Orientador: Maria Del Pilar Taboada Sotomayor / Banca: Luis Francisco Moreira Gonçalves / Banca Débora Gonçalves / Resumo: O desenvolvimento de métodos analíticos cada vez mais seletivos e sensíveis é de grande importância para uma melhor qualidade na determinação de espécies químicas, aumentando assim a confiabilidade dos resultados obtidos. Com isso, o emprego e otimização das etapas de separação/concentração se faz necessário. O emprego de polímeros molecularmente impressos (do inglês - "Molecularly Imprinted Polymers" - MIP) tem demonstrado ser uma eficiente ferramenta analítica com grande potencialidade para minimizar as limitações das técnicas de separação/concentração tradicionalmente empregadas. No caso deste trabalho, o template utilizado será o valerato de estradiol, principalmente pela sua importância inerente na reposição hormonal feminina e por se tratar de um disruptor endócrino (hormônio) lançado ao meio ambiente sem nenhum controle. Assim a síntese do polímero para identificação do valerato de estradiol foi realizada por diferentes rotas (bulk e precipitação) através de duas vias de polimerização (termopolimerização e fotopolimerização) variando o monômero funcional (MAA, acrilonitrila, 2-vinil-piridina e 1-vinilimidazol). O MIP e NIP foram colocados, um de cada vez, em sistema soxhlet contendo metanol/ácido acético na proporção 70/30 a 60º C, para remoção da molécula impressa, através de cinco lavagens sucessivas em períodos de 24 h. Essas águas de lavagem foram analisadas utilizando espectrofotometria UV/Vis. Posteriormente com os parâmetros otimizados o polímero que apresentou ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The development of selective and sensitive analytical methods has significant importance for an improvement at determination of chemical species; increasing the reliability of the results obtained. So, the use and optimization of separation and concentration steps is necessary. The use of Molecularly Imprinted Polymers (MIP) has been shown to be an efficient analytical tool with great potential to minimize the limitations of separation/concentration techniques traditionally employed. In the case of this study, the template used will be estradiol valerate, once of its inherent importance in female hormone replacement and because it is an endocrine disruptor (hormone) released into the environment without any control. Thus, the synthesis of the polymer for the identification of estradiol valerate was performed by different routes (bulk and precipitation) through two polymerization techniques (thermopolymerization and photopolymerization) by varying the functional monomer (MAA, acrylonitrile, 2-vinyl pyridine and 1- vinylimidazole). MIP and NIP were placed in a soxhlet system containing 70/30 methanol/acetic acid at 60 ° C for removal of the imprinted molecule through five successive washes in 24 h periods. These wash waters were analyzed using UV/Vis spectrophotometry. Subsequently, with the optimized parameters, the polymer that presented the best result was modified with magnetic nanoparticles (core@shell); being synthesized magnetic MIP (MMIP) for estradiol valerate, increas... (Complete abstract click electronic access below) / Mestre

Resumo: O objetivo deste estudo foi avaliar histológica, histometrica e imunoistoquimicamente os efeitos do laser (LLLT) e da terapia fotodinâmica (PDT) no tratamento periodontal de ratas ovariectomizadas com ou sem reposição hormonal. Duzentas e setenta ratas foram divididas em 3 grupos de 90 animais: (A) SHAM; (B) Ovariectomizadas; (C) Ovariectomizadas tratadas com reposição hormonal. Nos primeiros molares inferiores esquerdos, de todos os animais, a doença periodontal foi induzida por ligadura. Após 7 dias, esta foi removida e nestes dentes procedeu-se a raspagem e alisamento corono-radicular (RAR). A seguir foram dividos em