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The circadian clock is a molecular mechanism that synchronizes physiological changes with environmental variations. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, and cancer). Period 2 (Per2), a circadian protein, is at the center of the clock's function. The loss or deregulation of per2 has been shown to be common in several types of cancer including breast and ovarian [1, 2]. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERα) mRNA oscillates in a 24-hour period and, unlike Per2, ERα peaks during the light phase of the day. Because up regulation of ERα relates to tumor development, defining the mechanisms of ERα expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of ERα transcription. Transcriptional activation of ERα was measured using a reporter system in Chinese hamster ovary (CHO) cell lines. Data show that Per2 influences ERα transcription through a non-canonical mechanism independent of its circadian counterparts. Breast cancer susceptibility protein 1 (BRCA1) was confirmed to be an interactor of Per2 via bacterial two-hybrid assays, in accordance with previous studies [2]. BRCA1 is a transcriptional activator of ERα promoter in the presence of octamer transcription factor-1 (OCT-1) [3]. Our results indicate that the DNA binding domain of OCT-1, POU, to directly interact with Per2 and BRCA1, in vitro. Pull-down assays were used to map direct interaction of various Per2 and BRCA1 recombinant proteins and POU. Chromatin immunoprecipitation assays confirmed the recruitment of PER2 and BRCA1 to the estrogen promoter by OCT-1 and the recruitment of Per2 to the ERα promoter decreases ERα mRNA expression levels in MCF-7 cells. Our work supports a circadian regulation of ERα through the repression of esr1 by Per2 in MCF-7 cells. / Ph. D.

INTRODUÇÃO: Apesar de a endometriose profunda (EPF) ser a forma da doença de maior repercussão clínica, os estudos sobre a doença costumam ser baseados em lesões de endometriose ovariana (EOV) e peritoneal (EPT). A patogênese da EPF ainda é objeto de amplo debate, pois há poucos estudos feitos exclusivamente com lesões de EPF. O fator de transcrição codificado pelo gene homeobox A10 (HOXA10) regula a conferência de identidade tecidual de útero ao ducto paramesonéfrico indiferenciado durante o período embrionário. O gene mantém um padrão de expressão temporal e espacial bem definido e, durante a fase adulta, continua expresso no miométrio e endométrio. Sugere-se que HOXA10 esteja implicado na patogênese da endometriose, pois é expresso em EOV, EPT, endometriose pulmonar e endometriose retovaginal, um tipo de EPF. Possivelmente, o gene HOXA10 seja necessário para conferir identidade de endometriose a um tecido indiferenciado. O estradiol e a progesterona ativam a transcrição do gene HOXA10 e regulam diretamente sua ação. Esses hormônios estão envolvidos na patogênese da EPF, e suas atividades podem ser inferidas pelo estudo da expressão tecidual de seus receptores. A endometriose de reto-sigmoide (ERS) é um modelo representativo para o estudo da EPF. Neste estudo, avaliamos a expressão proteica do fator de transcrição HOXA10, das isoformas ? (ER-alfa) e beta (ER-beta) dos receptores de estrogênio, e do receptor de progesterona AB (PR-AB) e sua isoforma B (PR-B) na lesão (LES) e no tecido muscular liso perilesional (TMLP) de ERS de pacientes inférteis, durante as fases proliferativa e secretora do ciclo menstrual. MÉTODOS: amostras de LES e TMLP de ERS de 18 pacientes (9 operadas em cada fase do ciclo menstrual) foram agrupadas em blocos de microarranjos de tecidos (tissue microarray). As amostras foram coradas com anticorpos específicos para análise imunoistoquímica de cada uma das proteínas. Foram então avaliadas por microscopia ótica (MO) e pela análise das imagens digitalizadas das lâminas com por um software específico, a análise morfométrica (AM). RESULTADOS: HOXA10 foi expresso no estroma de LES de ERS durante a fase secretora, de acordo com a MO. ER-alfa e ER-betaforam expressos em glândulas e estroma de LES e TMLP de ERS durante ambas as fases do ciclo, de acordo com a MO e a AM. PR-AB e PR-B