secondaires. La séquence des promoteurs des gènes cibles a été criblée à l’aide d’une banque de 300 matrices modélisant les sites reconnus par divers facteurs de transcription. Les éléments de réponse aux œstrogènes (ERE) sont enrichis dans les promoteurs des gènes primaires. Les sites E2F sont enrichis dans les promoteurs des gènes cible secondaires. Un enrichissement similaire a été observé avec les régions liées par ERα et E2F1 en ChIP-on-chip pour chacune des catégories de gènes. La croissance des cellules de carcinome mammaire est inhibée par des traitements à l’acide rétinoïque (RA). L’analyse de micropuces d’ADN de MCF7 traitées avec RA a permis d’identifier de nombreux gènes cibles potentiels. Un enrichissement d’éléments de réponse à l’acide rétinoïque (RARE) est observable dans les promoteurs de ces gènes après avoir exclus les RARE se trouvant à l’intérieur d’éléments transposables. Des RARE présents dans des éléments transposables spécifiques aux primates sont aussi fixés in vivo dans les promoteurs de cibles connues de RA : BTG2, CASP9 et GPRC5A. Certains gènes cibles de RA dans les MCF7 sont aussi des cibles de E2, suggérant que le contrôle que ces molécules exercent sur la prolifération est en partie attribuable à des effets opposés sur un ensemble commun de gènes. / Two thirds of breast tumours depend on estrogens for their growth. The network of genes mediating the proliferative effect of estrogens is not fully characterized. Putative primary and secondary estrogen target genes were identified with microarray analysis of MCF7 breast cancer cells treated with estradiol (E2) in presence or absence of the protein synthesis inhibitor cycloheximide (CHX). The promoters of the target genes were screened for transcription factor binding sites with a collection of 300 matrix based DNA-binding profiles. Estrogen response elements (EREs) were enriched in the promoters of primary target genes. E2F binding sites were enriched in the promoters of secondary target genes. Similar enrichment was also observed in regions bounds by ERα and E2F1 in ChIP-on-chip experiments for each set of target genes. Retinoic acid (RA) treatment of mammary carcinoma cells inhibits their growth. Putative target genes were identified through microarray analysis of MCF7 cells treated with RA. Enrichment of retinoic acid response elements (RARE) was observed in their promoters after removing the elements found within transposable elements. Although transposable elements mask the enrichment, RARE within primate specific transposable elements are bound in vivo by retinoic acid receptors in the promoters of known target genes BTG2, CASP9 and GPRC5A. Some of the RA target genes in MCF7 cells are also target genes of E2 suggesting that these two molecules exert their effects on cell proliferation in part by opposite action on a common set of genes.

La grossesse est un état physiologique particulier où de nombreux changements fonctionnels et structuraux surviennent. Chez la rate, pour répondre aux besoins grandissants du fœtus, l’artère utérine se développe pour atteindre le double de son diamètre original avant parturition. Par conséquent, le débit sanguin utérin augmente d’environ vingt fois. Pour ce faire, les vaisseaux utérins sont l’objet d’un remodelage caractérisé par une hypertrophie et une hyperplasie des différentes composantes de la paroi. De plus, ce remodelage est complètement réversible après la parturition, par opposition au remodelage vasculaire « pathologique » qui affecte les artères systémiques, dans l’hypertension chronique, par exemple. La grossesse s’accompagne aussi de modifications hormonales importantes, comme les œstrogènes dont la concentration s’accroît progressivement au cours de cette période. Elle atteindra une concentration trois cents fois plus élevée avant terme que chez une femme non gravide. Cette hormone possède de multiples fonctions, ainsi qu’un mode d’action à la fois génomique et non génomique. Considérant l’ensemble de ces éléments, nous avons formulé l’hypothèse que l’œstradiol serait responsable de modifier la circulation utérine durant la grossesse, par son action vasorelaxante, mais aussi en influençant le remodelage de la vasculature utérine. Nous avons montré que le 17β-Estradiol (17β-E2) produit une relaxation due à un effet non génomique des artères utérines en agissant directement sur le muscle lisse par un mécanisme indépendant du monoxyde d’azote et des récepteurs classiques aux œstrogènes (ERα, ERβ). De plus, la relaxation induite par le 17β-E2 dans l’artère utérine durant la gestation est réduite par rapport à celle des artères des rates non