Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Philosophiae Doctor - PhD / Estrogens play an extraordinary role in the endocrine system regulation through the stimulation and regulation of endocrine pathways. 17β-estradiol is one of the final metabolites in estrogen regulation by hydroxylase enzymes that are well recognized for their metabolic role in hormone fragmentation and dissociation, through hydroxylation reactions that reversibly convert a series of androgens to estrogens (i.e. or one estrogen to the other). However, the 17β-estradiol hormone has been classified as one of the estrogenic endocrine disrupting compounds {i.e. EDC (s)} that show significant adverse effects in the estrogen pathways of male and female animal species. Estrogen receptor alpha (ER-α) is significantly activated by 17β-estradiol, which is a steroid hormone. A biosensor system for the determination of 17β-estradiol was developed based on the highly selective and specific physiological substrate level activation of the ER-α biomolecule by the (17β-estradiol) compound. The chemically-tuned tin selenide quantum dots capped with 3-mercaptopropionic acid were produced at room temperature and employed to capture the ER-α micro-molecule onto the electrode surfaces. These quantum dots possessed average particle size (APS) diameters between 4.6 ± 0.6 nm and an indirect band gap energy (Eg) of 3.14 eV. Surface modification on the quantum dots permitted the formation of efficient amide bonds between the capping molecules of the quantum dots and the estrogen receptor-alpha. The tin selenide quantum dots platform enhanced the surface bio-reactivity of the receptorsensor film. The receptorsensor’s sensitivity towards 17β-estradiol was 5.9 μA/μM associated with a response time (tResponse) of less than 1.2 s. The formal potential, Ep˚ˈ, of the receptorsensor-substrate complex was 149 mV. A detection limit (DL) of 1.9 nM was obtained for the electrochemical biosensing methodology. 17β-estradiol–receptorsensor response kinetics were also evaluated, where a dissociation rate (kd) of 7.6 μM/s, a 50 % inhibition concentration (IC50) value of 3.4 nM and a binding efficiency (Bmax) of 7 nM were obtained. Effective measure of 17β-estradiol concentrations as low as 3.8 nM present in surface waters have been reported to induce feminisation in male aquatic species. The receptorsensor’s dynamic linear range (DLR) nevertheless showed capability of screening a minimum of 0.2 nM to a maximum of 8 nM of the 17β-estradiol concentrations. Furthermore, during the estrogen replacement therapy (ERT), 17β-estradiol concentration levels are monitored at frequent phases, wherein 17β-estradiol concentrations from as low as 0.37 nM are recovered in the serum (i.e. this value was also evaluated to be within the receptorsensor’s-DLR), determining its future capability to be developed for; clinical-diagnosis screening of the 17β-estradiol.

No presente estudo, observamos aumentada expressão de iNOS e de proteínas s-nitrosiladas (S-NT) em aorta (AO) e concentração de NO no plasma de ratas diabéticas (DB), que foram corrigidas pelo tratamento com insulina (INS), que foi incapaz de normalizar a glicemia. O tratamento com o inibidor da iNOS L-NIL reduziu a expressão de S-NT e a concentração de NO. A contração induzida por agonistas adrenérgicos (ADRs), mas não por cloreto de potássio, que estava reduzida em anéis de AO sem endotélio de ratas DB, foi corrigida pelos tratamentos com INS e L-NIL. A expressão dos receptores de estrógeno ESR2 e GPER estava aumentada em AO de ratas DB e foi corrigida pelo tratamento com INS. Nossos resultados mostram que o aumento da expressão da iNOS/geração de NO é responsável pela redução da contração mediada por receptor ADR, mas não daquela induzida por despolarização direta da membrana. A INS modula negativamente a expressão da iNOS e dos receptores ESR2 e GPER em aorta de ratas DB, efeito que pode contribuir com a restauração da contração induzida por agonistas ADRs. / In the present study, we observed that in aortas (AO) of diabetic (DB) female rats have an increase in iNOS and S-nitrosilated (S-NT) proteins expression along with higher levels of plasmatic NO. Although insulin (INS) treatment did not normalize blood glucose levels, it corrected protein expression and NO concentrations. The iNOS inhibitor treatment reduced the altered expression of S-NT proteins and NO levels. The reduced adrenergic agonists (ADR)-induced contractions in DB without endothelium were corrected by INS and L-NIL treatments, such treatments did not affect the reduced vasoconstriction response to KCl in DB AO. The increase expression of estrogen receptors GPER and ESR2 found in DB AO was recovered by INS treatment. Our results showed that the increased expression of iNOS/NO generation is responsible for reducing ADR-induced contraction, but not for membrane depolarization-induced contraction. INS negatively modulates protein expression of iNOS, ESR2, and GPER receptors in DB AO; such effect may contribute to restore ADR-induced vascular contraction.

