Dissertations / Theses on the topic 'CYP199A4'

Thesis (M.A.)--Boston University
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Ovarian stimulation is frequently used in many assisted reproductive technologies (ART) procedures to increase the number of dominant follicles. However, achieving the optimal response to ovarian stimulation without complications can be difficult. In our study, we predict that future ovarian stimulation procedures will be more individually catered to each patient. A study that established a predictor model for ovarian stimulation showed that genetic single nucleotide polymorphisms (SNP) are good predictive factors (Fauser et al., 2007). Thus the objective of this study was to determine if there was any association between the various parameters that measure the success of ovarian stimulation and FSHR, CYP19, ESRJ and ESR2 singe nucleotide polymorphisms (SNP). A total of two hundred and four women undergoing either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) were genotyped to study the distribution of the different SNPs. Complete clinical information was available for a range of forty nine to seventy three women to study any association between genotype and basal FSH level (day 3), basal 17 -beta estradiol level, number of follicles, number of eggs and the level of 17-beta estradiol during hCG administration. The association was studied using the one way ANOVA test and linear regression model. There was a significant correlation between the CYP 19Al rs10046 genotypes and FSH level (p=0.004), number of follicles (p=0.03) and level of 17-beta estradiol during hCG administration (p=0.04). Also, FSHR rs2268263 genotype was significantly linked to FSH level between the G-allele group (A/G and G/G) and the homozygote A group (p=0.04). Taken all together, these results suggest that several genes-especially CYP 19AJ and FSHR rs2269263- play a significant role in determining the success of ovarian stimulation.
2031-01-01

Introdução:Até 60% das mulheres com endometriose apresentam como sintoma a infertilidade. Entretanto, os mecanismos envolvidos ainda permanecem não totalmente esclarecidos, especialmente quando não há distorção da anatomia pélvica. A etiologia multifatorial e comprometimento poligênico nesta doença têm sido amplamente aceitos. A aromatase é uma molécula das mais estudadas e há evidências de aumento da expressão do seu gene no endométrio eutópico e ectópico na endometriose. Esta enzima, codificada pelo gene CYP19A1, converte andrógenos a estrógenos e está presente normalmente nas células da granulosa, onde é fundamental para a produção esteroidogênica intrafolicular. Estudos in vitropor cultivo de células da granulosa, mostraram redução da atividade da aromatase em mulheres com endometriose. Devido à escassez de estudos que analisem a expressão do seu gene (CYP19A1) nessas células foi o que estimulou a proposta deste estudo. Este trabalho tem porobjetivo medir a expressão do gene da aromatase por PCR em tempo real nas células da granulosa luteinizadas murais de mulheres com endometriose submetidas a técnicas de reprodução assistida. Pacientes e Métodos: Estudo caso-controle, com 11 mulheres com endometriose e 11 com os fatores tubáreo ou masculino de infertilidade,submetidas à hiperestimulação ovariana controlada (HOC) para reprodução assistida, num total de 12 ciclos para o grupo com endometriose e 11 para o controle. Não houve diferença entre as características clínicas dos dois grupos quanto à idade e parâmetros do ciclo de HOC. As células da granulosa murais foram coletadas de folículos pré-ovulatórios maduros no dia da captação oocitária e isoladas. Posteriormente, procedeu-se à extração do RNA (clorofórmio/ isopropanol) e à transcrição reversa. A PCR em tempo real foi realizada para quantificar os níveis de RNA mensageiro produzidos para o gene da aromatase, normalizados aos produtos do gene endógeno, ß-actina (expressão relativa). Todos os experimentos foram realizados em duplicata. Resultados: não houve diferença na expressão do gene CYP19A1 nas células da granulosa luteinizadas murais de mulheres com endometriose quando comparadas ao grupo controle (p>0,05; Mann Whitney), mesmo na comparação combinada considerando-se separadamente os diferentes graus de endometriose (controle vs. endometriose mínima/leve vs. endometriose moderada/grave, p>0,05;Kruskall Wallis). Conclusão:Os resultados deste estudo sugerem que a aromatase apresenta um mecanismo complexo de controle para sua expressão gênica nas células da granulosa e, apesar de evidências prévias de sua reduzida atividade nessas células na endometriose, a expressão de seu gene parece não estar afetada pela doeça, de acordo com o presente estudo.