gestantes. Ceci serait attribuable à une diminution de monoxyde d’azote provenant de la synthase de NO neuronale dans les muscles lisses des artères utérines. Nos résultats démontrent que le récepteur à l’œstrogène couplé aux protéines G (GPER), la protéine kinase A (PKA) et la protéine kinase G (PKG) ne sont pas impliqués dans la signalisation intracellulaire associée à l’effet vasorelaxant induit par le 17β-E2. Cependant, nous avons montré une implication probable des canaux potassiques sensibles au voltage, ainsi qu’un rôle possible des canaux potassiques de grande conductance activés par le potentiel et le calcium (BKCa). En effet, le penitrem A, un antagoniste présumé des canaux potassiques à grande conductance, réduit la réponse vasoralaxante du 17β-E2. Toutefois, une autre action du penitrem A n’est pas exclue, car l’ibériotoxine, reconnue pour inhiber les mêmes canaux, n’a pas d’effet sur cette relaxation. Quoi qu’il en soit, d’autres études sont nécessaires pour obtenir une meilleure compréhension des mécanismes impliqués dans la relaxation non génomique sur le muscle lisse des artères utérines. Quant à l’implication de l’œstrogène sur le remodelage des artères utérines durant la gestation, nous avons tenté d’inhiber la synthèse d’œstrogènes durant la gestation en utilisant un inhibiteur de l’aromatase. Plusieurs paramètres ont été évalués (paramètres sanguins, réactivité vasculaire, pression artérielle) sans changements significatifs entre le groupe contrôle et celui traité avec l’inhibiteur. Le même constat a été fait pour le dosage plasmatique de l’œstradiol, ce qui suggère l’inefficacité du blocage de l’aromatase dans ces expériences. Ainsi, notre protocole expérimental n’a pas réussi à inhiber la synthèse d’œstrogène durant la grossesse chez le rat et, ce faisant, nous n’avons pas pu vérifier notre hypothèse. En conclusion, nous avons démontré que le 17β-E2 agit de façon non génomique sur les muscles lisses des artères utérines qui implique une action sur les canaux potassiques de la membrane cellulaire. Toutefois, notre protocole expérimental n’a pas été en mesure d’évaluer les effets génomiques associés au remodelage vasculaire utérin durant la gestation et d’autres études devront être effectuées. / Pregnancy is a peculiar physiological state in which many structural and functional changes occur. In rats, to meet the growing needs of the fetus, uterine artery expands to reach twice its original diameter before parturition. Therefore, uterine blood flow increases by about twenty times. To do this, the uterine vessels undergo substantial remodelling characterized by hypertrophy and hyperplasia of the various components of the wall. Furthermore, this remodelling is completely reversible after parturition, as opposed to “pathological vascular remodelling” that affects systemic arteries in chronic hypertension, for instance. Pregnancy is also accompanied by significant hormonal changes, such as estrogens whose concentration in the blood increases progressively during this period. It reaches concentrations three hundred times higher before term than in non-pregnant women. This hormone has multiple functions and mediates its effects by genomic and non-genomic pathways. Considering these elements, we hypothesized that estradiol would be responsible for remodeling the uterine circulation during pregnancy, by its vasorelaxant action, but also by influencing the remodeling of the uterine vasculature. We have shown that 17β-Estradiol (17β-E2) produced non-genomic relaxation of uterine arteries by acting directly on smooth muscle using a mechanism independent of nitric oxide and classical estrogen receptors (ERα, ERβ). Moreover, the relaxation induced by 17β-E2 in the uterine artery during pregnancy is reduced compared to that of arteries from non-pregnant animals. This is the result of decrease of nitric oxide derived from neuronal NO synthase in smooth muscle of uterine arteries. Our results also show that the estrogen receptor coupled to G proteins (GPER), protein kinase A (PKA) and protein kinase G (PKG) are NOT involved in intracellular signaling associated with the vasorelaxant effect induced by the 17β-E2. Moreover, we have shown a possible involvement of potassium channels sensitive to voltage and a possible involvement of large conductance calcium-activated potassium channels (BKCa). Indeed, the penitrem A, a suspected antagonist of BKCa, reduced the vasoralaxant responses of 17β-E2. However, some other actions of penitrem A are not excluded because iberiotoxin, known to inhibit the same channels, had no effect on this relaxation. However, further studies are