subgrupos de acordo com os tratamentos locais: I (n = 90) - Irrigação com 1 ml de soro fisiológico (RAR); II (n = 90) - Irrigação com 1 ml de soro fisiológico associado a aplicação de laser de baixa intensidade (LLLT) e III (n = 90) - Irrigação com 1 ml de azul de toluidina-O e, após 1 minuto, aplicação de laser em baixa intensidade (PDT). Dez animais de cada subgrupo foram sacrificados aos 7, 15 e 30 dias. Os animais do grupo A apresentaram perda óssea (PO) significativamente maior (p<0,01) no tratamento com RAR (1.11±0.26; 0.84±0.47) comparado à PDT (0.70±0.30; 0.42±0.20) nos períodos de 7 e 15 dias respectivamente. Os espécimes tratados com LLLT aos 30 dias demonstraram PO significativamente menor (p<0,01) no grupo A (0.35±0.18) comparado aos grupos B (0.82±0.21) e C (0.83±0.19). No tratamento com PDT, observou-se PO significativamente menor (p<0,01) no grupo A (0.42±0.20) comparado aos grupos B (0.95±0.20) e C (0.81±0.32) aos 15 dias. No entanto, no grupo C aos 30 dias a PDT demonstrou PO (0.52±0.23) em nível próximo ao observado nos espécimes do grupo A, no mesmo período (0.50±0.26). Na análise entre períodos, no mesmo grupo e tratamento, observou-se no grupo A, que o tratamento I apresentou PO significativamente maior... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate histological, histometrically and immunohistochemistry the influence of laser and photodynamic therapy (PDT) as an adjuvant treatment on the experimentally induced periodontitis in ovariectomy rats with or without replacement hormone. Two hundred and seventy females rats were divided into 3 groups of 90 animals. (A) Normal female rats; (B) Control - Ovarectomy; (C) Ovarectomy treated with replacement hormone. The periodontal disease was induced by ligation and after 7 days was removed and the animals divided into subgroups who received the treatments: I - scaling and root planing (SRP) and irrigation with saline; II - SRP and irrigation with saline solution and LLLT (Laser -AsGaAl - 685 nm); III - SRP, irrigation with 1ml of Toluidine Blue (TBO) and, after 1 minute, application of laser (685 nm), performing photodynamic therapy (PDT). Ten animals of each subgroup were sacrificed at 7, 15 and 30 days. The animals of the group A presented bony loss (BL) significantly larger (p <0,01) in the treatment with RAR (1.11±0.26; 0.84±0.47) compared to PDT (0.70±0.30; 0.42±0.20) at 7 and 15 days respectively. In the specimens treated with LLLT at 30 days showed BL significantly less (p <0,01) in the group A (0.35±0.18) compared at groups B (0.82±0.21) and C (0.83±0.19). In the treatment with PDT, it was observed BL significantly less (p <0,01) in the group A (0.42±0.20) than groups B (0.95±0.20) and C (0.81±0.32) at 15 days. However, in the group C at 30 days PDT showed BL (0.52±0.23) in close level that observed in the specimens of the group A, in the same period (0.50±0.26). In the analysis among periods, in the same group and treatment, it was observed in the group A, that the treatment I presented BL significantly larger (p <0,01) at 7 days (1.11±0.26); that the treatment II showed larger BL (p <0,01) in the period of 7 days (0.90±0.29)... (Complete abstract click electronic access below) / Orientador: Valdir Gouveia Garcia / Coorientador: Maria José Hitomi Nagata / Banca: Álvaro Francisco Bosco / Banca: Juliana Rico Pires / Mestre

