O presente estudo avaliou aspectos relacionados ao contexto inflamatório na hipertrofia cardíaca induzida pelos hormônios tiroideanos (HT) e o possível envolvimento do sistema renina-angiotensina (SRA) nesse processo, utilizando análises in vivo e com enfoque maior na abordagem in vitro. Os resultados mostraram algumas alterações em citocinas circulantes e cardíacas de animais tratados com HT. Além disso, as expressões de S100A8 e MyD88 foram aumentadas no coração de ratos submetidos ao hipertiroidismo e em cardiomiócitos em cultura estimulados com HT. S100A8 e MyD88 mediaram a ativação do fator nuclear NF-κB pelos HT, tendo papel crucial para o crescimento hipertrófico de cardiomiócitos tratados com HT. Por fim, a ação dos HT modulando a expressão de S100A8 e NF-κB foi mediada pelo SRA. Estes dados contribuem com o entendimento das bases moleculares da ação dos HT e da relação deste com o SRA na hipertrofia cardíaca. / The present study evaluated inflammation related aspects in cardiac hypertrophy induced by thyroid hormones (TH) and the possible involvement of the renin-angiotensin system (RAS) in this process, by using in vivo and in vitro analysis. The results showed alterations in circulating and cardiac cytokines from TH treated animals. The expression of S100A8 and MyD88 were increased in the heart of hyperthyroid rats and in cultured cardiomyocytes stimulated with TH. S100A8 and MyD88 mediated the nuclear factor NF-κB activation by TH and these factors presented crucial role to the hypertrophic growth of TH-treated cardiomyocytes. Finally, the action of TH on S100A8 and NF-κB expression was mediated by RAS. These data contribute to the knowledge of molecular basis of TH action and the relationship between TH and RAS in cardiac hypertrophy.

Poly- and perfluoroalkyl substances (PFAS) are ubiquitous contaminants across the globe, can bioaccumulate in aquatic taxa, and potentially biomagnify in food webs. Consequently, research examining the influence of PFAS on wildlife is warranted. Amphibians are sensitive to contaminants such as PFAS because of their porous skin and associations with aquatic habitats where contaminants accumulate. Because PFAS tend to bioaccumulate and can adversely affect the endocrine system, there is a need to examine uptake rates to inform ecotoxicology studies, as well as a need to examine sublethal effects. To address these knowledge gaps I conducted two experiments. First, I exposed larval northern leopard frogs (<i>Rana pipiens</i>), American toads (<i>Anaxyrus americanus</i>), and eastern tiger salamanders (<i>Ambystoma tigrinum</i>) to PFAS chemicals perfluorooctanoic acid (PFOA) or perfluorooctane sulfonate (PFOS) at concentrations of 10 or 1000 ppb for 10 days and sampled them every 48 hours during the exposure period. In the next experiment, I examined the effects of PFAS exposure via contaminated substrate on the survival and growth of post metamorphic amphibians of the same species. I found that, for all species, body burdens often reached steady state within 48 to 96 h of exposure. Steady-state body burdens of PFOA ranged from 3,819–16,481 ng/g dry weight among treatments and species (corresponding BCFs of 0.5 to 2.5), while PFOS body burdens ranged from 6,955–489,958 ng/g dry weight (corresponding BCFs of 47–259) among treatments and species. These data suggest that steady state is rapidly reached in larval amphibians exposed to PFAS, particularly regarding PFOS. This reflects a high potential for trophic transfer of PFAS within food webs because amphibians are often low in trophic position and are important prey for many aquatic and terrestrial species. In post-metamorphic amphibians, there was no influence of PFAS on survival or mass. However, significant effects on snout-vent length were observed in all species, and body condition differences were observed for two of my species. I found that all leopard frogs increased in scaled mass index (SMI) when exposed to a PFAS treatment, indicating an increased body condition. Toads exhibited a more variable SMI pattern across treatments, with no outstanding trends, and tiger salamanders did not differ significantly across treatments. These data suggest that sublethal effects vary greatly depending on the species, possibly due to life history traits. Future research examining biomagnification potential is warranted to determine the influence of PFAS on food webs. Additionally, there is a need to determine the physiological mechanisms underlying the observed effects of PFAS exposure.<br>

