京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13362号 / 論医博第2204号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 稲垣 暢也, 教授 渡邉 大, 教授 影山 龍一郎 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The nuclear receptor superfamily comprises a group of ligand-activated transcription factors that regulate the expression of target genes. They play a central role in diverse physiological pathways, and are therefore extremely important in the aetiology of various human disorders and as pharmaceutical therapeutic targets. This thesis describes molecular analyses of the thyroid hormone receptor (TR) and the peroxisome proliferator-activated receptor gamma (PPARγ), in disorders of thyroid hormone and insulin action respectively. The syndrome of Resistance to Thyroid Hormone (RTH), characterized by reduced tissue responsiveness to circulating thyroid hormones, is associated with diverse mutations in the ligand-binding domain of the thyroid hormone β receptor, localizing to three clusters around the hormone binding cavity. The first part of this thesis describes three novel RTH mutations (S314C, S314F, S314Y), due to different amino acid substitutions in the same codon, occurring in six separate families. Characterization of these mutant receptors showed marked differences in their functional impairment. In the second part of the thesis I report detailed functional studies of natural and synthetic receptor agonists with loss-of-function PPARγ mutants (P467L; V290M), previously identified in patients with severe insulin resistance, type 2 diabetes mellitus and hypertension. Both PPARγ mutants act as dominant negative inhibitors of wild type receptor (WT) action because of their failure to fully dissociate from corepressors. My results provide evidence that tyrosine-based rather than thiazolidinedione PPARγ agonists, may represent a more rational therapeutic approach to restoring mutant receptor function and ameliorating insulin resistance in our patients. Then, in an unrelated kindred a different, digenic mechanism of insulin resistance, with a combination of loss-of-function mutations in PPARγ and PPP1R3 (muscle-specific subunit of protein-phosphatase 1 mediating glycogen synthesis) is described. Functional characterisation of these mutant proteins provides unique insights into the complex interplay between this nuclear receptor and a second metabolic signalling pathway. Finally, three novel heterozygous mutations, in the ligand and DNA binding domains of PPARγ, identified in three unrelated subjects with partial lipodystrophy, severe insulin resistance, dyslipidaemia and hypertension are described. Their functional characterization suggests that they inhibit WT action via a novel, non DNA-binding interference mechanism.

Thyroid disruptions by xenobiotics have been associated with a broad spectrum of severe adverse human health effects, such as impaired brain development and metabolic syndrome. Ingestion of indoor dust and contact with industrial chemicals are two significant human exposure routes of thyroid hormone disrupting chemicals (THDCs), raising serious concerns for human health. However, it is a laborious and costly process to identify THDCs using conventional experimental methods, due to the number of chemicals in commerce and the varieties of potential disruption mechanisms. In this thesis, we are aimed at in silico identification of novel THDCs targeting transthyretin (TTR) and thyroid hormone receptor (THR) among dust contaminants and commonly used industrial chemicals. In vitro assays were used to validate the in silico prediction results. Co-crystallization and molecular dynamics (MD) simulations were applied to reveal binding modes of THDCs at the studied biological targets and to explain their intermolecular recognition. The main findings presented in this thesis are: 1. Over 144 environmental pollutants have been confirmed as TTR-binders in vitro and these cover a wide range of environmental pollutants and show distinct chemical profiles including a large group of halogenated aromatic compounds and a second group of per- and polyfluoroalkyl substances. (Paper I) 2. In total 485 organic contaminants have been reported to be detected in household dust. The developed QSAR classification model predicted 7.6% of these dust contaminants and 53.1% of their metabolites as potential TTR-binders, which emphasizes the importance of metabolic bioactivation. After in vitro validation, four novel TTR binders with IC50 ≤ 10 µM were identified, i.e. perfluoroheptanesulfonic acid, 2,4,2',4'-tetrahydroxybenzophenone (BP2), 2,4,5-trichlorophenoxyacetic acid, and 3,5,6-trichloro-2-pyridinol. (Paper II) 3. The development of a robust structure-based virtual screening (VS) protocol resulted in the prediction of 31 dust contaminants