needed to obtain a better understanding of the mechanisms involved in the relaxation non-genomics on the smooth muscle of uterine arteries. As for the involvement of estrogen on the remodeling of uterine arteries during pregnancy, we attempted to inhibit the synthesis of estrogen during pregnancy using an aromatase inhibitor. Several parameters were evaluated (plasma values, vascular reactivity, blood pressure) without significant changes between the control group and those treated with the inhibitor. The same observation was made for the determination of plasma estradiol, suggesting the uneffectiveness of aromatase blocking in these experiments. Thus, our experimental protocol failed to inhibit the synthesis of estrogen in rats during pregnancy and, in so doing, we cannot confirm or refute our hypothesis. In conclusion, we demonstrated that 17β-E2 acts on smooth muscle of the uterine arteries by a non-genomic pathway by apparently acting on potassium channels. However, our experimental protocol was not able to assess the genomic effects associated with uterine vascular remodeling during pregnancy and further studies are required to ascertain this aspect of our work.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Il apparaît, suite aux résultats de plusieurs études comportementales et d’imagerie cérébrale, que les hormones gonadiques peuvent moduler le fonctionnement cérébral chez la femme. Les asymétries cérébrales fonctionnelles (ACFs), en particulier, changeraient en fonction du niveau de progestérone et d’œstrogène. On a également observé que lorsque le taux d’œstrogène est bas, les performances aux tâches impliquant l’hémisphère droit sont améliorées. Par contre, les preuves de l’action physiologique de ces deux hormones sur le cerveau ne sont pas très nombreuses. Le peu d’études d’électrophysiologie cognitive qui ont porté sur les effets du cycle menstruel ont rapporté que la composante P300 y serait sensible. Aucune n’a cependant utilisé une tâche d’habileté spatiale ou de rotation mentale qui sont connues pour impliquer davantage l’hémisphère droit. Le but de la présente étude est de documenter les changements électrocorticaux reliés aux variations hormonales lors d’une tâche de rotation mentale. Notre hypothèse de départ est que le taux d’œstrogène influencera l’activité électrocorticale et la latéralisation. Les potentiels évoqués cognitifs ont été comparés chez les mêmes femmes (n=12) lors d’une tâche de rotation mentale, répétée à deux périodes du cycle menstruel. Nos résultats démontrent que la condition de rotation induit une latéralisation de l’activité pariétale, vers l’hémisphère gauche, quand le niveau d’œstrogène est bas. Par contre, lorsque le niveau d’œstrogène est élevé, il n’y a aucune latéralisation. Par ailleurs, nous avons observé une augmentation de l’amplitude de la P300 lors du niveau oestrogénique élevé. En conclusion, les fluctuations oestrogéniques du cycle menstruel ont un impact sur la latéralisation de l’activité électrocorticale, lors d’un effort de rotation mentale. / After many behavioral and some neuroimaging studies, it appears that the gonadic hormones can modulate the neuronal function of women's brain. In particular, the functional cerebral asymmetries can be affected by the level of progesterone and estrogens. It has been observed that when the level of estrogen is low, the performance at task that engages more the right hemisphere is enhanced. However, there is a lack of evidence for the physiological actions of these two hormones on the brain. The few event-related potential studies taking into account the menstrual cycle effects, had noticed that the component P300 can be affected. No electrophysiological study has used a mental rotation task or spatial ability tests which are known for their right hemisphere dominance. The aim of the present research is to document the effect of hormonal variations on the electrocortical activity, using a mental rotation task. Our hypothesis is that estrogen levels affect electrocortical activity and lateralization. The ERPs were compared in the same women (n = 12) during a mental rotation task, repeated over two periods of the menstrual cycle. Our results show a lateralization of the left parietal activity when estrogen levels are low and during the rotation. Whereas when the estrogen level is high, there is no lateralization. In addition, we observed an increase in the amplitude of P300 for this same high level. In conclusion, estrogens fluctuations associated with the menstrual cycle have an impact on the lateralization of electrocortical activity, when a mental rotation is needed.

