No sistema cardiovascular, os hormônios da tireoide exercem uma importante ação, influenciando a captação de cálcio, o inotropismo e o cronotropismo cardíaco e a resistência vascular periférica. No entanto, uma exacerbação destas ações, causada pelo aumento da secreção dos hormônios da tireoide, gera uma quebra desta homeostase e o desenvolvimento de hipertireoidismo. O hipertireoidismo leva ao aumento do consumo de oxigênio, gerando uma situação de estresse oxidativo. A exposição crônica ao estresse oxidativo leva à ativação de fatores de transcrição e citocinas, causando hipertrofia de cardiomiócitos e progressão para insuficiência cardíaca com inflamação e apoptose. Logo, o objetivo deste trabalho foi avaliar o papel dos hormônios da tireoide sobre a ativação de vias inflamatórias e apoptóticas mediada pelo estresse oxidativo. Neste estudo, nós avaliamos parâmetros de estresse oxidativo e algumas citocinas envolvidas com as vias de sinalização inflamatória e apoptótica. Para isso, utilizamos 60 ratos wistar, divididos em 2 grupos: Controle e Tratado (T4), com um n de 30 animais por grupo. O grupo T4 foi submetido à indução de hipertireoidismo através da adição de L-tiroxina (T4 – 12mg/L) na água de beber por 28 dias. O grupo controle não recebeu tratamento com L-tiroxina. Houve desenvolvimento de hipertireoidismo e indução de hipertrofia cardíaca no grupo T4. Verificamos também o aumento de H2O2 no coração e redução de -SH em eritrócitos no grupo T4. Houve aumento de LDH no grupo T4, indicando dano tecidual. Por fim, houve redução de PGC1-α, além de uma redução do p53 e de Bcl2 e aumento da razão Bax/Bcl2 no grupo T4. Os resultados apontam para a ocorrência de estresse oxidativo, o que com potencial dano induziu uma redução de PGC1-α e de p53, que podem estar relacionados à ativação de proteínas apoptóticas, como observado pelo aumento da razão Bax/Bcl2 no grupo tratado. / In the cardiovascular system, the thyroid hormones play an important action, influencing the uptake of calcium, the cardiac inotropy and chronotropy and peripheral vascular resistance. However, an exacerbation of these actions, caused by increased secretion of thyroid hormones, generates a breach of this homeostasis and could lead to development of hyperthyroidism. Hyperthyroidism leads to increased oxygen consumption, generating oxidative stress. Chronic exposure to oxidative stress leads to the activation of transcription factors and cytokines, causing cardiomyocyte hypertrophy and progression to heart failure with inflammation and apoptosis. Therefore, the aim of this study was to evaluate the role of thyroid hormones on the activation of inflammatory pathways and apoptotic mediated by oxidative stress. In this study, we evaluated some oxidative stress parameters and cytokines involved in inflammatory and apoptosis signaling pathways. For this, we used 60 Wistar rats, divided into 2 groups: control and treated (T4), with an n of 30 animals per group. The group T4 was subjected to hyperthyroidism induction by the addition of L-thyroxine (T4 - 12mg / L) in their drinking water for 28 days. The control group received no treatment with L-thyroxine. There was development of hyperthyroidism and induction of cardiac hypertrophy in the T4 group. We noticed the increase of H2O2 in the heart and reduced -SH in erythrocytes in the T4 group. There was LDH increase in the T4 group, indicating tissue damage. Finally, a reduction of PGC1-α, as well as a reduction of p53 and Bax/Bcl2 ratio increase in the T4 group. The results point to the occurrence of oxidative stress, which with potential damage induced a PGC1-α and p53 reduction, wich can be related to the activation of apoptotic proteins, as observed by increased Bax / Bcl2 ratio in the treated group.

Disfunções da glândula tireóide são, frequentemente, associadas a manifestações cardiovasculares e, em situações de hipertireoidismo, o coração hipertrofia. A hipertrofia cardíaca (HC) consiste em uma resposta adaptativa caracterizada pelo aumento de síntese de proteínas estruturais. O Sistema Ubiquitina Proteassoma (UPS) corresponde ao principal mecanismo de proteólise intracelular e crescentes evidências sugerem seu envolvimento no desenvolvimento da HC. O objetivo do presente estudo foi avaliar a modulação do UPS no tecido cardíaco de animais submetidos ao hipertireoidismo. Os resultados referentes ao aumento da atividade e expressão do proteassoma (PT) cardíaco apresenta-se mais contundente no grupo tratado por 7 dias, período em que a HC já encontra-se estável. Ao término de 14 e 21 dias, a modulação desse sistema tende à normalização. Os resultados obtidos atestam evidências da literatura que sugerem o aumento da atividade do PT cardíaco como resposta compensatória ao aumento de síntese proteica. / Thyroid gland disorders are often associated with cardiovascular events and hyperthyroidism state promotes cardiac hypertrophy (CH). CH consists in adaptive response characterized by increased synthesis of structural proteins. The Ubiquitin Proteasome System (UPS) is the major mechanism of intracellular proteolysis and increased evidences suggest its involvement in the development of CH. The aim of this study was to evaluate the modulation of UPS in cardiac tissue of animals subjected to hyperthyroidism. The results related to the increased proteasome (PT) activity and expression in the heart was more accentuated in the group treated for 7 days, when the CH process finds stable. At the end of 14 and 21 days of hyperthyroidism, the modulation of cardiac UPS achieves standard values. These results suggest an increased activity of cardiac PT as a compensatory response to protein synthesis induced by thyroid hormones.