foram expressos em glândulas e estroma de LES de ERS durante ambas as fases do ciclo, de acordo com a MO. PR-B foi mais expresso durante a fase secretora, independentemente do local de expressão, segundo a AM. A expressão de HOXA10 correlacionou-se diretamente com PR-AB e PR-B na ERS, segundo a AM. Não houve correlação entre ER-alfa e ER-beta com HOXA10, PR-AB ou PR-B em nenhuma fase do ciclo ou local de expressão de ERS. CONCLUSÕES: HOXA10 é expresso em ERS, um local fora do seu eixo espacial de expressão. A presença de HOXA10 pode ser necessária para conferir a identidade \"de novo\" na EPF, incluindo ERS. A progesterona pode ativar o gene HOXA10 e regular esta ação, possivelmente mediada por PR-B. O estradiol exerce sua ação mitógena na ERS através ER-alfa e ER-beta / INTRODUCTION: Although deep endometriosis (DE) is the major clinical form of endometriosis, studies regarding the disease are typically based on ovarian (OE) and peritoneal (PE) lesions. DE pathogenesis is still a matter of great discussion because there are few studies exclusively involving DE lesions. The transcription factor encoded by the homeobox gene A10 (HOXA10) regulates the identity imparted to the undifferentiated paramesonephric duct during embryogenesis. The gene is expressed in the myometrium and endometrium during adult life in a well-defined spatial and temporal mode. It has been suggested that HOXA10 plays a role in endometriosis pathogenesis because it is expressed in OE, PE, pulmonary endometriosis, and rectovaginal endometriosis, which is a clinical form of DE. Thus, HOXA10 may be necessary for \"de novo\" endometrial development from undifferentiated tissues. Both estradiol and progesterone activate HOXA10 transcription and directly regulate its action. These hormones are involved in DE pathogenesis, and therefore their activities could be assessed by studying the tissue expression of their receptors. Rectosigmoid endometriosis (RE) is a representative model for studying DE. In this study, we evaluated the protein expression of HOXA10, the estrogen receptor (ER) isoforms alfa (ER-alfa) and beta (ER-beta), the progesterone receptor AB (PR), and the PR isoform B (PR-B) in lesions (LES) and adjacent smooth muscle (SM) of RE from infertile patients during the proliferative and secretory phases of the menstrual cycle. METHODS: LES and SM samples from RE patients were grouped in tissue microarray blocks. Each of the proteins was analyzed by immunohistochemistry using regular optical microscopy (OM) and a software-assisted analysis of digitalized images as well as morphometric analysis (MA). RESULTS: HOXA10 was expressed in the stroma of the LES during the secretory phase based on OM. ER-alfa and ER-beta were expressed in the glands and stroma of LES and SM during both phases based on OM and MA. PR and PR-B were expressed in the glands and stroma of LES during both phases; however, PR-B had higher expression during the secretory phase, independent of its expression in the LES or SM. HOXA10 expression was directly correlated with PR and PR-B expression in RE. In addition, there was no correlation between the expression of ER-alfa and ER-beta with HOXA10, PR, or PR-B during any phase of the menstrual cycle or site of expression. CONCLUSIONS: HOXA10 is expressed in RE outside of its spatial domain of expression, and may be necessary for \"de novo\" development of DE, including RE. Progesterone might stimulate HOXA10 expression and regulate this action, which is most likely mediated by PR-B. Moreover, estradiol exerts its mitogenic effect in RE though ER-alfa and ER-beta