Recém-nascidos com sepse apresentam sintomas clínicos e alterações laboratoriais por tempo e gravidade variáveis. A sepse neonatal pode comprometer diversos tecidos e modificar a ação das enzimas, incluindo a desiodase tipo 1, responsável pela formação do T3 plasmático a partir do T4 nos tecidos periféricos. Além disso, em certos períodos da doença, pode haver uma ação reduzida do T4 em níveis teciduais. Estas alterações são identificadas como Doença Não Tireoidiana, e pouco se conhece sobre ela no período neonatal. Os objetivos deste estudo foram determinar os níveis séricos dos hormônios tireoidianos em recém-nascidos a termo durante a sepse e a convalescença, verificando a presença da Doença Não Tireoidiana e determinando seus padrões na sepse de curta duração, na sepse prolongada e no choque séptico. Foram estudados 28 recém-nascidos a termo com sepse, 12 com duração prolongada por mais de oito dias e 15 com choque séptico. Os recém-nascidos que tiveram sepse prolongada foram os que apresentaram maior perda de peso desde o nascimento até o início da doença, média de 21 dias, e que tiveram culturas positivas para fungos. Doença Não Tireoidiana foi encontrada em 60,7% dos casos, prevalente nos recém-nascidos com sepse de prolongada duração, dentre os quais a prevalência foi inversamente relacionada ao tempo do prolongamento da sepse. A Doença Não Tireoidiana não apresentou correlação com o choque séptico. A síndrome do T3 baixo, caracterizada por T3 baixo, TSH normal e T3 reverso geralmente aumentado, foi encontrada em 58,8% dos casos, sem diferença com a duração da sepse. O nível sérico de T3 foi mais baixo na sepse do que na convalescença sem diferença com o tempo de duração da doença. Não foi encontrada elevação de T3 reverso. A síndrome do T4 e T3 baixo, caracterizada por T4 e T3 baixo e TSH normal, foi encontrada em 29,5%, sem diferença com o tempo de duração da doença, apenas no choque séptico, retornando aos níveis normais na convalescença em ambos os grupos. A síndrome Mista que resulta da combinação de anormalidades foi encontrada em 11,7% dos casos, sem diferença com a duração da doença ou choque séptico. Doença Não Tireoidiana esteve presente nos recém-nascidos a termo com sepse, mais freqüente nos de prolongada duração. Síndrome do T3 baixo foi o padrão mais freqüente, porém sem elevação do T3 reverso; e a síndrome do T4 e T3 baixo só foi encontrada no choque séptico, embora sem relação com o mesmo. / Newborn infants with sepsis present clinical symptoms and laboratory alterations of varying lengths of time and degrees of severity. Neonatal sepsis may harm certain kinds of tissue and change the function of enzymes including Type 1 Deiodinase, which is responsible for the creation of Plasmatic T3 from T4 in peripheral tissues. Additionally, in certain periods of the illness there might be a reduced action of the T4 in tissue levels. These alterations are known as Nonthyroidal Illnesses. Little is known about Nonthyroidal Illnesses regarding these alterations during the neonatal period. The objective of this study was to determine the thyroidal hormone serum levels in full-term newborn infants during sepsis and convalescence, verifying the presence and determining the standards of the Nonthyroidal Illness in short-term sepsis, in prolonged sepsis and septic shock. 28 full-term newborn infants with sepsis were studied, along with 12 full-term newborn infants with prolonged sepsis in excess of eight days, and 15 with septic shock. The newborn infants who had prolonged sepsis were those who presented the greatest weight loss from birth through the start of the illness, 21 days on average, and whose cultures tested positive for bacteria. Nonthyroidal Illness was found in 60.7% of the cases; being most prevalent in newborn infants with prolonged sepsis, amongst whom the prevalence was inversely related to the prolonged time with sepsis. There was no correlation between Nonthyroidal Illness with septic shock. Low T3 syndrome, characterized by low T3, normal TSH and generally increased reverse T3, was found in 58.8% of the cases, with there being no difference in the length of the sepsis. The T3 serum level was lower in sepsis than in convalescence, with there being no difference in the duration of the illness. Elevated reverse T3 was not found. Low T4 and T3 syndrome, characterized by low T3 and T4, normal TSH was found in 29.5% of the cases, with there being no difference with the length of the illness duration and only found in septic shock, returning to normal levels in convalescence in both groups. The Mixed Syndrome, which results from the combination of abnormalities, was found in 11.7% of the cases, and there was no difference regarding length of illness or septic shock. Nonthyroidal Illness was present in full-term newborn infants with sepsis, and was most common in those of prolonged duration sepsis. Low T3 Syndrome was the most common standard. However, in the absence of elevated reverse T3 and T4 and T3 syndrome, it was only found during septic shock; even though it was unrelated to the same.