No sistema cardiovascular, os hormônios da tireoide exercem uma importante ação, influenciando a captação de cálcio, o inotropismo e o cronotropismo cardíaco e a resistência vascular periférica. No entanto, uma exacerbação destas ações, causada pelo aumento da secreção dos hormônios da tireoide, gera uma quebra desta homeostase e o desenvolvimento de hipertireoidismo. O hipertireoidismo leva ao aumento do consumo de oxigênio, gerando uma situação de estresse oxidativo. A exposição crônica ao estresse oxidativo leva à ativação de fatores de transcrição e citocinas, causando hipertrofia de cardiomiócitos e progressão para insuficiência cardíaca com inflamação e apoptose. Logo, o objetivo deste trabalho foi avaliar o papel dos hormônios da tireoide sobre a ativação de vias inflamatórias e apoptóticas mediada pelo estresse oxidativo. Neste estudo, nós avaliamos parâmetros de estresse oxidativo e algumas citocinas envolvidas com as vias de sinalização inflamatória e apoptótica. Para isso, utilizamos 60 ratos wistar, divididos em 2 grupos: Controle e Tratado (T4), com um n de 30 animais por grupo. O grupo T4 foi submetido à indução de hipertireoidismo através da adição de L-tiroxina (T4 – 12mg/L) na água de beber por 28 dias. O grupo controle não recebeu tratamento com L-tiroxina. Houve desenvolvimento de hipertireoidismo e indução de hipertrofia cardíaca no grupo T4. Verificamos também o aumento de H2O2 no coração e redução de -SH em eritrócitos no grupo T4. Houve aumento de LDH no grupo T4, indicando dano tecidual. Por fim, houve redução de PGC1-α, além de uma redução do p53 e de Bcl2 e aumento da razão Bax/Bcl2 no grupo T4. Os resultados apontam para a ocorrência de estresse oxidativo, o que com potencial dano induziu uma redução de PGC1-α e de p53, que podem estar relacionados à ativação de proteínas apoptóticas, como observado pelo aumento da razão Bax/Bcl2 no grupo tratado. / In the cardiovascular system, the thyroid hormones play an important action, influencing the uptake of calcium, the cardiac inotropy and chronotropy and peripheral vascular resistance. However, an exacerbation of these actions, caused by increased secretion of thyroid hormones, generates a breach of this homeostasis and could lead to development of hyperthyroidism. Hyperthyroidism leads to increased oxygen consumption, generating oxidative stress. Chronic exposure to oxidative stress leads to the activation of transcription factors and cytokines, causing cardiomyocyte hypertrophy and progression to heart failure with inflammation and apoptosis. Therefore, the aim of this study was to evaluate the role of thyroid hormones on the activation of inflammatory pathways and apoptotic mediated by oxidative stress. In this study, we evaluated some oxidative stress parameters and cytokines involved in inflammatory and apoptosis signaling pathways. For this, we used 60 Wistar rats, divided into 2 groups: control and treated (T4), with an n of 30 animals per group. The group T4 was subjected to hyperthyroidism induction by the addition of L-thyroxine (T4 - 12mg / L) in their drinking water for 28 days. The control group received no treatment with L-thyroxine. There was development of hyperthyroidism and induction of cardiac hypertrophy in the T4 group. We noticed the increase of H2O2 in the heart and reduced -SH in erythrocytes in the T4 group. There was LDH increase in the T4 group, indicating tissue damage. Finally, a reduction of PGC1-α, as well as a reduction of p53 and Bax/Bcl2 ratio increase in the T4 group. The results point to the occurrence of oxidative stress, which with potential damage induced a PGC1-α and p53 reduction, wich can be related to the activation of apoptotic proteins, as observed by increased Bax / Bcl2 ratio in the treated group.