CONTEXTO: A secreção de prolactina (PRL) e a expressão de seu gene são inibidas pela dopamina. Prolactinomas são os adenomas hipofisários funcionantes mais freqüentes, sendo que os agonistas dopaminérgicos são a primeira escolha para seu tratamento. No entanto, uma porcentagem dos pacientes é resistente aos agonistas dopaminérgicos. OBJETIVO: Como os mecanismos envolvidos na resistência aos agonistas dopaminérgicos não são totalmente compreendidos, o objetivo deste estudo foi obter mais informações no que diz respeito às alterações moleculares entre os prolactinomas sensíveis e resistentes aos agonistas dopaminérgicos. PACIENTES: O tecido tumoral de 22 pacientes com prolactinomas foram coletados e classificados como sensíveis ou resistentes (incluindo aqueles com crescimento tumoral) de acordo com sua resposta clínica e laboratorial aos agonistas dopaminérgicos. MÉTODOS: A expressão de 7 genes foi avaliada por Real Time polymerase chain reaction: gene do receptor de dopamina tipo 2 (DRD2), do fator de crescimento neural beta (NGF?) e de seu receptor (NGFR), dos receptores de estrógeno alfa (ESR1) e beta (ESR2), do pituitary tumor transforming gene (PTTG) e da metalotioneína 3 (MT3). RESULTADOS: A expressão mediana de DRD2 e de NGFR nos pacientes sensíveis foi significativamente maior quando comparada aos resistentes (p= 0.016 e p= 0.009, respectivamente). Além disso, ambas expressões estiveram significativamente correlacionadas positivamente com a redução da PRL durante o tratamento (r= ?0.66; p= 0.002 e r= 0.57; p= 0.017; respectivamente). Uma correlação positiva foi encontrada entre a mediana de expressão do NGF? e do DRD2 (r=0.53; p=0.023) e entre a mediana de expressão do PTTG e do ESR2 (r=0.66; p=0.008). também houve correlação entre a os valores de PRL sérica antes do tratamento e a mediana de expressão do gene do ESR2 (r=0.53, p=0.04). Não foi encontrada nenhuma correlação entre a expressão do gene da MT3 e a sensibilidade ou resistência aos agonistas dopaminérgicos. CONCLUSÕES: A expressão do gene do DRD2 e do NGFR estão relacionadas com a sensibilidade dos prolactinomas aos agonistas dopaminérgicos, enquanto a expressão do gene do PTTG e do ESR2 podem ter alguma relação com a agressividade tumoral. A resposta dos prolactinomas aos agonistas dopaminérgicos deve ser vista como um espectro variando do tumor mais sensível ao mais resistente com crescimento / CONTEXT: Prolactin (PRL) secretion and its gene expression are inhibited by dopamine. Prolactinomas are the most common secreting pituitary adenomas, with dopamine agonists being the first choice for their treatment. However, a subset of patients is resistant to dopamine agonists. OBJECTIVE: As the mechanisms involved in dopamine agonists resistance are not fully understood, the aim of this study was to get new insights regarding the molecular differences between prolactinomas responsive and resistant to dopamine agonists. PATIENTS: Tumor tissue of 22 patients harboring prolactinomas were collected and classified as responsive or resistant (including the ones with tumor growth) according to their clinical and laboratorial response to dopamine agonists. METHODS: The expression of 7 genes was evaluated by Real Time polymerase chain reaction: dopamine receptor type 2 (DRD2), nerve growth factor beta (NGF?) and its receptor (NGFR), estrogen receptor alpha (ESR1), beta (ESR2), pituitary tumor transforming gene (PTTG) and metallothionein 3 (MT3). RESULTS: Median DRD2 and NGFR expressions of responsive patients were significantly higher compared to the resistant ones (p= 0.016 and p= 0.009, respectively). Moreover, both expressions were positively correlated with PRL decrease during treatment (r= ?0.66; p= 0.002 and r= 0.57; p= 0.017; respectively). A positive correlation was found between NGFB and DRD2 (r= 0.53; p= 0.023) and PTTG and ESR2 expressions (r= 0.66; p= 0.008). There was also a correlation between serum PRL levels before treatment and ESR2 expression (r= 0.53, p= 0.04). It was not observed correlation between MT3 and responsiveness or resistance to dopamine agonists. CONCLUSIONS: DRD2 and NGFR expressions are related to prolactinoma responsiveness to dopamine agonists whereas PTTG and ESR2 may have a role in tumor aggressiveness. The response of prolactinomas to dopamine agonists should be view as a spectrum ranging from responsive to resistant with tumor growth