INTRODUÇÃO: Apesar de a endometriose profunda (EPF) ser a forma da doença de maior repercussão clínica, os estudos sobre a doença costumam ser baseados em lesões de endometriose ovariana (EOV) e peritoneal (EPT). A patogênese da EPF ainda é objeto de amplo debate, pois há poucos estudos feitos exclusivamente com lesões de EPF. O fator de transcrição codificado pelo gene homeobox A10 (HOXA10) regula a conferência de identidade tecidual de útero ao ducto paramesonéfrico indiferenciado durante o período embrionário. O gene mantém um padrão de expressão temporal e espacial bem definido e, durante a fase adulta, continua expresso no miométrio e endométrio. Sugere-se que HOXA10 esteja implicado na patogênese da endometriose, pois é expresso em EOV, EPT, endometriose pulmonar e endometriose retovaginal, um tipo de EPF. Possivelmente, o gene HOXA10 seja necessário para conferir identidade de endometriose a um tecido indiferenciado. O estradiol e a progesterona ativam a transcrição do gene HOXA10 e regulam diretamente sua ação. Esses hormônios estão envolvidos na patogênese da EPF, e suas atividades podem ser inferidas pelo estudo da expressão tecidual de seus receptores. A endometriose de reto-sigmoide (ERS) é um modelo representativo para o estudo da EPF. Neste estudo, avaliamos a expressão proteica do fator de transcrição HOXA10, das isoformas ? (ER-alfa) e beta (ER-beta) dos receptores de estrogênio, e do receptor de progesterona AB (PR-AB) e sua isoforma B (PR-B) na lesão (LES) e no tecido muscular liso perilesional (TMLP) de ERS de pacientes inférteis, durante as fases proliferativa e secretora do ciclo menstrual. MÉTODOS: amostras de LES e TMLP de ERS de 18 pacientes (9 operadas em cada fase do ciclo menstrual) foram agrupadas em blocos de microarranjos de tecidos (tissue microarray). As amostras foram coradas com anticorpos específicos para análise imunoistoquímica de cada uma das proteínas. Foram então avaliadas por microscopia ótica (MO) e pela análise das imagens digitalizadas das lâminas com por um software específico, a análise morfométrica (AM). RESULTADOS: HOXA10 foi expresso no estroma de LES de ERS durante a fase secretora, de acordo com a MO. ER-alfa e ER-betaforam expressos em glândulas e estroma de LES e TMLP de ERS durante ambas as fases do ciclo, de acordo com a MO e a AM. PR-AB e PR-B foram expressos em glândulas e estroma de LES de ERS durante ambas as fases do ciclo, de acordo com a MO. PR-B foi mais expresso durante a fase secretora, independentemente do local de expressão, segundo a AM. A expressão de HOXA10 correlacionou-se diretamente com PR-AB e PR-B na ERS, segundo a AM. Não houve correlação entre ER-alfa e ER-beta com HOXA10, PR-AB ou PR-B em nenhuma fase do ciclo ou local de expressão de ERS. CONCLUSÕES: HOXA10 é expresso em ERS, um local fora do seu eixo espacial de expressão. A presença de HOXA10 pode ser necessária para conferir a identidade \"de novo\" na EPF, incluindo ERS. A progesterona pode ativar o gene HOXA10 e regular esta ação, possivelmente mediada por PR-B. O estradiol exerce sua ação mitógena na ERS através ER-alfa e ER-beta / INTRODUCTION: Although deep endometriosis (DE) is the major clinical form of endometriosis, studies regarding the disease are typically based on ovarian (OE) and peritoneal (PE) lesions. DE pathogenesis is still a matter of great discussion because there are few studies exclusively involving DE lesions. The transcription factor encoded by the homeobox gene A10 (HOXA10) regulates the identity imparted to the undifferentiated paramesonephric duct during embryogenesis. The gene is expressed in the myometrium and endometrium during adult life in a well-defined spatial and temporal mode. It has been suggested that HOXA10 plays a role in endometriosis pathogenesis because it is expressed in OE, PE, pulmonary endometriosis, and rectovaginal endometriosis, which is a clinical form of DE. Thus, HOXA10 may be necessary for \"de novo\" endometrial development from undifferentiated tissues. Both estradiol and progesterone activate HOXA10 transcription and directly regulate its action. These hormones are involved in DE pathogenesis, and therefore their activities could be assessed by studying the tissue expression of their receptors. Rectosigmoid endometriosis (RE) is a representative model for studying DE. In this study, we evaluated the protein expression of HOXA10, the estrogen receptor (ER) isoforms alfa (ER-alfa) and beta (ER-beta), the progesterone receptor AB (PR), and the PR isoform B (PR-B) in lesions (LES) and adjacent smooth muscle (SM) of RE from infertile patients during the proliferative and secretory phases of the menstrual cycle. METHODS: LES and SM samples from RE patients were grouped in