Um dos mais importantes achados dos últimos anos foi o de que o remodelamento ósseo está sujeito ao controle do SNC, com o SNS agindo como efetor periférico. Um estudo do nosso grupo demonstrou que camundongos <font face=\"Symbol\">a2A/<font face=\"Symbol\">a2C-AR-/- apresentam um fenótipo de alta massa óssea, como também são resistentes à osteopenia induzida pelo excesso de hormônio HT. Com o intuito de verificar a participação do <font face=\"Symbol\">a2A-AR-/- nestes processos, tivemos como objetivos: caracterizar o fenótipo ósseo de camundongos <font face=\"Symbol\">a2A-AR-/- e avaliar o efeito do HT na estrutura óssea desses camundongos tratados. Pudemos observar que o comprimento longitudinal dos ossos dos animais <font face=\"Symbol\">a2A-AR-/- são menores do que dos animais selvagens e a análise por <font face=\"Symbol\">mCT do fêmur mostrou uma diminuição da porosidade da cortical. Com relação ao tratamento com hormônio tireoideano, os animais <font face=\"Symbol\">a2A-AR-/- tratados com T3 foram resistentes à diminuição do comprimento dos ossos causado pelo excesso de HT e vimos, ainda, que o osso trabecular dos animais <font face=\"Symbol\">a2A-AR-/- foi mais sensível aos efeitos deletérios da tirotoxicose, entretanto o osso cortical e parâmetros biomecânicos ósseos dos animais KOs foram menos sensíveis. Em conclusão, o presente estudo sugere que o <font face=\"Symbol\">a2A-AR está envolvido no processo de crescimento ósseo e que esse receptor possa mediar, pelo menos parcialmente, ações negativas do T3 nesse processo como também do HT no osso cortical. / One of the most important finds of the recent years is that bone remodeling is subject to the control of the CNS, with SNS acting as the peripheral effector. However, a recent study of our group showed that mice <font face=\"Symbol\">a2A/<font face=\"Symbol\">a2C-AR-/- have a high bone mass phenotype, even though are resistant to the thyroid hormone-induced osteopenia. In order to verify the role of <font face=\"Symbol\">a2A-AR-/- in these cases, we had as objectives to evaluate whether the isolated inactivation of <font face=\"Symbol\">a2A-AR interferes with the bone structure, and to evaluate the action of HT on these animals. We have observed that the longitudinal length of the bones of <font face=\"Symbol\">a2A-AR-/- animals are lower than those of wild type animals and the analysis of the femur by <font face=\"Symbol\">mCT showed a lower cortical porosity. With regard to treatment with thyroid hormone, we observed that <font face=\"Symbol\">a2A-AR-/- animals were resistant to the bone length decrease caused by thyroid hormone excess. We also noticed that the trabecular bone of <font face=\"Symbol\">a2A-AR-/- animals was more sensitive to the deleterious effects of thyrotoxicosis. Moreover, the cortical bone and bone biomechanical parameters KO animals were less sensitive. In conclusion, the findings of this study suggest that <font face=\"Symbol\">a2A-AR is involved in the process of bone growth and that this receptor may mediate at least partly, negative actions of T3 in this process as well as the HT in the cortical bone.

O presente estudo avaliou o papel do receptor AT1 de Angiotensina II no desenvolvimento da hipertrofia dos cardiomiócitos promovida pelo T3, bem como a participação dos mecanismos intracelulares deflagrados pelo receptor AT1 neste modelo de hipertrofia cardíaca. O silenciamento do receptor AT1 com RNA de interferência preveniu totalmente o desenvolvimento da hipertrofia dos cardiomiócitos induzida pelo T3. Os cardiomiócitos tratados com T3 demonstraram uma rápida ativação da via da Akt/GSK-3/mTOR, a qual foi atenuada ou prevenida pelo silenciamento do receptor AT1. Ainda, a expressão de Angiotensina I/II no lisado celular e a expressão do receptor AT1 foram rapidamente aumentados pelo T3. Esses dados demonstram pela primeira vez que o receptor AT1 é um mediador crítico da hipertrofia dos cardiomiócitos induzida pelo T3, bem como para a ativação da via da Akt, sugerindo que a via Ang I/II-AT1-Akt/GSK-3/mTOR corresponde a um potencial mediador dos efeitos tróficos exercidos pelo T3 nessas células. / The present study investigated the role of Angiotensin type 1 receptor (AT1R) in T3-induced cardiomyocyte hypertrophy, as well as the participation of the intracellular mechanisms mediated by AT1R in this cardiac hypertrophy model. The AT1R silencing using small interfering RNA totally prevented the development of T3-induced cardiomyocyte hypertrophy. The cardiomyocytes treated with T3 demonstrated a rapid activation of Akt/GSK-3/mTOR signaling pathway, which was attenuated or prevented by the AT1R silencing. In addition, local Angiotensin I/II (Ang I/II) levels and the AT1R expression were rapidly increased by T3 treatment. These data demonstrate for the first time that the AT1R is a critical mediator to the T3-induced cardiomyocyte hypertrophy, as well as to the activation of the Akt signaling, suggesting that the Ang I/II-AT1R-Akt/GSK-3/mTOR pathway corresponds to a potential mediator of the trophic effect exerted by T3 in cardiomyocytes.

