A vasodilatação promovida pela triiodotironina (T3) ocorre por sua ação direta sobre o relaxamento das células musculares lisas vasculares (CMLV), porém os mecanismos envolvidos são desconhecidos. Neste estudo mostramos que o T3 rapidamente relaxa as CMLV através da geração de óxido nítrico (NO), via óxido nítrico sintase neuronal e induzível (nNOS e iNOS), efeitos mediados pela sinalização PI3K/Akt. Ensaios funcionais em aortas sem endotélio, incubados com T3, mostraram menor resposta contrátil a Fenilefrina (FE), efeito este revertido pelo L-NAME, inibidor da NOS. Aortas de ratos hipertiroideos apresentaram aumento do receptor de Angiotensina II (AngII) do tipo 2 (AT2), acompanhado de diminuição de proteínas contráteis. In vitro o T3 diminui estas proteínas contráteis via AT2. Aortas sem endotélio dos ratos hipertiroideos apresentaram menor reatividade a AngII e maior relaxamento ao nitroprussiato de sódio (NPS), efeitos estes mediados via AT2. Por fim, observamos que o T3 é capaz de induzir produção de NO nas CMLV via PI3K/Akt, a qual é ativada pelo AT2 / 3,3\',5-triiodo-l-thyronine (T3) has been shown to induce vasodilation by its direct effect on vascular smooth muscle cells (VSMC). However, the mechanism by which T3 causes VSMC relaxation is still unknown. Here, we have shown that T3 causes rapid relaxation of VSMC via increased NO production from inducible and neuronal nitric oxide synthase (NOS). We further showed that these effects were mediated by PI3K/Akt signaling pathway. Vascular reactivity studies showed that endothelium-denuded aortas treated with T3 had a decreased response to phenylephrine which was reserved by L-NAME, NOS inhibitors. Aortas from hyperthyroid rats showed an upregulation of AT2 accompanied by decreased of contractile proteins. In vitro we observed that T3 decreases contractile proteins via AT2. Furthermore, endothelium-denuded aortas from hyperthyroid rats showed a decreased response to angiotensinII and augmented relaxation to sodium nitroprusside (SNP) via AT2 participation. Our data also suggests that PI3K/Akt signaling pathway is involved in T3-induced NO production in VSMC via AT2.

Os receptores dos hormônios da tireóide (TRs) são fatores de transcrição envolvidos na diferenciação celular, metabolismo e funções fisiológicas da maioria dos tecidos. Muitos estudos mostram que diversos efeitos farmacológicos mediados pelos TRs podem ser benéficos na farmacoterapia, especialmente aqueles mediados pelo TR que podem ser úteis em condições médicas importantes como obesidade, hipercolesterolemia e diabetes. Além disso, a descoberta que o TR é a isoforma predominante no coração, mediando a maioria dos efeitos cardiovasculares prejudiciais, estimulou a pesquisa por ligantes seletivos para o TR que poderiam ser utilizados em quadros clínicos importantes com perfil de segurança aceitável. Foi realizado um estudo das relações quantitativas entre a estrutura e atividade (QSAR) de um conjunto de compostos com atividade biológica descrita para TR e TR, que gerou modelos de Holograma QSAR com elevada consistência interna e externa, apresentando bom poder de correlação e predição das propriedades biológicas. Também foi realizado um minucioso estudo de triagem virtual, que propiciou a seleção de 7 compostos que foram adquiridos para terem suas atividades biológicas avaliadas. Ensaios de transfecção e gene repórter foram estabelecidos e utilizados na avaliação da atividade biológica dos compostos selecionados pelo ensaio virtual. Finalmente, um ensaio utilizando ressonância plasmônica de superfície (SPR) foi desenvolvido e utilizado para avaliar a atividade agonista desses compostos, e que pode ser útil para avaliar a atividade de novos ligantes. A técnica de SPR também foi empregada em um cuidadoso estudo da interação do TR com seus correguladores, que incluiu estudos cinéticos e termodinâmicos, propiciando a determinação das taxas cinéticas e parâmetros termodinâmicos para a interação do complexo TR-T3 com peptídeos derivados de dois de seus correguladores. Os resultados obtidos são relevantes e devem ser considerados no planejamento de futuros experimentos utilizando o LBD de TR e agonistas. / The thyroid hormone receptors (TRs) are transcriptional factors involved in cell differentiation, development, metabolism and physiological function of most tissues. Many lines of evidence show that several pharmacological actions of TRs might be beneficial in medical therapy, specially those mediated by TR that target important medical conditions like obesity, hypercholesterolemia and diabetes. Additionally, the findings that TR is the predominant isoform in the heart and mediates most of the TRs deleterious cardiovascular effects, stimulated the research for selective TR ligands which could address important medical needs with an acceptable safety profile. In this PhD thesis, studies of the quantitative structure-activity relationships (QSAR) of a dataset of compounds with reported biologic activity for both TR and TR were performed, and statistically significant Hologram QSAR models with good predictive ability for untested compounds were created. In parallel, a careful virtual screening procedure was executed, leading to the selection of 7 compounds which were purchased for the evaluation of their biological activities. Cell transfection and reporter gene assays were developed, validated and used to evaluate the biological activities of these compounds. Finally, a surface plasmon resonance (SPR) assay was developed and used to assess the agonistic activity of these compounds. The SPR technique was also employed in a careful study of the interaction between the ligand binding domain of TR and peptides derived from its coregulators, which included the determination of the kinetic and thermodynamic parameters for this interaction. The results suggest that flexibility plays an important role in the interaction between the receptor and its coregulators, and point out important aspects of experimental design that should be addressed when using TR LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TR ligands.

