Estudos recentes demonstram que o Hormônio Tiroideano (HT) é capaz de modular rapidamente o estado de fosforilação de proteínas quinases relacionadas ao processo de hipertrofia cardíaca. Evidências experimentais indicam que a proteína quinase ativada por AMP (AMPK) seja um alvo importante no controle do crescimento hipertrófico, uma vez que a ativação desta enzima determina ampla variedade de efeitos siológicos, incluindo o controle de proteínas relacionadas à síntese protéica. Dessa forma, os objetivos deste estudo foram os de avaliar os efeitos do HT sobre a modulação da via de sinalização da AMPK, além de verificar o possível envolvimento desta quinase no modelo de hipertrofia in vitro induzida pelo HT. Os resultados obtidos mostraram que rapidamente este hormônio ativa a AMPK e proteínas relacionadas a esta sinalização. Além disso, a estimulação farmacológica da AMPK atenua a hipertrofia de cardiomiócitos induzida pelo HT. Estes dados sugerem que a AMPK seja uma possível ferramenta terapêutica em doenças cardiovasculares como a hipertrofia cardíaca. / Some studies have shown that Thyroid Hormones (TH) are also able to rapidly modulate the phosphorylation state of protein kinases related to cardiac hypertrophy process by non-genomic actions. In this sense, experimental evidences indicate that AMP-activated protein kinase (AMPK) is an important target in the control of hypertrophic growth, since the activation of this enzyme determines wide variety of physiological effects, including the control of enzymes related to protein synthesis. Thus, the objective of this study was to evaluate the effects of TH on the modulation of AMPK signaling pathway and to check the possible involvement of this kinase in vitro model of hypertrophy induced by TH. The results showed that this hormone rapidly activates AMPK and related proteins to this signaling. Furthermore, pharmacological stimulation of AMPK attenuated cardiomyocyte hypertrophy induced by TH. These data suggest that AMPK may correspond to a possible therapeutic tool in cardiovascular disease.

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.

Introduction : Les séquelles érectiles de la prostatectomie totale pour cancer concernent la majorité des patients. Des protocoles de remplacement nerveux, prometteurs sur des modèles animaux, n’ont pas amélioré la récupération chez l’homme dont l’anatomie nerveuse pelvienne est plus complexe.Matériel et méthodes : Trois études expérimentales chez le rat ont évalué différentes stratégies, seules ou combinées, de réparation de nerfs caverneux lésés par écrasement : l’engainement synthétique ou autologue et l’adjonction locale ou systémique de traitements pharmacologiques. Deux études anatomiques ont consisté à mettre au point et à appliquer une technique de dissection assistée par ordinateur permettant une analyse morphologique et fonctionnelle de l’innervation péri-prostatique chez 7 fœtus masculins et 4 sujets cadavériques.Résultats : L’engainement synthétique du nerf caverneux lésé a permis une récupération érectile partielle. En y associant une délivrance locale de Triiodothyronine ou systémique de sildénafil, la récupération était complète. Un traitement par sildénafil seul permettait également une récupération complète. L’engainement autologue était inefficace. Les fibres nerveuses de la bandelette neuro-vasculaire se distribuaient en fibres antérieures destinées au sphincter urétral, antéro-latérales (nerfs caverneux) destinées aux corps caverneux et postéro-latérales (nerfs spongieux) destinées aux corps spongieux.Conclusion : Les nerfs destinés aux corps érectiles ont une position plus antérieure que celle décrite dans les traités chirurgicaux de référence. Ce trajet doit être pris en compte avant d’envisager des études cliniques de réparation nerveuse. Les stratégies de réparation du nerf caverneux devraient être adaptées au plan d’éxérèse envisagé et à la qualité de la préservation nerveuse réalisée. / Introduction : Erectile dysfunction occurs in more than half of the patients after a radical prostatectomy despite nerve-preserving surgical techniques. Nerve replacement protocols have been successful in animal models but they failed in improving the erectile function recovery in men where the pelvic nervous anatomy is more complex.Methods : Three experimental studies in rats have evaluated three different strategies, alone or in combination, to repair cavernous nerves after a crush-injury : guiding the nerve with an autologous or a synthetic disposal and adding local or systemic pharmacological treatments. Two anatomical studies have developped and applied a computer-assisted anatomical dissection technique to perform a morphological and a functional analysis of the peri-prostatic innervation in 7 male fetuses and 4 cadaveric subjects.Results : The erectile function recovery was partial after implantation of artificial conduits and complete with an additional local delivery of Triiodothyronine or systemic delivery of sildenafil. Sildenafil treatment alone also allowed full recovery. Autologous conduits were ineffective. The neurovascular bundles had distal divisions : anterior fibers innervated the urethral sphincter, antero-lateral fibers (cavernous nerves) innervated the corpora cavernosa and postero-lateral fibers (spongious nerves) innervated the corpora spongiosa.Conclusion : The position of the erectile fibers of the neurovascular bundles is more anterior than classically described. This pathway has to be considerated before any clinical study for cavernous nerve repair. For each patient, the planned type of nerve-preservation should determine the strategy for the nerve repair.

