Kaempferol Exhibits Progestogenic Effects in Ovariectomized Rats

OBJECTIVE: Progesterone (P4) plays a central role in women's health. Synthetic progestins are used clinically in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Unfortunately, synthetic progestins are associated with side effects, including cardiovascular disease and breast cancer. Botanical dietary supplements are widely consumed for the alleviation of a variety of gynecological issues, but very few studies have characterized natural compounds in terms of their ability to bind to and activate progesterone receptors (PR). Kaempferol is a flavonoid that functions as a non-steroidal selective progesterone receptor modulator (SPRM) in vitro. This study investigated the molecular and physiological effects of kaempferol in the ovariectomized rat uteri.

METHODS: Since genistein is a phytoestrogen that was previously demonstrated to increase uterine weight and proliferation, the ability of kaempferol to block genistein action in the uterus was investigated. Analyses of proliferation, steroid receptor expression, and induction of well-established PR-regulated targets Areg and Hand2 were completed using histological analysis and qPCR gene induction experiments. In addition, kaempferol in silico binding analysis was completed for PR. The activation of estrogen and androgen receptor signalling was determined in vitro.

RESULTS: Molecular docking analysis confirmed that kaempferol adopts poses that are consistent with occupying the ligand-binding pocket of PRA. Kaempferol induced expression of PR regulated transcriptional targets in the ovariectomized rat uteri, including Hand2 and Areg. Consistent with progesterone-l ke activity, kaempferol attenuated genistein-induced uterine luminal epithelial proliferation without increasing uterine weight. Kaempferol signalled without down regulating PR expression in vitro and in vivo and without activating estrogen and androgen receptors.

CONCLUSION: Taken together, these data suggest that kaempferol is a unique natural PR modulator that activates PR signaling in vitro and in vivo without triggering PR degradation.

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion. Repression of IGFBP2 is mediated by histone deacetylation at the IGFBP2 promoter and was reversed by treatment with histone deacetylase (HDAC) inhibitors. Our in vitro findings were confirmed in 50 invasive cancers and 79 cervical intra-epithelial neoplastic lesions caused by HPV16 infection, where IGFBP2 levels were reduced with increasing disease severity. In summary, the loss of IGFBP2 is associated with progression of premalignant disease, and sensitises cells to pro-invasive IGF signalling, and together with stromal derived factors promotes epithelial invasion.

Pharmacokinetics of melatonin in preterm infants

Aims: Preterm infants are deprived of the normal intra-uterine exposure to maternal melatonin and may benefit from replacement therapy. We conducted a pharmacokinetic study to guide potential therapeutic trials. Methods: Melatonin was administered to 18 preterm infants in doses ranging from 0.04-0.6μgkg^-1 over 0.5-6h. Pharmacokinetic profiles were analyzed individually and by population methods. Results: Baseline melatonin was largely undetectable. Infants receiving melatonin at 0.1μgkg^-1h^-1 for 2h showed a median half-life of 15.82h and median maximum plasma concentration of 203.3pgml^-1. On population pharmacokinetics, clearance was 0.045lh^-1, volume of distribution 1.098l and elimination half-life 16.91h with gender (P = 0.047) and race (P < 0.0001) as significant covariates. Conclusions: A 2h infusion of 0.1μgkg^-1h^-1 increased blood melatonin from undetectable to approximately peak adult concentrations. Slow clearance makes replacement of a typical maternal circadian rhythm problematic. The pharmacokinetic profile of melatonin in preterm infants differs from that of adults so dosage of melatonin for preterm infants cannot be extrapolated from adult studies. Data from this study can be used to guide therapeutic clinical trials of melatonin in preterm infants. © 2013 The British Pharmacological Society.