Evidências sugerem que as células da glia desempenham um papel importante na sinalização neuronal e na resposta inflamatória no Sistema Nervoso Central (SNC). Respostas inflamatórias crônicas, bem como a ativação da glia estão associadas com doenças neurodegenerativas, como Parkinson e Alzheimer. A inflamação crônica pode ser modulada por altas concentrações de espécies reativas de oxigênio (ERO) que potencializam esse quadro. O estrógeno (E2) é bem conhecido por suas ações neuroprotetoras que podem ser exercidas via receptores clássicos (ESR1, ESR2), não-classicos (GPER-1) ou ainda por sua ação antioxidante, proveniente da alta semelhança com as moléculas dos flavonóides. A ação do E2 no SNC é relevante uma vez que este hormônio está relacionado com a modulação da memória, neurogênese e plasticidade. Este trabalho tem como objetivo investigar o papel protetor do E2 em linhagem de células C6 de glioma de ratos em um modelo de estresse oxidativo que induz morte celular pela exposição a concentrações tóxicas de peróxido de hidrogênio (H2O2). Ensaios de PCR, Western Blot e de imunofluorescência confirmaram a presença e funcionalidade dos receptores ESR1, enquanto ensaios de PCR mostraram a presença do RNAm para o GPER-1 em células C6. Nossos resultados confirmaram que a H2O2 induz morte nas células C6 e o pré-tratamento com E2 (por 24 horas) e G1 (por 20 minutos) diminuiu a toxicidade da H2O2 de maneira dose-dependente, gerando aumento de viabilidade celular. Estes resultados destacam o envolvimento do E2 e seus receptores na prevenção do dano celular em células da glia. Além disso, eles também sugerem que o rápido efeito protetor do E2 parece estar associado com a sinalização rápida do E2 via GPER-1. Por Western blot e RT-PCR avaliamos a participação da via AKT-CREBBDNF frente aos tratamentos com E2, moduladores seletivos de estrógeno (SERMs) e G1, onde observamos que estes são capazes de modular a expressão da proteína AKT e os níveis de RNAm para BDNF. / Evidence suggests that glial cells play an important role in neuronal signaling and inflammatory responses in the central nervous system (CNS). Chronic inflammatory responses, as well as activation of glia, are associated with neurodegenerative disorders such as Parkinson´s and Alzheimer´s diseases. Chronic inflammation can be modulated by high concentrations of reactive oxygen species (ROS) that enhance this process. Estrogen (E2) is well known for its neuroprotective actions that can be performed via classical (ESR1, ESR2) and non-classical receptors (GPER-1) or by its antioxidant action due to its high similarity to flavonoids molecules. E2 action in the CNS is relevant as this hormone is associated to memory modulation, neurogenesis and plasticity. This work has as purpose to investigate the protective role of E2 in rat C6 glioma cell lines in a model of oxidative stress that induces cell death by exposure to toxic concentrations of hydrogen peroxide (H2O2). PCR, Western blot and immunofluorescence assays have confirmed the presence and functionality of the ESR1 receptor, while PCR assay has showed the presence of GPER-1 receptor mRNA in C6 cells. Our results confirmed that H2O2 induces cell death and pre-treatment with E2 (24 hours) and G1 (20 minutes) reduces H2O2 toxicity in a dose-dependent way, leading to increased cell viability. These results highlight the involvement of E2 and its receptors in preventing cell damage in glial cells. Moreover, they also suggest that the prompt E2 protective effect seems to be associated to the fast E2 signaling via GPER-1. We also evaluated the involvement of AKT-CREB-BDNF pathway when C6 cells were treated with E2, selective estrogen modulators (SERMs) and G1 by Western blot and RT-PCR assays, and we could notice that they can modulate the expression of AKT protein and BDNF RNAm levels.