Um dos grandes problemas da engenharia ambiental é a contaminação dos corpos hídricos. Os sanitaristas têm se preocupado com os hormônios sexuais, notadamente os estrógenos, compostos extremamente ativos biologicamente, que têm sido referidos como agentes etiológicos de feminilização e de vários tipos de cânceres. Os estrógenos naturais 17 \'beta\'-estradiol (E2), estriol (E3), estrona (E1) e o sintético 17 \'alfa\'-etinilestradiol (EE2), desenvolvidos para uso médico em terapias de reposição hormonal feminina e métodos contraceptivos, são os que despertam maiores preocupações, pela contínua introdução ao ambiente; hormônios que possuem a melhor conformação reconhecida pelos receptores que resultam respostas máximas, por isso são considerados responsáveis pela maioria dos efeitos disruptores desencadeados pela disposição de efluentes. A mudança de padrões quanto à atividade sexual dos jovens e a preocupação com o planejamento familiar, levaram ao grande consumo de contraceptivos que, através da urina, são levados pela rede coletora aos corpos de água. O indiscriminado uso desses hormônios na bovinocultura, suinocultura, avicultura e aqüicultura são responsáveis por parte considerável desse contaminante nos mananciais. Os hormônios excretados através da urina e fezes e agentes provenientes das indústrias de processamento de alimentos preocupam os sanitaristas porque o lançamento de efluentes in natura ou tratados, são as principais vias de contaminação do ambiente aquático, quer pelo déficit de infra-estrutura em saneamento, quer pela ineficiência tecnológica e/ou operacional na remoção desses compostos nas estações de tratamento de água ou de efluentes. Apesar de possuírem meia-vida relativamente curta, quando comparados a outros compostos orgânicos como praguicidas, os estrógenos naturais são continuamente introduzidos no ambiente, o que lhes confere caráter cumulativo. A proposta desta pesquisa foi verificar a presença de estrógenos na água bruta que chega à Estação de Tratamento de Água, após seu tratamento, em água tratada por osmose reversa e por tecnologia Milli Q (marca registrada). Para verificar e quantificar presença desses hormônios estrógenos em água de abastecimento de São Carlos-SP, foram realizadas exames através de imunoensaio quimiluminescente e radioimunoensaios. Os resultados apontaram que a ETA não possui solução eficiente para a remoção dos analitos de interesse dessa pesquisa, uma vez que na água tratada foram encontrados valores semelhantes aos da água bruta. / One of the major problems of environmental engineering is the water contamination. The sanitary persons have been concerned with the gonadal hormones, notably the estrogen, biologically active compounds extremely, which have been referred to as etiologic agents of feminization and several types of cancers. The natural estrogen 17 \'beta\'-estradiol (E2), estriol (E3), estrone (E1) and synthetic 17 \'alpha\'-ethinylestradiol (EE2), developed for medical use of hormone replacement therapy in women and contraceptive methods, are those that attract larger concerns by the continuous introduction into the environment; hormones that have the best conformation recognized by receptors that result answers maximum, so they are considered responsible for most of the effects disruptors triggered by the wastewater disposal. The change in patterns on the sexual activity of young people and the concern with family planning, led to the large consumption of contraceptives that, in the urine, are led by the distribution net to water. The indiscriminate use of these hormones in cattle, pigs, poultry and aquaculture are responsible for part of this contaminant in the source. The hormones excreted in the urine and feces and agents from the food processing industries in the sanitary concern that the launch of effluents in nature or treated, are the main routes of contamination of the aquatic environment, either by lack of infrastructure, sanitation, or by inefficiency technological and/or operating in the removal of these compounds in the treatment plants, water or effluent. Despite having relatively short stocking-lige when compared to other organics such as