Contribuição da ecografia cerebral transfontanelar para a avaliação do crescimento cerebral do recém-nascido pré-termo

Tese de doutoramento, Medicina (Pediatria), Universidade de Lisboa, Faculdade de Medicina, 2013

Timing and mode of delivery in twins : the ongoing controversy

Tese de doutoramento, Medicina (Ginecologia e Obstetrícia), Universidade de Lisboa, Faculdade de Medicina, 2014

Avaliação do potencial clínico de novos ligandos para o receptor estrogénico em diagnóstico e terapia de tumores da mama

Tese de doutoramento, Química (Química Inorgânica), Universidade de Lisboa, Faculdade de Ciências, 2015

Repressive desublimation and the great refusal in Bret Easton Ellis's fiction

In 1968, Herbert Marcuse believed that a Great Refusal was possible, one that would deny the exploitative power of corporate capitalism. Marcuse's vision was never realised. This essay argues that society today is in an advanced state of that which the Frankfurt School termed repressive desublimation and questions whether a liberationary praxis is still possible. It claims that Bret Easton Ellis's fiction choreographs an internalising of the forms of critique that marked 1968 and about which Marcuse writes. It is Ellis's act of double voicing that allows him to develop a duplicitous recalcitrant voice within the state of assimilation and it is double voicing which emerges as the key technique in Ellis's work that effects an ongoing critique in commodity society. Looking at Slavoj iek's recent revisionism of the notion of repressive desublimation, which connects Marxism and psychoanalysis, the essay considers how Ellis's novels, American Psycho, Glamorama and Lunar Park, function to address and reconfigure the relationship between the status of the Marxist fetishised object and the psychoanalytic phobic object in the present-day era of late capitalism. This essay seeks to illuminate how Ellis's fiction, through an involution of Marcuse's political theories, enacts a contemporary refusal from within the state of reification.

Predição precoce de risco de Pré-eclâmpsia na doença hipertensiva e diabetes na gravidez: Papel de fatores angiogénicos