tissue microarray blocks. Each of the proteins was analyzed by immunohistochemistry using regular optical microscopy (OM) and a software-assisted analysis of digitalized images as well as morphometric analysis (MA). RESULTS: HOXA10 was expressed in the stroma of the LES during the secretory phase based on OM. ER-alfa and ER-beta were expressed in the glands and stroma of LES and SM during both phases based on OM and MA. PR and PR-B were expressed in the glands and stroma of LES during both phases; however, PR-B had higher expression during the secretory phase, independent of its expression in the LES or SM. HOXA10 expression was directly correlated with PR and PR-B expression in RE. In addition, there was no correlation between the expression of ER-alfa and ER-beta with HOXA10, PR, or PR-B during any phase of the menstrual cycle or site of expression. CONCLUSIONS: HOXA10 is expressed in RE outside of its spatial domain of expression, and may be necessary for \"de novo\" development of DE, including RE. Progesterone might stimulate HOXA10 expression and regulate this action, which is most likely mediated by PR-B. Moreover, estradiol exerts its mitogenic effect in RE though ER-alfa and ER-beta

Clinical immunohistochemistry (IHC) techniques are not yet fully standardized. In this project, a standardization method was developed and tested for proficiency testing (PT) in external quality assurance (EQA) and quality control (QC) in clinical IHC laboratories. The breast cancer markers estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) were used as a model system. Digital image analysis (IA) was used in conjunction with new calibrated and standardized cell line microarrays (CLMA). CLMAs built from nine formalin-fixed paraffin-embedded (FFPE) breast cancer cell lines were used for both QC controls and PT samples, instead of traditionally used FFPE tissues, in the standardization of breast cancer IHC. IA was used for measurement of IHC results, and compared to evaluation by the traditional expert-assessment method. Laboratory Score: Reference Score Ratio (LSRSR) was derived from Histo-Scores (HScores) determined by IA. HScores and LSRSRs were examined statistically and evaluated as histograms and boxplots to summarize and rank participant laboratory EQA results, in comparison to a reference sample or reference laboratories in two consecutive Canada-wide EQA runs. LSRSR-derived reference ranges were highly sensitive in evaluating laboratory EQA performance in PT as well as for monitoring of controls for QC. Laboratory on-slide tissue and cell-line IHC QA controls were assessed using IA and Levey Jennings QC charts. These charts were determined to be an excellent way to observe trending in laboratory IHC staining over time, particularly when cell line controls were used. This approach also reduced the time and labor costs for PT evaluation. Overall, cell line calibration controls were functionally equivalent or better than tissue-based controls in QC and PT mainly because of cell line biological homogeneity and sample availability. This study identified an optimal design for preparation of IHC cell line controls and PT samples for breast cancer markers. Optimal, intermediate staining cell line IHC controls were identified for all three breast cancer markers. Using IA with LSRSR and cell line samples is recommended for standardization of IHC methodology. This approach advances QA for diagnostic IHC and when implemented will improve patient care

De nombreuses études expérimentales ou épidémiologiques suggèrent un rôle psycho- et neuro- protecteur des estrogènes que les résultats de certains essais cliniques ne confirment p as. L'objectif de cette thèse est d'étudier le rôle des estrogènes dans la dépression et le fonctionnement cognitif chez les femmes âgées en examinant les taux d'estrogènes sériques, l'exposition aux estrogènes au cours de la vie et l'impact des traitements hormonaux (TH) ou des récepteurs aux estrogènes. Les données sont issues de deux études longitudinales : le Melbourne Women's Midlife Health Project qui porte sur 438 Australiennes récemment postménopausées suivies 13 ans et l'étude des 