pesticides, natural estrogens are continuously released into the environment, which gives them character cumulative. The proposal of this research was to verify the presence of estrogen in the gross water that arrives at the water treatment plant, after the treatment, in water treated by reverse osmosis and by Milli Q technology. To verify and quantify presence of these hormones estrogen in water supply of San Carlos-SP, examinations were conducted through immunoassay chemiluminescent and radioimunoassays. The results showed that the ETA has no efficient solution for removal of interest analytes of this research, because in the treated water were found values similar to crude water.

Apesar do estudo exaustivo do transporte mucociliar nasal, ainda há dados controversos sobre a influência direta dos hormônios sexuais femininos nesse mecanismo. O presente estudo teve por objetivos: 1. avaliar o transporte mucociliar nasal de sacarina nos sexos masculino e feminino, comparando-o nas fases folicular, periovulatória e lútea de ciclos ovarianos consecutivos e 2. identificar a expressão e a localização dos receptores para estrógeno e progesterona na mucosa nasal humana em conchas nasais inferiores de indivíduos dos sexos masculino e feminino na idade reprodutiva. O transporte mucociliar nasal de sacarina foi avaliado prospectivamente em 14 voluntários não fumantes, sem queixas nasais, com idades entre 15 e 30 anos (7 homens e 7 mulheres, com média de idade 23,5 anos). Nas mulheres, o transporte mucociliar nasal de sacarina foi medido nas fases folicular, periovulatória e lútea durante dois ciclos ovarianos consecutivos (em cinco casos) ou três ciclos consecutivos (em dois casos). Nos homens, o transporte mucociliar nasal de sacarina foi avaliado em medidas repetidas aleatoriamente três vezes (em dois casos) ou seis vezes (em cinco casos). A expressão dos receptores para estrógeno e progesterona na mucosa nasal humana foi avaliada por método imunohistoquímico, de modo retrospectivo, em conchas nasais inferiores conservadas em formaldeído e fixadas na parafina, arquivadas após a cirurgia de turbinectomia parcial da concha inferior a que foram submetidos 20 pacientes da mesma faixa etária dos voluntários (10 pacientes do sexo masculino e 10 do sexo feminino, com idades entre 15 e 33 anos, média de idade 22,1 anos). Para a imuno-histoquímica utilizaram-se anticorpos monoclonais de camundongo contra receptores para estrógeno (clone 6F11, Novocastra) e para progesterona (clone 16, Novocastra) separadamente. Não houve diferenças significativas no transporte mucociliar nasal de sacarina entre as fases folicular, periovulatória e lútea em ciclos ovarianos consecutivos, nem entre os sexos (p=0,08). Entretanto, considerando-se apenas o primeiro ciclo ovariano, o transporte mucociliar nasal de sacarina foi mais rápido durante a fase folicular (p=0,03). Os receptores para estrógeno e progesterona foram encontrados no citoplasma das glândulas serosas da lâmina própria exclusivamente no sexo masculino (6/10 homens e 3/10 homens respectivamente). Concluindo, o estrógeno e a progesterona não influenciaram as medidas repetidas do transporte mucociliar nasal de sacarina em indivíduos sem queixas nasais. Contudo, os receptores para estrógeno e progesterona foram encontrados nas glândulas seromucosas da lâmina própria no sexo masculino, indicando que ambos os hormônios poderiam agir diretamente sobre a produção do muco nasal. / Although nasal mucociliary clearance has been thoroughly studied, there is controversial evidence that it is directly influenced by female sex hormones. This study focused on: 1. evaluating saccharin nasal mucociliary transport in both sexes and during the follicular, periovulatory and luteal phases of consecutive ovarian cycles, and 2. identifying the expression and localisation of estrogen and progesterone receptors in human nasal mucosa from inferior turbinates of patients in reproductive age. Saccharin nasal mucociliary transport was prospectively evaluated in 14 nonsmoking healthy volunteers aged 15 to 30 years (7 males and 7 females, mean age 23.5 years) who had no nasal complaints. In females, saccharin nasal mucociliary