Background: Up to 60% of women with endometriosis have infertility symptoms. However, mechanisms remain unclear, mainly when there is no distortion of pelvic anatomy. The multifactorial etiology and polygenic involvement of this disease have been widely accepted. Aromatase is one of the most studied molecules and there are evidences of increased expression of its gene (CYP19A1) on eutopic and ectopic endometrium in endometriosis. This enzyme, codified by the CYP19A1 gene, converts androgens to estrogens and is normally present in granulosa cells, where it plays an essential role for the intrafollicle steroidogenic production. In vitrostudies by granulosa cells culture have demonstrated reduced aromatase activity in women with endometriosis. The scarcity of studies assessing expression of the aromatase gene (CYP19A1) on these target cells stimulated the proposal of this research. The aim of this study is to quantify aromatase gene expression, by real-time PCR, in mural lutein-granulose cells of women with endometriosis undergoing assisted reproduction techniques. Patients and Methods: a case-control study was conducted on 11 women with endometriosis and 11 with male or tubal causes of infertility submitted to ovarian hyperstimulation (HOC), with a total of 12 cycles for endometriosis and 11 for the control group. There was no difference between the groups regarding age or HOC parameters. Mural lutein-granulosacells were harvested from pre-ovulatory follicles during oocyte retrieval and properly isolated. Later, RNA extraction (clorophorm/isopropanol) and reverse transcription were performed. Real-time PCR was run to quantify RNAm products of aromatase gene normalized to those from the control gene, beta-actin (relative expression). All experiments were carried out in duplicate. Results: there was no difference between the groups regarding the gene expression of CYP19A1 (aromatase) gene on mural lutein-granulosa cells (p>0.05, Mann Whitney), even if we consider separately the different stages of endometriosis (control vs. minimal/mild vs. moderate/severe, p>0.05, Kruskall Wallis). Conclusion: These results suggest that aromatase may have a complex control of its gene expression on granulosa cells and, despite of previous evidences showing its reduced activity on these target cells in endometriosis, the gene expression seems not affected by the disease, according to this study.

The enzyme Cytochrome P450 aromatase plays an essential role in the biosynthesis of estrogens, and its inhibition is an important target for the development of drugs for the treatment of breast cancer. The main purpose of the present thesis is to improve the understanding of the catalytic mechanism and the biochemistry of this enzyme from the standpoint of theoretical chemistry. The results of this thesis have been divided into three main sections: (1) Study of the reactive species of the enzyme aromatase: Compound I; (2) Study of the hydroxylation of the natural substrate androstenedione, during the first catalytic subcycle of the enzyme aromatase; and (3) Study of the hydroxylation of Exemestane, an esteroidal third generation aromatase inhibitor, currently used in hormone dependent breast cancer therapy.
La enzima citocromo P450 aromatasa juega un papel esencial en la biosíntesis de estrógenos, y su inhibición es un objetivo importante para el desarrollo de medicamentos para el tratamiento del cáncer de mama. El objetivo principal de la esta Tesis ha sido arrojar luz sobre el mecanismo catalítico y sobre la bioquímica de esta enzima, desde el punto de vista de la química teórica. Los resultados que se presentan en esta Tesis se han dividido en tres secciones principales: (1) Estudio de la especie reactiva de la enzima aromatasa: "Compound I"; (2) Estudio de la hidroxilación del substrato natural androstenediona, a lo largo del primer subciclo catalítico de esta enzima; y (3) Estudio de la hidroxilación del Exemestano, un inhibidor esteroideo de tercera generación de la enzima aromatasa, que se utiliza actualmente en el tratamiento del cáncer de mama hormonodependiente.

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The aromatase enzyme complex is responsible for converting androgens into estrogens in vertebrates. One of the key enzymes of this complex is the cytochrome P450arom gene expression and regulation of this enzyme is directly linked to sexual differentiation. Estrogen is essential for the development of the gonads and various physiological processes, ranging from normal growth to reproductive behavior. This study aimed to correlate the polymorphisms in the genes CYP19a CYP19b. In a first moment with three strains used in aquaculture (Chitralada, Supreme and GIFT) using thirty animals of each strain and, a second time only with the Supreme strain which has also the objective of analyzing the sex ratios found in both treatments at different temperatures of water and to establish possible relationships between them, analyzing the 122 animals treated at 25 ° C and 129 animals of treatment at 35 ° C. After extraction of DNA samples were subjected to PCR using primers designed to flank a region of interest. After confirmation of amplification samples were subjected to electrophoresis on Origins apparatus for identification of alleles. In the first study was identified that there is a polymorphism in the promoter region of CYP19b in the three strains. Were observed three alleles, ranging from 141 to 123 bp. Thus the primers for amplification of microsatellites in the regulatory region of the gene CYP19b were efficient, and therefore its use for identification of mutations in this region can be employed in the typical analysis of microsatellites. In the second study was identified that there is a polymorphism in the regulatory region of the gene 10 CYP19a, 5 alleles were observed. The proportion of females and males found in the treatment at 25 ° C was 1:2,3 1:2,88 and treatment at 35 ° C. However, it can be assumed that the polymorphism present in this portion of the regulatory region may be related to sex reversal found. Whereas the attempt to select individuals with polymorphisms in the regulatory region of the aromatase gene (and CYP19a CYP19b) is an essential study to evaluate the expression levels of both, so you can see how it is being expressed in individuals that will reverse or not , allowing for the reversal temperature with a higher proportion.
