Le kisspeptine est un peptide, encodé par le gène Kiss1, qui joue un rôle majeur dans le contrôle central de la fonction de reproduction en régulant la sécrétion du GnRH après la naissance. Les neurones exprimant Kiss1 apparaissent avant la naissance exclusivement dans le noyau arqué. Les objectifs de cette thèse étaient de décrire comment le système s‟organise avant la naissance en fonction du sexe à une période où il existe chez le mâle un pic prénatal de testostérone, puis de déterminer si cette organisation pouvait être influencée par les stéroïdes sexuels. Nos résultats ont permis 1) de caractériser précisément chez la souris la mise en place des cellules kisspeptine en fonction du sexe et d‟identifier un gradient de différenciation antéro-postérieur sexe-indépendant 2) de mettre en évidence une interaction réciproque avec les neurones à GnRH, chez les mâles et les femelles, suggérant ainsi une certaine maturité du système 3) de montrer la diversité des récepteurs aux stéroïdes sexuels déjà exprimés par ces cellules avant la naissance ainsi que l‟émergence d‟un dimorphisme sexuel 4) de démontrer la sensibilité et la réponse morphologique à l'oestradiol de ces cellules in vitro après la mise au point du premier modèle de culture primaire de cellules kisspeptine. / Kisspeptin neurons express the Kiss1 gene encoding kisspeptin, a potent neuropeptide secretagogue of gonadotropin releasing hormone (GnRH) that plays a fundamental role in regulation of reproductive life cycles after birth. Neurons expressing Kiss1 appear before birth only in the arcuate nucleus. The overall purpose of this study is to understand how kisspeptin neurons are set up during fetal development, if and how estrogen signaling in these cells could interfere with their development at a time of a prenatal testosterone peak in male. Our finding showed 1) precisely the placement of kisspeptin cells as a function of sex and to identify a sex-independent anteroposterior differentiation gradient 2) a reciprocal interaction with GnRH neurons, in males and females, thus suggesting a certain maturity of the system 3) the diversity of sexual steroids receptors already expressed by these cells before birth as well as the emergence of a sexual dimorphism 4) sensitivity and morphological response to estradiol of these cells in vitro after the development of the first primary culture model of kisspeptin cells.

Ethiopia maintains an extensive livestock population; however, reproductive performance of cattle and their breeding management are unsatisfactory. Currently, the sole diagnostic tool in the country is rectal palpation, which is inaccurate for early pregnancy in cattle. The study assessed reproductive performance and major reproductive problems using questionnaire survey, and evaluated simple, cost-effective alternative monitoring approaches using on-farm diagnostic tools to determine milk and serum progesterone (P4) and evaluate reproductive status. There were 177 dairy farms (range 1-115 cattle per farm) included in the questionnaire survey. Of these, 47 participated in the quantitative determination of P4 and estradiol profiles that used an enzyme-linked immunosorbent assay (ELISA) and in the on-farm diagnostic trial that used qualitative ELISA (Target P4 and Dipstick (P4 Rapid), and the reproductive status of 319 crossbred [Holstein Friesian (HF) X Zebu] dairy cattle was assessed. Questionnaires indicated that heifers in the study area reach puberty at older age and calve late, and cows have long postpartum estrus and calving interval. Anestrus, repeat-breeding, dystocia, retained fetal membranes (RFM), endometritis and abortion as the major reproductive problems in dairy cattle in the study area. Serum, milk, saliva and urine P4 and serum estradiol profiles of cattle at different reproductive status was determined using quantitative laboratory ELISA. High P4 levels was detected in pregnant and diestrus cattle than cattle that were anestrus and in-heat. Estradiol level was higher in cattle that were in-heat than cattle in other reproductive conditions. On-farm P4 ELISA indicated in-heat (estrus) 10 (3.1%), anestrus 77 (24.2%), repeater (follicular cyst) 9 (2.8%), normally cycling 69 (21.6%) and pregnant 154 (48.3%). The field P4 ELISA findings were validated using quantitative laboratory P4 ELISA, and similar results were obtained. The sensitivity and specificity of on-farm and laboratory P4 ELISA tests for diagnosing pregnancy were 88.6 & 99.4% and 98.1 & 100%, respectively. Once reproductive problems were identified using on-farm P4 ELISA and per rectal palpation, along with reproductive history, 122 cattle (75 cows and 47 heifers) were assigned to a 10-day Controlled Internal Drug Release (CIDR) in combination with prostaglandin F2-alpha (PGF2α) and equine chorionic gonadotrophin (eCG) based