La métamorphose des amphibiens est le processus rapide et irréversible par lequel un têtard aquatique se transforme en une grenouille respirant à la surface. Cette transition écologique, réminiscente de la période périnatale chez les mammifères, s'accompagne de changements spectaculaires (régime alimentaire, organes locomoteurs, système respiratoire...). Ces modifications morphologiques et physiologiques nécessitent la réponse concertée à un signal hormonal, les hormones thyroïdiennes (HT), de différents tissus vers des destin parfois opposés : apoptose (dans la queue), prolifération (dans les pattes), et remodelage (dans les intestins et le système nerveux central). Toutefois, la synchronisation de la réponse des différents tissus fait appel à d'autres signaux hormonaux, et notamment les glucocorticoïdes (GC). Ces derniers sont également les médiateurs principaux de la réponse au stress. Les processus endocriniens de la métamorphose et la réponse au stress sont fortement couplés. Les GC peuvent ainsi jouer le rôle d'interface permettant l'intégration de signaux environnementaux au niveau de réseaux de régulation. Dans le cadre de mon doctorat, j'ai analysé les transcriptomes des bourgeons de membres postérieurs et de l'épiderme caudal de têtards de Xenopus tropicalis traités ponctuellement avec des HT et / ou des GC. La comparaison de ces deux tissus a permis de caractériser la diversité des profils d'expression des gènes cibles des HT et des GC.Il en ressort plusieurs résultats majeurs. Tout d'abord, la diversité des profils d'interaction entre ces deux voies est limitée, et la majorité des types de profils sont communs aux deux tissus. Indépendamment du tissu, certains profils sont caractéristiques de fonctions biologiques spécifiques comme le remodelage de la matrice extracellulaire et le système immunitaire. Les gènes impliqués dans ces fonctions communes aux deux tissus sont cependant différents. Enfin, plusieurs facteurs impliqués dans la méthylation de l'ADN sont régulés par les deux hormones. / Amphibian metamorphosis is the rapid and irreversible process during which an aquatic tadpole transforms into an air breathing adult frog. This ecological transition, reminiscent of the mammalian perinatal period, comes with spectacular changes (diet, locmotor organs, respiratory system...). These morphological and physiological modifications necessitate the properly timed response to a single hormonal signal, the thyroid hormones (TH), in various tissues to lead them to sometimes opposite fates : apoptosis (in the tail), cell prolifération and differenciation (in the limbs) and remodeling (in the intestine and the central nervous system).However, TH do not act alone. In particular, glucocorticoids (GC) play important roles during this process. They also are the main mediator of the stress response. Endocrine processes of the metamorphosis and the stress response are deeply intertwined. GC can thus act as an interface to integrate environmental inputs into regulatory networks.During my doctorate, I analyzed the possible transcriptional crosstalks between TH and GC in two larval tissues : the tailfin (TF) and the hindlimb buds (HLB). Comparing these two tissues allowed me to caracterize the diversity of TH and GC target gene expression profiles. This resulted in several major results. First, the diversity of the profiles of crosstalk between these two pathways is limited, and the majority of the types of profiles is common to both tissues. Next, independently ofthe tissues, some profiles are caracteristic of spécific biological functions such as extracellular matrix remodeling and the immune system. Yet, the genes involved in these shared functions are different between the TF and the HLB. Finally, several factors involved in DNA methylation are subject to a crosstalk between the two hormones.