Sabe-se que a ativação do Sistema Nervoso Simpático (SNS) induz osteopenia via adrenoceptores b2 (b2-AR). Para investigar se o hormônio tireoideano (HT) interage com o SNS para regular a massa óssea, estudamos o efeito do HT em associação com isoproterenol ou propranolol (agonista e antagonista b-adrenérgicos) e avaliamos o efeito do HT em camundongos com elevado tônus simpático, devido à dupla inativação gênica do a2A-AR e a2C-AR (a2A/a2C-AR-/-), autorreceptores que inibem a liberação de noradrenalina. Vimos que esses animais apresentam um fenótipo de alta massa óssea, apesar do elevado tônus simpático e de intacta sinalização b2-adrenérgica, sugerindo que o a2A-AR e/ou a2C-AR, além do b2-AR, possam mediar ações do SNS no osso. O propranolol limitou e o isoproterenol acentuou os efeitos deletérios do HT no esqueleto, já os animais a2A/a2C-AR-/- apresentaram resistência à osteopenia induzida pela tireotoxicose, o que sugere que há interação entre SNS e o HT para regular a massa óssea, e que esta depende tanto do b2-AR como do a2A- e/ou a2C-AR. / It is known that the sympathetic nervous system (SNS) activation induces ostepenia, via b2-adrenoceptors (b2AR). To investigate if thyroid hormone (TH) interacts with the SNS to regulate bone mass, we studied the effect of TH in association with isoproterenol or propranolol (b-adrenergic agonist and antagonist) and evaluated the effect of TH in mice with a chronic elevated sympathetic tone, due to double disruption of a2A-AR and a2C-AR (a2a/a2c-AR-/-), autoreceptors that inhibit noradrenalin release. We showed that KO mice present a high bone mass phenotype in spite of an elevated sympathetic tone and of intact b2-adrenergic signaling, which suggests that a2A- and/or a2C-AR, besides b2-AR, may also mediate the SNS actions in the bone. Propranolol limited and isoproterenol accentuated the deleterious effects of TH in the skeleton, while a2A/a2C-AR-/- mice presented resistance to the T3-induced osteopenia, which suggest that there is an interaction between the SNS and TH to regulate bone mass, and that it is dependent on b2-AR and a2A-AR and/or a2C-AR signaling.

Recém-nascidos com sepse apresentam sintomas clínicos e alterações laboratoriais por tempo e gravidade variáveis. A sepse neonatal pode comprometer diversos tecidos e modificar a ação das enzimas, incluindo a desiodase tipo 1, responsável pela formação do T3 plasmático a partir do T4 nos tecidos periféricos. Além disso, em certos períodos da doença, pode haver uma ação reduzida do T4 em níveis teciduais. Estas alterações são identificadas como Doença Não Tireoidiana, e pouco se conhece sobre ela no período neonatal. Os objetivos deste estudo foram determinar os níveis séricos dos hormônios tireoidianos em recém-nascidos a termo durante a sepse e a convalescença, verificando a presença da Doença Não Tireoidiana e determinando seus padrões na sepse de curta duração, na sepse prolongada e no choque séptico. Foram estudados 28 recém-nascidos a termo com sepse, 12 com duração prolongada por mais de oito dias e 15 com choque séptico. Os recém-nascidos que tiveram sepse prolongada foram os que apresentaram maior perda de peso desde o nascimento até o início da doença, média de 21 dias, e que tiveram culturas positivas para fungos. Doença Não Tireoidiana foi encontrada em 60,7% dos casos, prevalente nos recém-nascidos com sepse de prolongada duração, dentre os quais a prevalência foi inversamente relacionada ao tempo do prolongamento da sepse. A Doença Não Tireoidiana não apresentou correlação com o choque séptico. A síndrome do T3 baixo, caracterizada por T3 baixo, TSH normal e T3 reverso geralmente aumentado, foi encontrada em 58,8% dos casos, sem diferença com a duração da sepse. O nível sérico de T3 foi mais baixo na sepse do que na convalescença sem diferença com o tempo de duração da doença. Não foi encontrada elevação de T3 reverso. A síndrome do T4 e T3 baixo, caracterizada por T4 e T3 baixo e TSH normal, foi encontrada em 29,5%, sem diferença com o tempo de duração da doença, apenas no choque séptico, retornando aos níveis normais na convalescença em ambos os grupos. A síndrome Mista que resulta da combinação de anormalidades foi encontrada em 11,7% dos casos, sem diferença com a duração da doença ou choque séptico. Doença Não Tireoidiana esteve presente nos recém-nascidos a termo com sepse, mais freqüente nos de prolongada duração. Síndrome do T3 baixo foi o padrão mais freqüente, porém sem elevação do T3 reverso; e a síndrome do T4 e T3 baixo só foi encontrada no choque séptico, embora sem relação com o mesmo. / Newborn infants with sepsis present clinical symptoms and laboratory alterations of varying lengths of time