Dans le thyrocyte normal, la TSH active une voie dépendante de l’adénylyl cyclase/AMPc, qui représente l’une des trois voies mitogéniques de la thyroïde. La cascade de signalisation de la TSH diffère des deux autres voies dans sa capacité à induire à la fois la prolifération et la différenciation, comprenant la synthèse et la sécrétion des hormones thyroïdiennes. Identifier les acteurs de cette cascade de signalisation, ainsi que les interactions entre effecteurs, est donc très important pour la compréhension de la fonction de la cellule thyroïdienne. C’est dans ce cadre que s’insère notre travail au cours duquel nous nous sommes intéressés au récepteur de la TSH ainsi qu’à une protéine récemment identifiée dans le laboratoire et dont l’expression est modulée en réponse à la TSH dans la thyroïde :C3VS. <p>C3VS est une protéine qui présente six motifs ankyrine et une tirette à leucine et dont la fonction était inconnue à l'époque. Dans un premier temps, nous avons contribué à l’obtention de la séquence codante complète du C3VS de chien, puis, l'identification des partenaires d'une protéine pouvant aider à caractériser sa fonction, nous nous sommes proposé de rechercher les partenaires potentiels de la région N-terminale de C3VS par la méthode double-hybride. Nous avons étudié la distribution tissulaire et la régulation par la TSH de différents partenaires isolés. Parmi eux, SUG1, une ATPase du protéasome 26S, a été étudiée plus avant mais l’interaction n’a pas pu être confirmée par "GST-pulldown assay". Simultanément, une remise en question de la position de la méthionine initiale de C3VS, couplée à une impossibilité d’exprimer la protéine en cellules COS par transfection mettait en péril le travail. En l’absence de plus d’arguments fonctionnels permettant d’orienter l’étude des positifs, cette partie du travail a été suspendue au profit de notre étude sur le récepteur de la TSH. L'activation de cascades différentes dans le thyrocyte humain et canin pouvant être due à l'action de protéines intracellulaires, nous avons tenté de rechercher par double-hybride des partenaires protéiques autres que les protéines G pour le récepteur de la TSH. Nous avons ainsi identifié PRA1 mais nous n’avons pas pu confirmer l'interaction entre les deux protéines par "GST-pulldown assay". Pour tenter de comprendre le rôle de cette interaction, nous avons réalisé des essais fonctionnels en transfectant des cellules pour évaluer l'implication de PRA1 sur la synthèse d’AMPc. Ces expériences ne nous ont pas permis de montrer un rôle pour PRA1 au niveau de la cascade, mais en revanche, nous avons mis en évidence le fait que la co-transfection de deux ADNc codant pour des protéines membranaires sature la machinerie de traduction et diminue l'expression du RTSH. <p>Dans une deuxième partie de notre travail, nous avons étudié la 5-phosphatase SHIP2, dont l’implication dans la cascade de réponse à l’insuline était suggérée, entre autres, par le travail d’Isabelle Vandenbroere qui avait montré l’interaction de cette protéine avec CAP et c-Cbl. Nous avons développé au laboratoire la culture de la lignée pré-adipocytaire 3T3-L1 et étudié la localisation de SHIP2 au niveau des rafts de ces cellules. Nous avons montré que SHIP2 n’y est pas recrutée. CAP et c-Cbl ne semblent pas non plus y être recrutées, tandis que nous y avons détecté le récepteur de l'insuline. La localisation de différentes protéines impliquées dans la cascade de l'insuline dans les rafts est une question controversée à l’heure actuelle et notre étude montre que l’implication fonctionnelle de SHIP2 dans la cascade de l'insuline n'est probablement pas dépendante des rafts.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Abstract The purpose of the study was to examine the cold-induced effects on pineal and thyroid hormones, as well as the associations of these hormones with psychological performance and to determine how psychological performance could be affected by demographic, anthropometric, physiological or biochemical measures during cold acclimatisation. The feasibility of urinary melatonin (MT), rather than 6-sulphatoxymelatonin (aMT6s), as an indicator of MT secretion was also examined. In the laboratory study, seasonal cold acclimatisation, its effects on hormones and their associations with mood and cognition were assessed in 15 young urban subjects exposed to cold in winter or summer in bright or dim light. In the field study, the associations of mood and cognition with demographic, anthropometric, physiological and biochemical measures were determined in healthy, euthyroid subjects (n = 133) in Antarctica in the beginning and at the end of summer and winter seasons. In both seasons, simple task performance was consistently impaired in the cold in experimental and field conditions. In complex tasks, negative, positive and mixed effects were observed. In the experimental study, serum MT and thyroid hormone levels were positively associated with mood. MT was negatively associated with simple task performance. Free triiodothyronine (T3) and thyrotropin (TSH) had mixed effects on simple task performance. TSH was positively associated with complex task performance. In the field study, higher age was associated with impaired cognition, especially in complex task performance. Total T3 was positively associated with mood and total thyroxine (T4) with complex task accuracy. Both urinary MT and aMT6s were good indicators of MT secretion, but the variation was smaller for MT. In conclusion, the associations of serum MT, TSH and thyroid hormone levels with mood and cognition found in experimental and field conditions are consistent with the psychological changes associated with the onset and consequent stages of the previously established polar T3 syndrome. In the field study, cognition and mood were associated with subject’s age and gender, which seemed to affect the physiological changes during acclimatisation to cold and darkness in Antarctica. / Tiivistelmä Tutkimuksen tarkoituksena oli selvittää kylmäaltistuksen aiheuttamia muutoksia käpy- ja kilpirauhasen hormonien tasoissa, hormonien yhteyttä psyykkiseen toimintakykyyn sekä demografisten, antropometristen, fysiologisten ja biokemiallisten tekijöiden yhteyttä psyykkiseen toimintakykyyn kylmäakklimatisaation aikana. Lisäksi verrattiin virtsan melatoniinin (MT) ja 6-sulfatoksimelatoniinin (aMT6s) soveltuvuutta MT-erityksen kuvaajina. Laboratoriotutkimuksessa selvitettiin talveen liittyvää kylmäsopeutumista, sen vaikutusta hormonitasoihin ja näiden yhteyttä mielialaan ja älylliseen toimintakykyyn 15 nuorella miehellä, jotka altistettiin kylmälle talvella ja kesällä sekä kirkkaassa että hämärässä valossa. Kenttäkokeessa Antarktiksella selvitettiin mielialan ja älyllisen toimintakyvyn sekä demografisten, antropometristen, fysiologisten ja biokemiallisten tekijöiden välistä yhteyttä terveillä henkilöillä (n =  133) kesä- ja talvikauden alussa ja lopussa. Suoriutuminen yksinkertaisista tehtävistä huononi kylmässä kesällä ja talvella sekä laboratorio- että kenttäoloissa. Kylmä vaikutti monimutkaisista tehtävistä suoriutumiseen vaihtelevasti. Laboratoriotutkimuksessa seerumin MT- ja kilpirauhashormonitasot korreloivat positiivisesti mielialan kanssa. MT korreloi negatiivisesti yksinkertaisista tehtävistä suoriutumisen kanssa. Vapaa trijodotyroniini (T3) ja tyrotropiini (TSH) korreloivat vaihtelevasti yksinkertaisista tehtävistä suoriutumisen kanssa. TSH korreloi positiivisesti monimutkaisista tehtävistä suoriutumisen kanssa. Kenttätutkimuksessa korkeampi ikä oli yhteydessä huonontuneeseeen älylliseen toimintakykyyn erityisesti monimutkaisissa tehtävissä. T3:n kokonaismäärä korreloi positiivisesti mielialan ja tyroksiinin (T4) kokonaismäärä monimutkaisista tehtävistä suoriutumisen kanssa. Sekä virtsan MT että aMT6s olivat hyviä MT-erityksen mittareita, mutta MT:ssa vaihtelu oli pienempää. Laboratorio- ja kenttäolosuhteissa havaitut MT:n, TSH:n ja kilpirauhashormonien yhteydet mielialaan ja älylliseen toimintakykyyn vahvistavat aiemmin havaittuja tuloksia polaarisen T3 -oireyhtymän synnystä ja oireyhtymän eri vaiheisiin liittyvistä psyykkisistä muutoksista. Kenttätutkimuksessa mieliala ja älyllinen toimintakyky olivat yhteydessä koehenkilön ikään ja sukupuoleen, jotka puolestaan voivat vaikuttaa fysiologisiin muutoksiin kylmään ilmastoon ja pimeyteen sopeutumisen aikana.

Abstract Maternal thyroid dysfunction and/or antibodies are present in 5–10% of pregnancies and may be associated with increased risks of adverse pregnancy and perinatal outcomes. In the present study maternal thyroid function and antibody status in the Northern Finland Birth Cohort 1986 was analyzed using early pregnancy serum samples. The impact of long-term storage on the stability of thyroid hormones and antibodies was studied and while TSH and thyroid hormone levels were not affected by storage time the concentrations of thyroid antibodies appeared to be significantly increased after 10 years of storage. Normal maternal thyroid function was evaluated by calculating thyroid hormone reference intervals in the thyroid antibody-negative population using a biobank of stored serum samples. Thyrotropin, free thyroxine and triiodothyronine reference intervals in the first and second trimester were 0.07–3.1 mU/L and 0.10–3.5 mU/L, 11.4–22.4 pmol/L and 11–18.9 pmol/L; and 3.4–7.0 pmol/L and 3.5–7.3 pmol/L, respectively, in this population (Abbott Architect method). Compared with thyroid antibody-negative mothers, antibody-positive mothers had significantly higher TSH and lower fT4 concentrations and an increased risk of experiencing death of an infant in the perinatal period with odds ratios (ORs) of 3.1 (95% confidence interval 1.4–7.1) for thyroid-peroxidase and OR 2.6 (1.1–6.2) for thyroglobulin antibody positivity. These infants were more often born very preterm, which could possibly explain these increased risks. Positive thyroid antibody status was not associated with preterm birth in this study. No other major pregnancy or perinatal complications were observed among mothers or newborns of mothers with thyroid dysfunction/antibodies. Mothers, who had hypothyroidism or thyroid antibodies during pregnancy, had a very high risk of subsequent thyroid disease: hazard ratio (HR) 17.7 (7.8–40.6) for overt hypothyroidism, 4.2 (2.3–7.4) for thyroid-peroxidase and 3.3 (1.9–6.0) for thyroglobulin antibody positivity. Mothers with hypothyroidism during pregnancy had increased risk of subsequent diabetes, (HR 6.0 [2.2–16.4]). Women at risk of thyroid dysfunction should be recognized and their prepregnancy counseling, blood sampling and treatment is probably beneficial. Whether universal screening of all pregnant women is