Abstract Prostate cancer is the most frequently diagnosed cancer in men in industrialized countries. Despite the substantial clinical importance of the disease, the mechanisms underlying the development and progression of prostate cancer are poorly understood. In the present study, fragment analysis of chromosome arm 16q was carried out with the aim of searching for sites of consistent chromosomal deletion, possibly uncovering the location of target genes that become inactivated in prostate carcinogenesis. The highest percentage of loss of heterozygosity (LOH) was found at chromosomal region 16q24.1-q24.2, including the gene for 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17HSD/KSR) type 2, HSD17B2. The data further indicated an association between loss of the most commonly deleted region and clinically aggressive features of the disease. A fragment analysis performed using sequential primary and locally recurrent prostate cancer specimens suggested the location of the genes related to prostate cancer progression to be at 16q24.3 and, further, gave rise to a hypothesis of the potential role of locus HSD17B2 as a prognostic marker for prostate cancer progression. Quantitative real-time polymerase chain reaction (PCR) revealed a decreased HSD17B2 gene copy number in prostate cancer specimens compared to their normal counterparts. A diminished HSD17B2 gene copy number was significantly associated with poor differentiation of the tumor. The progression of prostate cancer during androgen deprivation is a serious clinical problem, the molecular mechanisms of which largely remain to be clarified. The present data of enzyme activity measurements performed using high-performance liquid chromatography (HPLC) provided evidence of a substantial decrease in oxidative and an increase in reductive 17HSD/KSR activity during the transition of prostate cancer LNCaP cells into an androgen-independent state. Further, the changes detected in the activities largely coincided with the changes in the relative expression levels of genes for the potential 17HSD/KSR isoenzymes; 17HSD/KSR types 2, 5, and 7, as evidenced by relative quantitative reverse transcription PCR (RT-PCR). The data on the expression analysis of mRNA for 17HSD/KSR types 5 and 7 in prostate tissue specimens performed using in situ hybridization showed a moderately low but constitutive level for 17HSD/KSR7 mRNA in tissues of cancerous as well as hyperplastic origin. The expression of mRNA for 17HSD/KSR type 5, instead, varied considerably between different specimens, the highest expressions being strongly associated with aggressive and metastatic prostate cancer. Interestingly, furthermore, the intense expression of 17HSD/KSR5 was significantly associated with the androgen deprivation achieved either surgically or medically. Since 17HSD/KSRs critically contribute to the control of the bioavailability of active sex steroid hormones locally in the prostate, the variation in intraprostatic 17HSD/KSR activity might be crucially involved in the regulation of the growth and function of the organ.

Abstract The biosynthesis and metabolism of sex steroids are controlled by a series of steroidogenic enzymes. In the placenta and the breast, 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) is essential for the synthesis of all steroid hormones by catalyzing pregnenolone to progesterone (P) or dehydroepiandrosterone (DHEA) to androstenedione (A-dione). P450 aromatase (P450arom) converts androgens to estrogens and is therefore critical for estrogen production. 17β-hydroxysteroid dehydrogenases (17HSDs) are a group of enzymes responsible for the interconversion between low-activity 17-ketosteroids and high-activity 17β-hydroxysteroids, thus acting as key enzymes modulating the biosynthesis and metabolism of both estrogens and androgens. In situ hybridization assays showed that 3β-HSD1, P450arom and 17HSD1, 2, 5 and 7 are expressed in early and mid-gestation placentas. Abundant expressions of 3β-HSD1, P450arom and 17HSD1 were seen in syncytiotrophoblast (ST) cells. Signals of these three enzymes were also detected in some column cytotrophoblast (CCT) cells. 17HSD2 and 5 were located in intravillous stromal (IS) cells, whereas 17HSD7 mRNA was present in all types of placental cells. This suggests that the human placenta produces not only P and estrogens, but also androgens. Moreover, the placenta possesses a function, by the action of 17HSD2, to protect the fetus and the maternal body from excessive sex steroid influence. In tubal pregnancy, P450arom and 17HSD1 were found in ST cells, implying an estrogen biosynthesis mechanism similar to that in normal intrauterine pregnancy. In both JEG-3 choriocarcinoma cell line and cultured normal human cytotrophoblast (CTB) cells, retinoic acids were shown to promote the enzyme activity as well as mRNA expression of P450arom and 17HSD1, and hence their action on the biosynthesis of E2. The mRNA expressions of 17HSD1, 2 and 5 in 794 breast carcinoma specimens were analyzed and correlated with ERα, ERβ, PR, Ki67, c-erbB2 and clinical parameters. 17HSD1, 2 and 5 were detected in epithelial cells in normal and malignant breast tissues. In breast cancer specimens, the positive cases for 17HSD1, 2 and 5 were 16%, 25% and 65%, respectively. 17HSD1 was found to be an independent prognostic marker of the progression of breast cancer.