estrus synchronisation protocol to study the estrus response and conception rate. The overall estrus response and conception rates were 97.5% and 78.3%, respectively, with no significant differences in parity, pre-treatment reproductive status (anestrus or cycling (repeat-breeding/silent-estrus)) and farming system (smallholder vs organised commercial farms). The study has shown high estrus response and conception rate. Hence, this protocol is highly recommended to enhance fertility of dairy cattle in the study area and other regions. Finally, the study has determined the macronutrient composition of milk and assessed the effect of reproductive status, farm (nutritional) management, stages of lactation, parity and breed on milk composition in 246 dairy cows. This consisted of 184 crossbred cows from smallholder (n= 76: 36 non-pregnant and 40 pregnant) and organised commercial dairy farms (n= 108: 62 non-pregnant and 46 pregnant) in and around Mekelle, Ethiopia and by way of comparison, 62 HF cows (25 non-pregnant and 37 pregnant), either managed indoors or outdoors in a commercial farm in Edinburgh, United Kingdom. The mean milk fat, protein, lactose, total solid (TS) and solid-no-fat (SNF) recorded were 2.36%, 3.46%, 4.37%, 10.39% and 7.82%, in crossbred dairy cows, compared with 5.05%, 3.71%, 4.72%, 13.68% and 8.43%, in HF cows, respectively. Significantly lower (p < 0.05) level of macronutrient was recorded in milk from crossbred cows than HF cows. Milk fat in both breeds was affected by reproductive status, farm (nutritional) management and stages of lactation, but not by parity. In both cattle breeds, the milk fat content was significantly higher (p < 0.05) in pregnant than non-pregnant cows. Milk protein content was significantly (p < 0.05) affected by reproductive status (in crossbreds) and stages of lactation (in both breeds), but not by farm management or parity. Reproductive status (in crossbreds), stages of lactation (in both breeds) and parity (in crossbreds) affected lactose level; however, farm management had no effect on lactose level in both breeds. Milk fat was the most affected macronutrient content in both breeds. Low milk fat level in crossbred cows could be as a result of samples obtained from early milking coupled with nutritional management. In conclusion, the present study has determined the major reproductive problems in crossbred dairy cattle, assessed their actual reproductive status using rapid, cost effective, simple and applicable on-farm P4 tests, and established P4 and estradiol profiles at different reproductive status. The major breeding problem was poor estrus detection evidenced when animals reported anestrus were confirmed normally cycling using on-farm as well as laboratory P4 assays. These studies offer opportunities for establishing simple field reporting of reproductive status in these crossbred dairy cattle, which can have a major impact on breeding management and productivity.

I investigated two aspects of in vivo splicing that are poorly understood: spliceosome disassembly and recycling, and proofreading. To this end, I used the auxin-inducible degron (AID) to individually deplete several splicing factors in budding yeast and then I measured the effect on co-transcriptional spliceosome assembly through chromatin immunoprecipitation. In addition, using RNA next-generation sequencing, I measured the frequency of splicing errors following depletion or mutation of the fidelity factor, Prp22. I show that formation of the pre-spliceosome (the first stage of spliceosome assembly) is rapidly inhibited by global defects in late stages of spliceosome assembly. I demonstrate that this is due to the accumulation of arrested spliceosomes that sequester the splicing machinery and, as a result, causes a recycling defect. This suggests that spliceosomes that lack essential splicing factors are not always properly disassembled and recycled in vivo, and warns about potential systematic secondary effects when perturbing single components of the spliceosome. Secondly, I describe the development of a new version of the AID system for budding yeast, called the B-estradiol AID. To the best of my knowledge, an AID system for budding yeast that is fast-acting, tightly-controlled and gratuitous, was lacking until now. Lastly, I show that absence of Prp22 protein, which was previously proposed to play a role in splicing fidelity, correlates with more mistakes in 3’ss selection of many endogenous intron-containing transcripts in vivo. This provides indirect evidence to suggest that Prp22-dependent splicing proofreading is physiologically important. The data from this analysis will be useful in ongoing studies to try to identify common features that could improve our understanding of the mechanism of Prp22’s function in splicing proofreading.

