INTRODUÇÃO: O Câncer de Próstata (CaP) é a sexta neoplasia mais comum no mundo correspondendo a aproximadamente 10% do total de cânceres. No Brasil, o CaP é a neoplasia maligna não cutânea mais comum entre os homens. A hereditariedade é um dos principais fatores de risco do CaP, que se caracteriza pela herança de mutações em genes de susceptibilidade de alta penetrância que quando transmitidos aos descendentes aumentam o risco de desenvolvimento de tumores. Os andrógenos e estrógenos influenciam o desenvolvimento, maturação e manutenção da próstata, afetando a proliferação e a diferenciação. Isso tem despertado grande interesse no papel desses hormônios esteroides no desenvolvimento e manutenção tanto da próstata normal quanto maligna. Os Polimorfismos de Nucleotídeo Único (SNPs) são variantes de risco genéticos associados com uma série de doenças, incluindo o câncer. Considerando que a história familiar e que os componentes hormonais constituem fatores de risco para o desenvolvimento do CaP, acredita-se que a identificação de polimorfismos envolvidos nesses processos possa ter um papel relevante para auxiliar no desenvolvimento de ferramentas alternativas para a detecção precoce e para a definição do prognóstico desta neoplasia. OBJETIVOS: Analisar polimorfismos (SNPs) relacionados com histórico familiar e com fatores hormonais em amostras de sangue de pacientes com CaP e em homens saudáveis. Além disso, correlacionar os resultados da genotipagem com parâmetros clínico-patológicos. MÉTODOS: O estudo foi composto por 185 pacientes diagnosticados com CaP, sendo 97 casos esporádicos e 72 com histórico familiar (dois parentes de primeiro grau). O grupo controle foi composto por 70 amostras de sangue de indivíduos saudáveis, que comprovadamente não possuíam CaP e fazem acompanhamento com intuito preventivo. Foram selecionados 13 polimorfismos para análise: rs10486567, rs10993994, rs9364554, rs5945572, rs2735839, rs4430796, rs7501939, rs138213197, rs1271572, rs2987983, rs8072254, rs4919743 e rs3808330. A genotipagem foi realizada através da técnica de PCR em tempo real (qPCR) e correlacionada com o histórico familiar de CaP, PSA pré-operatório, graduação histológica de Gleason e estadiamento patológico. RESULTADOS: Analisamos a frequência dos polimorfismos selecionados e encontramos as seguintes correlações em nossos casos: os SNPs rs10486567 e rs9364554 aumentam a chance de desenvolvimento do CaP enquanto que o SNP rs8072254 diminui o risco. Com relação à hereditariedade, o SNP rs1271571 apresentou associação com o CaP esporádico. Na comparação com os fatores prognósticos encontramos que o SNP rs3808330 foi mais frequente em indivíduos que possuíam PSA < 10; o SNP rs7501939 foi mais frequente em indivíduos com menor escore de Gleason e ausência de recidiva e o SNP rs5945572 foi mais frequente em indivíduos com menor escore de Gleason na peça. CONCLUSÕES: De uma forma geral encontramos polimorfismos que parecem ter um papel relevante no desenvolvimento do CaP, na transmissão familiar e a fatores prognósticos. Estes importantes polimorfismos, ainda não haviam sido estudados na população brasileira e nosso trabalho identificou correlações ainda não demonstradas na literatura / BACKGROUND: Prostate Cancer (PCa) is the sixth most common cancer worldwide accounting for around 10% of all cancers. In Brazil, the PCa is the most common non-skin malignancy among men. Heredity is one of the main risk factors for PCa, which is characterized by mutations of heritage in highpenetrance susceptibility genes that when transmitted to offspring increase the risk of tumor development. Androgens and estrogens influence the development, maturation and maintenance of the prostate, affect proliferation and differentiation. This has aroused great interest in the role of these steroid hormones in the development and maintenance of both normal and malignant prostate. Single Nucleotide Polymorphisms (SNPs) are variants of genetic risk associated with a number of diseases including cancer. Considering that the family history and hormonal components are risk factors for the development of PCa, we believe that the identification of polymorphisms involved in these processes may have an important role to assist in the development of alternative tools for early detection and to define the prognosis of this cancer. OBJECTIVES: To analyze polymorphisms (SNPs) associated with family history and hormonal factors in patient blood samples with PCa and in healthy men. In addition, to correlate the results of genotyping with clinical-pathological parameters. METHODS: The study consisted of 185 patients diagnosed with PCa, divided into 97 sporadic cases and 72 with a family history. The control group consisted of 70 blood samples from healthy individuals who had no proven PCa and do it for preventive purposes. We selected 13 polymorphisms for analysis: rs10486567, rs10993994, rs9364554, rs5945572, rs2735839, rs4430796, rs7501939, rs138213197, rs1271572, rs2987983, rs8072254, rs4919743 and rs3808330. Genotyping was performed by PCR in real time (qRT -PCR) and correlated with family history of PCa, preoperative PSA, Gleason histologic grading and pathological staging. RESULTS: We analyzed the frequency of the selected polymorphisms and found the following correlations in our cases: SNPs rs10486567 and rs9364554 increase the chance of developing PCa while the SNP rs8072254 decreases the risk. Regarding heredity, the SNP rs1271571 presented association with sporadic PCa. In comparison with the prognostic factors we found that the SNP rs3808330 was more frequent in patients who had PSA < 10; SNP rs7501939 was more frequent in patients with lower Gleason score and no recurrence and the SNP rs5945572 was more frequent in subjects with lower Gleason score on the surgical specimen. CONCLUSIONS: In general we found that polymorphisms that appear to have a relevant role in the development of PCa in family transmission and in prognostic factors. These important polymorphisms had not yet been studied in the Brazilian population and our work has identified correlations yet not demonstrated in the literature