3 Cités/ESPRIT qui inclut 5644 Françaises plus âgées suivies 7 ans. Des modèles statistiques multivariés montrent que des facteurs hormonaux endogènes et exogènes présents à une période tardive de la vie reproductive (autour de la ménopause) peuvent diminuer le risque de dépression et qu'une diminution des taux d'estradiol sérique augmente ce risque. L'arrêt du TH ou la prise de TH "non-naturel" augmentent le risque de dépression tardive. Certains polymorp hismes des récepteurs aux estrogènes sont associés au risque de dépression et peuvent interagir avec le TH pour modifier le risque de dépression ou de décès. Une durée plus longue d'exposition aux estrogènes au cours de la vie ou un taux d'estradiol sérique élevé en fin de ménopause sont associés à de meilleures performances cognitives qui peuvent aussi varier avec certaines caractéristiques du TH. Le TH réduit aussi le risque de démence chez les femmes portant l'allèle ε4 de l'apolipoprotéine E. Ce travail suggère que la modulation des niveaux d'estrogènes pourrait avoir des applications thérapeutiques dans le traitement de la dépression ou des troubles cognitifs. Il montre que certains groupes de femmes ont une susceptibilité génétique accrue aux variations hormonales ou aux effets du TH suggérant l'existence de sous-types hormono-sensibles. / Experimental evidence suggests that estrogen can have psycho- and neuro-protective effects; however this has not been consistently supported by certain clinical trials and epidemiological studies. This thesis aimed to provide a detailed investigation of the role of estrogen in later-life depression and cognitive functioning by examining serum estrogen levels, estrogen exposure across the lifetime, characteristics of hormone treatment (HT) and the role of estrogen receptor polymorphisms. Data was obtained from two longitudinal population-based studies, the 13-year Melbourne Women's Midlife Health Project of 438 middle-aged postmenopausal women in Australia, and the seven-year Three City/ESPRIT study of 5644 older French women. Multivariate adjusted regression models showed that endogenous and exogenous hormonal characteristics late in the reproductive life can decrease the risk of late-life depression and a decline in serum estradiol levels incr eased the risk for recently postmenopausal women. Discontinuing HT increased the risk of depression for older women, as did certain "non-natural" forms of HT. Estrogen receptor polymorphisms were associated with late-life depression and can interact with HT to modify the risk of depression and mortality. Endogenous reproductive factors linked to higher lifetime estrogen exposure and high levels of estradiol in the early postmenopause were associated with better performance on certain cognitive tasks. Cognitive function also varied according to the characteristics of HT and HT reduced the risk of dementia in genetically susceptible women carrying the apolioprotein ε4 allele. This work brings some important new findings to this field of research, suggesting that the modulation of estrogen levels may be used as a possible therapeutic tool to reduce neuropsychiatric disorders and that certain subgroups of women may be genetically more susceptible to hormone modifications or to the effects of HT.

La ligne latérale postérieure embryonnaire du poisson-zèbre est composée d'un ensemble d'organes sensoriels, appelés neuromastes, qui permet au poisson de détecter les mouvements de l'eau. Le primordium qui génère la ligne latérale postérieure embryonnaire migre de la tête vers l'extrémité de la queue le long d'une piste de cellules sécrétrices de SDF-1, et dépose des groupes de cellules précurseurs des neuromastes. Cette migration dépend de la présence de CXCR4, le récepteur de SDF-1, dans la région en tête du primordium et de la présence du second récepteur de SDF-1, CXCR7, dans la région en queue du primordium. L'objectif de ma thèse est d'identifier des régulateurs de l'expression de cxcr4b au sein du primordium. Nous avons montré que l'inactivation du récepteur des strogènes ESR1 induit l'expression ectopique de cxcr4b dans les cellules de queue du primordium alors que sa