O complexo enzimático da aromatase é responsável pela conversão de andrógenos em estrógenos nos vertebrados. Uma das principais enzimas deste complexo é o citocromo P450arom e a regulação da expressão gênica desta enzima está diretamente ligada à diferenciação sexual. O estrogênio é essencial para o desenvolvimento das gônadas e diversos processos fisiológicos, que vão desde o crescimento normal até o comportamento reprodutivo. Este estudo teve como objetivo correlacionar o polimorfismo nos genes CYP19a e CYP19b. Em um primeiro momento com três linhagens utilizadas na piscicultura brasileira (Chitralada, Supreme e GIFT) utilizando trinta animais de cada linhagem e, em um segundo momento somente com a linhagem Supreme a qual tem-se o objetivo também de analisar as proporções de sexo encontrado em dois tratamentos com temperaturas diferentes da água e estabelecer possíveis relações entre eles, analisando 122 animais do tratamento a 25°C e 129 animais do tratamento a 35°C. Após a extração de DNA as amostras foram submetidas a PCR utilizando os primers desenhados para flanquear uma região de interesse. Após confirmação da amplificação as amostras foram submetidas à separação eletroforética no aparato Origins para identificação dos alelos. No primeiro estudo foi identificado que existe um polimorfismo na região promotora de CYP19b nas três linhagens. Foram observamos três alelos, variando entre 141 e 123 pb. Assim os primers para a amplificação de microssatélite na região regulatória do gene CYP19b foram 8 eficientes e, portanto, seu uso para identificação de mutações nesta região pode ser empregada na análise típica de microssatélite. No segundo trabalho foi identificado que há um polimorfismo na região regulatória do gene CYP19a, onde foram observados 5 padrões de bandas (supostamente alelos). A proporção de fêmeas e machos encontrada no tratamento a 25°C foi de 1:2,3 e de 1:2,88 no tratamento a 35°C. Contudo, supõe-se que o polimorfismo presente nesta porção da região regulatória pode estar relacionado à reversão sexual encontrada. Considerando a tentativa de selecionar indivíduos que apresentem polimorfismo na região regulatória dos genes da aromatase (CYP19a e CYP19b), é indispensável um estudo para avaliar os níveis de expressão de ambos, para que se possa observar como está sendo expressado em indivíduos que revertem ou não, possibilitando assim a reversão por temperatura com uma proporção mais elevada.

Plusieurs pathologies humaines sont associées à l'altération de l'expression ou de la fonction de facteurs de transcription GATA. L'hyperméthylation, l'hypoacétylation ou la surexpression des gènes GALA sont observées dans plusieurs types de cancers. Des mutations ponctuelles des gènes GATA sont aussi à l'origine de graves maladies congénitales. Plus de 60% des tumeurs du sein sont dépendantes des estrogènes et produisent elles-mêmes les hormones nécessaires à leur croissance. Cette production aberrante d'estrogènes est causée par la surexpression de l'enzyme aromatase, codée par le gène CYP19A1. L'utilisation ectopique dans les tumeurs du sein du promoteur PII du gène CYP19A1, normalement spécifique à l'ovaire et régulé par la voie de signalisation AMPc/PKA, est reconnue comme étant responsable de la surexpression de l'enzyme et de la production inappropriée d'estrogènes par les tumeurs. Les facteurs GATA sont impliqués dans la régulation du promoteur PII de CYP19A1 dans les gonades. J'ai démontré que GATA3 et/ou GATA4 sont exprimés dans plusieurs lignées cellulaires de cancer du sein. En réponse à l'activation de la voie de signalisation AMPc/PKA, GATA3/4 et le récepteur nucléaire LRH1 coopèrent de façon synergique pour moduler l'activité du promoteur PII dans les cellules de cancer du sein MCF-7. Ces résultats fournissent un nouveau mécanisme dépendant de GATA pour expliquer l'expression aberrante de l'enzyme aromatase dans les tumeurs du sein. Les mutations hétérozygotes connues de GATA4 ségrègent avec de graves défauts cardiaques congénitaux. GATA4 est un important régulateur du développement cardiaque mais aussi un marqueur précoce du développement gonadique. Toutefois, aucune des mutations connues de GATA4 n'affecte le développement ou la fonction gonadique des individus porteurs. L'effet des différentes mutations de ce facteur sur la régulation de l'expression de ses cibles gonadiques demeurait par contre inconnu. J'ai étudié cinq mutations différentes du facteur GATA4. J'ai démontré que certaines de ces mutations affectent significativement la fonction de GATA4 dans un contexte de régulation de l'expression de gènes gonadiques. Mes résultats démontrent que malgré l'absence d'un phénotype observable, la plupart des mutations de GATA4 réduisent significativement sa capacité d'activer l'expression de ses gènes cibles sans affecter sa capacité à se lier à l'ADN ou celle de recruter ses co-activateurs transcriptionnels. Dans l'ensemble, les résultats présentés dans ma thèse démontrent comment les facteurs GATA exprimés dans un contexte tumoral contribuent à l'expression aberrante de CYP19A1 dans les cellules cancéreuses du sein selon un mécanisme rappelant celui menant à l'expression gonadique de ce gène et d'autre part comment l'altération de la séquence peptidique du facteur GATA4, telle qu'observée dans de nombreuses malformations cardiaques congénitales, affecte la fonction de ce facteur de transcription dans la régulation de l'expression de ses cibles gonadiques.