Ainda existe controvérsia se a terapia hormonal estrogênica confere benefício ou dano cardiovascular. O dano potencial causado por doses padrão de estrogênio incluindo doença cardíaca coronariana e trombose venosa pode ser atenuada por uso de doses mais baixas. O objetivo deste estudo foi avaliar o efeito do tratamento com conjugado estrogênio equino, no leito mesentérico venular (LVM) de SHR ovariectomizadas (OVX), com dose padrão (SD) vs dose mínima (LD). Angiotensina II (AngII) foi perfundida de forma concentração-dependente. Hiperreatividade à Ang II, aumento na geração de EROS, redução da atividade da SOD e catalase e da biodisponibilidade do NO foram encontrados em OVX e SD vs controle e LD. Por outro lado, a resposta reduzida à AngII no LVM de ratas LD foi relacionado ao aumento da atividade da eNOS, redução na geração de EROS e aumento da expressão dos receptores AT2,ER<font face=\"Symbol\">&#945; e GPR30. Sugerimos que a dose mínima tem efeito protetor sobre o LVM das OVX e que a dose SD pode aumentar o risco para a doença venular induzindo alterações na reatividade venular. / Controversy still exists whether estrogen hormonal therapy confers cardiovascular benefit or harm. The potential harm caused by standard dosages of estrogen including coronary heart disease and venous thrombosis may be mitigated by use of lower doses. The aim of this study was evaluated the effect of conjugated equine estrogen (CEE) treatment, in isolated mesenteric venular bed (MVB) of ovariectomized SHR (OVX), at standard therapeutic (SD) vs low dose (LD). Angiotensin II (Ang II) was perfused in a concentration-dependent manner. Hyperreactivity to Ang II,augmented ROS generation, reduced SOD, catalase activity and NO availability were found in OVX and SD vs. control and LD. However, the reduced MVB response to Ang II in LD rats was related to increased endothelial NO synthase activity, reduced ROS generation and increased Ang II AT2, ER<font face=\"Symbol\">&#945; and GPR30 receptor expression. We suggest that CEE at a low dose has a protective effect in OVX mesenteric venular bed. The standard dose might increase the risk for venular disease by inducing alterations in venular reactivity.

It is well established that the 17-estradiol is involved in the development and homeostasis of reproductive and extra-reproductive tissues, particularly the mammary gland. Estradiol classically binds to Estrogen Receptor (ERα), which is a member of the nuclear receptor superfamily. ER mediates nuclear (transcription) and plasma membrane (signaling) ERα function. Interestingly, the membrane initiated steroid signaling (MISS) required a post translational modification of the receptor: palmitoylation of the human Cys-447 or the murine Cys-451 counterpart. The main objectives of my PhD thesis were to decipher the physiological role of membrane ERα in mammary gland development and to understand how the membrane ER signaling impact breast cancer. To do so, we used the transgenic mouse model C451A-ER in which the single point mutation (C451A) was introduced to abolish palmitoylation of ER (membrane addressing signal). We demonstrate that the point mutation of the palmitoylation site of ER alters the paracrine signaling of luminal epithelial cells and by consequence the repopulation properties of the mammary stem cells. We also studied the involvement of the membrane effects of the Estrogen Receptor ERα in the 17β-estradiol response dose of the mammary gland. Finally, by breeding the C451A-ER mice with the widely used transgenic mice model of tumorigenesis (PyMT), we provide the first evidence that the membrane ERα influences tumorigenesis. These findings pave the way on an unexpected role of non-genomic function of ERα in the mammary gland physiology and physiopathology. / It is well established that the 17-estradiol is involved in the development and homeostasis of reproductive and extra-reproductive tissues, particularly the mammary gland. Estradiol classically binds to Estrogen Receptor (ERα), which is a member of the nuclear receptor superfamily. ER mediates nuclear (transcription) and plasma membrane (signaling) ERα function. Interestingly, the membrane initiated steroid signaling (MISS) required a post translational modification of the receptor: palmitoylation of the human Cys-447 or the murine Cys-451 counterpart. The main objectives of my PhD thesis were to decipher the physiological role of membrane ERα in mammary gland development and to understand how the membrane ER signaling impact breast cancer. To do so, we used the transgenic mouse model C451A-ER in which the single point mutation (C451A) was introduced to abolish palmitoylation of ER (membrane addressing signal). We demonstrate that the point mutation of the palmitoylation site of ER alters the paracrine signaling of luminal epithelial cells and by consequence the repopulation properties of the mammary stem cells. We also studied the involvement of the membrane effects of the Estrogen Receptor ERα in the 17β-estradiol response dose of the mammary gland. Finally, by breeding the C451A-ER mice with the widely used transgenic mice model of tumorigenesis (PyMT), we provide the first evidence that the membrane ERα influences tumorigenesis. These findings pave the way on an unexpected role of non-genomic function of ERα in the mammary gland physiology and physiopathology.