Nucleotídeos extracelulares (ATP, ADP, AMP) e seu derivado adenosina são conhecidos sinalizadores do sistema cardiovascular podendo mediar vários processos fisiológicos entre eles a proliferação celular, agregação plaquetária, inflamação e o tônus vascular. Os níveis destas substâncias, localmente e na circulação sanguínea, são controlados pelas ecto-NTPDases em conjunto com a ecto-5’nucleotidase (ecto-5’-NT) que realizam a degradação completa do ATP até adenosina. Os hormônios tireoideanos tiroxina (T4) e triiodotironina (T3) e o hormônio esteróide sexual estradiol (E2) atuam ativamente no sistema vascular promovendo vasodilatação. Nosso objetivo foi investigar quais enzimas da família das NTPDases estão presente em células musculares lisas vasculares (CMLVs) e se a atividade destas enzimas pode ser influenciada pela ação desses hormônios, uma vez que seus substratos e produtos podem sinalizar processos de relaxamento/contração muscular. Para tanto, as CMLVs foram extraídas da artéria aorta de ratos Wistar adultos e cultivadas em meio de cultura DMEM. Após atingirem a confluência, as células foram tratadas com 50 nM de T3 ou T4 ou 1M de 17-estradiol por 72 horas. As atividades enzimáticas foram medidas pela liberação de fosfato inorgânico enquanto que a expressão das ectonucleotidases foi verificada por imunocitoquímica (proteína) e RT-PCR (RNAm). Os resultados deste trabalho mostram que as CMLVs expressam as NTPDases 1, 2, 3, 5 e 6 e a ecto-5’-NT, responsáveis pelo controle dos níveis de nucleotídeos e nucleosídeos extracelulares. O tratamento com os hormônios T3, T4 e E2 nestas células mostrou que a atividade da ecto-5’-NT foi aumentada pelos três hormônios. A análise do RT-PCR demonstrou que os tratamentos foram capazes de aumentar também a quantidade de RNAm da ecto-5’NT, indicando mecanismos de ação genômica dos hormônios estudados. Por outro lado, O tratamento com os hormônios tireoideos não alterou as atividades ATPásica e ADPásica, somente o estradiol foi capaz de aumentar a atividade ATPásica. Estes resultados também sugerem que, pela hidrólise aumentada do AMP, disponibilizem-se níveis maiores de adenosina, com importante potencial vasodilatador local. Entretanto, o fato de o estradiol ter aumentado a hidrólise de ATP, mas não a de ADP, nos permite pensar que o ADP, agregador plaquetário bem estabelecido, possa estar acumulando extracelularmente, contribuindo para o desenvolvimento de problemas circulatórios.

Camels are important husbandry animals which are also often bred in zoological gardens. Unfortunately, camels in European zoos are not usually trained and pregnancy diagnosis in a half-tamed camel is very difficult. Moreover, information of the maternal behaviour of camels is limited. This thesis reviewed current knowledge on camel husbandry, reproduction and behaviour and aimed to 1) examine non-invasive methods of heat and pregnancy diagnosis from urine and saliva in camels kept in zoological gardens; 2) explore their maternal and suckling behaviour; 3) describe experiences with artificial rearing of camel calves. The research into non-invasive pregnancy diagnosis was carried out from 2010 to 2012. Urine from 14 camel females kept in four European zoological gardens was collected and tested using two chemical tests -- the Cuboni reaction and barium chloride test. The Cuboni reaction was significantly (p < 0.01) affected by the pregnancy status of female camels, and its accuracy increased significantly (p < 0.05) in the period leading up to parturition. The barium chloride test did not provide reliable results. Next, the saliva of five adult female camels was sampled for more than one year and concentrations of progesterone (P4) and oestradiol (E2) were measured. The concentrations of P4 (n = 312) and E2 (n = 310) were both significantly (p < 0.0001) affected by the pregnancy status of the animals. Maternal and suckling behaviour was observed from 2003 to 2009 in six zoological gardens, and the presented study includes partial data from this period. Allosuckling (i.e. when a female nurses a non-filial offspring) was described for the first time in camels and it represented 8.58% of all suckling bouts. The non-filial calves suckled more often in the lateral position and preferably joined the filial calf when suckling, so the results support the 'milk theft' hypothesis (stealing of milk) as a main cause of this behaviour. Finally, calf rearing in the Prague zoological garden was summarised and two camel calves were successfully artificially reared. This PhD thesis concluded that 1) the Cuboni reaction with urine and salivary P4 and E2 measurements are suitable methods for pregnancy diagnosis in half-tamed female camels; 2) allosuckling is relatively common in captive Bactrian camels; 3) the artificial rearing of camel calves with a calf milk replacer can be successful.

