Studies in Europe, Japan, and North America have reported that wild fish exposed to treated sewage effluents can exhibit significant physiological and reproductive abnormalities consistent with exposure to hormonally active chemicals. The main objective of this research project was to examine the estrogenic and androgenic activity in treated sewage to determine the risk associated with treated sewage discharges in Australia and New Zealand. Several bioassays, including a sheep estrogen receptor and a rainbow trout androgen receptor binding assay, were set up and validated with model compounds. The assays were then used to measure the estrogenic and androgenic activity in sewage samples from 15 municipal sewage treatment plants (STP) utilizing a variety of treatment technologies. Raw sewage samples contained high levels of both estrogenic and androgenic activity, up to 185 ng/L estradiol equivalents (EEq) and up to 9330 ng/L testosterone equivalents (TEq), respectively. Secondary treatment processes such as activated sludge had the greatest impact on removal of biological activity from the wastewater. The estrogenic and androgenic activity in final treated effluents were <1 to 4.2 ng/L EEq and <6.5 to 736 ng/L TEq, respectively. Based on lowest observable effective concentrations reported in the literature, these levels are unlikely to induce biological effects in exposed fish in the short term. To examine potential long-term effects, resident mosquitofish chronically exposed to undiluted treated sewage were sampled. Several morphological biomarkers indicative of endocrine disruption were measured and compared with mosquitofish captured at a reference site. Mosquitofish captured in a constructed wetland for tertiary treatment of secondary treated sewage exhibited morphological differences such as elongated anal fins consistent with exposure to androgenic chemicals, although this effect was not measurable in fish collected at sites further downstream or at any of the other sites. Based on these results, it is unlikely that mosquitofish populations would be significantly affected by exposure to final treated sewage. A reverse transcription real-time polymerase chain reaction (RT-PCR) method to measure the production of a female-specific protein (vitellogenin) mRNA in adult male mosquitofish was developed, and this could be used as a rapid test to detect early changes in individuals exposed to estrogenic activity.

Dor pélvica crônica (DPC) afeta grande númerode mulheres e seu manejo ainda permanece complexo e insatisfatório. Estudos têm demonstrado um envolvimento do fator de crescimento neuronal (FCN) no processo de cronificação da dor. Participação hormonal neste processo também tem sido aventada, visto autores terem demonstrado influência estro/progestacional sobre nociceptores tanto direta quanto indiretamente através da influência exercida sobre os fatores neurotróficos. Foi objetivo deste estudo, verificar a associação entre a expressão do fator de crescimento neuronal (FCN), seu receptor (trk A) e os receptores de estrogênio e progesterona no peritôneo pélvico com a presença de dor pélvica crônica. Para tal foi realizado um estudo transversal incluindo um grupo de 22 mulheres com DPC, 8 com DPC e usuárias de anticoncepcional oral (DPC/ACO) e 7 sem dor. A dor foi analisada pela escala analógica visual (EAV) e questionário de McGill. Foi realizado imunohistoquímica para avaliar FCN e seu receptor trk A, receptores de estrogênio (RE) e progesterona (RP). A expressão de FCN teve media de 5, variando de 0 a 8, no grupo DPC, 5,5 no grupo DPC/ACO variando 3 a 8, e no grupo sem dor de 5 variando de 3 a 8 (p>0,05). A expressão de trk A apresentou media de 6, variandode 3 a 8, no grupo DPC, 6 no grupo DPC/ACO, variando de 4 a 8, e 6 no grupo sem dor variando de 4 a 6 (p>0,05). A expressão do RE apresentou média 4 no grupo DPC, variando de 0 a 8, 3,5 no grupo DPC/ACO variando de 0 a 8, e 7 no grupo sem dor, variando de 6 a 8 (p<0,05). A expressão do RP teve média 6,5 no grupo DPC, variando de 0 a 8, 5 no grupo DPC/ACO, variando de 0 a 7, e 7 no grupo sem dor, variando de 5 a 8 (p>0,05). Nossos resultados sugerem um papel anti-nociceptivo do estrogênio no peritôneo pélvico de mulheres no menacme, não mediado por expressão de FCN ou trk A. / Chronic pelvic pain (CPP) affects a great number of women and its management still remains complex and unsatisfactory. Studies have shown an involvement of the neuronal growing factor (NGF) in the process of permanence of pain. Hormonal participation in this process has also been put forward, as