and degrees of severity. Neonatal sepsis may harm certain kinds of tissue and change the function of enzymes including Type 1 Deiodinase, which is responsible for the creation of Plasmatic T3 from T4 in peripheral tissues. Additionally, in certain periods of the illness there might be a reduced action of the T4 in tissue levels. These alterations are known as Nonthyroidal Illnesses. Little is known about Nonthyroidal Illnesses regarding these alterations during the neonatal period. The objective of this study was to determine the thyroidal hormone serum levels in full-term newborn infants during sepsis and convalescence, verifying the presence and determining the standards of the Nonthyroidal Illness in short-term sepsis, in prolonged sepsis and septic shock. 28 full-term newborn infants with sepsis were studied, along with 12 full-term newborn infants with prolonged sepsis in excess of eight days, and 15 with septic shock. The newborn infants who had prolonged sepsis were those who presented the greatest weight loss from birth through the start of the illness, 21 days on average, and whose cultures tested positive for bacteria. Nonthyroidal Illness was found in 60.7% of the cases; being most prevalent in newborn infants with prolonged sepsis, amongst whom the prevalence was inversely related to the prolonged time with sepsis. There was no correlation between Nonthyroidal Illness with septic shock. Low T3 syndrome, characterized by low T3, normal TSH and generally increased reverse T3, was found in 58.8% of the cases, with there being no difference in the length of the sepsis. The T3 serum level was lower in sepsis than in convalescence, with there being no difference in the duration of the illness. Elevated reverse T3 was not found. Low T4 and T3 syndrome, characterized by low T3 and T4, normal TSH was found in 29.5% of the cases, with there being no difference with the length of the illness duration and only found in septic shock, returning to normal levels in convalescence in both groups. The Mixed Syndrome, which results from the combination of abnormalities, was found in 11.7% of the cases, and there was no difference regarding length of illness or septic shock. Nonthyroidal Illness was present in full-term newborn infants with sepsis, and was most common in those of prolonged duration sepsis. Low T3 Syndrome was the most common standard. However, in the absence of elevated reverse T3 and T4 and T3 syndrome, it was only found during septic shock; even though it was unrelated to the same.

Os hormônios tireoidianos (HTs) desempenham um papel importante em diversos processos, tais como o crescimento, desenvolvimento e metabolismo dos tecidos em geral. Embora estudos tenham demonstrado que os HTs agem diretamente nas células foliculares da tireoide reduzindo sua resposta ao TSH, pouco se sabe, a nível molecular, sobre essa e outras ações dos HTs na própria glândula tireoide. Nesse sentido, o objetivo do presente trabalho foi avaliar alterações no perfil de expressão gênica nas células foliculares tireoidianas (células PCCl3) em reposta ao tratamento com triiodotironina (T3). Após atingir confluência desejada, as células PCCl3 foram mantidas em meio depletado de HTs (grupo Hipo) por 24h. Após esse período, parte das células foi tratada com 10-7M de T3 (grupo T3) por 24 h. As células foram, então, lisadas para extração de RNA total para análise do transcriptoma, por RNAseq. Foi obtido como resultado, uma lista de genes diferencialmente expressos da qual foram selecionados cinco genes para validação in vitro (novo lote de células PCCl3 submetidas às mesmas condições descritas acima) e in vivo [ratos Wistar (250g), tratados por 4 semanas com T3 (1,5 &mu;g/100g de peso corporeo; grupo hipertireoideo) ou PTU (10 &mu;g/100g de peso corporeo; grupo hipotireoideo): o Slc16a1, que codifica o MCT8, responsável pelo transporte de T3 através da membrana, o Snrpd1, 9-March, Pfdn1 e Fam103a1, que codificam proteínas envolvidas no controle pós-transcricional e pós-traducional da expressão gênica. O tratamento com T3 estimulou a expressão dos genes Snrpd1, Pfdn1 e Fam103a1, enquanto reduziu a expressão de 9-March e Slc16a1. Juntos esses resultados demonstram a existência de um efeito autócrino exercido pelo T3 sobre o controle do seu próprio turnover proteico. / Thyroid hormones (HTs) play an important role in many processes, such as growth, development and metabolism of tissues in general. Although studies have shown that HTs act directly on follicular thyroid cells reducing their response to TSH, little is known, at the molecular level, about this and other actions of HTs in the thyroid gland itself. In this sense, the aim of the present