A partir des données de deux études de cohortes hospitalières françaises (MEVE etFARIVE), nous nous sommes intéressés aux facteurs de risque de récidive de maladieveineuse thromboembolique (MVTE).Nous avons confirmé un excès de risque de récidive de MVTE chez les hommescomparés aux femmes et montré que cette relation dépendait en partie de l’âge, de lamutation du FV Leiden et de la prise d'hormones au premier événement. Une analyse enfonction du sexe a mis en évidence que l’âge, l’obésité et des niveaux élevés de D-dimèresaugmentaient significativement le risque de récidive de MVTE chez les femmes. Par ailleurs,contrairement aux estrogènes oraux, les estrogènes transdermiques seuls ou combinés à laprogestérone micronisée n'exposaient pas les femmes ménopausées à un risque accru derécidive de MVTE. Chez les hommes, la mutation du facteur V Leiden, un antécédent familialde maladie artérielle et un premier événement idiopathique étaient des facteurs de risqueindépendants de récidive.L'identification de profils de risque différents en fonction du sexe pourrait permettreune meilleure stratification du risque de récidive de MVTE. Ces résultats devraientcontribuer à améliorer la prise en charge de la maladie par une évaluation individuelle de ladurée optimale du traitement anticoagulant. De plus, une bonne sécurité d’emploi desestrogènes transdermiques seuls ou combinés à la progestérone micronisée ouvre desperspectives cliniques intéressantes dans le traitement des troubles sévères de laménopause chez des patientes avec un antécédent personnel de MVTE.Mots / Based on data from two French hospital cohort studies, we investigated the riskfactors for recurrent venous thromboembolism (VTE).We confirmed the higher risk of recurrent VTE among men compared with womenand we suggested that this relation depended on age, Factor V Leiden mutation andhormone-related first event. A sex-specific analysis showed that advancing age, obesity andelevated D-dimer significantly increased the risk of recurrent VTE in women. Moreover, oralbut not transdermal estrogens, were associated with a higher risk of recurrent VTE amongpostmenopausal women. In men, factor V Leiden mutation, family history of arterial diseaseand an idiopathic first event were independent risk factors for VTE recurrence.The identification of sex-specific risk factors provides a new insight to riskstratification for VTE recurrence. These results could improve the prevention of this diseaseby an individual assessment of the optimal duration of anticoagulation therapy. In addition,our results provide first evidence supporting the safety of transdermal estrogens alone orcombined with micronized progesterone with respect to VTE recurrence risk. These datacould have important clinical implications for women with personal history of VTE whorequire hormone therapy for severe postmenopausal symptoms.

Η νόσος του Πάρκινσον χαρακτηρίζεται από την προοδευτική εκφύλιση της μελαινοραβδωτής ντοπαμινεργικής οδού που οδηγεί σε κινητικές διαταραχές. Θεωρείται πολυπαραγοντική νόσος, η αιτιολογία της οποίας παραμένει άγνωστη. Με δεδομένο ότι η διαθέσιμη φαρμακευτική αγωγή της νόσου στηρίζεται στη συμπτωματολογία της και έχει σοβαρές παρενέργειες, η νευροπροστασία από τα πρώϊμα στάδια της νόσου αποτελεί τομέα έντονης έρευνας. Τα τελευταία χρόνια, ένα ευρύ φάσμα παραγόντων ερευνήθηκε ως προς το νευροπροστατευτικό τους ρόλο σε νευροτοξικά μοντέλα οξείας ντοπαμινεργικής εκφύλισης. Ο μεταλλαγμένος μυς “weaver” αποτελεί ένα μοναδικό γενετικό μοντέλο μελαινοραβδωτής νευροεκφύλισης, η οποία λαμβάνει χώρα ενδογενώς και προοδευτικά, αρχίζοντας μετά την 7η μετεμβρυϊκή ημέρα (Ρ7) και προσεγγίζοντας το 50% την 21η μετεμβρυϊκή ημέρα (Ρ21). Στην παρούσα μελέτη, προκειμένου να διερευνηθούν νευροπροστατευτικοί μηχανισμοί κατά τα πρώτα στάδια της νευροεκφυλιστικής διαδικασίας στο μυ “weaver”, και να επιτευχθεί μία πλειοτροπική θεραπευτική δράση, χορηγήθηκαν τρεις φαρμακευτικοί νευροπροστατευτικοί παράγοντες με διαφορετικούς μηχανισμούς δράσης καθώς και ένα σχήμα συνδυασμού τους. Συγκεκριμένα, χορηγήθηκαν, μεμονωμένα και σε συνδυασμό, στους μυς “weaver” N-ακετυλοκυστεΐνη (ΝAC) (αντιοξειδωτική δράση), ασπιρίνη (αντιφλεγμονώδης δράση) και 17β οιστραδιόλη [Ε2] (αντιοξειδωτική, αντιαποπτωτική, νευροτροφική δράση) σε καθημερινή βάση από την Ρ1 μέχρι την Ρ21. Το νευροπροστατευτικό αποτέλεσμα αξιολογήθηκε με ανοσοϊστοχημικό προσδιορισμό των ντοπαμινεργικών νευρώνων της συμπαγούς μοίρας της μέλαινας ουσίας (SNpc) των μυών στους οποίους χορηγήθηκαν τα παραπάνω φάρμακα. Η χορήγηση των NAC και ασπιρίνης δεν επηρέασε την επιβίωση των ντοπαμινεργικών νευρώνων (DA) των weaver μυών. Αντίθετα η χορήγηση της 17β οιστραδιόλης οδήγησε σε σημαντική επιβίωση των DA νευρώνων της SNpc, της τάξης του 48%, στους weaver μυς που έλαβαν την αγωγή, συγκριτικά με τους weaver μυς που έλαβαν φυσιολογικό ορό. Επιπλέον η χορήγηση του συνδυασμού των τριών φαρμάκων (cocktail) προώθησε σε ακόμα μεγαλύτερο βαθμό την επιβίωση των DA νευρώνων της SNpc, σε ποσοστό 86%. Οι weaver μύες που έλαβαν το cocktail εμφάνισαν 26% περισσότερους DA νευρώνες σε σύγκριση με τους weaver μυς που έλαβαν μεμονωμένα 17β οιστραδιόλη προτείνοντας πιθανή συνεργιστική δράση μεταξύ 17β οιστραδιόλης και NAC. Η διερεύνηση του μηχανισμού της νευροεκφύλισης στην SNpc και της παρεχόμενης νευροπροστασίας από τη χορήγηση της 17β οιστραδιόλης και του cocktail πραγματοποιήθηκε σε δύο επίπεδα. Αρχικά με τον προσδιορισμό μιας σειράς δεικτών οξειδωτικού στρες όπως η υπεροξείδωση λιπιδίων και δείκτες της θειολικής κατάστασης του κυττάρου (GSH, GSSG, CSH NPSSC, PSH, PSSP, NPSH, NSPSSR). Ο προσδιορισμός της υπεροξείδωση λιπιδίων πραγματοποιήθηκε στο μεσεγκέφαλο και το ραβδωτό σώμα των φυσιολογικών και weaver μυών που έλαβαν φυσιολογικό ορό (saline +/+ και saline wv/wv), 17 β οιστραδιόλη (17β +/+ και 17β wv/wv) cocktail (cocktail +/+ και cocktail wv/wv). Τα επίπεδα της υπεροξείδωσης λιπιδίων, στο μεσεγκέφαλο, αυξήθηκαν περίπου κατά 98% στους saline wv/wv μυς συγκριτικά με τους saline +/+ δείχνοντας παρουσία έντονου οξειδωτικού στρες στην παθολογική κατάσταση των weaver μυών. Ήταν ενδιαφέρον όμως το γεγονός ότι η λιπιδική υπεροξείδωση ανεστάλη σε ποσοστό 27% στους 17β wv/wv ενώ επανήλθε στα φυσιολογικά επίπεδα στους cocktail wv/wv μύες. Από τους υπόλοιπους δείκτες που εξετάστηκαν μόνο το NPSSC έδειξε διαφορές