The cytochrome P450 enzyme CYP199A4 from Rhodopseudomonas palustris strain HaA2 is highly specific for the regioselective oxidation of para-substituted benzoic acids. A selection of these compounds was tested with the enzyme with the aim of investigating the mechanism of different P450-catalysed reactions. These studies revealed that the binding affinity and oxidative activity of CYP199A4 is influenced by the substituent at the para-position, and that to the enzyme’s known oxidative activities (demethylation, hydroxylation, heteroatom oxidation and desaturation) can be added alkene epoxidation, alkyne oxidation and aldehyde oxidation. The active oxidants involved in these CYP199A4-catalysed oxidations were investigated using two active site mutants at the conserved acid-alcohol pair, T252A CYP199A4 [CYP199A4 subscript] and D251N CYP199A4 [CYP199A4 subscript], which should disrupt different steps of the catalytic cycle. There was a general increase in hydrogen peroxide uncoupling in the T252A CYP199A4 [CYP199A4 subscript] mutant but significant levels of product formation were observed with each substrate. The D251N mutation reduced the activity of the enzyme dramatically in all but one case, suggesting that this mutation interferes with proton delivery as expected. The elevated rate of 4-ethynylbenzoic acid oxidation by T252A CYP199A4 [CYP199A4 subscript] when compared to the wild-type enzyme suggested the involvement of Cpd 0 in alkyne oxidation, while a reduction in activity with 4-methoxybenzoic acid implicated Cpd I in demethylation. Additionally, the notable increase in product formation and coupling efficiency of D251N CYP199A4 [CYP199A4 subscript] with 4-formylbenzoic acid suggested the involvement of the peroxo-anion in aldehyde oxidation. Larger cinnamic acids and closely related substrates were also investigated with CYP199A4. The binding affinity and oxidative activity of the enzyme decreased in the order 4-methoxybenzoic acid > 4-methoxycinnamic acid > 3-(4- methoxyphenyl)propionic acid > 4-methoxyphenylacetic acid, highlighting its selectivity for a planar, benzoic acid- or cinnamic acid-like framework. The exclusive oxidation of cinnamic acids and related derivatives at the para-position further demonstrated the high regioselectivity of CYP199A4. While CYP199A4 exhibited low oxidation activity towards para-methoxy substituted benzene derivatives, considerably higher levels of activity reminiscent of the demethylation of 4-methoxybenzoic acid were observed for the Ser244 → Asp244 (S244D) mutant of CYP199A4. The exclusive demethylation of the para-methoxy substituted benzenes by S244D revealed that the regioselectivity of CYP199A4 oxidation is maintained in this mutant. The regioselectivity of the S244D mutant was further investigated using a selection of methyl- and ethyl-substituted derivatives. The methyl analogues were exclusively oxidised at the para-position to a single α-hydroxylation product. α-Hydroxylation and Cα [α subscript] -Cᵦ desaturation products were generated in the turnovers of the ethyl derivatives. The alcohol was formed with high stereoselectivity. The electronic properties of the ethyl substrates were found to influence the ratio of hydroxylation/desaturation product, with the more electron donating substrates giving rise to a greater proportion of the latter. This suggested the involvement of a cationic intermediate in CYP199A4- catalysed desaturation.
Thesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2016.

A presente dissertação encontra-se inserida na unidade curricular intitulada Estágio do Mestrado Integrado em Ciências Farmacêuticas, no âmbito da qual realizei dois estágios referentes às componentes de farmácia hospitalar e comunitária, bem como um trabalho exploratório de investigação no campo da infertilidade feminina. O primeiro capítulo deste documento, é alusivo à componente laboratorial, desenvolvida no Centro de Investigação em Ciências da Saúde da Universidade da Beira Interior, sendo intitulado “Polimorfismos dos genes CYP19A1, GSTM1 e GSTT1 na infertilidade feminina”. A infertilidade assume-se como uma condição clínica de origem multifatorial, a qual deve ser interpretada como uma doença do casal e onde a componente genética assume um impacto amiúde. Deste modo, procurámos estudar a influência dos polimorfismos associados aos genes CYP19A1 (substituição de um triptofano por uma arginina no codão 39), GSTM1 (deleção) e GSTT1 (deleção) no desenvolvimento de infertilidade de etiologia feminina, bem como o impacto destes na resposta às técnicas de promoção da fertilidade caracteristicamente utilizadas. Delineámos ainda como objetivo do trabalho, estudar a associação entre os genótipos exibidos pelas mulheres e a causa de infertilidade potencialmente associada. O segundo capítulo desta dissertação, assume-se como uma análise crítica referente ao estágio realizado em farmácia hospitalar, o qual decorreu na Unidade Local de Saúde de Castelo Branco, tendo prefeito um total de trezentas e vinte horas. Nesta secção, encontra-se uma exposição pormenorizada da natureza da atuação farmacêutica em contexto hospitalar, bem como os aspetos legais inerentes à mesma. A última componente do relatório em epígrafe, aborda a componente de estágio em farmácia comunitária, a qual foi desenvolvida na farmácia Ferrer, em Castelo Branco, num total de quatrocentas e oitenta horas. Neste capítulo, figura uma apresentação holística da realidade farmacêutica no referido campo, bem como a atual conjuntura ética, legal e financeira inerente à mesma.