Der Monocarboxylattransporter 8 (Mct8) ist ein spezifischer Schilddrüsenhormon (SDH)-Transporter. MCT8-Mutationen führen zu einer psychomotorischen Retardierung in Kombination mit abnormalen SDH-Serumkonzentrationen. Das konstitutiv Mct8-defiziente Mausmodell repliziert den endokrinologischen, jedoch nicht den humanen neurologischen Phänotyp. Um die Hypothese eines stark beeinträchtigten T3-Transportes speziell in Neuronen als Ursache zu untersuchen, wurde das Neuron-spezifische Mct8-defiziente Mausmodell (CamK-Cre;Mct8fl/fl) generiert. Neben einer funktionalen, Mct8-exprimierenden Blut-Hirn-Schranke liegt eine funktionale Hypophysen-Hypothalamus-Schilddrüsen Achse vor. NMR-Analysen des zerebralen Energiestoffwechsels von CamK-Cre;Mct8fl/fl-Mäusen zeigen nach [1-13C] Glukoseinfusion verringerte Laktatintensitäten sowie eine reduzierte Laktatdehydrogenase-Aktivität. Zudem sind Astrozyten-spezifische Transporter und Enzyme des Neurotransmitterstoffwechsels und deren Biosynthese in ihrer Genexpression reduziert. Somit führt der neuronale Mct8-Verlust zu einem verlangsamten zerebralen Metabolismus sowie einer reduzierten neuronalen Aktivität. Die Rolle von Mct8 im Energiestoffwechsel wurde außerdem in primären Mct8-defizienten Astrozyten- und Neuronkulturen mittels Seahorse Flux Analyzer untersucht. In Mct8-defizienten Neuronen kommt es zu einer verringerten SDH-Aufnahme, was in einer verringerten Expression von OXPHOS-relevanten Proteinen sowie in einer verringerten Sauerstoffverbrauchsrate resultiert. Somit stützen die in vitro Daten die des CamK-Cre;Mct8fl/fl-Mausmodelles bezüglich einer reduzierten neuronalen Aktivität sowie eines verlangsamten zerebralen Stoffwechsels. Zusammenfassend zeigen die Ergebnisse, dass grundlegende Mechanismen des zerebralen Stoffwechsels bei neuronaler Mct8-Defizienz beeinträchtigt sind und die Rolle von Mct8 mit Hilfe weiterer konditioneller Mausmodelle (Astrozyten-spezifisch) und primären Ko-Kulturmodellen untersucht werden muss. / The monocarboxylate transporter 8 (Mct8) is the most specific thyroid hormone (TH) transporter. Mutations lead to a severe form of psychomotor retardation in combination with abnormal TH concentrations in sera. The global Mct8-deficient mouse model was intensively studied and it replicates the endocrine, but not the human neurological phenotype. To test the hypothesis, that a disturbed uptake of T3 especially into neurons is responsible for the phenotype, we generated a neuron-specific Mct8-deficient mouse model (CamK-Cre;Mct8fl/fl). CamK-Cre;Mct8fl/fl mice exhibit a functional Mct8-expressing blood-brain-barrier and a functional hypothalamus pituitary thyroid axis. NMR analyses of the cerebral energy metabolism of CamK-Cre;Mct8fl/fl mice after [1-13C] glucose injection revealed less enrichment of lactate and a reduced lactate dehydrogenase activity. Moreover, especially astrocyte-specific expressed transporter and enzymes of neurotransmitter metabolism and their biosynthesis are significantly reduced in comparison to control mice. These results point to a decelerated cerebral metabolism as well as a reduced neuronal activity caused by the neuronal loss of Mct8. In addition, we studied the impact of Mct8 on the energy metabolism in primary wildtype and Mct8-deficient astrocyte and neuron cultures by use of the Seahorse Flux Analyzer. Mct8-deficient neurons show a reduced uptake of TH, which results in a reduced expression of OXPHOS relevant proteins as well as a reduced oxygen consumption rate. Therefore, the in vitro raised data provide the observed changes of the CamK-Cre;Mct8fl/fl mice regarding a reduced synaptic activity as well as a reduced cerebral metabolism. Taken together, the data clearly shows that basic mechanisms of the cerebral metabolism are hampered in neuronal Mct8 deficiency. The role of Mct8 in this context needs further analyses with the help of conditional mouse models (astrocyte-specific) and primary co-culture models.