μεταξύ saline +/+ και saline wv/wv, ενώ οι GSSG, PSSP και PSH ακολούθησαν παρόμοια αύξηση στους cocktail +/+ και cocktail wv/w. Οι παρατηρήσεις αυτές δείχνουν ότι οι συγκεκριμένοι δείκτες από μόνοι τους δεν μπορούν να δώσουν σαφή εικόνα της οξειδωτικής κατάστασης, καθώς αποτελούν ταχέως μεταβαλλόμενα συστατικά αντιοξειδωτικών κύκλων. Στη συνέχεια διερευνήθηκε η έκφραση των γονιδίων Lasp1, Supt14h, Nr4a2 (nurr1), Dlg4 και του γονιδίου του μεταφορέα της σεροτονίνης (SERT), τα οποία φαίνονται να εμπλέκονται στα μονοπάτια της νευροεκφύλισης, στη μεσεγκεφαλική περιοχή και στο ραβδωτό σώμα των weaver μυών. Δεν παρατηρήθηκαν διαφορές στα επίπεδα έκφρασής τους με χρήση της τεχνικής RT-PCR σε καμία από τις υπό εξέταση περιοχές. Τα αποτελέσματα της παρούσας εργασίας οδηγούν στο συμπέρασμα ότι η 17β-οιστραδιόλη παρείχε σημαντική νευροπροστασία στους ντοπαμινεργικούς νευρώνες, για πρώτη φορά, ενός μοντέλου in vivo, ενδογενούς, προοδευτικής μελαινοραβδωτής νευροεκφύλισης, του μοντέλου weaver. Στο μηχανισμό της νευροπροστατευτικής δράσης της Ε2 φαίνεται να παίζει σημαντικό ρόλο η αντιοξειδωτική της δράση αφού η χορήγησή της αναστέλλει τη λιπιδική υπεροξείδωση. Επιπλέον η νευροπροστατευτική δράση της Ε2 ενδυναμώθηκε σημαντικά κατά τη συγχορήγηση του NAC, προτείνοντας την ύπαρξη συνέργειας μεταξύ της Ε2 και της GSH, για πρώτη φορά σε ένα in vivo μοντέλο νευροεκφύλισης. Η ενίσχυση του νευροπροστατευτικού αποτελέσματος από το cocktail δίνει ένα πρόσθετο επιχείρημα στην υπόθεση του αντιοξειδωτικού τρόπου δράσης της Ε2 αφού παράλληλα το cocktail επαναφέρει την υπεροξείδωση των λιπιδίων στα φυσιολογικά επίπεδα. Οι παρατηρήσεις αυτές προτείνουν την Ε2 ως μια μελλοντική υποψήφια φαρμακευτική αγωγή για νευροεκφυλιστικές καταστάσεις, όπως είναι η νόσος του Πάρκινσον, για τα θηλυκά βέβαια άτομα. Eπιπλέον προτείνουν ότι ο συνδυασμός της Ε2 και του NAC μπορεί να οδηγήσει σε εφαρμογή μικρότερων και κατά συνέπεια λιγότερο επιβαρυντικών, από άποψη παρενεργειών, δόσεων που θα οδηγεί σε ίδιο ή και μεγαλύτερο νευροπροστατευτικό αποτέλεσμα με τη μεμονωμένη χορήγηση της 17β-οιστραδιόλης. / Parkinson’s disease (PD) is characterized by the progressive degeneration of the nigrostriatal dopaminergic innervation that leads to motor disturbances. It is considered to be a multifactor disease, the etiology of which still remains unknown. Since currently available treatments are only symptomatic, having severe side-effects, neuroprotection from the early stages of the disease has been given much attention as a promising approach to PD management. Indeed, a broad range of agents has been investigated for their neuroprotective role in neurotoxical models of acute dopaminergic degeneration. “Weaver” mutant mouse represents a unique genetic model, in which the nigrostriatal neurodegeneration occurs endogenously and progressively, starting after postnatal day 7 (P7) and reaching 50% at P21. In the present study, aiming to identify neuroprotective mechanisms in the early progression of the “weaver” degenerative process and to achieve a potentially pleiotropic therapeutic action, we applied three pharmaceutical agents with different mechanisms of action, as well as a scheme combining them. Specifically, “weaver” mice were treated, individually and in combination, with N-acetylcysteine (NAC) (antioxidant), aspirine (anti-inflammatory) and 17b-estradiol [E2] (antioxidant, antiapoptotic, neurotrophic) daily, from P1 to P21. The neuroprotective effect was evaluated by immunohistochemical detection of dopaminergic (DA) neurons in the substantia nigra (SNpc) of treated animals. The administration of ΝΑC and aspirine did not influence the survival of (DA) neurons of weaver mice. On the contrary, the administration of 17b estradiol led to significant survival of DA neurons of SNpc, approximately 48%, in weaver mice that received E2, comparatively with weaver mice that received saline. Moreover the administration of the combination of the three drugs (cocktail) promoted the survival of DA neurons of SNpc, approximately 86% to a higher degree. Weaver mice that received cocktail had 26% more DA neurons compared to weaver mice that received individually 17b estradiol, proposing a possible synergistic action between 17b estradiol and NAC. The investigation of mechanism of neurodegeneration in SNpc and provided neuroprotection by 17b estradiol and cocktail, was realised in two levels. Initiall, by determination of oxidative stress markers, like lipid peroxidation and markers of cellular thiol redox (GSH, GSSG, CSH NPSSC, PSH, PSSP, NPSH, NSPSSR). The determination of lipid peroxidation was realised in the midbrain and striatum of normal and weaver mice that received saline (saline +/+ and saline wv/wv), 17 b estradiol (17b +/+ and 17b wv/wv) cocktail (cocktail +/+ and cocktail wv/wv). Lipid peroxidation levels in the midbrain were increased about 98% in saline wv/wv mice comparatively with the saline +/+, showing the presence of intense oxidative stress in the weaver mutant mouse. It was interesting, however, the fact that lipid peroxidation was inhibited approximately 27% in 17b wv/wv mice, while it was reverted at the normal levels in cocktail wv/wv mice. Regarding to the other oxidative markers that were examined, only NPSSC showed differences between saline +/+ and saline wv/wv, while the GSSG, PSSP and PSH followed similar increasement in both cocktail +/+ and cocktail wv/w animals. This observation indicates that these markers alone cannot give a clear figure of oxidative situation, as they constitute rapidly altered components of antioxidant cycles. Afterwards, we investigated the expression of genes Lasp1, Supt14h, Nr4a2 (nurr1), Dlg4 and serotonin transporter’s gene (SERT), which appear to be involved in neurodegeneration pathways, in the midbrain ant striatum of normal and weaver mice. There were not observed differences in their expression levels (using the RT-PCR technique) in both regions investigated. The results of the present study, lead to the conclusion that 17b-estradiol provided important neuroprotection in the DA neurons, for the first time, in a model of in vivo, endogenous, progressive dopaminergic degeneration, the weaver model. The mechanism of E2’s neuroprotective effect appears to be antioxidant as the administration of E2 suspends lipid peroxidation. Moreover the E2’s neuroprotective effect was strengthened significantly by the co-treatment of NAC, proposing the existence of synergy between E2 and GSH, for the first time in an in vivo model of neurodegeneration. The reinforced cocktail’s result gives an additional argument in the hypothesis of antioxidant mechanism of E2’s action, as cocktail, at the same time, restores lipid peroxidation in normal levels. These observations propose E2 as a future candidate pharmaceutic treatment for neurodegenerative situations, like PD, of course for female individuals. Moreover they propose that the combined treatment of E2 and NAC, can lead to the application of lower and, in consequence, less aggravating doses, concerning the side effects, that will lead to same or even higher neuroprotective result with the individual administration of 17b-estradiol