Baseado na hipótese de o que tratamento com 17b-Estradiol, reduz a modulação simpática, o estresse oxidativo e a participação do sistema renina angiotensina sobre o sistema cardiovascular, propomos estudar o balanço simpatovagal e o equilíbrio entre o óxido nítrico (NO) e as espécies reativas ao oxigênio (EROS) em ratas Wistar ovariectomizadas. Além disso, buscamos verificar a efetividade do 17b-Estradiol no controle da PA e na proteção cardiovascular contra os danos causados pelo estresse oxidativo usando doses menores do que as convencionais. Para isso, desenvolvemos três protocolos, usando ratas ovariectomizadas como um modelo experimental de menopausa, onde avaliamos parâmetros morfométricos, funcionais e bioquímicos que permitem a avaliação dos mecanismos relacionados ao objetivo deste trabalho de pesquisa. No primeiro protocolo (I) demonstramos o efeito do estrogênio, em uma dose alta, equivalente àquela praticada na clínica (5mg%), em melhorar o balanço autonômico para o sistema cardiovascular, provavelmente devido a uma melhora no equilíbrio entre as forcas vasodilatadoras, representadas pelo NO, e as forcas vasoconstritoras, representadas pelas EROS vasculares. O estudo também mostra uma forte associação entre o sistema nervoso simpático e a atividade da enzima NADPH oxidase neste modelo animal. No segundo protocolo (II) buscamos testar se 40% da dose convencional de estrogênio seria tão eficaz quanto a dose alta na melhora do controle da pressão arterial e estresse oxidativo cardíaco. Verificamos uma redução significativa na PA e nas concentrações de EROS, como ânion superóxido e peróxido de hidrogênio, um aumento das defesas antioxidantes não enzimáticas, avaliadas através do balanço redox e antioxidantes totais, e das enzimáticas, avaliadas pelas atividades das enzimas superóxido dismutase e catalase. Finalmente, realizamos o terceiro protocolo (III). Através deste testamos se uma dose de estrogênio ainda mais baixa, apenas 10% da dose convencional, seria tão eficaz quanto uma dose elevada para melhorar o controle da pressão arterial e diminuir o estresse oxidativo vascular. Verificamos que independente da dose usada, o estrogênio foi eficaz para reduzir a atividade da enzima NADPH oxidase, a concentração de peróxido de hidrogênio e a pressão arterial. Além de aumentar a concentração plasmática de nitritos totais e o estado redox. Dessa forma, demonstramos que uma dose baixa de estrogênio é tão eficaz como uma dose farmacológica para promover a proteção cardiovascular. / In this thesis was studied the sympathovagal and the nitric oxide (NO)/reactive oxygen species (ROS) balances in ovariectomized female rats. It was verified the effectiveness of 17b-estradiol in the blood pressure (BP) control and cardiovascular oxidative stress using low doses of estrogen. Then, we developed three protocols to evaluate morphometric, hemodynamic and biochemical parameters. In the first protocol (I), we demonstrate the estrogen effects with a pharmacological dose (5 mg%) to improve cardiovascular system autonomic balance. This result was probably due to an improvement in the balance between the vasodilator forces , represented by NO, and vasoconstrictive forces represented by vascular ROS. The study also showed a strong association between the sympathetic nervous system and the NADPH oxidase enzyme activity in this animal model. In the second protocol (II), we tested whether 40% of estrogen conventional dose would be as effective as a high dose in improving the control of blood pressure and oxidative stress. We found a significant reduction in BP and in ROS concentrations such as superoxide anion and hydrogen peroxide, an increase in non-enzymatic antioxidant defenses, as measured by the redox balance and total antioxidants, and enzymes, as assessed by the activities of superoxide dismutase and catalase. Finally, we performed the third protocol (III), which allowed us to test if a low estrogen dose (10% of conventional one), would be as effective as a high dose to improve the control of blood pressure and decrease the vascular oxidative stress. We found that estrogen was effective in reducing the activity of NADPH oxidase, the concentration of hydrogen peroxide and blood pressure in all dose used. Moreover, estrogen increased the total nitrite plasma concentration and redox state. Thus, we have demonstrated that a low dose of estrogen is as effective as a pharmacological dose to promote cardiovascular protection.