some authors have demonstrated estro/progestational influence under nociceptors direct or indirectly through their influence on neurotrofic factors. This study aimed to verify the association among the expression of neuronal growing factor (NGF), its receptor (TrKA) and the estrogen and progesterone receptors in the pelvic peritoneum with the presence of chronic pelvic pain. A transversal study was carried out including a group of 22 women with CPP, 8 with CPP and users of oral anticonceptional (CPP/OAC) and 7 without pain. The pain was analized by the visual analogic scale (VAS) and McGill\'s questionnaire. Imunehistochemical was performed to evaluate the NGF and its receptor TrKA, estrogen (ER) and progesteron (PR) receptors. The expression of NGF was an average of 5, varying from 0 to 8, in group CPP, 5,5 in group CPP/ OAC varying from 3 to 8, and in the group without pain varying from 3 to 8 (p>0,05). The expression of TrKA presented an average of 6, varying from 3 to 8, in the group CPP, 6 in the group CPP/OAC, varying from 4 to 8, and 6 in the group without pain varying from 4 to 6 (p>0,05). The expression of ER presented an average of 4 in the group CPP, varying from 0 to 8, 3,5 in group CPP/OAC varying from 0 to 8, and 7 in group without pain, varying from 6 to 8 (p<0,05). The expression of PR had an average 6,5 in the group CPP, varying from 0 to 8,5 in the group CPP/OAC, varying from 0 to 7, and 7 in the group without pain, varying from 5 to 8 (p>0,05). Our studies suggest an anti- nociceptive rule of estrogen in the pelvic peritoneum of women in menacme, not mediated by expression of NGF or TrKA.

Dor pélvica crônica (DPC) afeta grande númerode mulheres e seu manejo ainda permanece complexo e insatisfatório. Estudos têm demonstrado um envolvimento do fator de crescimento neuronal (FCN) no processo de cronificação da dor. Participação hormonal neste processo também tem sido aventada, visto autores terem demonstrado influência estro/progestacional sobre nociceptores tanto direta quanto indiretamente através da influência exercida sobre os fatores neurotróficos. Foi objetivo deste estudo, verificar a associação entre a expressão do fator de crescimento neuronal (FCN), seu receptor (trk A) e os receptores de estrogênio e progesterona no peritôneo pélvico com a presença de dor pélvica crônica. Para tal foi realizado um estudo transversal incluindo um grupo de 22 mulheres com DPC, 8 com DPC e usuárias de anticoncepcional oral (DPC/ACO) e 7 sem dor. A dor foi analisada pela escala analógica visual (EAV) e questionário de McGill. Foi realizado imunohistoquímica para avaliar FCN e seu receptor trk A, receptores de estrogênio (RE) e progesterona (RP). A expressão de FCN teve media de 5, variando de 0 a 8, no grupo DPC, 5,5 no grupo DPC/ACO variando 3 a 8, e no grupo sem dor de 5 variando de 3 a 8 (p>0,05). A expressão de trk A apresentou media de 6, variandode 3 a 8, no grupo DPC, 6 no grupo DPC/ACO, variando de 4 a 8, e 6 no grupo sem dor variando de 4 a 6 (p>0,05). A expressão do RE apresentou média 4 no grupo DPC, variando de 0 a 8, 3,5 no grupo DPC/ACO variando de 0 a 8, e 7 no grupo sem dor, variando de 6 a 8 (p<0,05). A expressão do RP teve média 6,5 no grupo DPC, variando de 0 a 8, 5 no grupo DPC/ACO, variando de 0 a 7, e 7 no grupo sem dor, variando de 5 a 8 (p>0,05). Nossos resultados sugerem um papel anti-nociceptivo do estrogênio no peritôneo pélvico de mulheres no menacme, não mediado por expressão de FCN ou trk A. / Chronic pelvic pain (CPP) affects a great number of women and its management still remains complex and unsatisfactory. Studies have shown an involvement of the neuronal growing factor (NGF) in the process of permanence of pain. Hormonal participation in this process has also been put forward, as some authors have demonstrated estro/progestational influence under nociceptors direct or indirectly through their influence on neurotrofic factors. This study aimed to verify the association among the expression of neuronal growing factor (NGF), its receptor (TrKA) and the estrogen and progesterone receptors in the pelvic peritoneum with the presence of chronic pelvic pain. A transversal study was carried out including a group of 22 women with CPP, 8 with CPP and users of oral anticonceptional (CPP/OAC) and 7 without pain. The pain was analized by the visual analogic scale (VAS) and McGill\'s questionnaire. Imunehistochemical was performed to evaluate the NGF and its receptor TrKA, estrogen (ER) and progesteron (PR) receptors. The expression of NGF was an average of 5, varying from 0 to 8, in group