as potential binders to THRβ1 including musk compounds, PFASs, and bisphenol A derivatives. The in vitro experiments confirmed four compounds as weak binders to THRβ1, i.e. 2,4,5-trichlorophenoxyacetic acid, bisphenol A (3-chloro-2-hydroxypropyl) (2,3-dihydroxypropyl) ether, 2,4,2',4'-tetrahydroxybenzophenone, and 2,4-dichlorophenoxyacetic acid. (Paper III) 4. We revealed the binding conformations of perfluorooctanesulfonic acid, perfluorooctanoic acid, and BP2 in the thyroxine binding sites (TBSs) of TTR by co-crystallizing TTR with the three compounds. A VS protocol was developed based on the TTR complex structures that predicted 192 industrial chemicals as potential binders to TTR. Seven novel TTR binders were confirmed by in vitro experiments including clonixin, 2,6-dinitro-p-cresol (DNPC), triclopyr, fluroxypyr, bisphenol S, picloram, and mesotrione. We further co-crystallized TTR with PBS, clonixin, DNPC, and triclopyr, and their complex structures showed that the compounds bind in the TBSs as proposed by the VS protocol. In summary, 13 indoor dust contaminants and industrial chemicals were identified as THDCs using a combination of in silico and in vitro approaches. To the best of our knowledge, none of these compounds has previously been reported to bind to TTR or THR. The identifications of these THDCs improve our understanding on the structure-activity relationships of THDCs. The crystal structures of TTR-THDC complexes and the information on THDC-Target intermolecular interactions provide a better understanding on the mechanism-of-actions behind thyroid disruption. The dataset compiled and in silico methods developed serve as a basis for identification of more diverse THDCs in the future and a tool for guiding de novo design of safer replacements.

Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Timothy G. Rozell / An increase in pre-mating dietary energy positively influences ovulation and lambing rates, and this practice is known as nutritional flushing. The mechanisms of flushing, however, are still unknown. Increasing dietary energy approximately two weeks before breeding likely increases the production of insulin-like growth factor I (IGF-I) within the ovary, which stimulates the synthesis of follicle stimulating hormone receptor (FSHR). Several alternatively spliced transcripts of the FSHR have been identified in sheep. Each variant form is believed to be produced according to the stage of follicle development. This study was carried out to evaluate expression patterns of the FSHR variant forms (FSHR-1, FSHR-2 and FSHR-3) in the sheep ovary in response to different flushing diets. For this experiment, yearling Rambouillet ewes (n=93) were allocated among 6 different energy type treatment diets, either prairie or alfalfa hay based, for at least two weeks in combination with the insertion of a controlled internal drug releasing device (CIDR). Two of the treatment groups had commercially available block supplements provided and two had rolled corn supplemented. Mid-ventral laparotomy was performed on each ewe 3.5 to 4 days after CIDR removal. Follicles 4 mm and greater were aspirated and categorized as either medium (M; 4 to 6 mm) or large (L; > 6 mm). Total RNA was extracted from granulosa cells (GC) and reverse transcribed followed by qPCR of the resulting cDNA using specifically designed primer sets for each variant of the FSHR and for the LH receptor. Changes in live weight were different (P < 0.01) between treatment diets but there were no statistical differences for NEFA concentrations between any of the treatments nor were there differences for body condition (mean = 3.0) or lambing rate. Therefore, it is likely a flushing response did not occur in this study. Expression of FSHR-1was different between M and L follicles (P < 0.01) and tended to be different for ewes fed alfalfa hay (P = 0.05). Overall mean expression of FSHR-3 was greater than expression of FSHR-1 or FSHR-2 (P < 0.01), although there was no difference between M and L follicles, or between treatment diets. The concentration of estradiol in follicular fluid was not different between the treatment diets or follicle sizes nor was expression of lutenizing hormone receptor (LHR), indicating that follicles were similar developmentally. The FSHR-1 form seemed to be the variant most likely to be involved in later stages of follicular development, and is potentially involved in follicle rescue. For all follicles, FSHR-3 was the more highly expressed form of the FSHR and may likely be essential throughout antral follicle development. Further research is required to determine the exact mechanism whereby initial energy status of ewes seems critical for the increased ovulation rate that occurs after energy supplementation (i.e. the flushing response).