O oócito e as células do cumulus apresentam uma comunicação bi-direcional através de projeções que atravessam a zona pelúcida, sendo fundamental no transporte de aminoácidos para o crescimento e maturação oocitária. O oócito regula diversas funções das células do cumulus que o rodeiam e é o responsável por mantê-las diferenciadas das demais. Desta forma, estas células têm se mostrado de grande utilidade para pesquisa, pois podem manter o estado menos indiferenciado in vitro, semelhantes às fases iniciais de desenvolvimento folicular e, portanto, ideais para estudos que visam entender melhor o processo de diferenciação celular durante a foliculogênese, bem como as interações hormonais que ocorrem neste período. Diversos trabalhos têm sugerido a ação sinérgica do FSH e do IGF-1 na esteroidogênese, através da estimulação das mesmas vias metabólicas, porém com grande variabilidade dos resultados entre as espécies. Neste trabalho, as células do cumulus foram coletadas durante o procedimento de FIV na Clínica Proser, e posteriormente cultivadas em meio DMEM modificado, na concentração final de 5x104 células/poço. Para ambos experimentos as células foram cultivadas em incubadora por 24h antes dos tratamentos específicos de cada grupo. Para o transporte de [14C]MeAIB foram feitos 3 grupos: 1) grupo controle; 2) grupo FSH; 3) grupo IGF-1+FSH. Todos os grupos foram cultivados por 24h em meio DMEM, sendo acrescido 25ng/mL de IGF-1 ao grupo IGF-1+FSH. Posteriormente, as células foram incubadas à 37ºC por 45 min em meio HBSS acrescido de 0,2 μCi/mL de [14C]MeAIB por amostra. Foi adicionado ao meio de incubação 75 mIU/ml de FSH nos grupos FSH e IGF-1+FSH. A reação foi encerrada com colocação das placas em gelo e o meio foi retirado da placa e congelado. As células foram lavadas com HBSS à 4ºC e 0,5 mL/poço de água foi adicionado antes de serem congeladas à -20°C. Para a contagem do radioativo, as células foram descongeladas, sonicadas e centrifugadas à 800g por 10min. Alíquotas de 100 μL foram retiradas de todas as amostras (meio interno e externo) e colocadas em 1,5mL de liquido de cintilação para a mensuração da radioatividade em espectrômetro de cintilação líquida LKB beta modelo 1215. A dosagem de proteína das amostras foi realizada segundo o método de Lowry. Os resultados foram expressos pela relação entre radioatividade das células e a radioatividade do meio de incubação. Para a dosagem de 17ß-estradiol as células foram cultivadas nas mesmas condições conforme o experimento anterior. Após 24 horas de cultivo, as células foram divididas em quatro grupos: 1)grupo controle: somente o meio de cultivo; 2) grupo FSH: foi adicionado 75mUI de FSH ao meio; 3) grupo IGF-1: foi adicionado na concentração de 25ng IGF-1/mL ao meio; 4) grupo FSH+IGF-1: foi adicionado FSH (75mUI/mL) e IGF-1 (25ng/mL) ao meio. Ao final de 24h de cultivo, o meio foi congelado à -20ºC. O meio foi diluído na proporção 1:10 no meio tampão do kit. Posteriormente, a mensuração do 17ß-estradiol foi feita por Elisa, utilizando-se o kit comercial 17ß-estradiol EIA kit. Foram utilizados os seguintes itens para correlação com os parâmetros experimentais: a idade e o número de oócitos MII que foram submetidos à ICSI. Para a análise estatística foram feitos os testes: ANOVA de uma via seguido de pósteste de Bonferroni, Shapiro-Wilk para avaliação da distribuição dos dados, Kruskal-Wallis e coefeciente de correlação de Pearson’s. As diferenças foram consideradas significativas quando P<0,05. Foi encontrado uma forte correlação negativa entre o número de oócitos MII e o transporte de [14C]MeAIB (n=5; P=0,03). Não foi encontrado correlação entre o transporte de [14C]MeAIB com a idade das pacientes, sendo o valore de P> 0,05. A incubação com FSH (75 mUI/mL) e o cultivo com IGF-1 (25ng/mL) durante 24h não estimularam o transporte de [14C]MeAIB nas células do cumulus humanas (n=5; P=0,620). O cultivo com a adição de FSH, IGF-1 ou ambos por 24h não aumentou a secreção de 17ß-estradiol (pg/mL) no meio de cultura, comparado ao grupo controle (n= 7; P = 0,855). Em relação à concentração de 17ßestradiol nas células do cumulus não tratadas, não foi encontrado nenhuma correlação entre os parâmetros avaliados de idade e número de oócitos MII (n=7) P> 0,05. Podemos concluir que o sistema A de transporte de aminoácidos está presente em células do cumulus humanas, sendo que a taxa basal é inversamente proporcional ao número de oócitos MII coletados. Provavelmente, o IGF-1 não ocasiona um aumento direto na expressão do FSHR, uma vez que quando adicionado ao meio de cutivo não estimulou os parâmetros analizados comparados aos grupos com somente FSH. Além disso, quando adicionado IGF-1 sozinho ao meio de cultura das células do cumulus, nenhuma alteração na produção de 17ß-estradiol foi observada, sugerindo que o IGF-1 não tenha um efeito direto na esteroidogênese destas células. Portanto, embora existam diversos trabalhos que tem auxiliado na compreensão da interação entre o IGF-1 e o FSH na diferenciação celular durante a foliculogênese, ainda faltam pontos cruciais neste processo em células humanas. Da mesma forma, são necessários mais estudos que caracterizem as células do cumulus, bem como a sua interação com o oócito, para que possamos aplicar estes conhecimentos com a finalidade