RESUMO: A pré-eclâmpsia tem elevada morbi-mortalidade materna e perinatal. A sua etiologia multi-fatorial tem sido objeto de investigação, não sendo ainda totalmente conhecida. Não se conhece também a razão da diferente suscetibilidade individual e das diferentes expressões da doença. A hipertensão crónica e a diabetes são fatores de risco reconhecidos, e o adiamento da maternidade contribui para que estas duas patologias sejam atualmente mais prevalentes entre as mulheres grávidas. Uma vez que o seu quadro fisiopatológico precede em meses o quadro clínico, tem-se investigado a possibilidade de serem encontrados marcadores precoces e indicadores de risco. Em Portugal, os estudos relativos à hipertensão na gravidez são escassos, bem como a investigação sobre fatores de risco e marcadores para a mesma. No sentido de avaliar possíveis marcadores de risco para o desenvolvimento de préeclâmpsia ou complicações hipertensivas foi colhida, para esta dissertação, uma amostra de 1215 mulheres que frequentaram a consulta de Hipertensão ou de Diabetes na gravidez de um centro terciário, entre 2004 e 2013. Optou-se pela realização de três estudos independentes, abrangendo os dois primeiros um leque temporal de 9 e de 2 anos respetivamente. O primeiro, centrado na hipertensão, pesquisou, em 521 mulheres com hipertensão na presente ou em anterior gravidez, fatores de risco capazes de influenciar a progressão para pré-eclâmpsia. O segundo, direcionado para a diabetes gestacional, considerou uma amostra de 334 grávidas, parte das quais tinha também hipertensão crónica e procurou identificar fatores que contribuíram para o aparecimento de complicações hipertensivas. O terceiro estudo, realizado em 2012 e 2013, em três coortes de grávidas com hipertensão crónica, com diabetes gestacional, e sem estas patologias - procurou avaliar no 1º trimestre o comportamento de dois marcadores placentares obtidos no 1º trimestre - proteína plasmática A associada à gravidez (PAPP-A) e o fator de crescimento placentar (PlGF) - e o seu papel, quer como bio-marcadores isolados, quer em associação aos fatores de risco encontrados nos anteriores estudos, na construção de um modelo preditivo de préeclâmpsia. No primeiro estudo, a nuliparidade, a hipertensão gestacional, a fluxometria das artérias uterinas com IP superiores ao P95 entre as 20-22 semanas e a existência de restrição de crescimento fetal, foram os fatores que contribuíram para a construção de um modelo preditivo de pré-eclâmpsia. No segundo estudo, a coexistência de diabetes e hipertensão crónica agravou o prognóstico, associando-se as complicações hipertensivas à multiparidade, obesidade, idade materna e etnia negra. No terceiro estudo verificou-se uma redução da PlGf e da PAPP-A no 1º trimestre nas duas primeiras coortes, comparativamente à coorte sem patologia; na análise separada de cada coorte, quando se verificaram complicações hipertensivas ou pré-eclâmpsia, as concentrações de PlGf e PAPP-A também foram inferiores. Contudo, na elaboração de um modelo preditivo de pré-eclâmpsia, em conjunto com marcadores encontrados, apenas a PlGf pode ser integrada no modelo preditivo, o que se verificou na coorte com hipertensão crónica. Os marcadores bioquímicos em estudo tiveram valores inferiores nas coortes com patologia hipertensiva, demonstrando uma deficiente produção destas proteínas placentares nestas situações, podendo ser importante a sua pesquisa. Contudo, neste estudo, apenas na coorte de hipertensão crónica a PlGf teve participação como fator de risco, na construção de um modelo preditivo de pré-eclâmpsia.--------------------------------------------------------------------------------------------------ABSTRACT: Preeclampsia is associated with a great maternal and perinatal morbimortality. Its multifactorial etiology has been under investigation and is still insufficiently understood. The reason why there are differences in individual susceptibility and differences in expressions of the disease is still unknown. Chronic hypertension and diabetes are known risk factors for preeclampsia and maternity delay contributes to the great prevalence of these pathologies among pregnant women. As the physiopathological signs antedate by months the clinical course of the disease, early risk factors and biological markers are object of clinical research. In Portugal, scarce clinical studies were devoted to hypertension in pregnancy and to risk factors and markers of this pathology. This dissertation inquires 1215 pregnant women who were treated for hypertension or diabetes in a tertiary care center between 2004 and 2013, in order to find risk markers for hypertensive complications or preeclampsia. We conducted three independent studies for this purpose. In the first one we investigated which risk factors could influence the progression to preeclampsia in 521 pregnant women with present or past history of hypertension. The second one was conducted to find what factors were associated to hypertensive complications, with a sample of 334 pregnant women with gestational diabetes, some also with chronic hypertension, addressing the identification of the factors contributing to hypertensive complications. The third study was conducted between 2012 and 2013 with three cohorts of pregnant women, with chronic hypertension, gestational diabetes, and in the third one, pregnant women had a low risk pregnancy. The objective of the study was to evaluate the behavior of two placental markers – PAPP-A and PlGf – obtained in the first trimester, and the role of these markers as isolated biomarkers or in association with other risk factors, in order to define a predictive model of early preeclampsia. In the first study, nuliparity, gestational hypertension, uterine arteries doppler with PI above P95 between 20-22 weeks of gestation and the presence of fetal growth restriction were the markers involved in a predictive model for preeclampsia. In the second study the cohort with the coexistence of diabetes and hypertension had registered worse result and hypertensive complications were associated to multiparity, obesity, maternal age and black ethnicity. In the third study there was a reduction of the PlGf and a PAPP-A concentration for the first trimester in the two first cohorts comparatively to the low risk cohort; the separate analysis of each cohort showed that plGf and PAPP-A concentrations were reduced when hypertensive complications appeared. However, when trying to find a preeclampsia predictive model, only plGf gave significant results for being considered in the model and this was only possible in the chronic hypertension cohort. The biochemical markers investigated in this study were reduced in the cohorts when high blood pressure complications occurred, showing a defective production of these placenta proteins, and suggesting that they should be investigated as first trimester biomarkers. Nevertheless, for this research, in the cohort of chronic hypertension only PlGf had a significant result, when multivariate analysis of all the risk factors was considered for the construction of a preeclampsia predictive model.

Patterns of oxygen consumption during establishment of cephalocaudal polarity in the early chick embryo.