It has been shown that thyroid hormones are important in development and growth in birds and further that thyrotropin (TSH) signaling regulated photoinduced seasonal reproduction. In addition to controlling the development of certain physiological traits, TSH can affect a wide range of phenotypes related to domestication such as behaviour, growth rate, more frequent reproductive cycle’s, pigmentation and also behaviour. Studies indicate that thyroid hormone physiology could potentially be responsible for differences in individual stress response as well as differences in social dominance. This project investigated behaviours expressed in the different genotypes on the Thyroid stimulating hormone receptor (TSHR) gene in chickens. Standard test such as Fear of human, Aerial predator, Tonic immobility and Social hierarchy were carried out with White leghorn (WL) as a domesticated species and Red Junglefowl (Rjf) as their wild counterpart; these were considered to be the control group. There was no significant result on genotype effect for the TSHR animals observed in those variables tested. The TSHR mutants were expected to mirror the White leghorn behavioural response and the TSHR wildtype the behaviour of Rjf. This was however not confirmed. There were a significant interaction between genotype effect and sex effect for TSHR for stand alert in the Aerial predator test which mirrored the results seen in the control groups. The male wildtype followed the male Rjf pattern however the mutant did not mirror the WL male. This study would benefit from more individuals to be tested, for stronger statistical results, plus also to have all genotypes represented to fully investigate the affect the TSHR mutation have on domesticated chickens and potentially the domestication process in a range of species. / TSHR

Thyroid hormones are known to be important in several processes in chicken, such as growth, metabolism and reproductive system. In previous studies the thyroid stimulating hormone receptor (TSHR)-gene has been identified as a target for a selective sweep in commercial breeds of chicken such as broiler and White Leghorn. The evolution of domesticated species can be split into three periods. The first is the natural selection in their natural habitat, the second the beginning of the domestication process, when humans started to tame and breed the wild animals and the third is when animals were bred for commercial interests such as egg laying properties and meat production in chicken. Landraces, which are domesticated but not commercially bred races, are a great resource for identifying during which period a specific gene, which differs between wild type and commercial bred breeds, were selected. In this study Swedish landrace chickens were used in order to analyze the importance of a mutation in the TSHR-gene in the domestication process. The results of this study gave that all, except two individuals from the Bohuslän-Dals svarthöna were homozygous for the mutation known from commercial breeds. The two individuals from Bohuslän-Dals svarthöna were both heterozygous for the mutation. These results suggest that the TSHR mutation is important for the domestication process and were already more or less fixed at the commencement of commercial breeding. The mutation is thought to be dominant and to have an inhibitory impact on the TSHR activity. This might result in hypothyroidism which would make alterations in the reproductive system. This is plausible because the constant availability of food in captivity makes the seasonal reproductive system no longer critical for survival of progeny.

