yes / Thyroid hormone (TH) is essential for adult brain function and its actions include several key roles in the hypothalamus. Although TH controls gene expression via specific TH receptors of the nuclear receptor class, surprisingly few genes have been demonstrated to be directly regulated by TH in the hypothalamus, or the adult brain as a whole. This study explored the rapid induction by TH of retinaldehyde dehydrogenase 1 (Raldh1), encoding a retinoic acid (RA)-synthesizing enzyme, as a gene specifically expressed in hypothalamic tanycytes, cells that mediate a number of actions of TH in the hypothalamus. The resulting increase in RA may then regulate gene expression via the RA receptors, also of the nuclear receptor class. In vivo exposure of the rat to TH led to a significant and rapid increase in hypothalamic Raldh1 within 4 hours. That this may lead to an in vivo increase in RA is suggested by the later induction by TH of the RA-responsive gene Cyp26b1. To explore the actions of RA in the hypothalamus as a potential mediator of TH control of gene regulation, an ex vivo hypothalamic rat slice culture method was developed in which the Raldh1-expressing tanycytes were maintained. These slice cultures confirmed that TH did not act on genes regulating energy balance but could induce Raldh1. RA has the potential to upregulate expression of genes involved in growth and appetite, Ghrh and Agrp. This regulation is acutely sensitive to epigenetic changes, as has been shown for TH action in vivo. These results indicate that sequential triggering of two nuclear receptor signalling systems has the capability to mediate some of the functions of TH in the hypothalamus.

No description available.

Orientador: Maria Cecília Rui Luvizotto / Coorientador: Heitor Flávio Ferrari / Banca: Daniela Bernadete Rozza / Banca: Livia Castanhas Breganó / Resumo: O tumor mamário canino (TMC) é a neoplasia que mais comumente acomete cadelas idosas não castradas. Na análise histopatológica, 41 a 53% dos TMCs são classificados como malignos. Os receptores de estrógeno α (REα) são receptores nucleares importantes na transcrição de fatores e transdução de sinais hormonais, considerados um dos fatores prognósticos para o tumor mamário humano, participam de forma ativa na carcinogênese mamária. Durante o desenvolvimento do TMC, macrófagos teciduais compõe o microambiente tumoral, com a função de estimular a angiogênese, aumentando a possibilidade de metástases, sendo fundamental para o prognóstico de neoplasia mamária na cadelae na mulher. O objetivo deste trabalho foi pesquisara expressão e a provável correlação de REα com a distribuição de macrófagos tumorais (TAMs) em TMCs por meio de imunohistoquímica. Foram analisados 40 tumores mamários malignos, classificados histologicamente em quatro grupos distintos e um grupo controle composto por 08 glândulas mamárias caninas sem alteração anatomopatológica e seus respectivos linfonodos regionais. Os resultados mostraram que a imunomarcação positiva ao REα variou de um a três casos dentre os grupos histológicos estudados, havendo nestes, menor número de metástase para linfonodo. A positividade para os TAMs mostrou associação com o tipo histológico, variando de moderada a acentuada nos carcinomas mamários de alto grau histológico que apresentaram necrose, invasão linfática e formação tubular. Não ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The canine mammary tumor (CMT) is the neoplasia that most commonly affects uncastrated elderly female dogs. On histopathological analysis, 41 to 53% of CMTs are classified as malignant. Estrogen receptors α (REα) are important nuclear receptors for transcription factors and signal transduction of hormones, considered prognosis factor for breast cancer in human, and recognized as actively involved in breast carcinogenesis. During the development of mammary cancer in canine species, macrophages composes the tumor microenvironment, stimulating angiogenesis, facilitating the local dissemination of neoplastic cells and increasing the possibility of metastasis, being fundamental for mammary neoplasia prognosis in dogs and women. This study aimed to evaluate the expression and the probable correlation of REα with the distribution of tumor associated macrophages (TAMs) in CMTs by immunohistochemistry. Forty CMTs were analyzed histologically in four distinct groups and a control group composed of eight canine mammary glands without alteration anatomopathological and their respective regional lymph nodes. The results showed that the REα positive immunostaining varied from one to three cases among the histological groups studied, and there was less lymph node metastasis. The positivity for TAMs showed a positive correlation with the histological type, ranging from moderate to severe in mammary carcinomas of high histological grade that presented necrosis, lymphatic invasion and tubular ... (Complete abstract click electronic access below) / Mestre