Estudos recentes demonstram que o Hormônio Tiroideano (HT) é capaz de modular rapidamente o estado de fosforilação de proteínas quinases relacionadas ao processo de hipertrofia cardíaca. Evidências experimentais indicam que a proteína quinase ativada por AMP (AMPK) seja um alvo importante no controle do crescimento hipertrófico, uma vez que a ativação desta enzima determina ampla variedade de efeitos siológicos, incluindo o controle de proteínas relacionadas à síntese protéica. Dessa forma, os objetivos deste estudo foram os de avaliar os efeitos do HT sobre a modulação da via de sinalização da AMPK, além de verificar o possível envolvimento desta quinase no modelo de hipertrofia in vitro induzida pelo HT. Os resultados obtidos mostraram que rapidamente este hormônio ativa a AMPK e proteínas relacionadas a esta sinalização. Além disso, a estimulação farmacológica da AMPK atenua a hipertrofia de cardiomiócitos induzida pelo HT. Estes dados sugerem que a AMPK seja uma possível ferramenta terapêutica em doenças cardiovasculares como a hipertrofia cardíaca. / Some studies have shown that Thyroid Hormones (TH) are also able to rapidly modulate the phosphorylation state of protein kinases related to cardiac hypertrophy process by non-genomic actions. In this sense, experimental evidences indicate that AMP-activated protein kinase (AMPK) is an important target in the control of hypertrophic growth, since the activation of this enzyme determines wide variety of physiological effects, including the control of enzymes related to protein synthesis. Thus, the objective of this study was to evaluate the effects of TH on the modulation of AMPK signaling pathway and to check the possible involvement of this kinase in vitro model of hypertrophy induced by TH. The results showed that this hormone rapidly activates AMPK and related proteins to this signaling. Furthermore, pharmacological stimulation of AMPK attenuated cardiomyocyte hypertrophy induced by TH. These data suggest that AMPK may correspond to a possible therapeutic tool in cardiovascular disease.

O hormônio tireoidiano (HT) é essencial para o desenvolvimento, maturação e metabolismo ósseos, enquanto que o sistema nervoso simpático (SNS) é, também, um potente regulador do remodelamento ósseo. Demonstrou-se que SNS regula negativamente a massa óssea, agindo via receptores ?2-adrenérgicos (?2-AR), expressos em osteoblastos. O nosso grupo demonstrou que os receptores ?2 adrenérgicos (?2-AR) também medeiam ações do SNS no esqueleto e que são expressos em osteoblastos, osteócitos, condrócitos e osteoclastos. Considerando-se que o HT interage com o SNS para regular uma série de processos fisiológicos, e que o excesso de HT e a ativação do SNS causam perda de massa óssea, levantamos a hipótese de que há interação entre o HT com o SNS para regular a massa óssea. Estudos do nosso grupo vêm sustentando essa hipótese, uma vez que camundongos com inativação gênica dos receptores beta2-AR apresentam resistência à osteopenia induzida por doses tóxicas de HT. Considerando-se, ainda, que a interação do HT com o SNS em vários tecidos e/ou órgãos depende da sinalização beta2 adrenérgica, o presente estudo teve como objetivo avaliar se a interação do HT com o SNS para regular a morfofisiologia óssea envolve o beta2-AR. Para tanto, estudamos o efeito de 10x e 20x a dose fisiológica de triiodotironina (3,5ug ou 7.0ug de T3/100g de massa corporal/dia, respectivamente), por 90 dias, na microaquitetura óssea e em parâmetros biomecânicos do fêmur de camundongos com inativação gênica do beta2-AR (beta2-AR-/-), e nos seus respectivos Selvagens (Selv), os camundongos da linhagem FVB. Como esperado, o tratamento com T3 promoveu efeitos deletérios na microarquitetura trabecular das fêmeas Selv, enquanto alguns desses efeitos foram mais brandos ou inexistentes nos animais beta2-AR-/-, revelando resistência do osso trabecular dos animais knockout (KO) aos efeitos deletérios da tireotoxicose. Em contraste, a microarquitetura femoral dos camundongos machos beta2-AR-/- se mostrou mais sensível aos efeitos deletérios da tireotoxicose, em relação aos respectivos Selv. Quanto ao