This dissertation is part of a curricular unit named Internship of Integrated Master of Pharmaceutical Sciences, in which I performed to internships on hospital and community pharmacy. We can also find a research work referent to female infertility. The first chapter of this document, named “Polymorphisms of CYP19A1, GSTM1 and GSTT1 genes on female infertility” refers to the laboratory component, held at the Health Sciences Investigation Center of University of Beira Interior. Infertility is a clinical condition with multifactorial origin, and it should be interpreted as a couple’s disease, where genetic factors have huge impact. This way, we studied the influence of polymorphisms of CYP19A1 (substitution of a tryptophan by and arginine on codon 39), GSTM1 (deletion) and GSTT1 (deletion) genes on the development of female infertility and its impact on the response of fertility treatments. We also studied the association between genotypes and the cause of infertility potentially involved. The second chapter of this dissertation is a critical analysis of the internship held in hospital pharmacy. It took place on Unidade Local de Saúde de Castelo Branco, in a total of three hundred and twenty hours. On this section, we may find in a detailed way, pharmaceutical operations on hospital pharmacy and all legal aspects related to it. The last component of this report, refers to the internship on community pharmacy, which was held on Ferrer pharmacy, in Castelo Branco, in a total of four hundred and eighty hours. On this chapter, we may find an holistic presentation of pharmaceutical reality on this field, as well as all ethical, legal and financial matters related to it.

博士
國立陽明大學
生化暨分子生物研究所
98
Zebrafish is a popular model for genetic and developmental studies, yet very little is known about the mechanism of zebrafish sex determination due to the lack of discernible sex chromosomes and the difficulty of distinguishing the sex of juvenile fish. To alleviate these problems, we generated fish with predictable sex trait by methyltestosterone treatment followed by natural mating. Histology and gene expressions analysis in these juvenile fish with known gender was characterized. Our genetic selection scheme matches the prediction that female-dominant genetic factors are required to determine zebrafish sex. We also found the first sign of zebrafish sex differentiation to be ovarian gonocyte proliferation and differentiation at 10-12 days post-fertilization (dpf). Sex dimorphic expression of cyp19a1a and sox9a, the female and male somatic cell markers, respectively, was also observed at three weeks of age before the appearance of testicular morphology. Besides, we found cyp19a1b was expressed in radial glial cells just peripheral to the brain ventricles in ventral telencephalon, preoptic area, and hypothalamus. Estrogens could upregulate the expression of cyp19a1b in larval brain. We have generated transgenic zebrafish lines expressing GFP driven by cyp19a1b promoter, which exhibited fluorescence signals in adult radial glial cells and in estrogen induced fish larvae. This line provides a tool to further characterize radial glial progenitor cells as well as evaluation of xenoestrogen effects.

L’oestradiol joue un rôle important dans la reproduction en général, particulièrement dans la croissance folliculaire chez la vache. La production de l’œstradiol nécessite l’expression du gène CYP19A1 suite à la stimulation des cellules de granulosa par l’hormone folliculostimulante (FSH) ou le facteur de croissance insulinique de type 1 (IGF-1). Chez la vache, il existe six promoteurs (1.1 ; 1.2 ; 1.3 ; 1.4 ; 1.5 et 2) qui dirigent la transcription du gène CYP19A1 dans les cellules de la granulosa. Le principal promoteur qui dirige la transcription au niveau de l’ovaire (cellules de granulosa) est le promoteur 2 (P2). Cependant, l’effet de la FSH et de l’IGF-1 sur l’activation de ces promoteurs d’aromatase demeure mal connu. De plus, la demi-vie du transcrit CYP19A1 est très courte avec une région 3’UTR relativement longue. L’analyse de la séquence 3’UTR montre la présence des motifs ARE (séquence riche en AU), des études antérieur montrent que ces séquences impliquent