Orientador: Nelci Fenalti Hoehr, Joyce M. Anichino-Baizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T11:11:48Z (GMT). No. of bitstreams: 1 Alessio_AlineMorandi_M.pdf: 5240817 bytes, checksum: f759b9c995ffcb5198175f672d2a40aa (MD5) Previous issue date: 2005 / Resumo: o estrógeno atua na hemostasia promovendo efeitos pró e antitrombóticos, sua ação ocorre através de sua ligação a receptores nucleares específicos, denominados receptores 'alfa' e 'beta' do estrógeno (RE). A presença de polimorfismos nos genes que codificam o RE poderia modular a resposta do estrógeno, assim como a transcrição de genes e conseqüentemente a expressão. Existem fatores de risco adquiridos para a trombose venosa profunda (TVP) que estão associados a alterações do nível de estrógeno como: gravidez, puerpério, uso de anticoncepcional oral (ACO) e a terapia de reposição hormonal. Os objetivos deste estudo foram de associar polimorfismos nos genes dos RE 'alfa' e 'beta' em controles e mulheres e homens com TVP e avaliar a influência deles na atividade da proteína C (PC), proteína S (PS), antitrombina e concentração de fibrinogênio. Nas mulheres sob o uso de ACO, o genótipo AA do polimorfismo G1730A e, nas mulheres grávidas, os genótipos AA e GA do polimorfismo G1082A, ambos no gene do RE-~, podem ter um efeito protetor para o risco de TVP. Porém, o genótipo GG do polimorfismo G1730A tem uma influência no aumento da atividade da PS nas mulheres do grupo controle total e nas mulheres sob o uso de ACO; e os genótipos AA e AG do polimorfismo A351G no gene do RE-'alfa' , e o genótipo GG do polimorfismo G1082A tem um efeito no aumento da concentração de fibrinogênio nas mulheres grávidas. Contudo, a mutação G20210A no gene da protrombina, o fator V de Leiden e os polimorfismos do RE- foram fatores preditores de trombose venosa. Apesar da miscigenação brasileira, a PC apresentou diminuída em mulheres AITo-descendente. Nossos resultados sugerem que polimorfismos no gene dos RE-'beta' têm uma influência maior no risco para TVP. A contribuição deste trabalho é de grande importância, pois nenhum estudo associou o risco de TVP com os polimorfismos dos RE-'beta' / Abstract: Estrogen acts in haemostasis promoting pro and antithrombotic effects. Its action occurs through linking the specific nuclear receptors called 'alfa' and 'beta' estrogen receptor (ER). Polymorphisms in the genes that codify the ER a and ~could modulate estrogen response, as well as gene transcription and expression. There are acquired risk factors for deep venous thrombosis (DVT) that can alter the estrogen levels. These factors are oral contraceptives (OC), hormonal replacement therapy, pregnancy, and puerperium. The objectives of this study were to associate the polymorphisms in the genes of the a and ~ER in controls, women and rnen with DVT, and to evaluate the influence of these polyrnorphisms in protein C, protein S (PS), in antithrombin activity and fibrinogen concentration. Women under OC use with the AA genotype ofthe G1730A polyrnorphism, and pregnant women with the AA and GA genotypes of the G1082A polymorphism, both in the ER-~ gene, could have a protective effect for DVT risk. However, GG genotype of the G1730A polymorphisrn has an influence in the increase of the PS activity in women of the total control group and women under OC use; and the AA and AG genotypes of the A351G polymorphism in the ER-a gene, and the GG genotype of the G1O82A polymorphism have an effect in the increase of the fibrinogen leveI in pregnant women. However, the G20210A rnutation in the prothrombin gene, the Leiden V factor and the polymorphisms in the ER-'alfa' gene were predictor factors of venous thrombosis. Despite Brazilian miscegenation, PC activity was lower in Afro-descendent women. Our results suggest that the polymorphisrns in the ER-'beta' gene have a higher influence for DVT risk. The contribution of this study is of great importance, as no study has associated DVT risk with the ER polymorphisms yet / Mestrado / Patologia Clinica / Mestre em Ciências Médicas