Receptores nucleares formam uma superfamília de proteínas responsáveis pela regulação da expressão de genes. Estruturalmente, são formados por três domínios: um domínio N-terminal bastante variável, um domínio altamente conservado de ligação com o DNA e um domínio C-terminal, menos conservado, denominado domínio de ligação com o ligante (LDB). Diversos experimentos mostram que a interação com o ligante afeta a estrutura e a mobilidade da hélice C-terminal dos receptores nucleares (hélice 12 do domínio de ligação com o ligante), sendo o principal mecanismo de ativação e repressão da transcrição. As primeiras estruturas de LBDs de receptores nucleares revelaram importantes diferenças entre estruturas contendo ligantes (holo) e estruturas apo, principalmente no que diz respeito a posição da hélice 12: em estruturas apo, foi observada a H12 em uma conformação aberta, expondo o sítio de ligação com o ligante, enquanto que em estruturas holo, foi observada a H12 em uma conformação fechada, dobrada sobre o corpo do LBD e envolvendo completamente o ligante. Essa diferença sugeriu um mecanismo para a entrada e saída de ligantes do sítio de ligação denominado modelo da ratoeira, entretanto, esse modelo apresenta diversas inconsistências e tem sido desacreditado. Estudos experimentais e teóricos recentes mostram que a hélice 12 é mais móvel na ausência de ligantes, entretanto, esses estudos não fornecem evidencias de que o aumento da mobilidade da está associado com o deslocamento da H12 em relação ao corpo do LBD, como sugerido pelo modelo da ratoeira. Embora esteja claro que a hélice 12 é mais móvel na ausência de ligantes, a dimensão da variação conformacional sofrida pela hélice 12 ainda não está clara. Nesse trabalho buscamos a construção de um modelo capaz de dimensionar a mobilidade da hélice 12 através da comparação direta entre simulações de dinâmica molecular e experimentos de anisotropia de fluorescência resolvida no tempo. Utilizando simulações de dinâmica molecular reproduzimos experimentos de anisotropia de fluorescência acoplando a sonda cys-flúor a hélice 12 do PPARγ para estudar sua mobilidade. Mostramos que as observações experimentais só podem ser explicadas por conformações onde a sonda fluorescente permanece presa a superfície do LBD. Foi mostrado também que curvas de anisotropia com decaimentos comparáveis com os decaimentos experimentais estão associados a pequenas variações conformacionais de hélice 12. Simulações para dois modelos de apo-PPARγ com a H12 aberta em relação ao corpo do LBD e para as estruturas cristalográficas de apo-RXR e apo-ER, onde a H12 também adota uma conformação aberta, revelaram curvas de anisotropia com decaimentos mais rápidos que os experimentais. Esses resultados implicam em um modelo onde a H12 sofre alterações conformacionais locais, não apresentando variações tão dramáticas como o proposto pelo modelo da ratoeira. / Nuclear Hormone Receptors comprise a protein superfamily responsible for regulation of gene expression. Structurally, they are composed by three domains: a variable N-terminal domain, a highly conserved DNA-binding domain (DBD), and a less conserved C-terminal domain, known as ligand binding domain (LBD). Many experiments have shown that the interaction with ligands affects the structure and the mobility of nuclear receptors C-terminal helix (LBDs Helix 12), being the main mechanism of transcription activation and repression. The first nuclear receptor LBDs structures revealed important differences between ligand bound (holo) and apo-structures concerning the position of the H12: in apo structures, H12 adopted an open conformation, exposing the ligand binding pocket, whereas in holo structures, the H12 was closed, packed over the body of the LBD, burying completely the ligand. This difference suggested a mechanism for ligand entry and exit from the binding pocket called mouse-trap model, however this model has several inconsistencies and has been discredited. Recent experimental and theoretical studies have shown that H12 is more labile in the absence of ligand, but these studies dont provide evidences that the increase in the mobility is associated with the detachment of H12 from the body of the LBD as suggested by the mouse-trap model. Although its clear that H12 is more flexible in the absence of ligands, the size of the conformational changes undergone by H12 is not yet clear. In this work we seek to construct a definitive model for the range of motions that H12 may undergo in the presence or absence of ligand using molecular dynamics simulations. Through direct comparison between molecular dynamics simulations and time-resolved fluorescence anisotropy experiments, we show that experimental observation can only be explained by conformations where the fluorescent probe is interacting with the surface of the PPARγ surface. We also show that simulations with anisotropy decay rates comparable to the experimental decay are associated with small helix 12 conformational changes. Simulations with two models of apo-PPARγ with H12 detached from the body of the LBD and with crystallographic structures of apo-RXR and apo-ER, where the H12 also is in an open conformation, display anisotropy decay rates significantly faster than the experimental ones. These results imply a model for the molecular mobility of the LBD where H12 undergoes local conformational changes and should exhibit dynamic properties less dramatic than proposed by the mouse trap model.

