by Tsui Kai Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 100-112). / Abstract --- p.i / Acknowledgements --- p.iii / List of Abbreviations --- p.iv / Contents --- p.v / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- The Effect of In Vivo Methimazole Treatment on the Functions of Macrophages --- p.10 / Introduction --- p.10 / Materials and Methods --- p.14 / Results --- p.27 / Discussion --- p.38 / Chapter Chapter 3 --- The Effect of In Vivo Methimazole Treatment on the Function of Lymphocytes --- p.41 / Introduction --- p.41 / Materials and Methods --- p.44 / Results --- p.50 / Discussion --- p.60 / Chapter Chapter 4 --- The Lack of Demonstrable Effect of In Vitro Methimazole Treatment on the Functions of Macrophages and Lymphocytes --- p.62 / Introduction --- p.62 / Materials and Methods --- p.63 / Results --- p.66 / Discussion --- p.74 / Chapter Chapter 5 --- Effect of Thyroid Hormone Replacement on the Immune Response of Methimazole-Treated Mice --- p.76 / Introduction --- p.76 / Materials and Methods --- p.78 / Results --- p.82 / Discussion --- p.91 / Chapter Chapter 6 --- General Discussion --- p.95 / References --- p.99

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-27T15:36:18Z No. of bitstreams: 1 VANDREZA CARDOSO BORTOLOTTO.pdf: 2120802 bytes, checksum: d9c6e128c892e469fe3f24a3ebad9f86 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-27T15:36:32Z (GMT) No. of bitstreams: 1 VANDREZA CARDOSO BORTOLOTTO.pdf: 2120802 bytes, checksum: d9c6e128c892e469fe3f24a3ebad9f86 (MD5) / Made available in DSpace on 2018-09-27T15:36:32Z (GMT). No. of bitstreams: 1 VANDREZA CARDOSO BORTOLOTTO.pdf: 2120802 bytes, checksum: d9c6e128c892e469fe3f24a3ebad9f86 (MD5) Previous issue date: 2017-07-17 / A glândula tireoide é uma das maiores glândulas do corpo, responsável pela produção de triiodotironina (T3) e tiroxina (T4), hormônios responsáveis pela homeostase do organismo. A redução na produção destes hormônios leva a um quadro de hipotireoidismo. O hipotireoidismo é uma desordem endócrina, mais prevalente no sexo feminino, e que pode causar uma série de alterações comportamentais e neurológicas, dentre elas a depressão. O flavonoide crisina, presente no maracujá do mato, própolis e mel de abelha, vem sendo estudado a alguns anos, sendo relatados seus efeitos antioxidantes, anticancerígenos, antihiperglicêmicos, ansiolíticos, e atualmente tem-se demonstrado sua atividade antidepressiva. O objetivo deste estudo foi investigar a ação terapêutica da crisina em modelo tipo depressivo induzido pelo hipotireoidismo em camundongos fêmeas C57BL/6 adultas. Primeiramernte os animais foram divididos em dois grandes grupos (n=20): controle e hipotireoidismo. O hipotireoidismo foi induzido por exposição contínua ao fármaco antitireoideo metimazol (MTZ) 0,1% + 0,475% de sucralose, durante 31 dias na água. No 31º dia foi retirado sangue da veia caudal e determinado os níveis de T3 e T4. Após os animais foram separados em quatro grupos (n=10): controle, hipotireoidismo, crisina, hipotireoidismo + crisina. A crisina (20mg/kg) foi administrada diariamente por 28 dias, via oral. Ao final do tratamento, os animais passaram por testes comportamentais de campo aberto (TCA), nado forçado (TNF) e suspensão de cauda (TSC), após realizou-se eutanásia nos animais, e coletou-se o sangue por punção cardíaca para análise bioquímica de T3 e T4, e retirou-se as estruturas cerebrais hipocampo e córtex pré frontal, para análises neuroquímicas de serotonina (5-HT), dopamina (DA), norepinefrina (NA). Nossos resultados demonstraram que os animais com hipotireoidismo apresentaram um aumento no tempo de imobilidade nos testes de TNF e TSC e a crisina foi capaz de reverter este tempo em ambos os testes. Demonstrou-se também que a crisina foi capaz de restaurar os níveis de neurotransmissores: 5-HT em ambas