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

As relações sociais são construídas e mantidas a partir da interação entre os indivíduos. A ocitocina (OT), vasopressina (AVP), estrogênios (EST) e dopamina (DOPA), bem como seus respectivos receptores estão envolvidos na modulação do comportamento sexual de fêmeas. Estruturas do sistema nervoso central (SNC), tais como o bulbo olfatório (BO), o hipotálamo (HPT), o córtex pré-frontal (CPF) e o hipocampo (HPC) exercem importantes funções relacionadas à motivação sexual, reconhecimento de odores, memória e respostas emocionais. Em camundongos fêmeas, a OT é essencial para o comportamento de lordose e para estabelecer a preferência pelo parceiro. Este estudo teve como objetivo analisar o impacto do nocauteamento no gene da OT (OTKO) no comportamento sexual de camundongos fêmeas, na síntese hipotalâmica de AVP e na expressão gênica dos receptores de OT (OTR), AVP (AVPR1a), EST alfa (ERα), EST beta (ERβ) e DOPARD2 no BO, HPT, CPF e HPC. Foram utilizados 11 camundongos fêmeas (C57BL/6J) para o grupo controle (WT) e o mesmo número para o grupo OTKO. A detecção do transgene no genoma foi realizada pela técnica de reação em cadeia da polimerase (PCR) e os animais foram classificados como: WT (OT+/+), heterozigoto (OT+/-) e OTKO (OT-/-). O teste do comportamento sexual da fêmea foi realizado na noite do proestro e os parâmetros comportamentais avaliados foram: frequência, duração e latência de lordose, além da frequência de montas, bem como as posturas não receptivas e a locomoção. A coleta das estruturas encefálicas (BO, HPT, CPF e HPC) aconteceu na manhã seguinte do teste comportamental. Os cDNAs foram sintetizados por transcrição reversa seguida de reação em cadeia da polimerase (RT-PCR). A expressão gênica de cada receptor foi calculada a partir da fórmula 2-ΔΔCt. Os dados do registro comportamental e da expressão gênica foram expressos pela média, erro padrão da média (±EPM), analisados pelo teste de Mann-Whitney e o nível de significância aceito foi de p<0,05. Nossos resultados mostraram aumento significativo da latência e diminuição significativa da frequência e da duração do comportamento de lordose em fêmeas OTKO. Em relação às posturas não receptivas, fêmeas OTKO apresentaram diminuição significativa da latência e aumento significativo da frequência e da duração destas posturas. No que concerne à expressão gênica dos diferentes receptores, fêmeas OTKO apresentaram diminuição significativa da expressão gênica do OTR apenas no HPC em relação ao grupo WT. Verificamos que o grupo OTKO apresentou diminuição significativa da expressão gênica do AVPR1a apenas no HPT, mas aumento da expressão no HPC quando comparadas ao grupo WT. Também, fêmeas OTKO apresentaram diminuição significativa da expressão gênica do ERα e ERβ apenas no CPF quando comparadas ao grupo WT. Não foram encontradas diferenças significativas na expressão gênica do DOPARD2 em nenhuma das estruturas estudadas quando comparados os dois grupos estudados. No que diz respeito ao nocauteamento no gene da OT, nossos resultados mostraram que este não promove diferença significativa na síntese de RNAm de AVP no HPT do grupo OTKO quando ao grupo WT. Nossos principais achados nos permitem inferir que a ausência da OT dentro do SNC, bem como a importante alteração da expressão dos genes estudados (OTR, AVPR1a, ERα e ERβ) principalmente no CPF estão relacionados com a diminuição do comportamento sexual observada nas fêmeas OTKO. / Social relations are built and maintained from the interaction between individuals. Oxytocin (OT), vasopressin (AVP), estrogens (EST), dopamine (DOPA) and their receptors are involved in the modulation of sexual behavior in females. Structures of the central nervous system (CNS), such as the olfactory bulb (OB), hypothalamus (HPT), medial amygdale, prefrontal cortex (PFC) and hippocampus (HPC) have important functions related to sexual motivation, odor recognition, memory, and emotional responses. In mice, OT is essential for lordosis behavior and to establish the female preference for her partner. The experimental model using knockout animals for OT allows evaluating the physiological and behavioral changes generated from this genetic manipulation. This study aimed to analyze the impact of OT gene knockout (OTKO) in sexual behavior of female mice, in the synthesis hypothalamic of AVP and gene expression of OT receptors (OTR), AVP (AVPR1a), EST alpha (ERα), EST beta (ERβ) and DOPARD2 in OB, HPT, PFC and HPC. We used 11 female mice (C57BL/6J) for the control group (WT) and the same number for the OTKO group. Detection of the transgene in the genome was performed by polymerase chain reaction (PCR) and the animals were classified as: WT (OT+/+), heterozygous (+/-OT) and OTKO (OT-/-). The female sexual behavior test was performed on the evening of proestrus and these behavioral parameters were evaluated: frequency, duration and latency lordosis, frequency of mounts, as well as non-receptive positions and locomotion. The collection of brain structures (OB, HPT, PFC and HPC) happened the next morning the behavioral test. The CDNAs were synthesized by reverse transcription followed by polymerase chain reaction (RT-PCR). The gene expression of each receptor was calculated from the 2-ΔΔCt formula. Data from the behavioral record and gene expression were expressed as mean, standard error of the mean (±SEM) and analyzed by the Mann-Whitney test. In all cases, P<0.05 was considered statistically