de melhorar as taxas de MIV oocitária. / Oocyte and the cumulus cells have a bi-directional communication through projections that cross the zona pellucida, being fundamental for the transport of amino acids necessary for gamete growth and maturation. Oocyte plays a dominant role in establishing the heterogeneity of the granulosa cells found in preovulatory follicles by preventing the differentiation of the cumulus granulosa cells. Thus, culturing cumulus cells from preovulatory follicles is a suitable approach to study granulosa cell differentiation as well as the hormonal interactions that occur in folliculogenesis. Several studies have suggested the synergic action of FSH and IGF-1 in steroidogenesis, through the stimulation of the same metabolic pathways, but with great variability of results among species. We evaluate the basal transport and the transport stimulated by FSH [14C]MeAIB in human cumulus cells, observing whether the addition of IGF-I to the culture medium alters this parameter. Cumulus cells were collected during the IVF procedures at Proser Assisted Reproduction Center, and cultured in modified DMEM, at a final concentration of 5x104 cells/well. For both experiments, cells were cultured in an incubator for 24h before the specific treatment of each experimental group. For the transport of [14C]MeAIB 3 groups were made: 1) control group; 2) FSH group; 3) IGF-1 + FSH group. All groups were further cultured for 24h. Twenty five mg/mL of IGF-1 were added to the to the culture medium of the IGF-1 + FSH group. Cells were incubated at 37°C for 45 min in HBSS medium plus 0,2 μCi/mL of [14C] MeAIB per sample, wherein FSH and FSH + IGF-1 groups had 75 mIU/mL of FSH added to the incubation medium. The reaction was terminated by placing the plates on ice and the medium was removed from the plate and freezed. Cells were washed with HBSS at 4°C and 0.5 mL/well of water were added before being frozen at -20°C. For radioactive counting, cells were thawed, sonicated and centrifuged at 800g for 10min. Aliquots of 100 uL were taken from all samples (internal and external medium) and placed in 1.5 mL of scintillation liquid for measurement of radioactivity in a liquid scintillation spectrometer LKB beta 1215 model. Protein dosage of the samples was performed according to the method of Lowry. Results were expressed by the ratio between the radioactivity of cells and the radioactivity of the incubation medium. For the 17ß-estradiol dosage, cells were cultured under the same conditions as the previous experiment. After 24 hours of culture, the cells were divided into four groups: control group = only the culture medium; FSH group = it was added 75mUI of FSH to the medium; IGF-1 group = it was added IGF-1 at a concentration of 25ng/mL to the medium; FSH + IGF-1 group = it was added FSH (75mUI/mL) and IGF-1 (25ng/mL) to the medium. After of 24 hours of cultivation, the medium was frozen at -20°C. The medium was diluted in a 1:10 ratio in the kit buffer medium. Afterwards, measurement of 17ß-estradiol was made by ELISA using a 17ß-estradiol EIA commercial kit. The following items were used for correlation with the experimental parameters: the age of the patients and the number of MII oocytes that underwent ICSI. For statistical analysis two tests were used: One-way ANOVA followed by Bonferroni post-test, Shapiro-Wilk for evaluation of data distribution, Kruskal-Wallis and Pearson's correlation coefficient. Differences were considered significant when P <0.05. A strong negative correlation was found between the number of MII oocytes and the transport of [14C]MeAIB (n = 5; p = 0.03). No correlation was observed between the transport of [14C]MeAIB and the age of patients (P>0.05). The incubation with FSH (75 mIU/mL) and the cultivation with IGF-1 (25ng/mL) for 24 hours did not stimulate the transport of [14C]MeAIB in human cumulus cells (n = 5; P = 0.620). The culture with the addition of FSH, IGF-1, or both for 24 hours did not increase the secretion of 17ß-estradiol (pg/mL) in the culture medium compared to the control group (n = 7; P = 0.855). Regarding the 17ß-estradiol concentration in the untreated cumulus cells, it was not found any correlation between the evaluated parameters of age and number of MII oocytes (n = 7) P>0.05. We conclude that the A system amino acid transport is present in human cumulus cells, and that the basal rate is inversely proportional to the number of MII collected oocytes. Probably, IGF-1 does not cause a direct increase in FSHR expression, once when added to the culture medium it did not stimulate the parameters analyzed compared to the groups with FSH alone. Moreover, when IGF-1 is added alone to the culture medium of cumulus cells, no change in 17ß-estradiol production was observed, suggesting that IGF-1 has not a direct effect on these cells steroidogenesis. Therefore, although there are several studies that have assisted in understanding the interaction between IGF-1 and FSH in cell differentiation during folliculogenesis, there are still crucial unknown points in this process in human cells. Likewise, more studies are needed to characterize the cumulus cells, as well as their interaction with the oocyte, so we can apply this knowledge to improve oocyte IVM rates.