dans la régulation de la stabilité ou la dégradation de l’ARNm, ce qui est fort probable que la courte demi-vie de l’ARNm CYP19A1 est sous le contrôle post-transcriptionel. L’objectif de la thèse visait à étudier la régulation de l’expression du gène CYP19A1 chez la vache. Il y a deux thèmes soit étude de la régulation transcriptionnelle ciblant le promoteur et soit étude de la régulation post-transcriptionnelle impliquant la région 3’non traduite (3’UTR). Le premier objectif vise à étudier la régulation transcriptionnelle du gène CYP19A1. Nous avons étudié l'activité du promoteur ovarien bovin dans deux modèles de cellules de la granulosa, les cellules lutéinisées et nonlutéinisées in vitro, suite à une stimulation des cellules par la FSH ou IGF-1. Nous avons également évalué la voie de signalisation impliquée dans la régulation des différents promoteurs en utilisant un RT-PCR et un gène rapporteur (les différents promoteurs d’aromatase ont été insérés dans le vecteur pGL3promoter en amont du gène exprimant la luciférase). Les résultats de RT-PCR démontrent que la FSH et l’IGF-1 augmentent les concentrations d’ARNm provenant des deux promoteurs 2 et 1.1 dans les cellules de la granulosa non lutéinisées. Des expériences subséquentes ont montré que la FSH stimule le promoteur 2 via la voie PKA tandis que l'IGF-1 stimule le promoteur 2 via la voie PKC. La FSH et l’IGF-1 stimulent l’expression du promoteur 1.1 via la voie PI3K. L’analyse de l’activité luciférase démontre que dans les cellules de granulosa lutéinisées, la FSH stimule le promoteur 1.1 de façon dose dépendante et ne semble y avoir aucun effet significatif sur le promoteur 2. Nous avons donc comparé l’activité du promoteur PII/P2 humain, du rat, de la chèvre et de la vache dans les cellules de granulosa bovine lutéinisées. Le résultat le plus significatif est que le promoteur 2 bovine (et caprine) dépend de plusieurs facteurs de transcription (NR5A2, FOXL2) comparé au promoteur PII humain et celui du promoteur proximal du rat qui dépendent principalement de l'AMPc. En effet, nos résultats ont démontré une expression raisonnablement robuste du P2 bovine lorsque les cellules sont traitées à la forskoline, NR5A2 et FOXL2. Le facteur FOXL2 semble déterminer l'activité du promoteur 2 chez le ruminant. Le deuxième objectif vise à étudier la régulation post-transcriptionnelle du gène CYP19A1. Pour ce faire, nous avons déterminé la séquence minimale de l'ARNm CYP19A1 requise pour la régulation de sa demi-vie. Différents séquences de la région 3’UTR ont été insérés dans le vecteur pGL3promoter en aval du gène exprimant la luciférase ou soit dans le vecteur pGEMTeasy. Le vecteur pGL3promoter a été transfecté dans les cellules de granulosa lutéinisées pour évaluer l'impact de la séquence 3'UTR sur l'expression du gène rapporteur de la luciférase, alors que le vecteur pGEMTeasy a été utilisé pour la transcription in vitro afin de générer de l’ARNm. Ce dernier sera utilisé en réaction croisée au UV avec des extraits protéiques pour démontrer l’association du complexe ARNm/protéine. L’analyse de l’activité luciférase a permis d’identifier une séquence de 200 pb située entre 926 et 1134 pb de la région 3'UTR de l’ARNm CYP19A1 qui a réduit significativement l’activité de la luciférase. Selon les analyses de la réaction croisée au UV, une ou plusieurs protéines de 66 et 80 kDA se lient spécifiquement à la séquence de 200 pb qui réduit l’activité de luciférase. Cette protéine s'exprime dans les cellules de granulosa, mais n’a pas été détectée dans d'autres tissus comme le foie et le cœur. Par ailleurs, l’utilisation du gène rapporteur sensible à la FSH a suscité l’intérêt d'une compagnie pharmaceutique qui vend de l’equine chorionic gonadotropin (eCG) pour lui permettre de distinguer facilement l’eCG ayant une forte activité FSH et donc, avoir un produit commercial plus efficace et de meilleure qualité. Dans cette étude, nous avons développé un système de bioessai à la FSH basé sur la transfection des cellules avec un récepteur à la FSH et un gène rapporteur colorimètrique qui permet d’estimer l’activité de la FSH dans le sérum de la jument et qui pourrait être applicable au niveau de la ferme/industrie.