3-Jodthyronamin (3-T1AM) und Thyronamin (T0AM) sind endogene Signalmoleküle, die eine große strukturelle Ähnlichkeit zu Schilddrüsenhormonen aufweisen, allerdings die klassischen Wirkungen des aktiven Schilddrüsenhormons 3,5,3’-Trijodthyronin (T3) antagonisieren. In der vorliegenden Arbeit wurde untersucht, ob Thyronamine (TAMs) Substrate von Dejodasen (Dio1, Dio2, Dio3) sind. Die TAMs wurden mit isozymspezifischen Dio-Präparationen inkubiert. Die Dejodierungsprodukte wurden mittels Hochleistungsflüssigkeitschromatographie und Tandemmassenspektrometrie (LC-MS/MS) analysiert. Mit Präparationen der Dio1 wurden Dejodierungen von 3,3’,5’-Trijodthyronamin, 3’,5’- und 3,3’-Dijodthyronamin am phenolischen Ring sowie Dejodierungen von 3,5,3’-Trijodthyronamin und 3,5-Dijodthyronamin am Tyrosylring beobachtet. Dio2 haltige Präparationen katalysierten ebenfalls Dejodierungen von 3,3’,5’-Trijodthyronamin und 3’,5’-Dijodthyronamin am phenolischen Ring. Mit Dio3 haltigen Präparationen wurden alle TAMs mit jodiertem Tyrosylring dejodiert. In Kompetitionsversuchen inhibierten ausschließlich die TAMs, die als Substrate von Dio Isozymen identifizierten wurden, eine etablierte Dejodierungsreaktion eines bekannten Substrats. Im Gegensatz dazu interferierten TAMs, die in den LC-MS/MS Experimenten als Substrate der Dio Isozyme ausgeschlossen wurden, nicht mit der genannten etablierten Dejodierungsreaktion. Zusammenfassend wurde in der vorliegenden Arbeit gezeigt, dass TAMs Substrate aller drei Dio Isozyme sind und jedes Isozym eine eigene Substratspezifität aufweist. Diese Befunde weisen darauf hin, dass Dio Isozyme an der Biosynthese von TAMs beteiligt sein könnten. Ferner wurden die Biosynthesewege für 3-T1AM und T0AM eingegrenzt. Desweiteren gestatten die Ergebnisse neue Einblicke in die generellen strukturellen Voraussetzungen für Dio Substrate, da TAMs die bisher einzigen endogenen Dio Substrate darstellen, deren Seitenkette am Tyrosylring eine positive Ladung aufweist. / 3-iodothyronamine (3-T1AM) and thyronamine (T0AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T3) actions. The present study investigated whether thyronamines (TAMs) are substrates of three Dio isozymes (Dio1, Dio2 and Dio3). TAMs were incubated with isozyme specific Dio preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3’,5’-triiodothyronamine, 3’,5’- and 3,3’-diiodothyronamine as well as tyrosyl ring deiodinations of 3,5,3’-triiodothyronamine and 3,5-diiodothyronamine were observed with preparations containing Dio1. Preparations of Dio2 also deiodinated 3,3’,5’-triiodothyronamine and 3’,5’-diiodothyronamine at the phenolic rings. All TAMs with tyrosyl ring iodine atoms were deiodinated by Dio3 containing preparations. In functional competition assays, the newly identified TAM substrates inhibited an established iodothyronine deiodination reaction. By contrast, TAMs which had been excluded as Dio substrates in LC-MS/MS experiments, failed to show any effect in the competition assays, thus verifying the former results. In summary, all three Dio isozymes catalyzed TAM deiodination reactions with each isozyme exhibiting a unique substrate specificity. These data support a role for Dio isozymes in TAM biosynthesis and contribute to confining the biosynthetic pathways of 3-T1AM and T0AM. Furthermore, they provide new insights into the structural requirements for Dio substrates in general since TAMs represent the only endogenous Dio substrates described, so far, which possess a positively charged tyrosyl ring side chain.