[Truncated abstract] It is becoming increasingly obvious that cell signalling pathways are more complicated than we originally perceived. Research is revealing that, not only is there a multitude of new proteins involved in signalling cascades, but also that previously identified proteins may have additional, alternate roles in intracellular trafficking. Gonadotropin-releasing hormone (GnRH) in conjunction with its receptor (GnRHR), the primary regulator of reproduction in all species, is no exception. In the past few years it has become readily accepted that the classic linear GnRHR-Gαq/11 signalling pathway is not universal and that this receptor is involved in a far greater range of cellular activities than was previously considered. In particular, it is widely accepted that continuous administration of GnRH analogs results in an inhibition of growth of a number of reproductive-derived tumours and that this may, in part, be mediated by direct activation of GnRHs expressed on these cells. However, it is not fully understood how the GnRHR mediates these growth effects or whether such effects are unique to reproductive-derived cancer cells. Research within this thesis aimed to determine how the presence or absence of this receptor in different cell types might affect the ability of GnRH to directly mediate growth effects. We demonstrate that continuous treatment with a GnRH agonist (GnRHA) induces an anti-proliferative effect in a gonadotropederived cell line (LβT2) and also in HEK293 cells stably expressing either the rat or human GnRHR. The anti-proliferative effect was time- and dose-dependent and was specifically mediated via the GnRHR, as co-treatment of the GnRHRexpressing cell lines with a GnRH antagonist blocked the growth suppressive effect induced by GnRHA treatment. Cell cycle analysis revealed that the GnRHA treated HEK/GnRHR cell lines induced an accumulation of cells in the G2/M phase while a G0/G1 arrest was observed in LβT2 cells. Previous identification by our group of a potential interaction between the GnRHR and the transcription factor E2F4, an integral cell cycle regulatory protein, prompted further investigation as to the nature of this interaction. Bioluminescence energy transfer (BRET) was utilised to demonstrate that the GnRHR also interacts with E2F5, another member of the E2F family of cell cycle proteins that shares a high level of homology to E2F4. In addition, it was determined that the interaction between human GnRHR and E2F4, detected using BRET, was influenced by cell density.

Orientador: Cesar Cabello dos Santos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T00:11:00Z (GMT). No. of bitstreams: 1 Morais_SirleiSiani_M.pdf: 4527732 bytes, checksum: 211f9fdc8eb9af5035b14ae6f421798e (MD5) Previous issue date: 2009 / Resumo: Objetivo: Comparar a capacidade de predição de estados dos receptores hormonais do modelo de lógica fuzzy em relação ao modelo de Gail em mulheres saudáveis que após cinco anos desenvolveram câncer de mama. Métodos: Este estudo retrospectivo avaliou os dados referentes ao perfil de 341 mulheres cinco anos antes do diagnóstico de câncer de mama, atendidas no ambulatório de mama do CAISM no período de janeiro de 2006 a junho de 2008. A resposta a quimioprevenção está diretamente associada ao estado dos receptores hormonais. Estudos de quimioprevenção utilizam em sua maioria, mulheres com alto risco de câncer de mama, calculado através do modelo de Gail. Utilizou-se um modelo desenvolvido através da lógica fuzzy, para classificar o risco do estado dos receptores hormonais, tendo sido avaliado a sua capacidade de predição, e comparando-se aos valores do risco do modelo de Gail, classificado em alto e baixo risco. O modelo de lógica fuzzy considerou idade, idade à menarca, número de biópsias prévias, o número de familiares afetados por câncer de mama e idade ao primeiro parto. Resultados: O modelo de lógica fuzzy apresentou uma sensibilidade de 89,3% (IC 95% = 85,5-93,1) e acurácia de 72,1% superiores ao modelo de Gail (49,6% e 50,7% respectivamente p < 0,0001), porém uma menor especificidade (23,6%; IC 95% = 14,8-32,4) em relação ao modelo de Gail (53,9%). Conclusão: A lógica fuzzy foi mais eficaz na predição do estado dos receptores hormonais em comparação ao modelo de Gail / Abstract: Background: To compare the capability of prediction