The perinate is particularly risk-prone to chemical species which have the potential of inducing neuronal apoptosis or necrosis and thereby adversely altering development of the brain, to produce life-long functional and behavioral deficits. This paper is an overview for many substances of abuse, but the purview is much more broadened by the realization that even elevated levels of estrogens and corticosteroids in the pregnant mother can act as neuroteratogens, by passing via the placenta and altering neural development or inducing apoptosis in the perinate. Finally, therapeutic risks of anesthetics are highlighted, as these too induce neuronal apoptosis in the neonate by either blocking N-methyl-D-aspartate receptors or by acting as gamma-aminobutyric acid agonists. By understanding the mechanisms involved it may ultimately be possible to interrupt the mechanistic scheme and thereby prevent neuroteratological processes.

Twelve university women students served as experimental subjects in a study of the serum lipids and urinary estrogens of healthy nonpregnant menstruating young women, who were living under their usual conditions. The subjects maintained constant weight on their ordinary diets during the entire study period. Antecubital blood and 24-hour urine specimens were collected on certain days which represented different stages of the menstrual cycle. Quantitative analyses were made on serum total cholesterol, lipid phosphorus (phospholipids), triglycerides and total lipids. Gas-liquid chromatographic analysis of the fatty acid composition of each serum lipid component was also made. Urinary estrone, 17β- estradiol and estriol were separated and quantitatively determined by chromatographic and spectrophotometric techniques. Basic data on serum lipid levels , composition of the fatty acids of cholesterol esters, phospholipids and triglycerides and urinary estrogens were obtained on these young women . Findings included the following: 1. Mean values of serum total cholesterol, phospholipids, triglycerides and total lipids were 162, 165, 105 and 544 mg per cent, respectively. The interindividual variation was greater than intraindividual variation. The values of triglycerides were more variable than those of cholesterol and phospholipids. 2. The major fatty acids in lipid fractions were palmitic, stearic, oleic and linoleic. The highest amounts of fatty acid in cholesterol esters, phospholipids and triglycerides were linoleic, 51; palmitic, 28; and oleic, 33 per cent, respectively. Inter- and intra-individual variations were high. 3. The urinary estrogen values showed that 17 B-estradiol (E 2 ), was usually present in the least and estriol (E 3 ), in the greatest amounts. The mean values of E 1 (estrone), E 2 , E 3 and Et (total) were as follows: 8. 7, 4. 8, 16. 4, and 29. 9 μg per 24-hour urine. 4. The menstrual cycle did affect the urinary excretion of estrogens which showed the lowest values during the first week and then rose to a peak which occurred on or about the time of ovulation or mid-cycle. Then it fell and rose again between the third and fourth week of the cycle. The second peak was usually lower than the first one. 5. Cyclical changes of the concentrations of serum total cholesterol, phospholipids and total lipids have been observed. These changes appeared to be influenced by the estrogenic hormonal activity of the menstrual cycle. The increased excretion of urinary estrogens with a decreased (negative correlation) concentration of serum lipids was recognized. 6. Linoleic acid in cholesterol esters, as well as palmitic acid in phospholipids were found in cyclic changes. The patterns were quite similar to those of serum lipids.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18655号 / 工博第3964号 / 新制||工||1610(附属図書館) / 31569 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 伊藤 禎彦, 教授 米田 稔 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

No description available.