transport was measured in the follicular, periovulatory and luteal phases during two consecutive ovarian cycles (five cases) or three consecutive cycles (two cases). In males, the saccharin nasal mucociliary transport was randomly repeated three times (two cases) or six times (five cases). Estrogen and progesterone receptor expression in human nasal mucosa was retrospectively assessed by immunohistochemistry in archival, formalin-fixed, paraffin-embedded inferior nasal conchae from 20 patients submitted to partial inferior turbinectomy whose ages were matched to their of the volunteers (10 male and 10 female patients aged 15 to 33 years, mean age 22.1 years). Immunohistochemistry used mouse monoclonal antibodies against estrogen receptor (6F11 clone, Novocastra) and progesterone receptor (16 clone, Novocastra) separately. There were no significant differences in saccharin nasal mucociliary transport among follicular, periovulatory and luteal phases in consecutive ovarian cycles, nor between sexes (p=.08). Even though, considering the first ovarian cycle only, saccharin nasal mucociliary transport was faster during the follicular phase (p=.03). Estrogen and progesterone receptors were found in the cytoplasm of serous glands of the lamina propria exclusively in males (6/10 males and 3/10 males respectively). In conclusion, estrogen and progesterone did not influence repeated measures of saccharin nasal mucociliary transport in males and females with no nasal complaints. Nevertheless, estrogen and progesterone receptors were found in seromucous glands of the lamina propria in males, indicating that both hormones might act directly over nasal mucus production

Spurenanalytische Untersuchungen von Umweltproben erfolgen meist standardmäßig mittels Festphasenextraktion (SPE) und anschließender GC/MS. Das Ziel der vorliegenden Arbeit bestand darin, alternative Methoden für die spurenanalytische Bestimmung von Estrogenen aus Umweltproben aufzuzeigen. Als Analyte wurden die natürlichen Estrogene Estron (E1), Estradiol (E2) und Estriol (E3), sowie das synthetische Ethinylestradiol (EE2) ausgewählt. Da als Haupteintragspfad dieser Substanzen in die Umwelt die Kläranlagen anzunehmen sind, erfolgte die Methodenentwicklung ausgehend von gereinigtem Abwasser. Als Probenvorbereitungstechniken kamen neben der Festphasenextraktion (SPE), die klassische Flüssig-Flüssig-Extraktion (LLE), sowie die relativ neue Methode der Festphasenmikroextraktion (SPME) zum Einsatz. Die analytische Bestimmung der angereicherten Extrakte erfolgte mittels HPLC/UV und HPLC/ELCD bzw. mittels GC/FID und GC/MS. Zur Entwicklung einer Screening-Methode wurde die adsorptive Anreicherung der Estrogene an Kohlenstoffelektroden sowie das Cathodic Stripping von EE2 an Quecksilber erprobt. Die Verfahrensschritte wurden zunächst anhand dotierter Proben optimiert, und die Methoden dann anhand der erzielten Bestimmungsgrenze, Selektivität und Präzision verglichen. Die Eignung der Verfahren wurde abschließend durch die Messung realer Abwasserproben überprüft. / Trace analytical environmental investigations are usually done by solid phase extraction (SPE) and GC/MS. The aim of the present thesis was the development of alternative methods for the trace analysis of estrogens in the environment. Estrone (E1), Estradiol (E2) and Estriol (E3) as well as the synthetic estrogen Ethynylestradiol (EE2) were chosen for this investigation. These estrogens enter the environment mainly via sewage treatment plants. Therefore the method development was launched on purified waste water. Solid phase extraction (SPE), liquid liquid extraction (LLE) and the relatively new solid phase micro extraction (SPME) were used for sample preparation. The analytical determination of the extracted analytes was done by HPLC/UV and HPLC/ELCD or GC/FID and GC/MS. Adsorptive enrichment of estrogens on different carbon electrodes and cathodic stripping of EE2 on mercury were tested as possible screening methods. The established methods were optimized using spiked samples. After comparing their quantitation limits, selectivity and precision, they were applied to real sewage water samples for showing qualification.