Oestradiol plays an important role in reproduction in general, particularly during follicular growth. Production of estradiol requires the expression of CYP19A1 following stimulation of granulosa cells by follicle-stimulating hormone (FSH) and insulin like growth factor-1 (IGF-1). In cows, there are six promoters (1.1, 1.2, 1.3, 1.4 and 1.5 and 2) that direct transcription of CYP19A1, and promoter 2 (P2) is the major promoter used in granulosa cells. However the effect of FSH and IGF-1 on the activation of these promoters of aromatase remains unclear. Further, the CYP19A1 gene has a very short half-life and a long 3' non-translated region (3'UTR) that suggests post-transcriptional as well as transcriptional regulation. The aim of my PhD project is to study the regulation of the CYP19A1 gene in the cow. This summary is divided into two parts, the transcriptional regulation involving the promoter region and the post-transcriptional regulation involving the 3'UTR. The first part of my project was to study the transcriptional regulation of CYP19A1 gene; we measured the expression of the different promoters in luteinized or nonluteinized bovine granulosa cells following stimulation of cells with FSH or IGF-1. The results of RT-PCR showed that FSH and IGF-1 increases mRNA levels from both promoters 2 and 1.1 in non luteinized granulosa cells. Subsequent experiments showed that FSH stimulates the promoter 2 via the PKA pathway and IGF-1 stimulated promoter 2 via the PKC pathway. FSH and IGF-1 stimulate the expression of 1.1 via the PI3K pathway. In subsequent studies in luteinized cells with luciferase reporter genes driven by the specific CYP19A1 promoters, FSH stimulated promoter 1.1 in a dose dependent manner but that promoter 2 was weakly activated and not responsive to FSH. We then compared the activity of human, rat, goat and bovine promoters in luteinised bovine granulosa cells. The most significant result is that the bovine (and caprine) P2 depends on several transcription factors (NR5A2, FOXL2) whereas the human and rat promoters largely depend on cAMP. In fact, these data demonstrate a reasonably robust expression of the bovine P2 when treated with forskolin, NR5A2 and FOXL2. FOXL2 appears to be a determinant of promoter activity in ruminants. The second part of my project was to study the post-transcriptional regulation of the CYP19A1 gene. The objective was to identify the elements required for the regulation of the half-life of CYP19A1 mRNA. To do so, we generated and inserted different fragments of the 3'UTR region of CYP19A1 mRNA in the pGL3promoter vector downstream of the luciferase gene, which was then transfected into luteinized granulosa cells to assess the impact of the 3'UTR sequence on the expression of luciferase reporter gene. We identified a sequence of 200 bp between 926 and 1134 bp of the 3'UTR region of CYP19A1 mRNA that significantly reduced luciferase activity. The same fragments were inserted into the pGEMTeasy vector for in vitro transcription and the generation of mRNA for UV crosslinking with protein extracts to demonstrate the presence of mRNA/protein complexes. We detected protein complexes of 66 and 80KDA that specifically bound to the 200 pb probe. This protein is expressed in granulosa cells but not in other tissues such as the liver and heart. The use of reporter gene attracted the interest of a company producing equine chorionic gonadotropin (eCG), and an interest was expessed in developing this system to measure the FSH-like bioactivity in eCG, and therefore have a more effective commercial product. In this study, we developed a FSH bioassay system based on the transfection of cells with an the FSH receptor and a colorimetric reporter gene to estimate the activity of FSH in the serum of the mare ; these results may be applicable at the farm / industry.

Tesis (Grado Doctor en Ciencias Biológicas)--Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Lugar de Trabajo: Cátedra de Diversidad Animal II. Facultad de Ciencias Exactas, Físicas y Naturales. Universidad Nacional de Córdoba. 2013- 151 h. con Apéndices + CD. tabls.; ils.; grafs. Contiene Referencia Bibliográfica, y Copia de Publicaciones Derivadas de la Tesis. Abstract en español e inglés.
El objetivo general de la presente Tesis Doctoral fue realizar un análisis integral, que contemple el efecto de la exposición a contaminantes ambientales sobre biomarcadores de diferentes niveles de organización (moleculares, histológicos y somáticos) en peces. Para ello se evaluó la respuesta de los genes cyp19a1 (aromatasa), la histología de las gónadas y los índices somáticos en machos de la especie íctica autóctona Jenynsia multidentata tanto en condiciones de laboratorio como a campo. En bioensayos se analizaron los cambios en los biomarcadores frente a la exposición a 17β‐estradiol, 17α‐etinilestradiol, 4n‐nonilfenol y 17α‐metiltestosterona; mientras que a campo se realizó una caracterización de la calidad del agua y de los niveles de expresión de los genes cyp19a1, y se compararon las respuestas de los biomarcadores entre dos sitios con diferente calidad de agua de la cuenca del Río Suquía. Los resultados obtenidos en esta tesis demuestran que la aromatasa cerebral responde como un biomarcador sensible frente a la exposición a compuestos que presentan actividad estrogénica en condiciones de laboratorio, respondiendo incluso ante la concentración más baja de estos compuestos.El análisis histológico de los testículos reveló un gran impacto de la exposición a los diferentes químicos sobre este órgano. A diferencia de lo observado a nivel molecular y tisular, los índices somáticos (Factor de Condición, Índice Hepatosomático e Índice Gonadosomático) no mostraron marcados cambios frente a la exposición a compuestos estrogénicos. A campo se observaron fluctuaciones de la expresión de la aromatasa cerebral a lo largo del año, siendo máxima durante la estación reproductiva en ambos sitios de muestreo, sugiriendo su implicancia en el control del ciclo reproductivo. Se observó un desfasaje en el incremento de la expresión del gen entre los sitios, registrándose el aumento con un mes de retraso en el sitio contaminado respecto al de referencia,lo cual podría estar relacionado con desfasajes en el comienzo del ciclo reproductivo. Los índices somáticos revelaron efectos adversos debidos a la exposición a poluentes ambientales y la histología de los testículos se vio severamente afectada en el sitio contaminado,evidenciando lesiones que incluyeron la desorganización del tejido testicular, alteraciones severas de las células germinales, presencia de células vacuolares, desincronización del proceso de espermatogénesis y necrosis. Las concentraciones de los compuestos utilizados para los ensayos de laboratorio se encuentran dentro del rango detectado en aguas superficiales de Argentina,demostrando que los peces están expuestos a estos niveles de contaminantes en el ambiente. Los cambios deletéreos observados en J. multidentata frente a la exposición crónica a contaminantes ambientales (tanto en condiciones de laboratorio como a campo), podrían afectar su capacidad reproductiva y representar una amenaza para la especie.