osso cortical femoral, vimos que o tratamento com T3 aumentou o perímetro endosteal e a área medular nos animais Selv machos e fêmeas, mas não nos animais beta2-AR-/-, o que sugere que o T3 promove reabsorção óssea endosteal no osso cortical, em um mecanismo que depende da via de sinalização do beta2-AR. Vimos, ainda, que o tratamento com T3 causou reduções significativas na carga máxima, tenacidade, rigidez e resiliência do fêmur dos camundongos fêmeas Selv. Em contraste, nenhum desses parâmetros biomecânicos foi afetado pelo tratamento com T3 no fêmur das fêmeas KO, evidenciando, mais uma vez, uma resistência desses animais aos efeitos deletérios da tireotoxicose no tecido ósseo. Por outro lado, os camundongos machos Selv e KO se mostraram resistentes aos efeitos deletérios do tratamento com T3 sobre os parâmetros biomecânicos do fêmur, sugerindo a participação de fatores sexuais na interação do HT com o SNS para regular a morfofisiologia óssea. Em conjunto, os achados do presente estudo corroboram a hipótese de que o HT interage com o SNS através da via dos receptores beta2 adrenérgicos para regular a morfofisiologia óssea, especialmente em fêmeas e no osso cortical / Thyroid hormone (TH) is essential for bone development, maturation and metabolism, while the sympathetic nervous system (SNS) is also a potent regulator of bone remodeling. SNS has been shown to negatively regulate bone mass, acting via beta2-adrenergic (beta2-AR) receptors expressed in osteoblasts. Our group demonstrated that alpha2-adrenergic (alpha2-AR) receptors also mediate SNS actions in the skeleton and are expressed in osteoblasts, osteocytes, chondrocytes and osteoclasts. Considering that TH interacts with the SNS to regulate a series of physiological processes, and that the excess of TH and the activation of the SNS cause loss of bone mass, we hypothesize that there is interaction between TH and the SNS to regulate the bone mass. Studies of our group have supported this hypothesis, since mice with gene inactivation of alpha2-AR present resistance to the osteopenia induced by toxic doses of TH. Considering that the TH-SNS interaction in various tissues and/or organs depends on beta2-adrenergic signaling, the present study aimed to evaluate whether the interaction of TH with the SNS to regulate the bone morphophysiology involves beta2- AR. Therefore, we studied the effect of 10x and 20x the physiological dose of triiodothyronine (3.5ug or 7.0ug of T3/100g body mass/day, respectively), for 90 days, in the bone microarchitecture and biomechanical parameters of the femur mice with beta2-AR gene inactivation (beta2-AR-/-), and of their respective Wild-type (WT) controls, the FVB lineage mice. As expected, T3 treatment promoted deleterious effects on the trabecular microarchitecture of the WT females, while some of these effects were milder or nonexistent in beta2-AR-/- animals, revealing trabecular bone resistance of knockout (KO) animals to the deleterious effects of thyrotoxicosis. In contrast, the femoral microarchitecture of the male beta2-AR-/- mice was more sensitive to the deleterious effects of thyrotoxicosis, in relation to the respective WT animals. Regarding to the femoral cortical bone, we saw that T3 treatment increased the endosteal perimeter and the medullary area both male and female WT animals, but not in the beta2-AR-/- mice, suggesting that T3 promotes endosteal bone resorption in the cortical bone, in a mechanism that depends on the alpha2-AR signaling pathway. We also found that treatment with T3 caused significant reductions in the maximum load, tenacity, stiffness and resilience of femurs of the WT female mice. In contrast, none of these biomechanical parameters was affected by T3 treatment in the KO females, demonstrating again resistance of these animals to the deleterious effects of thyrotoxicosis on bone tissue. On the other hand, WT and KO male mice were resistant to the deleterious effects of T3 treatment on the biomechanical parameters of the femur, suggesting the participation of sexual factors in the interaction of HT with the SNS to regulate bone morphophysiology. Taken together, the findings of the present study corroborate the hypothesis that TH interacts with the SNS through the beta2 adrenergic receptor pathway to regulate bone morphophysiology, especially in females and cortical bone