O câncer de mama é a doença maligna mais comum e a principal causa de morte entre as mulheres. Sua complexa etiologia envolve múltiplos fatores de risco, a maioria deles relacionada aos níveis cumulativos de exposição da mama aos estrógenos. A maioria de suas ações é mediada pela ligação a seus receptores ER e ER que são fatores de transcrição. Outro fator que exerce um controle extraordinário no comportamento celular, regulando a transcrição gênica e influenciando diversos processos biológicos, e que, quando alterado, é associado ao processo de tumorigênese da mama é a matriz extracelular. A laminina, um dos principais componentes da matriz extracelular, interage com as células através das integrinas e está relacionada ao fenótipo maligno, atuando na adesão, migração, proliferação, diferenciação e sobrevivência celular. Nosso grupo identificou diversos genes diferencialmente expressos em células de câncer de mama ER+ na presença ou ausência de uma monocamada de laminina utilizando a técnica DDRT-PCR. Dois dos genes identificados, DDEF2 e PHLDA1, estão associados à adesão; DDEF2 envolvido na sinalização das integrinas e PHLDA1 relacionado com apoptose por perda de adesão. Nosso objetivo foi investigar os efeitos do 17-estradiol e da laminina na regulação da expressão dos genes DDEF2 e PHLDA1 nas linhagens celulares MCF-7, MDA-MB-231 e posteriormente S30, utilizando a técnica RT-PCR em tempo real. O gene PHLDA1 foi induzido pelo E2 via ER nas células MCF-7 e pela laminina nas células S30, e o gene DDEF2 foi reprimido pelo E2 e induzido pela laminina nas células S30 / The breast cancer is the most common malignant disease and the leading cause of death among women. Its complex etiology involves multiple risk factors, most of them related to the levels of cumulative breast exposure to estrogen. Most of its actions is mediated by binding to its receptor ER and ER that are transcription factors. Another factor that has a tremendous control in cell behavior, regulating the gene transcription and influencing various biological processes, which when altered, is attached to the process of tumorigênese of the breast is the extracellular matrix (ECM). The laminin, one of the main components of the ECM, interacts with the cells through integrins and is related to the malignant phenotype, acting in adhesion, migration, proliferation, differentiation and cell survival. Our group identified several genes diferentialy expressed in breast cancer cells ER + in the presence or absence of a laminin monolayer using the technique DDRT-PCR. Two of the genes identified, DDEF2 and PHLDA1, are associated with adhesion; DDEF2 is involved in the integrins signaling and PHLDA1 is related with apoptosis by loss of adhesion. Our goal was to investigate the effects of 17-estradiol and laminin in regulating the expression of the genes DDEF2 and PHLDA1 in MCF-7, MDA-MB-231 and later S30 cell lines, using the real time RT-PCR technique. The gene PHLDA1 was induced by E2 via ER in MCF-7 cells and the laminin in S30 cells, and the gene DDEF2 was suppressed by E2 and induced by laminin in S30 cells