justified is still under debate. / Tiivistelmä Kilpirauhasen toimintahäiriö tai ainoastaan kilpirauhasvasta-aineita (tyreoideaperoksidaasi- tai tyreoglobuliinivasta-aineita) esiintyy 5–10 % raskaana olevista naisista ja ne mahdollisesti lisäävät riskiä raskausajan ja vastasyntyneisyyskauden ongelmiin. Tässä väitöskirjatyössä tutkittiin Pohjois-Suomen syntymäkohorttia vuodelta 1985–1986. Äitien kilpirauhasen toimintaa tutkittiin alkuraskauden verinäytteiden avulla. Selvitimme pitkäaikaisen (20 vuotta) pakkassäilytyksen vaikutusta kilpirauhaslaboratoriokokeisiin. Tutkimuksessamme pakkassäilytyksellä ei ollut vaikutusta kilpirauhashormonien pitoisuuksiin, mutta kilpirauhasvasta-aineiden pitoisuudet olivat merkittävästi lähtötasoa korkeampia 10 säilytysvuoden jälkeen. Äitien normaali kilpirauhasen toiminta arvioitiin laskemalla aineistosta kilpirauhashormonien viitevälit kilpirauhasvasta-ainenegatiivisille naisille raskauden ensimmäiselle ja toiselle kolmannekselle käyttäen Abbott Architect metodia. Viitearvot olivat: tyreotropiinille 0.07–3.1 mU/l ja 0.10–3.5 mU/l, vapaalle tyroksiinille 11.4–22.4 ja 11–18.9 pmol/l sekä vapaalle trijodotyroniinille 3.4–7.0 ja 3.5–7.3 pmol/l. Äidin kilpirauhasen toimintahäiriöt eivät liittyneet vaikeisiin raskausajan tai vastasyntyneisyyskauden ongelmien, kuten ennenaikaisuuden ja kohtukuolemien esiintymiseen. Äidin kilpirauhasvasta-aineiden esiintyminen, mikä osoittaa kroonista autoimmuunityreoidiittia, lisäsi riskiä lapsen kohtukuolemaan ja ensimmäisen elinviikon kuolemaan; riski oli jopa kolminkertainen tyreoideaperoksidaasivasta-ainepositiivisten äitien vastasyntyneillä. Nämä vastasyntyneet olivat usein syntyneet hyvin ennenaikaisina (ennen 28. raskausviikkoa), mikä voi selittää tätä riskiä. Äidin kilpirauhasvasta-aineet eivät kuitenkaan lisänneet ennenaikaisten synnytysten riskiä tässä tutkimuksessa. Äideillä, joilla oli todettu kilpirauhasen vajaatoiminta tai kilpirauhasvasta-aineita, itsellään oli korkea, jopa 17-kertainen, riski sairastua myöhempiin kilpirauhasen sairauksiin, ja kilpirauhasen vajaatoiminta kuusinkertaisti sokeritautiin sairastumisriskin. Olisi tärkeää tunnistaa jo ennen raskautta ne naiset, joilla on riski sairastua kilpirauhasen vajaatoimintaan. Raskauden aikaisesta yleisestä seulonnasta ei vielä ole yksimielisyyttä.

Abstract Maternal gestational thyroid dysfunction has been associated with adverse neuropsychological development in children. This study investigated the effects of maternal thyroid dysfunction in early pregnancy and/or antibodies on the thyroid function and antibody status of children, as well as their association with the offspring’s ADHD symptoms, scholastic performance and sensory development. The study population consisted of the Northern Finland Birth Cohort of 1986. The mothers’ TSH, fT4 and TPO-Ab concentrations were evaluated in early pregnancy and in their offspring at 16 years of age. Data on the mothers and their families, their child’s health, development, behavior and scholastic performance were collected via parental questionnaires conducted in early pregnancy and when the children were 7-8 and 16 years old. Their teachers evaluated the children’s behavior and scholastic performance at 8 years of age, and at 16 years old the adolescents evaluated themselves. Maternal gestational thyroid dysfunction associated with adolescents’ increased odds of having the same thyroid dysfunction type. Adolescents of TPO-Ab-positive mothers had increased odds of being TPO-Ab-positive themselves. TPO-Ab-positive children had increased odds of having thyroid dysfunction. Increasing maternal TSH concentrations increased a child’s odds of having ADHD symptoms (OR 1.4 [95% CI 1.1-1.8]). Children of hypothyroxinemic mothers had increased odds of repeating a class at school (OR 3.5 [1.1-11.5]), and those of hyperthyroid mothers had increased odds of Finnish language learning difficulties (1.6 [1.03-2.4]). Furthermore, thyroid dysfunction in adolescents increased their odds of learning difficulties. No association was observed between maternal thyroid dysfunction and a child’s diagnosed intellectual deficiency and sensory development. Maternal thyroid dysfunction during pregnancy associated with thyroid dysfunction in the offspring. Maternal thyroid dysfunction may have a mild impact on her offspring’s neuropsychological development, but it had no effect on a child’s risk of diagnosed intellectual deficiency or sensory development. Children have compensatory mechanisms for overcoming early developmental thyroid hormone insufficiencies. Randomized trials for screening and treating maternal thyroid dysfunction during pregnancy are needed to evaluate the benefits to offspring. / Tiivistelmä Äidin raskauden aikaiset kilpirauhasen toimintahäiriöt on yhdistetty lapsen neuropsykologisen kehityksen ongelmiin, mutta aiempi tutkimustieto aiheesta on ristiriitaista. Tämän vuoksi tutkimme äidin raskauden ajan kilpirauhasen toimintahäiriöiden ja/tai vasta-aineiden vaikutusta nuoren kilpirauhastoimintaan ja vasta-ainestatukseen, ja näiden molempien vaikutusta lapsen ADHD-oireisiin, koulumenestykseen ja aistien kehitykseen. Tämän väitöskirjatyön aineistona oli väestöpohjainen Pohjois-Suomen syntymäkohortti 1986, johon kuuluu yli 99 % alueen raskaana olevista naisista. Äitien TSH, T4-V ja TPO-Ab – mittaukset tehtiin alkuraskaudessa ja