Os receptores tireoidianos (TRs) são proteínas envolvidas em várias funções fisiológicas importantes para os organismos, pois são potentes reguladores do desenvolvimento, divisão e diferenciação celular, metabolismo e homeostase. Eles são responsáveis pela regulação da transcrição de genes-alvo específicos, mediando efeitos pleiotrópicos de hormônios lipofílicos nas células. Na ausência de ligantes essas proteínas estão complexadas a correpressores, impedindo a transcrição de genes por elas regulados. Por outro lado, a presença do ligante induz à transcrição através da ligação a elementos responsivos do DNA e coativadores. Nesse trabalho alguns aspectos do TR foram evidenciados, permintindo-se um melhor conhecimento acerca do funcionamento e estrutura desse receptor. Os experimentos de oligomerização revelaram a presença dos tetrâmeros do TR, os quais estavam restritos ao Receptor X Retinóico, sugerindo mecanismos novos na regulação do receptor. Os ensaios de raios-X a baixos ângulos resultaram nos primeiros modelos estruturais de baixa resolução de construções maiores do TR, demonstrando o correto posicionamento de seus domínios em sua estrutura geral, o que forneceu informações importantes sobre sua estrutura geral. Os experimentos de fluorescência avaliaram a ligação desses receptores a diversos elementos responsivos, em termos de constantes de dissociação e seletividade para cada um deles. E, por fim, os experimentos de troca de hidrogênio por deutério revelaram a movimentação que ocorre no domínio de ligação do ligante do receptor antes e após a adição do ligante T3. Esses resultados ampliam um pouco mais os conhecimentos sobre os mecanismos de ação e sobre a estrutura quaternária dos TR, promovendo um melhor entendimento dos conceitos básicos envolvidos na atuação dessas macromoléculas, as quais estão inseridas em redes complexas de regulação e interação com outras proteínas. / The thyroid receptors (TRs) are proteins, which are involved in diverse and important physiological functions in the organisms, since they are regulators of development, cell divison and differentiation, metabolism and homeostasis. They are responsible by the regulation of specific gene transcription, through pleiotropic effects of lipophilic hormones in the cells. In the absence of the ligand these proteins are complexed to correpressors and block the transcription of genes that are regulated by them On the other hand, in the presence of the ligand transcription is induced through the binding of the receptors to DNA response elements and coactivators. New findings about TR described in this study helped to improve the understanding of the function and structure of the receptor. This was accomplished by: oligomerization experiments which showed the presence of TR tetramers, a quarternary structure described before only for the Retinoid Receptor X, and suggested new regulation mechanisms for the receptors; the small angle X-ray scattering assays which resulted in the first low resolution structural models of bigger constructions of TR, showing the correct position of TR domains and providing important information about the global TR structure; the anisotropy fluorescence experiments which evaluated the binding of these receptors to diverse response elements, in terms of dissociation constants and selectivity for each one of the HREs tested; and finally, the hydrogen/deuterium experiments which revealed the ligand binding domain mobility before and after the ligand addition. In summary, we can say that these results all together extended the knowledge about the TR action mechanisms and its quarternary strucuture, providing better understanding of the basic concepts involved in these macromolecules behavior, which are inserted into a complex network of regulation and interaction with other proteins.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Infertility is defined as not being able to conceive after 1 year of regular intercourse without use of contraception. Unexplained infertility is a diagnosis given to couples where the reason to infertility cannot be clarified even after the routine examination. Undefined infertility is a common and growing problem because most people are not aware of the fact that fertility decreases after the age of 35. Hyper- and hypothyroidism has been known to affect the menstrual cycle as well as increased risk of miscarriage. However, the specific effect of thyroid hormones on infertility has not yet been clarified. This study aims to compare the gene expression of two thyroid hormone receptors DIO2 and MOT8 in human endometrium and fallopian tube tissue from two phases of the menstruation cycle, follicular phase and lutheal phase. The methods used were RT-qPCR and immunohistochemistry, which showed a statistically significant difference in the expression of DIO2 and MOT8 between fallopian tube tissue and endometrium, but not between follicular and lutheal phase. However, MOT8 seemed to have a tendency to be down-regulated in the follicular phase but the results need to be validated with different endogenous controls and larger study groups.

Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T18:46:34Z : No. of bitstreams: 1 nogueira_mfg_dr_botfmvz.pdf: 892396 bytes, checksum: 471e1cec0bdbf86073d92bc94c87f460 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Em células da teca e da granulosa, de folículos bovinos, foram detectados quatro transcritos alternativos do receptor do hormônio luteinizante (LHR). Apenas dois deles são traduzidos em proteínas funcionais com afinidades distintas em relação aos ligantes. Em humanos e símios, a isoforma completa (full-length) tem afinidade pelo LH e hCG, enquanto que a isoforma que apresenta deleção do exon 10 tem afinidade somente pelo hCG. Além disso, isoformas com deleção do exon 3 foram observadas em ratos, embora nenhum outro estudo tenha investigado essa região do gene bovino. Objetivouse com este trabalho caracterizar o padrão da expressão do gene do LHR nas células da teca e da granulosa de folículos antrais bovinos. Ovários foram coletados em matadouro, os folículos (5-14 mm) foram dissecados e as células da teca e da granulosa separadas para extração de RNA total com Trizol. As concentrações de esteróides no fluido folicular foram determinadas por radioimunoensaio (RIE). A expressão gênica do LHR foi mensurada por RTPCR semiquantitativo com oligonucleotídeos iniciadores (primers) específicos para amplificar o fragmento entre o final da região extracelular e o final da intracelular (LHRBC; primers posicionados nos exons 9 e 11). A ocorrência de transcritos alternativos oriundos do início da região extracelular (LHRA; primers posicionados nos exons 2 e 9) foi investigada mediante amplificação por RT-PCR. Como controle interno no PCR, utilizou-se a expressão da GAPDH. Paralelamente, células da granulosa cultivadas in vitro foram tratadas com 1 ou 10 ng de FSH no meio de cultura. Mediante RT-PCR, foi investigada a expressão das isoformas do LHRBC nas células da granulosa cultivadas e tratadas com FSH... / Growth of dominant follicle in the absence of circulating FSH and the events following the LH surge that culminate in ovulation, are dependent on the interaction between LH and its receptor (LHR). Four LHR alternative transcripts were described in theca and granulosa cells from bovine follicles. Only two of them can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands. In humans and marmosets, the full-length isoform has affinity to both LH and hCG molecules, whereas the isoform with deletion of only exon 10 has affinity to hCG exclusively. Additionally, isoforms with deletion of exon 3 were observed in rats, although no previous report have investigated this region of the bovine gene. The objective of this study was to characterize the pattern of gene expression of the LHR in theca and granulosa cells from bovine antral follicles. Additionally, LHR expression was determined in cultured granulosa cells under FSH treatment. From ovaries collected in abattoir, antral follicles were dissected (5 to 14mm of diameter), and samples of theca and granulosa cells were obtained to total RNA extraction (Trizol protocol). Steroids concentrations in the follicular fluid were determined by RIA. Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify part of extracellular region (LHRA; primers annealing on exons 2 and 9) and the fragment from the end of extracellular region, including the transmembrane domain and finishing near the end of intracellular region (LHRBC; primers annealing on exons 9 and 11). As internal control of the PCR, it was used GAPDH expression. Cultured granulosa cells were treated with 0, 1 or 10 ng of FSH (3 replicates each dose). As in vivo and positive control, theca cell sample was utilized to comparison... (Complete abstract, access undermentioned electronic address)

Orientador: Ciro Moraes Barros / Resumo: Em células da teca e da granulosa, de folículos bovinos, foram detectados quatro transcritos alternativos do receptor do hormônio luteinizante (LHR). Apenas dois deles são traduzidos em proteínas funcionais com afinidades distintas em relação aos ligantes. Em humanos e símios, a isoforma completa ("full-length") tem afinidade pelo LH e hCG, enquanto que a isoforma que apresenta deleção do exon 10 tem afinidade somente pelo hCG. Além disso, isoformas com deleção do exon 3 foram observadas em ratos, embora nenhum outro estudo tenha investigado essa região do gene bovino. Objetivouse com este trabalho caracterizar o padrão da expressão do gene do LHR nas células da teca e da granulosa de folículos antrais bovinos. Ovários foram coletados em matadouro, os folículos (5-14 mm) foram dissecados e as células da teca e da granulosa separadas para extração de RNA total com Trizol. As concentrações de esteróides no fluido folicular foram determinadas por radioimunoensaio (RIE). A expressão gênica do LHR foi mensurada por RTPCR semiquantitativo com oligonucleotídeos iniciadores ("primers") específicos para amplificar o fragmento entre o final da região extracelular e o final da intracelular (LHRBC; "primers" posicionados nos exons 9 e 11). A ocorrência de transcritos alternativos oriundos do início da região extracelular (LHRA; "primers" posicionados nos exons 2 e 9) foi investigada mediante amplificação por RT-PCR. Como controle interno no PCR, utilizou-se a expressão da GAPDH. Paralelamente, células da granulosa cultivadas in vitro foram tratadas com 1 ou 10 ng de FSH no meio de cultura. Mediante RT-PCR, foi investigada a expressão das isoformas do LHRBC nas células da granulosa cultivadas e tratadas com FSH... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Growth of dominant follicle in the absence of circulating FSH and the events following the LH surge that culminate in ovulation, are dependent on the interaction between LH and its receptor (LHR). Four LHR alternative transcripts were described in theca and granulosa cells from bovine follicles. Only two of them can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands. In humans and marmosets, the full-length isoform has affinity to both LH and hCG molecules, whereas the isoform with deletion of only exon 10 has affinity to hCG exclusively. Additionally, isoforms with deletion of exon 3 were observed in rats, although no previous report have investigated this region of the bovine gene. The objective of this study was to characterize the pattern of gene expression of the LHR in theca and granulosa cells from bovine antral follicles. Additionally, LHR expression was determined in cultured granulosa cells under FSH treatment. From ovaries collected in abattoir, antral follicles were dissected (5 to 14mm of diameter), and samples of theca and granulosa cells were obtained to total RNA extraction (Trizol protocol). Steroids concentrations in the follicular fluid were determined by RIA. Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify part of extracellular region (LHRA; primers annealing on exons 2 and 9) and the fragment from the end of extracellular region, including the transmembrane domain and finishing near the end of intracellular region (LHRBC; primers annealing on exons 9 and 11). As internal control of the PCR, it was used GAPDH expression. Cultured granulosa cells were treated with 0, 1 or 10 ng of FSH (3 replicates each dose). As in vivo and positive control, theca cell sample was utilized to comparison... (Complete abstract, access undermentioned electronic address) / Doutor

L'épithélium intestinal est caractérisé par un renouvellement et une différenciation continus dépendant des cellules souches somatiques situées au fond des cryptes. Le renouvellement rapide est assuré par plusieurs réseaux de voies signalisation dont la dérégulation peut être à l'origine de l'initiation et/ou de la progression tumorale. Mon laboratoire d'accueil a décrit l'implication des hormones thyroïdiennes et de leur récepteur nucléaire TRa1 dans le contrôle de l'homéostasie de l'épithélium intestinal via la régulation de la voie Wnt/b-caténine d'une part et l'implication de TRa1 dans l'induction de tumeurs intestinales grâce à des souris surexprimant TRa1 d'autre part. De plus, dans un contexte APC muté, l'expression transgénique de TRa1 accélère la progression tumorale et favorise la dissémination métastatique. Des analyses transcriptomiques mettent en évidence une forte activation de la voie Wnt par TRa1. Ces résultats ont été confirmés chez l'homme en étudiant la régulation de la voie Wnt par TRa1 dans des carcinomes colorectaux (CRC). Nous avons confirmé la surexpression de TRa1 dans les tumeurs humaines et validé son impact sur la voie Wnt tant dans les tumeurs humaines que dans des lignées cellulaires et sur leur agressivité. L'ensemble des données montre une forte implication de TRa1 dans la tumorigenèse chez la souris et chez l'homme et ouvrent des portes pour des recherches visant TRa1 comme cible de traitement thérapeutique contre le cancer / The intestinal epithelium is characterized by constant renewal and differentiation due to the presence of stem cells located at the bottom of the crypts. This permanent renewal depends on the crosstalk between several signaling pathways whose alteration can lead to tumor initiation and progression. Our team demonstrated the implication of the thyroid hormones and their nuclear receptor TRa1 in the control of the intestinal epithelium homeostasis through the regulation of the Wnt pathway. Moreover, the overexpression of TRa1 in the intestinal epithelium of mice is sufficient to promote tumor initiation, and in an APC loss of function context, it accelerates tumor progression highlighting its oncogenic potential. Through gene expression profiling, we highlighted an activation of the Wnt pathway activity by TRa1 during tumor progression. We next confirm these results in human patient samples by demonstrating that high TRa1 expression in tumors invariably is associated with an increased Wnt pathway activity. In addition, in CRC cell lines, TRa1-associated WNT pathway activation enhances their aggressiveness. Altogether these results showed the implication of TRa1 in intestinal carcinogenesis and open avenues for new therapeutic treatment against TRa1 targeting TRa1