Fertilization is a unique biological event where male and female gametes fuse together to produce a new organism. Before the gametes are able to fuse, however, they must undergo a series of controlled changes. For the male gamete, capacitation and acrosome reaction (AR) must occur, which take place during the sperm migration through the female genital tract. Unfortunately, while the process of capacitation has been known for over half a century, the molecular basis and influential factors behind it are not fully understood. Although estrogens have been considered mainly female reproductive hormones, there is increasing evidence suggesting that these steroids have an important role also in regulation of male reproductive functions. Sperm come into the contact with estrogens during their formation in the male and female genital tract, indicating that the hormone may play an important role in sperm maturation. In this study, we examined the importance of three endogenous estrogens (E1 -estron, E2 - 17β estradiol, E3 - estriol) and one synthetic estrogen (EE2 - 17α ethinylestradiol) on sperm maturation during capacitation and AR. Stimulatory effect were observed with all tested estrogens on both capacitation and zona pellucida induced AR. Moreover, we have determinied that the stimulatory effect on...

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) ≫ MCF-7 (57%) > Ishikawa (51%) ≫ HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERα + ERβ + for MCF-7, HCC-1806 and Ishikawa; and ERβ only for HEC-1B. HeLa cells were also tested and found to express ERβ, but not ERα. A small percentage of both ERs were surface-exposed and functionally active. The endometrium- predominant ERβ isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERβ + and - isogenic HEC-1B cells.

Background: Environmental exposures acting through different mechanisms have been linked with a number of cancers. Perfluoroalkyl chemicals (PFCs) are endocrine disrupting chemicals affecting estrogen homeostasis. Objectives: We examined the association between PFCs and a group of estrogen related cancers and explored if increased non-occupational exposure was associated with increased odds of developing these cancers. We also explored which of these chemical exposures was more correlated with each cancer. Methods: Using data from the National Health and Nutrition Examination Survey (NHANES), we selected participants ≥ 20 years of age. Our outcome variable was presence or absence of breast, prostate, ovarian and uterine cancer (yes/no); our exposure variables were serum PFCs. Logistic regression models were used in investigating the association between PFCs and cancer types and between quartiles of PFCs exposure concentrations and presence or absence of cancer while adjusting for covariates. Discriminant analysis was used to assess the correlation between individual PFCs compounds and individual cancer types. Results: PFCs were associated with increased odds of ovarian cancer; PFOA: 1.02(1.01, 1.02), PFOS: 1.01 (1.012, 1.013), PFHS 1.031 (1.030, 1.033), PFDE: 1.29(1.27, 1.30) and increased odds of breast cancer; PFOA: 1.089(1.089, 1.09), PFOS: 1.011(1.011, 1.011), PFNA: 1.031(1.030, 1.033), PFHS: 1.02 (1.02, 1.02), PFDE: 1.19(1.18, 1.19). PFCs were not associated with increased odds of prostate or uterine cancers. Comparing the odds in quartile 4 to quartile 1 for ovarian cancer, PFOA: 1.77(1.75,1.79), PFOS: 2.25(2.22, 2.28), PFHS: 1.86(1.84, 1.88), PFDE: 2.11(2.09, 2.14). For breast cancer, PFOA: 2.30(2.28, 2.31), PFOS: 1.47(1.46, 1.48), PFNA: 1.04(1.03, 1.05), PFHS:7.07(6.97,7.17), PFDE: 1.38(1.37, 1.39). PFOA was more correlated with breast cancer (0.7) and PFHS was more correlated with ovarian cancer (0.9). Discussion: PFCs were associated with increased odds of ovarian and breast cancers with a positive dose-response relationship. PFOA was more correlated with breast cancer and PFHS more with ovarian cancer.