kohortin lasten mittaukset 16-vuotiaana. Molempien kohdalla käytettiin väestöpohjaisia viitevälejä toimintahäiriön määrittämiseksi. Tietoja raskaudesta, äidin ja muun perheen sairastavuudesta, elintavoista ja sosioekonomisista tekijöistä ja lapsen terveydestä, kehityksestä, koulumenestyksestä ja käyttäytymisestä kerättiin kyselylomakkeilla raskauden aikana, 7-8-vuotiaana ja 16-vuotiaana. Myös luokanopettajat arvioivat lapsen koulumenestystä ja käyttäytymistä, ja nuoret itse arvioivat koulumenestystään 16-vuotiaina. Äidin raskauden aikainen kilpirauhasen toimintahäiriö nosti nuoren riskiä saada sama kilpirauhasen toimintahäiriö kuin äidillään. Äidin TPO-vasta-aine-positiivisuus nosti nuoren riskiä vasta-ainepositiivisuuteen. Nuoren positiiviset vasta-ainepitoisuudet nostivat riskiä poikkeaville kilpirauhasarvoille. Äidin nouseva TSH-pitoisuus yhdistyi lapsen suurempaan riskiin saada ADHD oireita 8-vuotiaana, mutta selkeää raja-arvoa sille ei löytynyt. Äidin hypo- tai hypertyreoosi eivät nostaneet lapsen ADHD-oireiden riskiä. Äidin kilpirauhastoimintahäiriöt nostivat hieman nuoren riskiä oppimisvaikeuksille ja luokan kertaamiselle. Myös nuoren oma kilpirauhastoiminta vaikutti vähäisessä määrin oppimiseen ja keskittymiseen. Äidin kilpirauhastoiminnalla ei ollut vaikutusta lapsen matalaan älykkyysosamäärään tai aistien kehitykseen Äidin raskaudenaikainen kilpirauhasen toimintahäiriö vaikutti lapsen neuropsykologiseen kehitykseen lievästi, mutta löydösten kliininen merkitys on vähäinen. Lasten keskushermoston korjaavat mekanismit todennäköisesti kompensoivat varhaiskehityksen kilpirauhashormonien vajetta. Randomoidulla tutkimuksella voitaisiin selvittää, hyötyisivätkö lapset äidin kilpirauhassairauden seulomisesta ja hoitamisesta alkuraskaudessa.

Thyroid hormones (THs; T4 and T3), secreted from thyroid gland, play an important role in human growth and development. T3 (3,5,3′-triiodothyronine) is the active hormone and the conversion of T4 (3,3′,5,5′-tetraiodothyronine) to T3 in cells is mediated by iodothyronine deiodinases enzymes (DIOs). DIOs are selenocysteine containing enzymes and are classified into three types (DIO1, DIO2 and DIO3). DIO1 catalyzes the outer-ring deiodination (ORD; T3 formation) and inner-ring deiodination (IRD; rT3 formation) reactions, involving in the activation (T4 to T3 conversion) and inactivation (T4 to rT3 conversion), respectively. DIO2 and DIO3 catalyse the ORD and IRD reactions, respectively. This homeostasis is regulated tightly and any deviation would lead to diseases like hyperthyroidism or hypothyroidism. Recently it is of interest to many research groups to develop iodothyronine deiodinase mimics and we have developed naphthalene-based peri-substituted thioselenol pair at 1,8-positions (1.25), which remove iodine selectively from inner-ring of T4. When selenium atom is substituted in place of sulfur (selenol-selenol pair; 1.26), the deiodination activity was ca. 90 times faster than with 1.25. This thesis deals with various aspects of the effect of substituents on the naphthalene-1,8-diselenol and solvent effect on the thyroid hormone deiodination by naphthalene-based iodothyronine deiodinase mimics. Figure 1. (A) Deiodination reactions by DIOs. (B) Chemical structure of 1.25 and 1.26. The thesis consists of five chapters. The first chapter provides a general overview about sialoproteins, thyroid hormone biosynthesis, thyroid hormone metabolism, halogen bonding, iodothyronine deiodinase mimics and proposed mechanisms for the deidoination of thyroid hormones. This chapter also introduces peri-naphthalene-1,8-diselenol (1.26), which is the key compound in this thesis and discusses about proposed mechanism for the deiodination of thyroxine involving co-operative halogen bonding and chalcogen bonding mechanism. Figure 2. (A) TH action. (B) Proposed mechanism for the deiodination of T4 by 1.26 involving cooperative halogen bonding and chalcogen bonding. Chapter 2 discusses about the synthesis, characterization and deiodination activity of a series of naphthalene-based peri-substituted-1,8-diselenols (Figure 3). These diselenols regioselectivity remove iodine from inner ring of thyroxine and other thyroid hormones, (T3 and 3, 5-T2). Substitution with different groups on the naphthalene ring did not change the regioselectivity of deiodination, indicating that the deiodination activity does not depend on the nature of substituents. Secondary or tertiary amine side chain group attached at the 2nd position of the naphthalene ring showed better activity. It is due to the secondary interaction, which facilitates the iodine removal. It was further confirmed with the substitutions at the 4th position of the ring to discriminate the possibility of electronic effect. The higher deiodination rate owing to the t-butyl group at second position of the ring also suggests that the steric effect may also play a role in the deiodination reaction (Figure 4). It is proposed that peri substituted naphthalene-1,8-diselenols remove iodine from thyroid hormones through halogen bonding-chalcogen bonding mechanism (Figure 2). The investigation of Se···Se bond distance from the crystal structures and through DFT calculation and NMR experiment showed that the stronger chalcogen bond could be the reason for the increase in the reactivity observed with substituted