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Ambiental, Florianópolis, 2014 / Made available in DSpace on 2015-05-19T04:02:36Z (GMT). No. of bitstreams: 1 333386.pdf: 2765398 bytes, checksum: 958506392e40a3a664199cb4d989de75 (MD5) Previous issue date: 2014 / Os estrogênios, presentes no meio aquático como contaminantes e classificados como desreguladores endócrinos, são substâncias com comprovado efeito maléfico aos ecossistemas aquáticos e aos seres vivos, portanto, torna-se necessária a remoção destes poluentes dos esgotos sanitários. Com isto, neste trabalho foi avaliada a remoção simultânea dos hormônios estrona (E1), 17ß-estradiol (E2) e 17a-etinilestradiol (EE2) em condições aeróbias. Neste contexto, foram realizados ensaios de bancada aeróbios de curta duração, conduzidos em diferentes condições operacionais (três concentrações de sólidos suspensos voláteis e da fonte de carbono, três tipos de meio aquoso, lodo inativado e lodo ativado proveniente de três plantas de tratamento diferentes), com o intuito de verificar a relação destas variáveis com a remoção dos hormônios. Além disso, um reator de enriquecimento foi monitorado, para verificar a degradação dos estrogênios a partir da microbiota, sendo que a única fonte de carbono externa adicionada ao sistema foram os hormônios (E1, E2 e EE2). Por fim, dois sistemas piloto de tratamento de esgoto sanitário, lodos ativados (LA) e biorreator a membrana (MBR), foram monitorados para observar a degradação ou a remoção da fase aquosa dos poluentes de interesse. O E2 foi degradado mais facilmente em todos os casos avaliados. Nos experimentos com o lodo inativado, o composto sintético EE2 foi removido mais rapidamente, pelo processo de adsorção, do que os compostos naturais. Um consórcio bacteriano foi obtido através do enriquecimento, e a sua capacidade de degradação foi comprovada. A diversidade da comunidade bacteriana presente em cada amostra de lodo foi estudada, possibilitando a comparação da diversidade das bactérias dominantes, existentes em cada um dos sistemas. Os reatores LA e MBR apresentaram eficiência de remoção dos hormônios bastante semelhante, sugerindo que a presença da membrana como forma de retenção da biomassa no sistema, pode ser substituída por sistemas mais simplificados como os lodos ativados operados em batelada sequencial. A ocorrência do processo de nitrificação foi o principal fator relacionado à eficiência de remoção dos hormônios observada neste trabalho.<br> / Abstract: The estrogens, present in the aquatic environment as contaminants and classified as endocrine disrupting compounds, are substances with proven harmful effects to the aquatic ecosystems, therefore, it becomes necessary to remove these pollutants from sewage. Thus, the simultaneous removal of the hormones estrone (E1), 17ß-estradiol (E2) and 17a-ethinylestradiol (EE2) under aerobic conditions were evaluated in this work. In this context, aerobic batch tests of short duration, conducted under different operating conditions (three concentrations of volatile suspended solids and carbon source, three types of aqueous media, deactivated sludge and activated sludge from three different pants) were performed, in order to verify the relationship between these variables with the estrogens removal. Moreover, an enrichment reactor was monitored, to check the degradation of estrogens from the microbiota, whereas the hormones were the only external carbon source added to the system. Finally, two systems of wastewater treatment, activated sludge (AS) and membrane bioreactor (MBR), were monitored to observe the degradation or elimination from the aqueous phase of the pollutants of interest. The E2 was more easily degraded in all cases evaluated. In experiments with the sludge inactivated, the synthetic compound EE2 was more quickly removed by adsorption than the natural compounds. A bacterial consortium was obtained by enrichment, and its degradation capacity was proven. The structure of bacterial communities present in each sample was studied, allowing comparison of the diversity of dominant bacteria present in each system. The reactors AS and MBR showed removal efficiency hormones quite similar, suggesting that the presence of the membrane retains the biomass in the system, can be replaced by a more simplified system such as activated sludge sequencing batch reactor. The occurrence of the nitrification process was the main factor related to the removal efficiency of hormones observed in this work.