significant. Our results showed significant increase in latency and decrease in the frequency and duration of lordosis behavior in OTKO females. For non-receptive postures, OTKO females were significantly reduced latency and increased frequency and duration of these postures. Regarding the gene expression of different receptors, OTKO females showed significant decrease in OTR gene expression only in the HPC compared to WT group. We found that the OTKO group showed a significant decrease in gene expression of AVPR1a only in HPT, but increased expression in HPC as compared to WT group. Also, OTKO females showed significant decrease in gene expression of ERα and ERβ only the PFC when compared to the WT group. There were no significant differences in gene expression DOPARD2 in any of the studied structures when comparing the two groups. Regarding knockout in OT gene, our results showed that this does not promote significant difference in AVP mRNA synthesis in HPT of OTKO group when the WT group. Our main findings allow us to infer that the absence of OT within the CNS, as well as a significant changes in expression of the genes studied (OTR, AVPR1a, ERα and ERβ) mainly in the PFC are related to decreased sexual behavior observed in OTKO females.

Prostate cancer increases its incidence with age after men in their fifth decade as the ratio of estrogen to androgen rises. Epidemiological studies indicated that high levels of estrogens are associated with the high-risk ethnic groups for prostate cancer. Therefore, estrogens may be involved in prostatic carcinogenesis. It is widely believed that the actions of estrogens are mediated by estrogen receptors. However, expression of estrogen receptor in normal prostate and lesions of the gland was controversial. With the recent discovery of second estrogen receptor (ER-β), this issue became more complicated and it needs to be readdressed. In addition, the biological involvement of ER-β in human prostate remains to be investigated. In this study, we demonstrated that human normal prostate epithelial cells express ER-β but not ER-α, suggesting that estrogens act directly on these epithelial cells via ER-β. Using RT-PCR analysis, the transcripts of ER-β were detected in our primary human prostatic epithelial cell cultures that were derived from the ultrasound-guided peripheral zone biopsies and the cells express two estrogen-regulated genes such as progesterone receptor (PR) and pS2. Moreover, we had developed an ER-β antibody with fully characterizations and used it for immunohistochemistry. Results indicated that ER-p protein is expressed in the basal compartment of prostatic epithelium of the gland. Our findings lead to a new hypothesis that estrogens directly act on human prostatic epithelial cells to modulate its biological functions. To investigate expression of ERs in prostate cancer, RT-PCR analysis was used. We found that all three human prostate metastatic cancer cell lines, DU145, PC-3 and LNCaP, express ER-β transcripts while ER-α mRNA expression only in PC-3 cells. Expressions of PR and pS2 in these cell lines are various. LNCaP cells express both PR and pS2 mRNAs but DU145 cells with only PR and PC-3 cells with only pS2. Our immunohistochemical results on prostatic lesions revealed down-regulation of ER-β expression in high-grade of dysplasia and carcinoma of peripheral zone of the prostate compared to their low-grade lesions. This down-regulation in high-grade carcinoma was verified in transcriptional level by RT-PCR analysis on micro dissected normal epithelium and lesion samples of the gland. In the metastasis, ER-β was found to be reactivated as we observed ER-β mRNA expression in prostate cancer cell lines. Recent evidence suggests that ER-β may be antiproliferative factor for a protective effect against the mitogenic activity of estrogens in breast and androgens in prostate. Activation of the receptor may exhibit cell growth inhibition. We demonstrated that antiestrogens [ICI-182,780 (ICI) and 4-hydroxytamoxifen], raloxifene and phytoestrogen (resveratrol), but not estrogens (17β-estradiol and diethylstilbestrol), inhibit growth of DU145 cells which express only ER-β while PC-3 cells with both ERs showed growth inhibition in response to estrogen and antiestrogen treatments. In DU145 cells, the ICI-induced cell growth inhibition was prevented by blockade of ER-β expression using antisense oligonucleotide. It indicated that the inhibition is mediated via ER-p associated pathway. Using flow cytometry, we found that ICI-treatment could induce accumulation of cells at GO-G1 phase of cell cycle. Similarly, this GO-G1 cell accumulation was also induced by raloxifene in DU145 cells. For resveratrol, the treatment exhibited dual effects on cell cycle distribution in DU145 cells. In the early treatment, resveratrol induced cell cycle arrests at GO-G1phase. The prolonged treatment leads to S-phase cell cycle arrest. To study the molecular mechanism of this ER-p associated cell growth inhibition, real-time RT-PCR analysis was used to semi-quantitate the transcript levels of tentative ER-β regulated genes such as telomerase reverse transcriptase (TERT), survivin and thymidylate synthase (TS) in the treated cells compared to those in control. Results demonstrated that the treatment of ICI could down-regulate TERT and survivin mRNA expressions with dose-dependent fashion. As the ICI-treatment, resveratrol downregulated expression levels of TERT, survivin and TS in DU145 cells. Down-regulation of TS may be related to the S-phase cell cycle arrest observed in the prolonged treatment of resveratrol. Taken together, our findings support the concept that ER-β participates in cell cycle regulation in normal and malignant prostatic epithelial cells. Presence of ER-β in basal cells of the prostate acini indicates that the direct actions of estrogens may be involved in the normal physiology of the gland. Loss of this receptor in primary prostate cancer and its re-expression in metastasis suggests the roles of ER-β in the cancer progression. Activation of the receptor by antiestrogen and phytoestrogen induced cell growth inhibition in prostate cancer cells. The mechanism may be mediated by reduction of cell survival factors and eventually decrease in cell viability and induction of cell cycle arrests.