Das Schilddrüsenhormon Triiodthyronin (T3) ist ein essenzieller Regulator physiologischer Prozesse der Entwicklung, des Wachstums und im Intermediärstoffwechsel. Täglich werden zahlreiche natürliche und synthetische Stoffe aufgenommen, die mit dem endokrinen System interferieren und deshalb als Endokrine Disruptoren (ED) bezeichnet werden. Zur Untersuchung einer direkten Interferenz von ED mit den Schilddrüsenhormonrezeptoren (TR) und ihrer transkriptionellen Aktivität wurde im Rahmen dieser Arbeit ein neues Luziferase-basiertes T3 Reportergensystem mit TRalpha1-transfizierten humanen Leberzellen aufgebaut. Durch Validierung mit dem synthetischen TR-Agonisten GC-1 und dem Antagonisten NH-3 konnte nachgewiesen werden, dass dieser Assay ein hoch-sensitives System zur Analyse von agonistischen sowie antagonistischen Effekten von Testsubtanzen darstellt. Zur Bestimmung der endokrinen Aktivitäten einiger humanrelevanter Vertreter aus den Stoffkategorien der Nahrungsmittel, Kosmetika, Pestizide und Industriechemikalien wurden Dosis-Wirkungskurven in Aktivierungs- und T3-Kompetitionsexperimenten ermittelt. In mikromolaren Konzentrationen wirkten von insgesamt 21 Testsubstanzen einige als reine Agonisten oder Antagonisten während andere gemischt agonistische/antagonistische Effekte hatten. Aufgrund ihrer hier beobachteten Effekte und der gegebenen Humanexposition wird eine eingehendere Analyse von 4-Methylbenzyliden Campher, 4-Nonylphenol, Acetochlor, Benzophenon 2, Benzophenon 3, Bisphenol A, Genistein, Octylmethoxycinnamat, Tetrabromobisphenol A und Xanthohumol empfohlen. Außerdem erwiesen sich einige Metaboliten von Schilddrüsenhormonen als potente Agonisten im T3-Reportergenassay und bedürfen weiterer Aufmerksamkeit. Für die molekulare Charakterisierung der Einflüsse solcher Substanzen auf die T3-regulierte Transaktivierung konnte mit dem hier etablierten Bioassay ein zuverlässiges neues Testsystem für reproduzierbare Screeningserien geschaffen werden. / Triiodothyronine (T3) is a crucial regulator of many physiological processes during development, growth and metabolism. A variety of natural and synthetic substances, which are collectively termed endocrine disrupters (ED) due to their interference with the endocrine system, is taken up on a daily base. A novel luciferase-based T3-responsive reporter gene system employing a human liver cell line transfected with thyroid hormone receptor (TR) alpha1 was established in this work to elucidate the potential molecular interference of certain ED with TR and their transcriptional activity. This assay was validated to be a highly sensitive and reliable tool for analyzing agonistic and antagonistic effects of test compounds using the synthetic TR agonist GC-1 and the antagonist NH-3. Dose-response data of test compounds contained in food, cosmetics, pesticides, plasticizers and other industrial chemicals were obtained after applying the substances alone in activation assays or in combination with T3 in competition assays. In total 21 test compounds were screened of which some acted as pure agonists or antagonists while others were mixed agonists/antagonists in the micromolar concentration range and only one was without effect. Follow-up studies are recommended for some of these substances with regard to their effects as determined in this bioassay and in light of information known on human exposure, i.e., 4-methylbenzyliden camphor, 4-nonylphenol, acetochlor, benzophenone 2, benzophenone 3, bisphenol A, genistein, octylmethoxycinnamate, tetrabromobisphenol A and xanthohumol. In addition some endogenous metabolites of thyroid hormones were surprisingly potent agonists in the T3 reporter gene assay and merit further attention. The novel bioassay established here represents a reliable tool for the screening and molecular characterization of substances interfering with T3-mediated transactivation of gene expression.

Foi estudado o ritmo de filtração glomerular (RFG) de pacientes com câncer bem diferenciado da tireoide submetidos à radioiodoterapia (RIT). O estudo avaliou o RFG durante estímulo do hormônio estimulador da tireoide (TSH) por suspensão da reposição hormonal tireoidiana (RHT) ou no uso do hormônio estimulador da tireoide recombinante humano (TSHrh), correlacionou o RFG com o perfil hormonal tireoidiano, avaliou o RFG durante e na semana após a RIT, avaliou o RFG e a dose efetiva de radiação para corpo inteiro e correlacionou métodos de estimativa de RFG. Vinte e oito pacientes incluídos em estudo clínico randomizado não cego foram divididos em dois grupos de 14 pacientes, sendo o grupo A (GA) submetido à suspensão da RHT e o grupo B (GB) ao uso do TSHrh. Os pacientes tiveram antes e após o estímulo do TSH a determinação do RFG por 51Cr-EDTA e coletas séricas do perfil hormonal tireoidiano e creatinina, albumina e ureia, e, após a RIT, colheram exames séricos de creatinina, albumina e ureia, e tiveram estimadas suas doses efetivas de corpo inteiro. Os exames de creatinina, albumina e ureia foram utilizados para estimar o RFG pelas equações de creatinina sérica, Modified Diet in Renal Disease (MDRD), e Cockcroft-Gault. O GA apresentou, pelo 51Cr-EDTA, variação de -18,5% do RFG de 94,4±18,6 mL/min antes da