Oxygen uptake was studied during the establishment of cephalocaudal polarity in the very early chick embryo, i.e., 10 hr before (stage VI) and at laying (stage X). Oxygen fluxes in minute regions of the intact blastoderms were measured in vitro by scanning microspectrophotometry in the presence or absence of glucose. The oxygen consumption of the whole blastoderm remained constant (6 nmol O2 X hr-1) throughout the period studied, although the number of cells increased more than twofold. The regional oxygen fluxes varied from 0.41 to 1.13 nmol O2 X hr-1 X mm-2 at stage VI and from 0.42 to 0.70 nmol O2 X hr-1 X mm-2 at stage X. At stage VI, the oxygen flux in the center of the blastoderm was significantly higher than that in its periphery. This pattern remained evident when the values were corrected for cell number or for cytoplasmic volume. At stage X, there was a tendency for the oxygen fluxes to decrease from the posterior to the anterior regions of the area pellucida. Thus the pattern of oxidative metabolism in the late uterine embryos seems to change from radial to bilateral. This change of symmetry probably reflects the process of formation of the embryonic axis. In addition, the fact that the oxygen uptake was similar in the presence or absence of glucose suggests that early chick embryos metabolize essentially intracellular stores.

Notch tumor suppressor function

Cancer development results from deregulated control of stem cell populations and alterations in their surrounding environment. Notch signaling is an important form of direct cell-cell communication involved in cell fate determination, stem cell potential and lineage commitment. The biological function of this pathway is critically context dependent. Here we review the pro-differentiation role and tumor suppressing function of this pathway, as revealed by loss-of-function in keratinocytes and skin, downstream of p53 and in cross-connection with other determinants of stem cell potential and/or tumor formation, such as p63 and Rho/CDC42 effectors. The possibility that Notch signaling elicits a duality of signals, involved in growth/differentiation control and cell survival will be discussed, in the context of novel approaches for cancer therapy

Inter- and intra-individual variation of oxytocin secretion in oestrous mares exposed to stallions

The neuro-peptide hormone oxytocin regulates several reproductive mechanisms in mammals, such as uterine contractions during parturition and milk ejection in the lactating mammary gland. Oxytocin may also influence behavior and behavioral strategies, e.g. pair bonding, social recognition, maternal behavior, trust building, or anxiety. Teasing oestrous mares by a stallion provokes the release of oxytocin. We therefore tested whether such elevated oxytocin levels reveal possible mate preferences as determined in typical preference tests.

Mortality among 24,865 workers exposed to polychlorinated biphenyls (PCBs) in three electrical capacitor manufacturing plants: a ten-year update.

The objective of this analysis was to evaluate mortality among a cohort of 24,865 capacitor-manufacturing workers exposed to polychlorinated biphenyls (PCBs) at plants in Indiana, Massachusetts, and New York and followed for mortality through 2008. Cumulative PCB exposure was estimated using plant-specific job-exposure matrices. External comparisons to US and state-specific populations used standardized mortality ratios, adjusted for gender, race, age and calendar year. Among long-term workers employed 3 months or longer, within-cohort comparisons used standardized rate ratios and multivariable Poisson regression modeling. Through 2008, more than one million person-years at risk and 8749 deaths were accrued. Among long-term employees, all-cause and all-cancer mortality were not elevated; of the a priori outcomes assessed only melanoma mortality was elevated. Mortality was elevated for some outcomes of a priori interest among subgroups of long-term workers: all cancer, intestinal cancer and amyotrophic lateral sclerosis (women); melanoma (men); melanoma and brain and nervous system cancer (Indiana plant); and melanoma and multiple myeloma (New York plant). Standardized rates of stomach and uterine cancer and multiple myeloma mortality increased with estimated cumulative PCB exposure. Poisson regression modeling showed significant associations with estimated cumulative PCB exposure for prostate and stomach cancer mortality. For other outcomes of a priori interest--rectal, liver, ovarian, breast, and thyroid cancer, non-Hodgkin lymphoma, Alzheimer disease, and Parkinson disease--neither elevated mortality nor positive associations with PCB exposure were observed. Associations between estimated cumulative PCB exposure and stomach, uterine, and prostate cancer and myeloma mortality confirmed our previous positive findings.