As relações sociais são construídas e mantidas a partir da interação entre os indivíduos. A ocitocina (OT), vasopressina (AVP), estrogênios (EST) e dopamina (DOPA), bem como seus respectivos receptores estão envolvidos na modulação do comportamento sexual de fêmeas. Estruturas do sistema nervoso central (SNC), tais como o bulbo olfatório (BO), o hipotálamo (HPT), o córtex pré-frontal (CPF) e o hipocampo (HPC) exercem importantes funções relacionadas à motivação sexual, reconhecimento de odores, memória e respostas emocionais. Em camundongos fêmeas, a OT é essencial para o comportamento de lordose e para estabelecer a preferência pelo parceiro. Este estudo teve como objetivo analisar o impacto do nocauteamento no gene da OT (OTKO) no comportamento sexual de camundongos fêmeas, na síntese hipotalâmica de AVP e na expressão gênica dos receptores de OT (OTR), AVP (AVPR1a), EST alfa (ERα), EST beta (ERβ) e DOPARD2 no BO, HPT, CPF e HPC. Foram utilizados 11 camundongos fêmeas (C57BL/6J) para o grupo controle (WT) e o mesmo número para o grupo OTKO. A detecção do transgene no genoma foi realizada pela técnica de reação em cadeia da polimerase (PCR) e os animais foram classificados como: WT (OT+/+), heterozigoto (OT+/-) e OTKO (OT-/-). O teste do comportamento sexual da fêmea foi realizado na noite do proestro e os parâmetros comportamentais avaliados foram: frequência, duração e latência de lordose, além da frequência de montas, bem como as posturas não receptivas e a locomoção. A coleta das estruturas encefálicas (BO, HPT, CPF e HPC) aconteceu na manhã seguinte do teste comportamental. Os cDNAs foram sintetizados por transcrição reversa seguida de reação em cadeia da polimerase (RT-PCR). A expressão gênica de cada receptor foi calculada a partir da fórmula 2-ΔΔCt. Os dados do registro comportamental e da expressão gênica foram expressos pela média, erro padrão da média (±EPM), analisados pelo teste de Mann-Whitney e o nível de significância aceito foi de p<0,05. Nossos resultados mostraram aumento significativo da latência e diminuição significativa da frequência e da duração do comportamento de lordose em fêmeas OTKO. Em relação às posturas não receptivas, fêmeas OTKO apresentaram diminuição significativa da latência e aumento significativo da frequência e da duração destas posturas. No que concerne à expressão gênica dos diferentes receptores, fêmeas OTKO apresentaram diminuição significativa da expressão gênica do OTR apenas no HPC em relação ao grupo WT. Verificamos que o grupo OTKO apresentou diminuição significativa da expressão gênica do AVPR1a apenas no HPT, mas aumento da expressão no HPC quando comparadas ao grupo WT. Também, fêmeas OTKO apresentaram diminuição significativa da expressão gênica do ERα e ERβ apenas no CPF quando comparadas ao grupo WT. Não foram encontradas diferenças significativas na expressão gênica do DOPARD2 em nenhuma das estruturas estudadas quando comparados os dois grupos estudados. No que diz respeito ao nocauteamento no gene da OT, nossos resultados mostraram que este não promove diferença significativa na síntese de RNAm de AVP no HPT do grupo OTKO quando ao grupo WT. Nossos principais achados nos permitem inferir que a ausência da OT dentro do SNC, bem como a importante alteração da expressão dos genes estudados (OTR, AVPR1a, ERα e ERβ) principalmente no CPF estão relacionados com a diminuição do comportamento sexual observada nas fêmeas OTKO. / Social relations are built and maintained from the interaction between individuals. Oxytocin (OT), vasopressin (AVP), estrogens (EST), dopamine (DOPA) and their receptors are involved in the modulation of sexual behavior in females. Structures of the central nervous system (CNS), such as the olfactory bulb (OB), hypothalamus (HPT), medial amygdale, prefrontal cortex (PFC) and hippocampus (HPC) have important functions related to sexual motivation, odor recognition, memory, and emotional responses. In mice, OT is essential for lordosis behavior and to establish the female preference for her partner. The experimental model using knockout animals for OT allows evaluating the physiological and behavioral changes generated from this genetic manipulation. This study aimed to analyze the impact of OT gene knockout (OTKO) in sexual behavior of female mice, in the synthesis hypothalamic of AVP and gene expression of OT receptors (OTR), AVP (AVPR1a), EST alpha (ERα), EST beta (ERβ) and DOPARD2 in OB, HPT, PFC and HPC. We used 11 female mice (C57BL/6J) for the control group (WT) and the same number for the OTKO group. Detection of the transgene in the genome was performed by polymerase chain reaction (PCR) and the animals were classified as: WT (OT+/+), heterozygous (+/-OT) and OTKO (OT-/-). The female sexual behavior test was performed on the evening of proestrus and these behavioral parameters were evaluated: frequency, duration and latency lordosis, frequency of mounts, as well as non-receptive positions and locomotion. The collection of brain structures (OB, HPT, PFC and HPC) happened the next morning the behavioral test. The CDNAs were synthesized by reverse transcription followed by polymerase chain reaction (RT-PCR). The gene expression of each receptor was calculated from the 2-ΔΔCt formula. Data from the behavioral record and gene expression were expressed as mean, standard