suspensão da RHT para 76,2±15,7 mL/min (p=0,0002) e o GB apresentou pelo 51Cr-EDTA variação de 4% do RFG de 90,8±18,4 mL/min antes do TSHrh para 92,6±15,2 mL/min (p=0,64). O RFG variou significativamente só no GA, sem apresentar proporcionalidade entre as variações do hormônio tireoidiano e do RFG. Não houve correlação do RFG com elevação do TSH. Por equações baseadas em creatinina, houve, no GA, queda do RFG durante toda a suspensão da RHT e estabilidade após o retorno da RHT, e, no GB, houve estabilidade do RFG durante todo o estudo. A dose efetiva de corpo inteiro não apresentou diferenças significativas entre os grupos (p=0,76). Na comparação entre o 51Cr-EDTA e as equações para estimativa de RFG, a correlação de Pearson foi de 0,78 para creatinina sérica, 0,79 para MDRD e 0,66 para CockcroftGault, e a comparação das variações do RFG observadas no GA entre o 51Cr-EDTA e a equação por creatinina sérica foram estatisticamente diferentes. Concluiu-se que o RFG apresenta redução na suspensão da RHT, relacionado ao hipotireoidismo e não à elevação de TSH, voltando a estabilizar após retorno da RHT, e que não varia no uso do TSHrh, que a dose efetiva de corpo inteiro não varia entre os grupos proporcionalmente ao RFG, e que a melhor correlação foi do 51Cr-EDTA com a equação MDRD / Glomerular filtration rate (GFR) was studied in well differentiated thyroid cancer patients referred for radioiodine therapy (RIT). The study evaluated GFR during thyroid stimulating hormone (TSH) stimulation after thyroid hormone withdrawal (THW) or after recombinant human thyroid stimulating hormone (rhTSH), correlated GFR with thyroid hormone profile, evaluated GFR during and in the week after RIT, evaluated GFR and whole body radiation effective dose, and correlated different methods for GFR determination. 28 patients were included in a non-blinded randomized clinical trial and divided in two groups of 14 patients, being group A (GA) stimulated by THW and group B (GB) stimulated by rhTSH. Patients had GFR determined by 51Cr-EDTA, as well as serum thyroid hormone profile, creatinine, albumin and urea before and after TSH stimulation, and after RIT had determined their serum creatinine, albumin and urea and whole body radiation effective dose. Creatinine, albumin and urea were used to estimate GFR by serum creatinine, Modified Diet in Renal Disease (MDRD), and Cockcroft-Gault equations. GA presented a -18,5% GFR variation by 51CrEDTA varying from 94,4 ± 18,6 mL/min before THW to 76,2±15,7 mL/min after THW (p=0,0002) while GB presented a 4% GFR variation by 51Cr-EDTA varying from 90,8 ± 18,4 mL/min before TSHrh to 92,6 ± 15,2 mL/min after rhTSH (p=0,64). GFR significantly varied only in GA without presenting proportionality with thyroid hormone variation. There was no correlation between rise in TSH levels and GFR. Creatinine equations demonstrated a sustained reduction in GFR during THW and GFR stability after thyroid hormone reposition, while GB presented stable GFR during the whole study. Whole body radiation effective dose didn\'t present significant differences between the two groups (p=0,76). Comparing 51Cr-EDTA and GFR estimative equations presented Pearson correlation score of 0,78 for serum creatinine, 0,79 for MDRD and 0,66 for Cockcroft-Gault, while comparison between variances in GA between 51Cr-EDTA e serum creatinine equation was significantly different. In conclusion GFR presents a reduction during THW related to hypothyroidism and not to TSH rise and stabilizing after thyroid hormone therapy, GFR does not vary during rhTSH, whole body radiation effective dose does not vary between the two groups proportionally to GFR, and that MDRD equation had the best correlation with 51Cr-EDTA

Ainda não é claro o papel dos hormônios tireoideanos (HT) no desenvolvimento do esqueleto fetal. Para responder a questão, induzimos hipotireoidismo materno e fetal em camundongos prenhes através da administração de metimazol e perclorato de sódio. O esqueleto fetal apresentou discretas morfológicas até 16,5 dias de idade embrionária (E). Apenas no final da gestação, em 18,5E, foram observadas a redução significativa da zona hipertrófica, do número de condrócitos hipertróficos, desorganização e diminuição da quantidade dos condrócitos proliferativos, além da redução da expressão do colágeno I, X e osteocalcina. Os TRs, assim como LAT1, LAT2 e MCT8 foram detectados em todas as idades estudadas. A alta expressão gênica da D3, principal inativadora do hormônio tireoideano, em 14,5E e sua redução significativa durante o desenvolvimento, até atingir níveis indetectáveis no período pós-natal indicam que a D3 seja responsável por manter baixos níveis de HT no esqueletono início da gestação, garantindo um desenvolvimento ósseo normal. / Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice and fetuses were made hypothyroid. The skeleton morphology was preserved up to 16.5 embryonic days (E). Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrofic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly at E18.5, and even more after birth. The expression levels of D3 gene during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that its expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.