Adiponectin is a protein secreted by adipose tissue which possesses insulin-sensitivity and anti-atherosclerotic properties. In this work, we will study if high molecular weight adiponectin is a key molecule in the association between energy metabolism, thyroid axis and bone metabolism in children. To this end, we studied a population of 234 asymptomatic Caucasian children. A sample of blood was also collected to quantify HMW adiponectin, thyroid hormones, bone markers, vitamin D and sFASN. We also obtained 6 biopsies of visceral adipose tissue. The results derived from this study show a positive association of high molecular weight adiponectin with free T4 and soluble FASN and a negative association with carboxylated OC. And provide further evidence of the important role of HMW adiponectin as a biomarker of the common regulation between adipose tissue, the thyroid axis and bone metabolism in healthy children. / L'adiponectina és una proteïna secretada pel teixit adipós que té propietats insulinosensibilitzants i anti-arterioscleròtiques. L’objectiu d’aquest estudi és estudiar a l’adiponectina d’alt pes molecular com a molècula clau en la interacció entre el metabolisme energètic, l’eix tiroïdal i el metabolisme ossi. Per a tal fi, s’ha dissenyat un estudi clínic descriptiu transversal en una mostra de nens sans. A tots ells se'ls va quantificar l’adiponectina d'APM, les hormones tiroïdals, marcadors ossis, la vitamina D i FASN soluble. Addicionalment, es van obtenir 6 biòpsies de teixit adipós visceral.Els resultats derivats d’aquest estudi mostren una associació positiva de l’adiponectina d’alt pes molecular amb la T4 lliure i la FASN soluble i negativa amb la OC carboxilada. I aporten noves evidències de l'important paper de l'adiponectina d'APM com a marcador biològic de la regulació comuna entre el teixit adipòs, l’eix tiroïdal i el metabolisme ossi en edat pediàtrica.

Obesity is one of the most important public health problems facing the world today. Gene expression studies applied to fat depots from obese subjects have provided important clues about the pathophysiology of adipose tissue. The data collected in this thesis show that the synthesis of fatty acids (lipogenesis) is decreased in the adipose tissue of obese subjects, and describe the behavior of a new lipogenic factor. We also demonstrate that subcutaneous fat (beneath the skin of the buttocks, thighs and abdomen) is characterized by a greater responsiveness to thyroid hormones than the visceral (around the omentum, the intestines and the perirenal areas), and describe the increased activity of enzymes that activate thyroid hormones in adipose tissue of obese patients and the effects on the metabolism. According to these results, the local activation of thyroid hormone and the ability to synthesize fat are altered in adipose tissue from obese patients, and indicate significant differences between visceral and subcutaneous adipose tissue depots. / La obesidad es uno de los problemas de salud pública más importante. Los estudios de expresión aplicados a los depósitos de grasa en sujetos obesos han aportado importantes indicios sobre la fisiopatología del tejido adiposo. Los datos recogidos en esta tesis doctoral demuestran que la síntesis de grasa (lipogénesis) está disminuida en el tejido adiposo del paciente obeso, y describen el comportamiento de un nuevo factor lipogénicos. Se demuestra además que el tejido adiposo subcutáneo (situado bajo la piel de las nalgas , muslos y abdomen) está caracterizado por una mayor capacidad de respuesta a las hormonas tiroideas respecto al adiposo visceral (alrededor del epiplón, los intestinos y las áreas perirrenal) y se describe un incremento en la actividad de las enzimas que activan las hormonas tiroideas en el tejido adiposo del paciente obeso y los posibles efectos de esta eventualidad sobre el metabolismo. Según los resultados recopilados en esta tesis doctoral, la activación