error of the mean (±SEM) and analyzed by the Mann-Whitney test. In all cases, P<0.05 was considered statistically significant. Our results showed significant increase in latency and decrease in the frequency and duration of lordosis behavior in OTKO females. For non-receptive postures, OTKO females were significantly reduced latency and increased frequency and duration of these postures. Regarding the gene expression of different receptors, OTKO females showed significant decrease in OTR gene expression only in the HPC compared to WT group. We found that the OTKO group showed a significant decrease in gene expression of AVPR1a only in HPT, but increased expression in HPC as compared to WT group. Also, OTKO females showed significant decrease in gene expression of ERα and ERβ only the PFC when compared to the WT group. There were no significant differences in gene expression DOPARD2 in any of the studied structures when comparing the two groups. Regarding knockout in OT gene, our results showed that this does not promote significant difference in AVP mRNA synthesis in HPT of OTKO group when the WT group. Our main findings allow us to infer that the absence of OT within the CNS, as well as a significant changes in expression of the genes studied (OTR, AVPR1a, ERα and ERβ) mainly in the PFC are related to decreased sexual behavior observed in OTKO females.

A deprivação de estrogênios tem sido descrita como uma das causas da redução da complacência arterial no organismo feminino. Por outro lado, exercícios físicos aeróbios são amplamente indicados para o tratamento de diversas desordens do sistema cardiovascular e para redução do enrijecimento de artérias centrais em mulheres na fase de pós-menopausa. Este estudo investigou as características morfoquantitativas da matriz extra-celular da túnica média da aorta ascendente de fêmeas de Rattus norvegicus da linhagem Wistar submetidas a exercício físico aeróbio e à ovariectomia. Foram utilizadas 20 fêmeas com seis meses de idade. Os animais foram distribuídos em 4 grupos de 5 animais cada: GIS (grupo intacto sedentário); GIT (grupo intacto treinado); GOS (grupo ovariectomizado sedentário) e GOT (grupo ovariectomizado treinado). O exercício físico constituiu de uma hora diária de corrida em esteira ergométrica, a 60% da velocidade máxima, durante 12 semanas consecutivas. A preparação do material foi feita com técnicas convencionais de histologia. As colorações utilizadas foram: Hematoxilina-Eosina, Hematoxilina de Verhoeff, Resorcina de Weigert, Resorcina de Weigert com prévia oxidação e Picro-sirius, para a quantificação do volume ocupado pelas túnicas média e íntima e da luz da aorta, da densidade de volume das lamelas elásticas, das fibras elaunínicas, das fibras oxitalânicas e do tecido colágeno, respectivamente. A túnica média da aorta foi estudada por meio de métodos estereológicos em microscopia de luz. A análise morfoquantitativa revelou que a ovariectomia associada à prática de exercício físico aeróbio não interferiu no volume ocupado pelas túnicas media e íntima, e pela luz da aorta; e nem na razão entre esses parâmetros. O grupo GIT apresentou densidade de volume significativamente maior das lamelas elásticas, das fibras oxitalânicas e do colágeno. O grupo GOT apresentou densidade de volume significativamente menor do tecido colágeno. Com os resultados e as análises deste estudo, pode-se supor que o exercício físico aeróbio, mesmo se iniciado na fase adulta do animal, pode ser um recurso para evitar a perda da elasticidade da aorta ascendente de ratas Wistar em situações de carência de estrogênios. / Low levels of estrogens have been related to arterial stiffness in female women. On the other hand, physical exercises are indicated for treatment of cardiovascular deseases and for reducing central arterial stiffness in postmenopausal women. The aim of this research was studied the features of extracellular matrix of ascending aorta tunica media in female rats submitted to ovariectomy and aerobic physical exercises training. Twenty six-months-old female Wistar rats were divided in four groups (n=5): sedentary intact (sed-int), exercise-trained intact (ex-int), sedentary ovariectomized (sed-ovx) and exercise-trained ovariectomized (ex-ovx). Exercise protocol was performed on a motor treadmill in a map speed for 12 weeks five times per week. The samples were preparated by convencional histology technics. The stains used were: Hematoxylin-Eosin, Verhoeff stain, Weigerts Resorcin, Weigerts Resorcin after oxidation and Picrossirus, for quantification the volume tunica media and intima and the volume lumen of aorta, volume density of lamellae, elauninic fibers, oxytalan fibers and collagenous, respectively. Aorta tunica media was studied by stereology methods in light microscopy. The results showed the ovariectomy and aerobic physical exercises did not interfere the volume of aorta tunica media and intima, the volume of lumen neither volume tunica media and intima-to-volume lumen ratio of aorta. Ex-int showed volume density significantly major of lamellae, oxytalan fibers and collagenous. Ex-ovx showed volume density significantly minor of collagenous. In conclusion, we can suppose aerobic physical exercises can avoid the ascending aorta stiffness in Wistar rats in low levels of estrogens situations.