INTRODUÇÃO: As proteínas desacopladoras de prótons (UCPs: uncoupling proteins) pertencem à família dos transportadores mitocondriais H+/ácidos graxos e têm distribuição diferenciada nos tecidos. Sabe-se que a UCP1 é responsável pela termogênese, mas o exato papel fisiológico da UCP2 e UCP3 ainda não está completamente estabelecido. Os hormônios tireoideanos (T3 e T4) estimulam a expressão da UCP3 em músculo cardíaco e esquelético, no entanto o mecanismo pelo qual exercem esse efeito não é conhecido. Este projeto visa avaliar se as alterações na expressão gênica da UCP3 são relacionadas a efeito primário do T3 ou são secundárias à estimulação do sistema renina-angiotensina ou do sistema ?-adrenérgico. MÉTODOS: Para a realização do estudo, criou-se um modelo animal de hipertireodismo, em ratos machos Sprague-Dawley, através da 3 administrações de 100 ?g/100 g peso corpóreo de LT3, em dias alternados, associado ou não à captopril (1 mg/100 g de peso corpóreo), ?-bloqueador propranolol (1 mg/100g de peso corpóreo) ou ?2-agonista clenbuterol (0,04 mg/100 g de peso corpóreo). A expressão do mRNA da UCP3 foi semi-quantitativamente determinada por Northern blot em amostras de músculo ventricular cardíaco e músculo esquelético (gastrocnemius e soleus). A expressão da proteína UCP3 foi avaliada por Western blot em músculo esquelético (quadríceps). Os resultados foram expressos em unidades arbitrárias de densitometria óptica. RESULTADOS: O tratamento com LT3 resultou em aumento estatisticamente significativo do conteúdo de mRNA da UCP3 em miocárdio (~3 vezes) e músculo esquelético (~8 vezes) (p<0,05) e esse efeito não foi alterado por nenhuma das medicações usadas concomitantemente. Não houve efeito sinergístico ou aditivo sobre a expressão do mRNA da UCP3 quando o LT3 foi administrado conjuntamente ao ?2-agonista. O aumento na quantidade de mRNA da UCP3, em músculo esquelético, foi associado à aumento na expressão da proteína UCP3. CONCLUSÃO: O efeito do LT3 sobre a expressão da UCP3, nos tecidos analisados, não são dependentes da angiotensina II, nem do sistema ?-adrenérgico, provavelmente refletindo uma ação direta do LT3 sobre a expressão do gene UCP3 / Thyroid hormones (T3 and T4) stimulate UCP-3 expression in skeletal muscle. Here, we examined whether thyroid hormone-induced changes in UCP-3 mRNA expression are related to directs effects of T3 or reflect secondary effects of the hormone through stimulation of renin-angiotensin or ?-adrenergic systems. Hyperthyroidism was produced by three injections of 100 ?g T3/100 g body weight on alternate days with or without concomitant treatment with either captopril (an ACE inhibitor), propranolol (a ?-blocker) or clenbuterol (a ?2-agonist). The relative abundance of UCP-3 mRNA was measured in ventricular myocardium and skeletal muscle (gastrocnemius and soleus). T3 resulted in a significant increase in the relative abundance of UCP-3 in heart and skeletal muscle (P < 0.05), and the effect was not altered by captopril or propanolol; the inhibitors alone had no effect of UCP-3 mRNA content. There was no synergistic or additive effect of T3 and clenbuterol on UCP-3 mRNA expression in skeletal muscle. Increased UCP-3 mRNA levels were associated with increased UCP-3 protein expression in skeletal muscle. We conclude that the effect of T3 on UCP-3 expression in cardiac and skeletal muscle is not dependent on either angiotensin II or the ?-adrenergic system and probably reflects a direct action of the hormone on UCP-3 gene expression

L'hormone thyroïdienne (T3) contrôle à la fois les processus développementaux et physiologiques. Elle agit via les récepteurs de l'hormone thyroïdienne (TR), membres de la famille des récepteurs hormonaux nucléaires. Ils agissent comme des facteurs de transcription dépendants du ligand. La méthylation de l'ADN en position 5 de la cytosine est une modification épigénétique importante qui affecte la structure de la chromatine et l'expression des gènes. Des études récentes ont établi un rôle important des protéines de la famille TET (Ten-eleven translocation) dans la régulation de la dynamique de la méthylation de l'ADN. Elles convertissent la 5-méthyl-cytosine (5mC) en 5-hydroxyméthylcytosine (5hmC). D’autres études ont démontré que les protéines TET (TET1, TET2 et TET3) possèdent des fonctions de régulation transcriptionnelle dépendantes et indépendantes de leur activité catalytique. Notre étude a identifié TET3 comme une nouvelle protéine interagissant avec TR. Le domaine AF2 de TR ainsi que le domaine catalytique et le domaine CXXC de TET3 sont responsables de cette interaction. Celle-ci permet la stabilisation de TR lié à la chromatine, entraînant une potentialisation de son activité transcriptionnelle. L'effet