surexpression induit une réduction du domaine d'expression de cxcr4b, suggérant que ESR1 agit comme un répresseur de cxcr4b. Cette découverte expliquerait pourquoi les strogènes diminuent la capacité métastatique des cellules du cancer du sein strogéno-dépendants. L'inactivation de ESR1 conduit aussi à l'extinction de l'expression de cxcr7b dans les cellules de queue du primordium, cet effet étant toutefois indirect et induit par la signalisation ectopique SDF-1/CXCR4 dans ces cellules. L'inactivation et la surexpression de ESR1 provoquent toutes deux une migration défectueuse du primordium, confirmant l'importance de ce récepteur dans le contrôle de la migration dépendante de SDF-1. Nous avons aussi montré qu'un effecteur majeur de la signalisation Wnt canonique, LEF-1, contribue au contrôle de l'expression de cxcr4b et de cxcr7b dans les cellules en tête du primordium. Nous montrons que la prolifération cellulaire, qui assure une taille constante du primordium en dépit des dépositions successives de cellules, est réduite en absence de LEF-1, et que cela conduit à une ligne latérale postérieure incomplète. / The zebrafish embryonic posterior lateral line is componed by a set sense organs, called neuromasts, allowing the fish to detect the water movements. The primordium that generates the embryonic posterior lateral line of zebrafish migrates from the head to the tip of the tail along a trail of SDF-1-producing cells, and deposits cell groups that will become the neuromasts. This migration critically depends on the presence of the SDF-1 receptor CXCR4 in the leading region of the primordium and on the presence of a second SDF1 receptor, CXCR7, in the trailing region of the primordium. The aim of my thesis is to identify regulators of the cxcr4b expression within the primordium. Here we show that inactivation of the estrogen receptor ESR1 results in ectopic expression of cxcr4b throughout the primordium, whereas ESR1 overexpression results in a reciprocal reduction in the domain of cxcr4b expression, suggesting that ESR1 acts as a repressor of cxcr4b. This finding could explain why estrogens significantly decrease the metastatic ability of ESR-positive breast cancer cells. ESR1 inactivation alsoleads to extinction of cxcr7b expression in the trailing cells of the migrating primordium; this effect is indirect, however, and due to the down-regulation of cxcr7b by ectopic SDF-1/CXCR4 signaling in the trailing region. Both ESR1 inactivation and overexpression result in aborted migration, confirming the importance of this receptor in the control of SDF-1-dependent migration. We also showed that a major effector of the canonical Wnt signaling, LEF-1, contributes to the control of both cxcr4b and cxcr7b expression in the leading cells of the primordium. We show that cell proliferation, which ensures constant primordium size in spite of sucessive rounds of cell deposition, is reduced upon lef1 inactivation, leading in a truncated posterior lateral line.

<p>Adenokarcinom prostate (PCa) je najče&scaron;ći karcinom u mu&scaron;karaca. Intraepitelne prostatične neoplazme visokog gradusa (HGPIN) su lezije koje prethode nastanku invazivnog karcinoma i podrazumevaju kompletno odsustvo bazalnih ćelija i invaziju strome malignim acinusima. Estrogeni receptor &beta; (ER&beta;) se nalazi u jedrima bazalnih i sekretornih ćelija acinusa i delimično u stromalnim ćelijama. Cilj istraživanja je da prikaže i lokalizuje ER&beta; u različitim morfolo&scaron;kim lezijama prostate: hiperplaziji (BHP), PINu i u PCa sa različitim Gleason scorom. Pretpostavlja se da prekancerozne lezije u svojim različitim fazama evolucije ne koreliraju u potpunosti sa ekspresijom ER&beta;. LGPIN pokazuje ekspresiju, dok u HGPINu nema ekspresije. Takođe je pretpostavka da ekspresija ER&beta; postoji u većine srednje diferentovanih PCa, te da se ekspresija posmatranog receptora gubi sa porastom Gleason scora. Ispitivano je pet grupa bolesnika:&nbsp; kontrolna grupa sa BHP i četiri eksperimentalne grupe (PIN i 3 različite grupe PCa). Studija je sprovedena na mu&scaron;karcima različite starosti u periodu 2010&ndash;2012. Nijedan pacijent nije prethodno