local de hormonas tiroideas y la capacidad para sintetizar acidos grasos del tejido adiposo del paciente obeso están alteradas, e indican importantes diferencias entre los depósitos de grasa visceral y subcutáneo. / L’obesitat és un dels problemes de salut pública més important. Els estudis d'expressió aplicats als dipòsits de greix en subjectes obesos han aportat importants indicis sobre la fisiopatologia del teixit adipós. Les dades recollides a aquesta tesi doctoral demostren que la síntesis de greix (lipogènesis) està disminuïda al teixit adipós del pacient obès, i descriuen el comportament d'un nou factor lipogènic. Es demostra, a més a més, que el teixit adipós subcutani (situat sota la pell de les natges, cuixes i abdomen) està caracteritzat per una major capacitat de resposta a les hormones tiroidees respecte a l'adipós visceral (al voltant de l’epipló, els intestins i las àrees perirenals) i es descriu un increment en l’activitat dels enzims que activen les hormones tiroidees al teixit adipós del pacient obès i els possibles efectes d'aquesta eventualitat sobre el metabolisme. Segons els resultats recopilats a aquesta tesi doctoral, l’activació local d'hormones tiroidees i la capacitat per sintetitzar greixos del teixit adipós del pacient obès estan alterades, i indiquen importants diferències entre els dipòsits de greix visceral i subcutani.

Thyroxine (T4), the main secretory hormone of the thyroid gland, is produced on thyroglobulin by thyroid peroxidase (TPO)/hydrogen peroxide/iodide system. The synthesis of T4 by TPO involves two independent steps: iodination of tyrosine and phenolic coupling of the resulting iodotyrosine residues. The prohormone T4 is then converted to its biologically active form T3 by a selenocysteine-containing iodothyronine deiodinase (ID-I), which is present in highest amounts in liver, kidney, thyroid and pituitary. The 5'-deiodination catalyzed by ID-I is a ping-pong, bisubstrate reaction in which the selenol (or selenolate) group of the enzyme (E-SeH or E-Se-) first reacts with thyroxine (T4) to form a selenenyl iodide (E-SeI) intermediate. Subsequent reaction of the selenenyl iodide with an as yet unidentified intracellular cofactor completes the catalytic cycle and regenerates the selenol. Although the deiodination reactions are essential for the function of thyroid gland, the activation of thyroid stimulating hormone (TSH) receptor by auto-antibodies leads to an overproduction of thyroid hormones. In addition, these antibodies stimulate ID-I and probably other deiodinases to produce relatively more amount of T3. Figure 1. Synthesis of thyroid hormones by heme-containing Thyroid Peroxidase(TPO)(Refer PDF File) As these antibodies are not under pituitary feedback control system, there is no negative influence on the thyroid activity and, therefore, the uncontrolled production of thyroid hormones leads to a condition called “hyperthyroidism”. Under these conditions, the overproduction of T4 and T3 can be controlled by specific inhibitors, which either block the thyroid hormone biosynthesis or reduce the conversion of T4 to T3. A unique class of such inhibitors is the thiourea drugs, methimazole (1, MMI), 6-n-propyl-2-thiouracil (3, PTU), and 6-methyl-2-thiouracil (5, MTU). Although these compounds are the most commonly employed drugs in the treatment of hyperthyroidism, the detailed mechanism of their action is still not clear. According to the initially proposed mechanism, these drugs may divert oxidized iodides away from thyroglobulin by forming stable electron donor-acceptor complexes with diiodine, which can effectively reduce the thyroid hormone biosynthesis. It has also been proposed that these drugs may block the thyroid hormone synthesis by coordinating to the metal center of thyroid peroxidase (TPO). After the discovery that the ID-I is responsible for the activation of thyroxine, it has been reported that PTU, but not MMI, reacts with the selenenyl iodide intermediate (E-SeI) of ID-I to form a selenenyl sulfide as a dead end product, thereby blocking the conversion of T4 to T3 during the monodeiodination reaction. The mechanism of