de modulation de TET3 sur TR présenté ici est indépendant de son activité hydroxylase de TET3. Ainsi, cette étude met en évidence un nouveau mode d'action de TET3 en tant que régulateur non classique de TR, modulant sa stabilité et son accès à la chromatine plutôt que son activité de transcription intrinsèque. Des mutations du gène codant pour TRα provoquent le symptôme RTHα dont la gravité varie en fonction de la mutation. Les différentes capacités d’interaction des mutants TRα, pertinents pour la maladie de RTHα humaine, avec TET3 pourraient expliquer les différences d’effet dominant négatif. La fonction de régulation de TET3 pourrait s’appliquer plus généralement aux facteurs de transcription des récepteurs nucléaires, car différents membres de la superfamille des récepteurs nucléaires présentent la même interaction avec TET3, tels que AR (récepteur des androgènes), ERR (récepteur des œstrogènes) et RAR (récepteur de l'acide rétinoïque). L'interaction entre TET3 et RAR implique le domaine de liaison ADN de RAR. La pertinence fonctionnelle de l'interaction TET3 / RAR a été étudiée plus en détail dans les cellules souches embryonnaire (cellules ES). L’absence combinée des trois TET a entraîné la diminution de 5hmC et la dérégulation des gènes impliqués dans la différenciation des cellules ES. Parmi les gènes dérégulés, nous avons identifié un sous-ensemble de gènes cibles de l’acide rétinoïque, suggérant que les RAR (récepteurs d'acide rétinoïque) et les TET pourraient travailler ensemble pour réguler la différenciation des cellules ES. Une étude supplémentaire a révélé que les protéines TET peuvent jouer un rôle dans la facilitation du recrutement de RAR aux régions promotrices de ses gènes cibles. En outre, nos résultats montrent un rôle potentiel de l'activité hydroxylase des protéines TET dans la modulation de l'activité transcriptionnelle des RAR. En conclusion, notre travail a identifié les protéines TET comme nouveaux régulateurs des récepteurs nucléaires. Les mécanismes exacts impliqués doivent être étudiés plus avant. / Thyroid hormone (T3) controls both developmental and physiological processes. Its nuclear receptors, thyroid hormone receptors (TRs), are members of the nuclear hormone receptor family which act as ligand-dependent transcription factors. DNA methylation at the fifth position of cytosine is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (TET) family proteins in regulating DNA methylation dynamics by converting 5-methyl-cytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Studies demonstrated that TETs proteins (including TET1, TET2 and TET3) possess catalytic activity dependent and independent transcriptional regulatory functions. Our study identified TET3 as a new TR interacting protein. The AF2 domain of TR and the catalytic domain and CXXC domain of TET3 are responsible for their interaction. This interaction allows the stabilization of chromatin bound TR, resulting in a potentiation of its transcriptional activity. The modulation effect of TET3 on TR presented here is independent of its hydroxylase activity. Thus this study evidences a new mode of action for TET3 as a non-classical regulator of TR, modulating its stability and access to chromatin rather that its intrinsic transcriptional activity. Mutations in TR cause the RTH symptom which severity varies with the particular mutation. The differential ability of different TRα mutants, relevant for the human RTHα disease, to interact with TET3 might explain their differential dominant negative activity. The regulatory function of TET3 might be more general towards the nuclear receptor transcriptional factors since different members of the superfamily present the same interaction with TET3, such as AR (androgen receptor), ERR (Estrogen-related receptor) and RAR (retinoic acid receptor). The interaction between TET3 and RAR involves the DNA binding domain of RAR. The functional relevance of TET3/RAR interaction was further studied in ES cells. Combined deficiency of all three TETs led to depletion of 5hmC and deregulation of genes involved in ES differentiation. Among the deregulated genes, a subset of RA response genes was identified, suggesting that RARs (retinoic acid receptors) and TETs might work together to regulate ES cell differentiation. Further dissection revealed that TET proteins may have a role in facilitating RAR recruitment to the promoter regions of these RAR target genes. Moreover, our results indicated a potential role of the hydroxylase activity of TET proteins in modulating RAR transcriptional activity. Altogether, our work identified TET proteins as new regulators of NR (Nuclear Receptors). The exact mechanisms involved need to be further studied.