primio hormonsku terapiju. Sekstant biopsije prostate su bojene na ER&beta; (Novocastra). Lokalizacija i intenzitet ER&beta; ekspresije prikazani su kroz skor: 0 = nula; 1 = &lt;1%; 2 = 1&ndash;10%; 3 = 11&ndash;33%; 4 = 34&ndash;66%; 5 = &gt; 66%. Pozitivni fibroblasti i endotelne ćelije su kori&scaron;ćene za poređenje. Smanjena ekspresija ER&beta; primećena je kod malignih i premalignih lezija prostate naspram BHP. Ekspresija ER&beta; u epitelnim ćelijama acinusa bila je najslabija u dobro diferentovanim PCa. Kod BHP i dobro diferentovanih PCa bila je veća ekspresija ER&beta; u bazalnim ćelijama nego u sekretornim. Lo&scaron;e diferentovani PCa prikazali su smanjenje ekspresije ER&beta; u bazalnim ćelijama. Ukupna ćelijska ekspresija ER&beta; predstavlja složen i ponekad moguće paradoksalan nalaz, na osnovu čega primarni PCa zadržava ekspresiju ovog receptora, ali ipak značajno nižu u poređenju sa benignim epitelom i premalignim lezijama. Ovaj nalaz podupire stanovi&scaron;te o antiproliferativnoj ulozi ER&beta; u tkivu prostate.</p> / <p>Adenocarcinoma of the prostate (PCa) is the most common cancer in men. High-grade prostatic intraepithelial neoplasia (HGPIN) are lesions that precede to invasive carcinomas and include complete absence of basal cells and stromal invasion by malignant acini. Estrogen receptor &szlig;(ER&szlig;) is located in the nuclei of basal and secretory cells and partly in stromal ones.The aim of the research is to describe and localize ER&szlig; in different morphological lesions: prostate hyperplasia (BPH), PIN and PCa with different Gleason score. It is assumed that pre-cancerous lesions in different stages of their evolution not correlate completely with the expression ER&szlig;. LGPIN shows expression, while there is no expression in HGPIN. It is also an assumption that the expression ER&szlig; exists in most medium differentiated PCa, and that the expression of this receptor loses with increasing of Gleason score. Five groups of patient were investigated: control group with BPH and four experimental groups (PIN and 3 different groups of PCa). The study was conducted on men of different ages in the period 2010-2012. None of the patients received prior hormonal therapy. Sextant prostate biopsy were stained on ER&szlig; (Novocastra). ER&szlig; expression is shown through the score: 0 = zero; 1 = &lt;1%; 2 = 1-10%; 3 = 11-33%; 4 = 34-66%; 5 =&gt; 66%. Positive fibroblasts and endothelial cells were used for comparison. Reduced expression was observed in malignant and premalignant lesions of the prostate versus BPH. ER&szlig; expression in the epithelial cells of acini was the weakest in well-differentiated PCa. In BPH and well differentiated PCa was greater expression in the basal cells than in secretory ones. Poorly differentiated PCa showed a decreased ER&szlig; expression in basal cells. Total cellular expression of ER&szlig; is a complex and sometimes paradoxical finding on the basis of which the primary PCa retains expression of this receptor, but significantly lower compared to benign epithelium and premalignant lesions. This finding supports the antiproliferative role of ER&szlig; in prostate tissue.</p>

A cardiomiopatia chagásica crônica é a consequência mais grave da Doença de Chagas, quadro infeccioso humano causado pelo protozoário Trypanosoma cruzi. Uma vez que os cardiomiocitos podem ser invadidos pelo T. cruzi, é nosso interesse averiguar se esta população estrutural reconhece o parasita in vivo através dos TLRs (Toll Like receptors). Visto que a maioria dos TLRs sinaliza através da molécula adaptadora MyD88, no presente trabalho temos estudado a participação deste elemento transdutor. Para isto fomos examinar a infecção pelo T. cruzi em camundongos F2 (Mer / MyD88flox+/+), modelo animal no qual o tratamento com a droga Tamoxifeno deve eliminar a molécula MyD88 exclusivamente nos cardiomiócitos. Resultados: em um estudo prévio, constatamos que cardiomiócitos tumorais murinos HL-1, em repouso ou após infecção pelo T. cruzi, transcrevem a molécula MyD88. A seguir, validamos o modelo experimental in vivo, ao mostrar que o tratamento com tamoxifeno dos animais F2 