O cancro da mama é uma patologia de grande impacto na população feminina, devido à grande incidência, prevalência e morbimortalidade a que está associada. O património genético representa um papel importante nesta entidade nosológica. No entanto, mutações que conferem alto risco para esta doença, apenas explicam uma diminuta porção dos casos. Genes de baixa penetrância evidenciam um importante papel na suscetibilidade e têm sido alvo de estudo nas diversas populações. Trabalhos desenvolvidos pelo nosso grupo, na população que o Centro Hospitalar Cova da Beira abrange, apontaram para uma associação entre polimorfismos de genes de baixa penetrância e cancro da mama. São eles “CYP19A1 Trp39Arg (T/C)”, “GSTT1 null” e “GSTM1 null”. Realizámos um trabalho do tipo caso-controlo, em que abordámos uma amostra de 37 mulheres pertencentes à população do Centro Hospitalar Cova da Beira. Foi estudado se os 3 polimorfismos (“CYP19A1 Trp39Arg (T/C)”, “GSTT1 null” e “GSTM1 null”) apresentam diferentes níveis de expressão, entre mulheres com diagnóstico de cancro da mama, mas com prognóstico distinto. Focámo-nos assim em dois principais parâmetros, “Estadio clínico” e “Envolvimento ganglionar regional”. Neste trabalho de investigação foram utilizadas amostras de tecido de mama fixadas e incluídas em parafina. A técnica usada para quantificar a expressão génica foi PCR em tempo real. Para a análise estatística recorreu-se ao teste de qui-quadrado de Pearson e a testes não paramétricos. Para calcular as diferenças na expressão foi utilizado o método comparativo de CT, 2-??CT. Os resultados obtidos, para os polimorfismos de GSTT1 e GSTM1, foram influenciados pela diminuta quantidade de RNA extraída do tecido em parafina. Contudo, apesar da difícil extração de RNA, obteve-se uma elevada eficácia nas metodologias laboratoriais que dependeram deste procedimento, cerca de 81,1%. No que concerne ao polimorfismo CYP19A1 Trp39Arg, detetou-se expressão em cerca de 36,7% dos casos estudados. Após validarmos os resultados obtidos verificámos que a expressão entre 2 grupos de diferente estadio clínico é distinta. O grupo com “Estadio III” expressa 4,33 vezes mais CYP19A1 Trp39Arg que o grupo no “Estadio I e II”, sendo que a presença ou ausência dos Recetores de estrogénio tem que ser considerada para a correta interpretação dos resultados. Especulamos que a expressão deste polimorfismo da aromatase poderá ter algum tipo de impacto na determinação do prognóstico do cancro da mama, nesta população estudada.
Breast cancer is a pathology with great impact in female population, because it is associated with considerable incidence, prevalence, morbidity and mortality. Genetic heritage represents an important role in this nosological entity. However, mutations that confer high risk of developing this disease only explain a small proportion of the cases. Low penetrance genes are an important susceptibility factor and are being the target of several studies in populations. Research accomplished by our group within the population that Centro Hospitalar Cova da Beira covers, pointed to an association between low penetrance genes polymorphisms and the risk of breast cancer. These polymorphisms are “CYP19A1 Trp39Arg (T/C)”, “GSTT1 null” and “GSTM1 null”. We conducted a case-control study in a sample of 37 women belonging to the population covered by Centro Hospitalar Cova da Beira. Our main goal was to determine if the 3 polymorphisms (“CYP19A1 Trp39Arg (T/C)”, “GSTT1 null” and “GSTM1 null”) have different expression levels, between women diagnosed with breast cancer, but with distinct prognoses. Our focus was based in two main parameters, “Clinical staging” and “Regional lymph node involvement”. In this research work we used paraffin embedded tissues of breast. Expression quantification was accomplished by Real Time PCR. Statistical analysis was performed recurring to the Pearson’s chi-squared test and nonparametric tests. To estimate expression levels differences we used the CT comparative method, 2-??CT. GSTT1 and GSTM1 results were influenced by small RNA quantities extracted from the paraffin embedded tissues. Nevertheless, despite difficult RNA extraction, we obtained a high efficacy in the laboratory methodologies depending on this procedure, about 81.1%. Regarding CYP19A1 Trp39Arg expression, 36.7% of the studied cases were detected. After result validation we noticed that this expression was different between two groups of distinct clinical staging. “Stage III” expresses 4.33 more CYP19A1 Trp39Arg than “Stages I and II”, being aware that the presence or absence of the Estrogen receptor has to be considered to interpret the results correctly. We speculate that this aromatase polymorphism expression may have some kind of impact in the determination of breast cancer prognoses in the studied population.