CPP, 5,5 in group CPP/ OAC varying from 3 to 8, and in the group without pain varying from 3 to 8 (p>0,05). The expression of TrKA presented an average of 6, varying from 3 to 8, in the group CPP, 6 in the group CPP/OAC, varying from 4 to 8, and 6 in the group without pain varying from 4 to 6 (p>0,05). The expression of ER presented an average of 4 in the group CPP, varying from 0 to 8, 3,5 in group CPP/OAC varying from 0 to 8, and 7 in group without pain, varying from 6 to 8 (p<0,05). The expression of PR had an average 6,5 in the group CPP, varying from 0 to 8,5 in the group CPP/OAC, varying from 0 to 7, and 7 in the group without pain, varying from 5 to 8 (p>0,05). Our studies suggest an anti- nociceptive rule of estrogen in the pelvic peritoneum of women in menacme, not mediated by expression of NGF or TrKA.

La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance. Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles. / It has long been appreciated that estrogenic signaling plays a critical role in the development of hormone-dependent cancers such as breast cancer. Two-thirds of breast cancers express estrogen receptor (ER) which has been demonstrated to play an irrefutable role in tumour development and progression. However the acquisition of endocrine resistance has become a major obstacle in the treatment of hormone-dependent cancers that have acquired a hormone-independent state. Hormone-independent cancers emerge from an array of pathways involving ER activation in the absence of estrogen, hypersensitivity of ER to low serum levels of estrogen and activation by estrogen antagonists. The activity of ER is critically influenced by the cellular environment such as growth factor signaling pathways, availability of coregulatory proteins and the promoter sequence of target genes. The mechanisms studied have mostly considered the role of ERα, however with the discovery of the second subtype, ERβ, the understanding on the diversity of potential mechanisms involving ER-dependent responses have improved. Hormonal-independent activation of ER can occur in estrogen-dependent breast tumours, with concomitant rise in kinase signaling pathways, resulting in the acquisition of a therapeutic resistant phenotype in treated women. Our knowledge is relatively limited on which pathways trigger ER signaling and how these phosphorylation-coupled events affect ER activity. ERα is considered the dominant subtype and correlates with most of the prognostic factors in breast cancers. Conversely the role of ERβ remains unclear. The results presented in this thesis were carried out with the objective of gaining a better understanding of ERβ’s role in cellular proliferation by examining the behavior of ERβ and ERα during the activation of growth factor signaling pathways by cell-surface receptor-tyrosine kinases. We demonstrate here that the activation of cell surface receptors of the ErbB family, specifically ErbB2/ErbB3, inhibits the transcriptional activity of ERβ despite the presence of the coactivator CBP, yet activated ERα. Furthermore the inhibition of ERβ was attributed to a specific serine residue located within the hinge region, not present in ERα. Additional studies of ErbB2/ErbB3-initiated signaling revealed that it triggered the activation of the PI3K/Akt pathway which targeted the serine residue within the hinge region of ERβ. In fact, phosphorylation of ERβ by the PI3K/Akt pathway led to an increase in receptor ubiquitination which promoted its degradation by the ubiquitin-proteasome system which was subtype specific. Interestingly, proteasomal degradation required the presence of the coactivator CBP, which is normally involved in assisting nuclear receptor transcriptional activity. Although the activation of the PI3K/Akt pathway correlated with a decrease in the expression of ERβ target genes it led to an increase in the proliferation of breast cancer cells. Inhibiting the degradation of ERβ reduced the enhanced proliferation of breast cancer cells brought about by the treatment of ErbB3’s ligand, Hrgβ1. Increasing evidence indicates that growth factor signaling pathways can selectively regulate the transcriptional activity of ER subtypes, and the ratio of ERα/ERβ expression in breast tumours is becoming a popular prognostic factor to evaluate the severity of the tumour. Therefore the molecular characterization of the coupling between growth factor signaling and ER function should provide improved therapeutical approaches to overcome or delay the onset of resistance to endocrine therapy in hormone-dependent cancers.