anti-thyroid activity is further complicated by the fact that the gold-containing drugs such as gold thioglucose (GTG) inhibit the deiodinase activity by reacting with the selenol group of the native enzyme. Recently, the selenium analogues 2 (MSeI), 4 (PSeU) and 6 (MSeU) attracted considerable attention because these compounds are expected to be more nucleophilic than their sulfur analogues and the formation of an –Se–Se– bond may occur more readily than the formation of an –Se–S– bond with the ID-I enzyme. However, the data derived from the inhibition of TPO by selenium compounds show that these compounds may inhibit the TPO activity by a different mechanism. Therefore, further studies are required to understand the mechanism by which the selenium compounds exert their inhibitory action. Our initial attempts to isolate 2 were unsuccessful and the final stable compound in the synthesis was characterized to be the diselenide (8). In view of the current interest in anti-thyroid drugs and their mechanism, we extended our approach to the synthesis and biological activities of a number of sulfur and selenium derivatives bearing the methimazole pharmacophore. The thesis consists of five chapters. The first chapter gives a general introduction to thyroid hormone synthesis and anti-thyroid drugs. In this chapter, the biosynthesis of thyroid hormones, structure and function of heme peroxidases, activation of thyroid hormones by iodothyronine deiodinases are discussed. This chapter also gives a brief introduction to some common problems associated with the thyroid gland, with a particular emphasis on hyperthyroidism. The structure and activity of some commonly used anti-thyroid drugs and the role of selenium in thyroid are discussed. The literature references related to this work are provided at the end of the chapter. The second chapter deals with the synthesis and characterization of the selenium analogue (MSeI) of anti-thyroid drug methimazole and a series of organoselenium compounds bearing N-methylimidazole pharmacophore are described. The clinically employed anti-thyroid drug, methimazole (MMI), exists predominantly in its thione form, which is responsible for its anti-thyroidal activity. The selenium analogue MSeI, on the other hand, is not stable in air and spontaneously oxidizes to the corresponding diselenide (MSeIox). Experimental and theoretical studies on MSeI suggest that this compound exists in a zwitterionic form in which the selenium atom carries a large negative charge. The structure of MSeI was studied in solution by NMR spectroscopy and the 77Se NMR chemical shift shows a large upfield shift (-5 ppm) in the signal as compared to the true selones for which the signals normally appear in the downfield range (500-2500 ppm). This confirms that MSeI exists predominantly in its zwitterionic form in solution. Our theoretical studies show that the formation of the diselenide (MSeIox) from selenol tautomer is energetically more favored than the formation of the disulfide (MMIox) from the thiol tautomer of MMI. This study also shows that the replacement of the N−H group in MSeI by an N-methyl or N-benzyl substituent does not affect the nature of C−Se bond. In the third chapter, the inhibition of lactoperoxidase-catalyzed oxidation of ABTS by anti-thyroid drugs and related derivatives is described. The commonly used anti-thyroid agent methemazole (MMI) inhibits the lactoperoxidase (LPO) with an IC50 value of 7.0 µM which is much lower than that of the other two anti-thyroid drugs, PTU and MTU. The selenium analogue of methimazole (MSeI) also inhibits LPO with an IC50 value of 16.4 µM, which is about 4-5 times lower than that of PTU and MTU. In contrast to thiones and selones, the S- and Se-protected compounds do not show any noticeable inhibition under identical experimental conditions. While the inhibition of LPO by MMI cannot be reversed by increasing the hydrogen peroxide concentration, the inhibition by MSeI can be completely reversed by increasing the peroxide concentration. Some of the selenium compounds in the present study show interesting anti-oxidant activity in addition to their