peri-naphthalene-1,8-diselenols. Figure 3. peri-substituted naphthalene-1,8-diselenols used for the study. Figure 4. Relative deiodinase activity of substituted-peri-naphthalene-1,8-diselenols with T4. In Chapter 3, we have discussed about the effect of chalcogen atom substitution in a series of deiodinase mimics on the deiodination of thyroid hormones. Moving from thiol-selenol pair (1.25) to selenol-selenol pair (1.26) in naphthalene based peri-substituted mimics, an increase in the activity was observed. In this chapter, we have shown that substituting with tellurium, as tellurium-thiol pair (3.3) and ditellurol (3.4) increases the reactivity of deiodination to several times and also regioselectivity of deiodination is changed from IRD in the case of 1.26 to both IRD and ORD for 3.3 and 3.4. The presence of two tellurol moieties (3.4) or a thiol-tellurol pair (3.3) can mediate sequential deiodination of T4, to produce all the possible thyroid hormone derivatives under physiologically relevant conditions (Figure 5). This study provided the first experimental evidence that the regioselectivity of the thyroid hormone deiodination is controlled by the nucleophilicity and the strength of halogen bond between the iodine and chalcogen atoms. Figure 5. (A) HPLC chromatograms of deiodination reaction of T4 with 3.3 and 3.4. (B) Chemical structure of 3.3 and 3.4. (C) Sequential deiodination reaction of T4 by 3.3 and 3.4. Chapter 4 describes the effect of alkyl conjugation at 4′-OH position of THs on the deiodination by iodothyronine mimics. In addition to the deiodination, iodothyronines undergo conjugation with sulfate and glucuronic acid group at 4′-hydroxyl position. Conjugation alters the physico-chemical properties of iodothyronines. For example, it is known that sulfate conjugation increases the rate of deiodination to a large extend. We have conjugated alkyl group at 4′-hydroxyl position of iodothyronines and investigated the deiodination reactions with reported peri-substituted naphthalene-1,8-diselenols. We observed that similar to sulfated thyroid hormones O-methylthyroxine also undergoes both phenolic and tyrosyl ring deiodination reactions and overall the rate of deiodination is increased at least by 5 times as compared with T4 under identical conditions. The phenolic iodine removal is favored by conjugation as compared to the tyrosyl ring iodine, which is similar to the observation made for T4S. Interestingly, when the acetamide group is conjugated at 4′-OH position, the regioselectivity of deiodination is changed exclusively to 5′-iodine. DFT calculations show that the positive potential on the iodine increase upon conjugation, which leads to stronger halogen bonding interaction with selenol, might be the reason for the change in the regioselectivity of deiodination. Figure 6. (A) HPLC chromatogram of deiodination reaction of T4(Me) with 1.26. (B) Initial rate comparison of T4 and T4(Me).(C) HPLC chromatogram of deiodination reaction of T4(AA) with 1.26 showing the formation of T3(AA) (ORD product). (D) Electron potential map of T4, T4(Me) and T4(AA) showing the increase in electro positive potential on 5′-iodine upon conjugation. Chapter 5 deals with the solvent effect on the deiodination reactions of THs by iodothyronine deiodinase mimics. As discussed in the earlier chapters, the deiodination reaction of thyroxine by naphthalene based-1,8-diselenols under physiological conditions produce, rT3 (IRD) as the only observable products. Surprisingly, when the deiodination reaction was performed in DMF or DMSO in the presence of 1.26, the regioselectivity of reaction was changed and the formation of both T3 (ORD) and rT3 was observed. In DMF or in DMSO, the deiodination reactivity of 1.26 was found to be 1000 fold higher than the reaction performed in phosphate buffer at pH 7.4. Figure 7. (A) HPLC chromatogram for the deiodination reaction of T4 in DMF by 1.26 showing both IRD and ORD. (B) A comparison of initial rate for the deiodination reactions of T4, T3 and 3,5-T2 in DMF and in DMSO by 1.26. (C) HPLC chromatograms for the deiodination reaction of T4 in DMF by 1.26 in the presence of TEMPO, showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. (D) HPLC chromatograms for the deiodination reaction of T4 in DMSO by 1.26 in the presence of TEMPO showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. 3,5-DIT was not denominated under physiological conditions, however, in DMF and in DMSO, 3,5-DIT was deiodinated by 2.4 to produce 3-MIT. We also observed that the control reactions in DMF or DMSO also showed a little deiodination activity. The very high reactivity observed in the presence of DMF or DMSO implied that the mechanism of denomination in these solvents may be different. It has been reported that DMSO or DMF radicals can be formed with small amounts of a base. Reaction mixture consisting of NaBH4 (for generating selenol from diselenide) and NaOH (T4 solution) may facilitate the radical formation. We also performed the reaction in the presence of TEMPO (free radical scavenger) and observed the inhibition of deiodination reaction. However, it is not clear whether the radical pathway could be one of the possible mechanisms of deiodination in these solvents by compounds 1.26 and 2.4. Further studies are required to propose a radical mechanism in different solvents such as DMF and DMSO.