resulta na diminuição de MyD88 no coração, mas não no baço. Ainda, constatamos que a transcrição de MyD88 é mais intensa na aurícula do que no ventrículo, sendo igualmente abolida dos animais F2 pelo tratamento com tamoxifeno. Por outro lado, verificamos que o tamoxifeno determina um aumento da parasitemia em ambos os animais F2 e controle (MerCreMer+/+), não se observando diferenças significativamente entre estes. Finalmente, estudos preliminares mostraram que a eliminação de MyD88 nos cardiomiócitos dos animais F2 não altera significativamente o quadro de patologia (parasitismo e infiltração leucocitária) aos 10 ou 28 dias de infecção pelo T. cruzi, quando comparado ao de animais controle (MerCreMer+/+) igualmente tratados com tamoxifeno e infectados. / Chronic Chagas cardiomyopathy is the most serious consequence of Chagas Disease, caused by Trypanosoma cruzi. Cardiomyocytes are invaded by T. cruzi, it is our interest to see if this structural population in vivo recognizes the parasite through TLRs. Since most TLRs signal through MyD88, we studied in vivo participation of Myd88 in cardiomyocyte response. Treatment with tamoxifen (Tam) eliminate the MyD88 molecule exclusively from cardiomyocytes in F2 mice, which we used to understand its role on T. cruzi infection. Results: HL-1 cells, a murine cardiomyocyte tumor line, in infection with T. cruzi, transcribe the MyD88 molecule. Transcription of MyD88 is more intense in the atria than in ventricle. Tam treatment of F2 mice eliminates the MyD88 at the heart completely, but not at spleen. Tam caused increased parasitaemia, no differences were observed in the parasitaemia curve of infected F2 and MerCreMer+/+ (control) mice. Finally, MyD88 elimination in cardiomyocytes of F2 mice does not alters the pathology frame at days 10 and 28 post infection.

The tetrahydroisoquinoline (THIQ) core structure is explored as a steroidomimetic nucleus with attractive pharmaceutical properties. A library was synthesised employing Pomeranz-Fritsch, Pictet-Spengler, Bischler-Napieralski strategies yielding 77 final targets, substituted at every position, for biological evaluation. Complementary strategies overcame synthetic difficulties, sometimes yielding two products in a single cyclisation. Three compounds were initially tested against a panel of 19 nuclear receptors (NRs) and exhibited broad substitution-dependent activity. 2-(4-Chlorophenyl)-1-isopropyl-1,2,3,4-tetrahydroisoquinolin-6-ol fully inhibited every NR at 100 µM, confirming the THIQ as a lead for optimisation. Compounds were evaluated for cytotoxicity against 60 cell lines by the NCI (USA), exhibiting moderate to insignificant cytotoxicity. Three compounds showed ca. 30-90% of average growth inhibition and were selected for a five dose test. Off-target evaluation highlighted compounds with activity against glucagon-like peptide 1 secretion, calcitonin gene-related peptide receptor antagonism and with >100% inhibition against the metabotropic glutamate receptor 2. Estrogen receptor-related receptor α (ERRα), a constitutively active orphan NR, is a hormone-dependent cancer target and diethylstilboestrol (DES), a known inverse agonist, possesses similarities to THIQs. THIQs tested against ERRα revealed no general SAR rules, but showed a lower degree of efficacy in a commercial TR-FRET assay, with 1-benzyl-2-(4-chlorophenyl)-4-methyl-1,2,3,4-tetrahydroisoquinolin-6-ol showing 79% efficacy at 100 µM as an inverse agonist, being more active than DES (64% at 100 µM). Inhibition of steroidogenic enzymes like 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is an emerging approach for the treatment of HDBC, compared to other current clinical strategies. THIQs evaluated against 17β-HSD1 showed good activity in both whole cell and cell lysate assays, with the best inhibitor, 2-(4-chlorophenyl)-4-isopropyl-1,2,3,4-tetrahydroisoquinolin-6-ol, possessing an IC50 value of 336 nM. The value of THIQ as a drug-like steroidomimetic scaffold is thus established and this work reveals straightforward strategies to optimise potency and selectivity for a range of potential targets by structural and stereochemical iteration.