inhibition propertities. In the presence of glutathione (GSH), MSeI constitutes a redox cycle involving a catalytic reduction of H2O2 and thereby mimics the glutathione peroxidase (GPx) activity in vitro. These studies reveal that the degradation of the intracellular H2O2 by the selenium analogues of anti-thyroid drugs may be beneficial to the thyroid gland as these compounds may act as antioxidants and protect thyroid cells from oxidative damage. Because the drugs with an action essentially on H2O2 can reversibly inhibit thyroid peroxidase, such drugs with a more controlled action could be of great importance in the treatment of hyperthyroidism. Figure 2. (A) Concentration-inhibition curves for the inhibition of LPO-catalyzed oxidation of ABTS by MMI and MSeI at pH 7.0 and 30 °C. (B) Plot of initial rates (vo) for the LPO-catalyzed oxidation of ABTS vs concentration of H2O2. (a) Control activity, (b) 40 µM of MSeI, (c) 40 µM of MSeIox, (d) 80 µM of PTU, (e) 80 µM of MTU, (f) 40 µM of MMI. The incubation mixture contained 6.5 nM LPO, 1.4 mM ABTS, 0.067 M phosphatebuffer(pH7).(Refer PDF File) The fourth chapter describes the inhibition of lactoperoxidase (LPO)-catalyzed iodination of L-tyrosine by anti-thyroid drug methimazole (MMI) and its selenium analogue (MSeI). These inhibition studies show that MSeI inhibits LPO with an IC50 value of 12.4 µM, which is higher than that of MMI (5.2 µM). The effect of hydrogen peroxide on the inhibition of LPO by MMI and MSeI is also discussed. These studies also reveal that the inhibition of LPO-catalyzed iodination by MSeI can be completely reversed by increasing the peroxide concentration. On the other hand, the inhibition by MMI cannot be reversed by increasing the concentration of the peroxide. To under stand the nature of compounds formed in the reactions between anti-thyroid drugs and iodine, the reactions of MSeI with molecular iodine is described. MSeI reacts with I2 to produce novel ionic diselenides, and the nature of the species formed in this reaction appears to be solvent dependent. The formation of ionic species (mono and dications) in the reaction is confirmed by UV-Vis, FT-IR and FT-Raman spectroscopic investigations and single crystal x-ray studies. The major conclusion drawn from this study is that MSeI reacts with iodine, even in its oxidized form, to form ionic diselenides containing iodide or polyiodide anions, which might be possible intermediates in the inhibition of thyroid hormones. Dication X-ray crystal structure of the monocation X-ray crystal structure of the dication In the fifth chapter, the synthesis and characterization of several thiones and selones having N,N-disubstituted imidazole moiety are described. Experimental and theoretical studies were performed on a number of selones, which suggest that these compounds exist as zwitterions in which the selenium atom carries a large negative charge. The structures of selones were studied in solution by NMR spectroscopy and the 77Se NMR chemical shifts for the selones show large upfield shifts in the signals, confirming the zwitterionic structure of the selones in solution. The thermal isomerization of some S- and Se-substituted methyl and benzyl imidazole derivatives to produce the thermodynamically more stable N-substituted derivatives is described. A structure–activity correlation was attempted on the inhibition of LPO-catalyzed oxidation and iodination reactions by several thiouracil compounds, which indicates that the presence of an n-propyl group in PTU is important for an efficient inhibition. In contrast to the S- and Se-substituted derivatives, the selones produced by thermal isomerization exhibited efficient inhibition, indicating the importance of reactive selone (zwitterionic) moiety in the inhibition. The inhibition data on another well-known anti-thyroid agent carbimazole (CBZ) support the assumption that CBZ acts as a prodrug, requiring a conversion to methimazole (MMI) for its inhibitory action on thyroid peroxidase. (Refer pdf file/original thesis)

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 86 pages. Includes vita. Includes bibliographical references.