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Introdução: Os sintomas psíquicos e vasomotores são altamente prevalentes na transição menopáusica e na pós-menopausa, e estão relacionados ao hipoestrogenismo decorrente da falência ovariana que ocorre na mulher na meiaidade. Sua exata fisiopatogenia é desconhecida, porém alterações de neurotransmissores, como a serotonina e a noradrenalina, parecem estar relacionados ao aparecimento dessa sintomatologia. A terapia estrogênica geralmente é efetiva em aliviar esses sintomas, no entanto muitas mulheres não podem ou não desejam este tipo de tratamento, por isso, diversas alternativas têm sido estudadas. Objetivos: esta tese teve como objetivo avaliar, em um modelo experimental de climatério em ratas, parâmetros fisiológicos, comportamentais e bioquímicos, visando testar duas novas terapias: a cetamina, um antagonista não competitivo do receptor N-metil-D-aspartato (NMDA) e a eletroestimulação transcraniana de corrente contínua (ETCC) respectivamente, para o comportamento do tipo depressivo e a disfunção termorregulatória. Métodos: Ratos Wistar fêmeas adultas (200 a 250 g) foram randomizadas pelo peso e submetidas a modelo de menopausa por meio de ovariectomia bilateral ou a procedimento sham (falsa cirurgia). No primeiro experimento, os animais foram submetidos ao teste do nado forçado para avaliar comportamento do tipo depressivo e posteriormente receberam uma dose de cetamina 10mg/kg de peso intraperitoneal. No segundo experimento, os animais foram avaliados quanto à disfunção termorregulatória e tratados com ETCC catódica. Resultados: No primeiro experimento, as ratas em estado hipoestrogênico apresentaram comportamento do tipo depressivo que foi revertido pela cetamina. As ratas sham apresentaram um quadro de menopausa precoce indexado por citologia vaginal, provavelmente decorrente da manipulação de anexos. Além disso, as ratas ovariectomizadas apresentaram comportamento tipo ansioso. No entanto, não houve alteração da atividade locomotora e exploratória entre os grupos. No segundo experimento, as ratas ovariectomizadas apresentaram aumento da temperatura retal que foi parcialmente revertido pela eletroestimulação transcraniana de corrente contínua; as ratas ovariectomizadas apresentaram níveis elevados de interleucina 8 no soro, em relação às não ovariectomizadas, sem diferença nos níveis hipotalâmicos; houve aumento dos níveis séricos e diminuição dos níveis hipotalâmicos de BDNF nas ratas ovariectomizadas e interação do modelo com a ETCC em relação aos níveis corticais de BDNF. Nos testes nociceptivos, as ratas ovariectomizadas apresentaram diminuição do tempo de latência de resposta no teste da placa quente e alodinia mecânica no teste Von Frey, parcialmente revertida pela ETCC, no entanto, não houve diferença entre os grupos no teste tail flick. Conclusão: Nossos estudos demonstram que o modelo de ovariectomia utilizado foi eficaz em reproduzir os sintomas apresentados no período perimenopausa tendo, portanto, potencial translacional. Adicionalmente, sugerem que, além dos sistemas serotoninérgico e noradrenérgico, outros sistemas parecem estar associados ao aparecimento de sintomas na transição menopáusica como, por exemplo, o sistema glutamatérgico. Demonstram ainda que a cetamina e a ETCC podem ser eficazes como adjuvantes no tratamento dos sintomas do climatério, respectivamente, farmacológico (nos sintomas depressivos) e não farmacológico (nos “fogachos”). / Introduction: Psychological and vasomotor symptoms have a high prevalence in women during menopause transition and post menopausal years. Though these symptoms are associated to decline of estrogen levels due to ovarian failure, their exact pathophysiology is unknown. Variations on neurotransmitters such as serotonin and norepinephrine seem to be responsible by great amount of these symptoms. Estrogen therapy is usually effective in relieving these symptoms. However, many women have contraindications or do not wish to use this kind of treatment, thus several therapeutic alternatives have been studied. Objectives: This thesis was designed to evaluate, in an experimental model of menopause in rats, physiological, behavioral and biochemical parameters, aiming to test two new therapies: ketamine, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and transcranial direct current stimulation (tDCS) respectively for the depressive-like behavior and thermoregulatory dysfunction. Methods: Female adult Wistar rats (200- 250g) were randomized by weight and subjected to a menopause model through bilateral ovariectomy or sham (false surgery) procedure. In the first experiment, the animals were subjected to the forced swimming test to assess depressive-like behavior and subsequently received a dose of 10mg/kg of weight of intraperitoneal ketamine. In the second experiment, the animals were evaluated for thermoregulatory dysfunction and treated with cathodal tDCS. Results: In the first experiment, the rats in hypoestrogenic state showed depressive-like behavior that was reversed by ketamine. The sham rats presented a precocious menopause indexed by vaginal cytology, probably due to the surgical handling of the tubes and ovaries. Moreover, the ovariectomized rats showed anxiety-like behavior. However, there was no change in locomotor activity between groups. In the second experiment, the ovariectomized rats showed an increase in rectal temperature that was partially reversed by tDCS. Moreover, the ovariectomized rats showed elevated serum levels of interleukin-8, compared to non-ovariectomized rats, with no difference in hypothalamic levels; there was an increase in serum levels and decreased hypothalamic BDNF levels in ovariectomized rats, and there was interaction of ovariectomy and ETCC in relation to cortical BDNF levels. In the nociceptive tests, the ovariectomized rats presented decreased response latency in the hot plate test and mechanical alodinia in the von Frey test; however, there was no difference between groups in the tail flick test. Conclusion: Our studies demonstrate that the ovariectomy model used was effective in reproducing the symptoms that women present during perimenopause and therefore has translational potential. Additionally, these data suggest that, beyond the serotonergic and noradrenergic systems, other systems seem to be associated with the onset of symptoms in menopausal transition such as the glutamatergic system. Our data also demonstrate that ketamine and ETCC can be effective adjuvant therapeutic tools to the relief of climacteric symptoms, respectively, pharmacological (on depressive symptoms) and non pharmacological (on "hot flashes").