Foi estudado o ritmo de filtração glomerular (RFG) de pacientes com câncer bem diferenciado da tireoide submetidos à radioiodoterapia (RIT). O estudo avaliou o RFG durante estímulo do hormônio estimulador da tireoide (TSH) por suspensão da reposição hormonal tireoidiana (RHT) ou no uso do hormônio estimulador da tireoide recombinante humano (TSHrh), correlacionou o RFG com o perfil hormonal tireoidiano, avaliou o RFG durante e na semana após a RIT, avaliou o RFG e a dose efetiva de radiação para corpo inteiro e correlacionou métodos de estimativa de RFG. Vinte e oito pacientes incluídos em estudo clínico randomizado não cego foram divididos em dois grupos de 14 pacientes, sendo o grupo A (GA) submetido à suspensão da RHT e o grupo B (GB) ao uso do TSHrh. Os pacientes tiveram antes e após o estímulo do TSH a determinação do RFG por 51Cr-EDTA e coletas séricas do perfil hormonal tireoidiano e creatinina, albumina e ureia, e, após a RIT, colheram exames séricos de creatinina, albumina e ureia, e tiveram estimadas suas doses efetivas de corpo inteiro. Os exames de creatinina, albumina e ureia foram utilizados para estimar o RFG pelas equações de creatinina sérica, Modified Diet in Renal Disease (MDRD), e Cockcroft-Gault. O GA apresentou, pelo 51Cr-EDTA, variação de -18,5% do RFG de 94,4±18,6 mL/min antes da suspensão da RHT para 76,2±15,7 mL/min (p=0,0002) e o GB apresentou pelo 51Cr-EDTA variação de 4% do RFG de 90,8±18,4 mL/min antes do TSHrh para 92,6±15,2 mL/min (p=0,64). O RFG variou significativamente só no GA, sem apresentar proporcionalidade entre as variações do hormônio tireoidiano e do RFG. Não houve correlação do RFG com elevação do TSH. Por equações baseadas em creatinina, houve, no GA, queda do RFG durante toda a suspensão da RHT e estabilidade após o retorno da RHT, e, no GB, houve estabilidade do RFG durante todo o estudo. A dose efetiva de corpo inteiro não apresentou diferenças significativas entre os grupos (p=0,76). Na comparação entre o 51Cr-EDTA e as equações para estimativa de RFG, a correlação de Pearson foi de 0,78 para creatinina sérica, 0,79 para MDRD e 0,66 para CockcroftGault, e a comparação das variações do RFG observadas no GA entre o 51Cr-EDTA e a equação por creatinina sérica foram estatisticamente diferentes. Concluiu-se que o RFG apresenta redução na suspensão da RHT, relacionado ao hipotireoidismo e não à elevação de TSH, voltando a estabilizar após retorno da RHT, e que não varia no uso do TSHrh, que a dose efetiva de corpo inteiro não varia entre os grupos proporcionalmente ao RFG, e que a melhor correlação foi do 51Cr-EDTA com a equação MDRD / Glomerular filtration rate (GFR) was studied in well differentiated thyroid cancer patients referred for radioiodine therapy (RIT). The study evaluated GFR during thyroid stimulating hormone (TSH) stimulation after thyroid hormone withdrawal (THW) or after recombinant human thyroid stimulating hormone (rhTSH), correlated GFR with thyroid hormone profile, evaluated GFR during and in the week after RIT, evaluated GFR and whole body radiation effective dose, and correlated different methods for GFR determination. 28 patients were included in a non-blinded randomized clinical trial and divided in two groups of 14 patients, being group A (GA) stimulated by THW and group B (GB) stimulated by rhTSH. Patients had GFR determined by 51Cr-EDTA, as well as serum thyroid hormone profile, creatinine, albumin and urea before and after TSH stimulation, and after RIT had determined their serum creatinine, albumin and urea and whole body radiation effective dose. Creatinine, albumin and urea were used to estimate GFR by serum creatinine, Modified Diet in Renal Disease (MDRD), and Cockcroft-Gault equations. GA presented a -18,5% GFR variation by 51CrEDTA varying from 94,4 ± 18,6 mL/min before THW to 76,2±15,7 mL/min after THW (p=0,0002) while GB presented a 4% GFR variation by 51Cr-EDTA varying from 90,8 ± 18,4 mL/min before TSHrh to 92,6 ± 15,2 mL/min after rhTSH (p=0,64). GFR significantly varied only in GA without presenting proportionality with thyroid hormone variation. There was no correlation between rise in TSH levels and GFR. Creatinine equations demonstrated a sustained reduction in GFR during THW and GFR stability after thyroid hormone reposition, while GB presented stable GFR during the whole study. Whole body radiation effective dose didn\'t present significant differences between the two groups (p=0,76). Comparing 51Cr-EDTA and GFR estimative equations presented Pearson correlation score of 0,78 for serum creatinine, 0,79 for MDRD and 0,66 for Cockcroft-Gault, while comparison between variances in GA between 51Cr-EDTA e serum creatinine equation was significantly different. In conclusion GFR presents a reduction during THW related to hypothyroidism and not to TSH rise and stabilizing after thyroid hormone therapy, GFR does not vary during rhTSH, whole body radiation effective dose does not vary between the two groups proportionally to GFR, and that MDRD equation had the best correlation with 51Cr-EDTA