study was to evaluate changes in the gene expression profile in the thyroid follicular cells (PCCl3 cells) in response to triiodothyronine (T3) treatment. After reaching 60% confluence, PCCl3 cells were maintained in HT depleted medium (Hypo group) for 24h. After this time, part of the cells was treated with 10-7M T3 (T3 group) for 24 h. Cells were then lysed for total RNA extraction for transcriptome analysis by RNAseq. As a result, a list of differentially expressed genes from which five genes for in vitro validation (PCCl3 cells under the same conditions described above) and in vivo [Wistar rats (250g), treated for 4 weeks with T3 (1.5 &um;g / 100 g body weight, hyperthyroid group) or PTU (10 &um;g / 100 g body weight; hypothyroid group): Slc16a1, which encodes MCT8, responsible for the transport of T3 through the membrane, Snrpd1, 9-March, Pfdn1 and Fam103a1, which encode proteins involved in post-transcriptional and post-translational control of gene expression. T3 treatment stimulated the expression of the Snrpd1, Pfdn1 and Fam103a1 genes, while reducing the expression of 9-March and Slc16a1. Together these results demonstrate the existence of an autocrine effect exerted by T3 on the control of its own protein turnover.

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.

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Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. There is also increasing evidence that changes in the phosphoproteome influence TH action. In addition, the disruption of TH-action by endocrine-disrupting compounds (EDC) is an emerging field and the developmental process of metamorphosis is a target as well as a model system for this research. This work identifies components of the proteome and phosphoproteome in TH-sensitive tadpole tissues that are altered during the initiation stages of TH-induced metamorphosis prior to the overt remodeling of the tissues. Proteomic analyses included two-dimensional (2D) gel electrophoresis for the assessment of differential protein/phosphoprotein expression, combined with mass spectrometry (MS) protein analysis for protein identification. Initial proteomic approaches in Xenopus laevis identified a number of proteins that are differentially expressed in the tadpole tail within 48 h of exposure of premetamorphic tadpoles to 3,5,3’-triiodothyronine (T3). Additionally, a time-course analysis of brain tissue within this 48 h period revealed alterations in phosphoproteins. The importance of phosphoproteome modulation in the process of metamorphosis was further revealed in the TH-induced tail of Rana catesbeiana tadpoles, where the inhibition of cyclin-dependent kinase activity which prevents tail regression, altered the tail phosphoproteome profile. Failure to identify the phosphoproteins involved in these initial studies led me to develop and apply new proteomic approaches. To this end, subcellular and protein fractionation methods were developed and combined with 2D gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using MS. Here components of the proteome and phosphoproteome were identified in the tail fin that changed within 48 h of exposure of premetamorphic R. catesbeiana tadpoles to 10 nM T3. This approach allowed the identification of and led to the cloning of a novel Rana larval type I keratin, RLK I, which is a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript level was reduced during T3-induced and natural metamorphosis, consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, was changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success in identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. This study demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which limited genome information is currently available. The early appearance of caspase-cleaved RLK I in the TH-induced process led to its investigation as a contributor to apoptosis. Furthermore, the caspase-cleavage product of RLK I was used as a biomarker in the development of an assay for the detection of disruptors of TH-action based on ex-vivo multi-well culturing of R. catesbeiana tail fin biopsies. This assay was able to detect perturbations in TH-signalling within 48h of exposure demonstrating that it has utility as a novel system for screening of TH disrupting chemicals. The present study identified proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole metamorphosis prior to overt tissue remodeling and provided important insight into the molecular mechanisms of this postembryonic development. In particular, I have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone and can be used for the detection of EDCs of TH-action in an ex vivo assay.