of the state of hormonal receptors used in the fuzzy logic in relation to Gail's model for healthy women that after five years developed breast cancer. Methods: This retrospective study has avaliated the data concerning the profile of 341 women five years before the breast cancer diagnosis who were attend in the breast clinic at the Women's Integral Healthcare Center of Unicamp, between June 2006 and June 2008. The reaction to the chemotherapy is directly associated to the state of the hormonal receptors. Studies concerning chemotherapy as a preventive alternative use in most of the cases women with high risk of having breast cancer and the calculation is made through Gail's method. This study was used the model developed through Fuzzy logic in order to classify the risk concerning the state of the hormonal receptors, evaluating their capacity of prediction and comparing them to the values of risk in Gail's model- which is classified in high and law. In the Fuzzy logic model it was considered the age, the age at menarch, the number of previous biopsies, the number of relatives affected by breast cancer and the age of firt live birth. Results: the Fuzzy logic model presented a sensibility of 89.3% (CI 95% = 85.5- 93.1) e accuracy of 72.1%, higher to Gail's model (49.6% and 50.7% respectively for p < 0.0001), however with a smaller specificity (23.6%; CI 95% = 14.8-32.4) in relation to Gail's model (53.9%). Conclusion: the fuzzy logic was more efficient in predicting the state of hormonal receptors in comparison to Gail's model / Mestrado / Tocoginecologia / Mestre em Tocoginecologia

O CÃncer do colo uterino, permanece sendo uma patologia de grande incidÃncia (16,3/100 mil) e mortalidade (1.627/100 mil) no nordeste brasileiro; altamente correlacionado ao HPV (98%, por PCR). A relaÃÃo entre estÃgios evolutivos da neoplasia do colo uterino e receptores hormonais ainda à obscura. Verificou-se a expressÃo imunohistoquÃmica dos receptores hormonais RE e RP em diferentes estÃgios evolutivos do cÃncer do colo uterino em comparaÃÃo ao ciclo menstrual e idade. Em 144 amostras da cÃrvice uterina, analisaram-se 62 casos de LIEBG, 41 casos de LIEAG, e 41 casos de CEC. A expressÃo imunohistoquÃmica dos receptores hormonais foi avaliada em escores (1 a 9): intensidade da reaÃÃo (1 a 3) e dispersÃo celular (1 a 3), tanto no epitÃlio quanto no estroma.A correlaÃÃo entre os parÃmetros foi avaliada pelo Teste exato de Fisher-Yates. Houve diminuiÃÃo da expressÃo nuclear de RE e RP à medida da progressÃo tumoral. No estroma a expressÃo nuclear do RE e RP tendem a aumentar de LIEBG para LIEAG e CEC, embora nestes os escores fossem menores. Com relaÃÃo ao ciclo menstrual nÃo houve alteraÃÃes significativas entre a primeira e segunda fase do ciclo menstrual. As anÃlises prà e pÃs-menopausa nÃo evidenciaram variaÃÃes significativas na expressÃo dos receptores hormonais estudados. / Cervical cancer is associated with a very high incidence (16.3/100.000) and mortality (1,627/100.000) in northeastern Brazil; strongly correlated with HPV (98% by PCR). However, the relation between the stages of cervical cancer and the hormonal receptors ER and PR remains unclear. This study presents an immunohistochemical analysis of the morphological expression of the hormonal receptors ER and PR in different stages of cervical cancer. One hundred forty-four samples were obtained for analysis from the cervices of patients at a cancer screening service in northeastern Brazil in the years 2001 and 2002. Following gynecological examination colposcopically guided biopsies revealed apparently abnormal epithelia. Subsequently the specimens were classified in scores (1 to 9) according to hormonal receptor expression: intensity reaction (1 to 3) versus cell-marked dispersion (1 to 3).Sixty-two cases of LSIL, 41 cases of HSIL and 41 cases of ISCC were compared with regard to menstrual cycle and age. In SIL nuclear reactivity to ER decreased gradually from the lower to the higher stages, including that of ISCC. Stromal marked nuclei were seen in SIL and tended to increase in ISCC in spite of displaying lower scores. There were no significant differences for ER and PR expression related to menstrual cycle phases or menopausal status. Pre- and post-menopausal analyses showed no significant variation in the expression of either ER or PR.