Tamoxifen is an effective anti-estrogen for treatment of women with hormonedependent breast cancer but acquired drug resistance limits its therapeutic benefit. We have previously reported that expression of active Akt3 in MCF-7 breast cancer cells results in estrogen-independent tumors that are actually stimulated to grow after tamoxifen treatment. We hypothesize that this tamoxifen resistance may be attributed to binding of different co-regulator proteins and/or different binding affinity of these proteins to the estrogen receptor in M CF-7 cells overexpressing active Akt3 as compared to parental MCF-7 cells. We have immuno-precipitated the estrogen receptor along with bound co-regulator proteins in both cells lines after tamoxifen, estradiol, or vehicle treatment. After 2-D gel electrophoresis separation of these immuno-precipitated proteins and comparing them using PDQuest 2-D analysis software, we identified protein spots that were statistically different under the treatment conditions between the two cell lines. The isolated protein spots were subjected to MALDI-TOF mass spectrometry. By searching protein databases through the MASCOT website for protein identification, we have identified estrogen receptor co-regulator proteins that may play a potential role in tamoxifen resistance. Current studies are focused on addressing the role of differential protein binding as a possible mechanism of tamoxifen resistance in Akt3 over-expressing breast cancer cells.

The maternal brain experiences a high level of plasticity during pregnancy which is understood to be largely mediated by changes to the maternal immune, endocrine and nervous systems to prepare for offspring care. Given the prevalence of infections during pregnancy, the effect of gestational immune activation (GIA) on these systems and the maternal brain have not been fully characterized. In this work, GIA is used with respect to immune activation in the dams, instead of the maternal immune activation (MIA) terminology that is typically used for offspring exposure. In addition to this, the protective effects of environmental enrichment against biological stressors such as an infection have only been investigated within the context of MIA offspring. This study examines the effects of lipopolysaccharide (LPS) induced GIA and housing conditions on the postpartum maternal hippocampus, a region of the brain that plays an in-direct role in supporting maternal behaviours. This is achieved through examining the number of ER-α positive cells and active microglia (Iba-1 positive cells) in the different layers of the postpartum maternal rat hippocampus. Nulliparous female Sprague-Dawley rats, housed in either animal care control (ACC) or environmental enrichment (EE) conditions, were treated with intraperitoneal injections of 100μg/kg lipopolysaccharides (LPS) or a pyrogen-free saline solution (VEH). Mothers were sacrificed on postpartum day 22 and brains were collected. Brains were preserved and later prepared for immunohistochemical labelling of ER-α and Iba-1 positive cells in the layers of the hippocampus (CA1, CA3 and Dentate Gyrus (DG): GrDG, MoDG, and PoDG). This project found a statistically significant effect of LPS treatment on the number of ER-α positive cells in the CA3 and in the PoDG layers of the hippocampus; LPS served to reduce the overall number of estrogen receptors in these areas. These findings support the potential for immune challenges to disrupt the adaptations made to maternal neuroendocrine pathways that prepare for post-pregnancy offspring care.