La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance. Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles. / It has long been appreciated that estrogenic signaling plays a critical role in the development of hormone-dependent cancers such as breast cancer. Two-thirds of breast cancers express estrogen receptor (ER) which has been demonstrated to play an irrefutable role in tumour development and progression. However the acquisition of endocrine resistance has become a major obstacle in the treatment of hormone-dependent cancers that have acquired a hormone-independent state. Hormone-independent cancers emerge from an array of pathways involving ER activation in the absence of estrogen, hypersensitivity of ER to low serum levels of estrogen and activation by estrogen antagonists. The activity of ER is critically influenced by the cellular environment such as growth factor signaling pathways, availability of coregulatory proteins and the promoter sequence of target genes. The mechanisms studied have mostly considered the role of ERα, however with the discovery of the second subtype, ERβ, the understanding on the diversity of potential mechanisms involving ER-dependent responses have improved. Hormonal-independent activation of ER can occur in estrogen-dependent breast tumours, with concomitant rise in kinase signaling pathways, resulting in the acquisition of a therapeutic resistant phenotype in treated women. Our knowledge is relatively limited on which pathways trigger ER signaling and how these phosphorylation-coupled events affect ER activity. ERα is considered the dominant subtype and correlates with most of the prognostic factors in breast cancers. Conversely the role of ERβ remains unclear. The results presented in this thesis were carried out with the objective of gaining a better understanding of ERβ’s role in cellular proliferation by examining the behavior of ERβ and ERα during the activation of growth factor signaling pathways by cell-surface receptor-tyrosine kinases. We demonstrate here that the activation of cell surface receptors of the ErbB family, specifically ErbB2/ErbB3, inhibits the transcriptional activity of ERβ despite the presence of the coactivator CBP, yet activated ERα. Furthermore the inhibition of ERβ was attributed to a specific serine residue located within the hinge region, not present in ERα. Additional studies of ErbB2/ErbB3-initiated signaling revealed that it triggered the activation of the PI3K/Akt pathway which targeted the serine residue within the hinge region of ERβ. In fact, phosphorylation of ERβ by the PI3K/Akt pathway led to an increase in receptor ubiquitination which promoted its degradation by the ubiquitin-proteasome system which was subtype specific. Interestingly, proteasomal degradation required the presence of the coactivator CBP, which is normally involved in assisting nuclear receptor transcriptional activity. Although the activation of the PI3K/Akt pathway correlated with a decrease in the expression of ERβ target genes it led to an increase in the proliferation of breast cancer cells. Inhibiting the degradation of ERβ reduced the enhanced proliferation of breast cancer cells brought about by the treatment of ErbB3’s ligand, Hrgβ1. Increasing evidence indicates that growth factor signaling pathways can selectively regulate the transcriptional activity of ER subtypes, and the ratio of ERα/ERβ expression in breast tumours is becoming a popular prognostic factor to evaluate the severity of the tumour. Therefore the molecular characterization of the coupling between growth factor signaling and ER function should provide improved therapeutical approaches to overcome or delay the onset of resistance to endocrine therapy in hormone-dependent cancers.