Size regulation of double and half embryos in the mice musculus

Genetic chimeras made by aggregating early mouse embryos have many uses in developmental biology and have also provided insights into embryonic growth regulation. There is an indication that the embryo can regulate for an increase in size because although aggregation chimeras are twice as big as normal embryos when made, they are born of normal size. Upward regula..... tion of size reduced embryos is also possible. Half embryos made by the isolation or destruction of one of the blastomeres of a 2-cell embryo are also born of normal size. Little is known about the timing or the mechanism of this size regulation. In this study, the timing of size regulation in double and half embryos was clearly established by comparison of cell numbers derived from serial reconstruction of light microscope sections of control and experimental embryos. It was shown that size regulation in double embryos occurred around 6dl6h and in half embryos by 7dOh. Size regulation occurred in all tissues at the same time indicating a single control mechanism for the entire embryo. More detailed examination of the growth of double embryos revealed that size regulation occurred by alteration in cell cycle length~ No excessive cell death was found in double embryos compared to the controls and continuous labelling with [3H] thymidine showed no large non-dividing cell population in double embryos. However, a comparison of the mitotic index of double and control embryos after colcemid treatment, revealed a large difference between the two around 5dl6h to 6d16h. During this period, control embryos underwent a proliferative burst not shown by the double embryos. The mechanism for cell cycle control is not clear; it may be intrinsic to the embryo or determined by the uterine environment. Evidence was found suggesting that differentiation in the postimplantation embryo was cell number dependent. The timing of differentiative events was examined in half, double and control embryos. Proamnion formation, which occurs prior to size regulation, occurs at the same cell number but at different times in the three groups of embryos. However mesoderm which appears after size regulation was seen at the same time in all grollps of embryos.

EARLY LIFE EXPOSURE TO GENISTEIN AND DAIDZEIN DISRUPTS STRUCTURAL DEVELOPMENT OF REPRODUCTIVE ORGANS IN FEMALE MICE

In mice, exposure to isoflavones (ISO), abundant in soy infant formula, during the first 5 d of life alters structural and functional development of reproductive organs. Effects of longer exposures are unknown. The study objective was to evaluate whether exposure to a combination of daidzein and genistein in the first 10 compared to 5 d of life results in greater adverse effects on ovarian and uterine structure in adult mice. Thirteen litters of 8–12 pups were cross-fostered and randomized to corn oil or ISO (2 mg daidzein + 5 mg genistein/kg body weight/d) for the first 5 or 10 d of life. The 10-d protocol mimicked the period when infants are fed soy protein formula (SPF) but avoids the time when suckling pups can consume the mother’s diet. Body and organ weights and histology of ovaries and uteri were analyzed. There were no differences in the ovary or uterus weight, number of ovarian follicles, number of multiple oocyte follicles, or percent of ovarian cysts with 5 or 10 d of ISO intervention compared to respective controls. The 10-d ISO group had higher body weights from 6 d to 4 mo. of age and a higher percent of hyperplasia in the oviduct than the respective control. Lower numbers of ovarian corpus lutea and a higher incidence of abnormal changes were reported in the uteri of both ISO groups compared to their respective controls. Five- and 10-d exposure to ISO had similar long-lasting adverse effects on the structures of ovaries and uterus in adult mice. Only the 10-d ISO exposure resulted in greater body weight gain at adulthood.

Associations Between Personal Cancer History and Lung Cancer Risk

The study aim was to investigate the relationship between factors related to personal cancer history and lung cancer risk as well as assess their predictive utility. Characteristics of interest included the number, anatomical site(s), and age of onset of previous cancer(s). Data from the Prostate, Lung, Colorectal and Ovarian Screening (PLCO) Cancer Screening Trial (N = 154,901) and National Lung Screening Trial (N = 53,452) were analysed. Logistic regression models were used to assess the relationships between each variable of interest and 6-year lung cancer risk. Predictive utility was assessed through changes in area-under-the-curve (AUC) after substitution into the PLCOall2014 lung cancer risk prediction model. Previous lung, uterine and oral cancers were strongly and significantly associated with elevated 6-year lung cancer risk after controlling for confounders. None of these refined measures of personal cancer history offered more predictive utility than the simple (yes/no) measure already included in the PLCOall2014 model.