estruturas cerebrais e DA no hipocampo dos animais com hipotireoidismo, corroborando com os resultados dos testes comportamentais, nos quais o TNF está relacionado com o sistema serotoninérgico e o TSC com o sistema dopaminérgico. Em conclusão, nossos resultados demonstram pela primeira vez que a crisina é capaz de reverter o estado tipo depressivo induzido pelo hipotireoidismo, possivelmente por normalizar os níveis de 5-HT e DA. / The thyroid gland is one of the largest glands in the body, it produces triiodothyronine (T3) and thyroxine (T4), these hormones are responsible for body homeostasis. The reduction in the production of these hormones leads to hypothyroidism. The hypothyroidism is an endocrine disorder, more prevalent in females, which can cause a number of behavioral and neurological changes, including depression. The chrysin flavonoid present in the passion fruit of the bush, propolis and bee honey, has been studied for some years, being reported your antioxidant effect, anticancer, antihyperglycemic, anxiolytic, and currently your antidepressant activity was demonstrated. The objective of this study was to investigate the therapeutic action of chrysin in a model of like-depression induced by hypothyroidism in adult C57BL/6 female mice. First, the animals were divided into two groups (n=20): control and hypothyroidism. Hypothyroidism was induced by continuous exposure to the antithyroid drug methimazole (MTZ) 0.1% + 0.475% sucralose for 31 days on water. On the 31st day blood was drawn from the caudal vein and T3 and T4 levels were determined. After the animals were separated into four groups (n=10): control, hypothyroidism, chrysin, hypothyroidism + chrysin. The treatment of chrysin (20mg/kg) was administered daily for 28 days, orally. At the end of treatment the animals they passed for behavioral tests of open field test (OFT), forced swimming test (FST) and tail suspension test (TST), performed euthanasia in the animals, and collected the blood by cardiac puncture for biochemical analyze of T3 and T4, and the hippocampus and prefrontal cortex brain structures were removed for neurochemical analyzes of serotonin (5-HT), dopamine (DA), norepinephrine (NA). Our results demonstrated that animals with hypothyroidism showed an increase in the time of immobility in the tests of FST and TST and the chrysin was able to reverse this time in both tests. It was also demonstrated that the chrysin was able to restore the levels of neurotransmitters: 5-HT in both structures cerebral and DA in the hippocampus of animals with hypothyroidism, corroborating with the results of behavioral tests, in which FST is related to the serotonergic system and the TST with the dopaminergic system. In conclusion, our results demonstrate for the first time that chrysin is able of reversing the depressive-induced state induced by hypothyroidism, possibly by normalizing 5-HT and DA levels.

The flavin-containing monooxygenases (FMO / E.C.1.14.13.8) are microsomal NADPH and oxygen-dependent flavoprotein enzymes that catalyze the oxidation of a wide variety of xenobiotics, including drugs and environmental toxicants. Nucleophiles containing nitrogen, sulfur, phosphorus and selenium heteroatoms are the substrates of FMO. Bovine liver microsomal FMO enzyme activity was characterized using methimazole as substrate, which is a highly specific substrate for FMO. From 12 different bovine liver samples, microsomes were prepared and the average specific activity of bovine liver microsomal FMO was found to be 2.37 &amp / #61617 / 0.30 nmol/min/mg (Mean &amp / #61617 / SE, n=12). The rate of reaction was linear up to 0.5 mg of bovine liver microsomal protein. The maximum FMO enzyme activity was detected at 37 &amp / #61616 / C and at pH 8.0. Effects of detergents / Triton X-100 and Emulgen 913, on FMO activity were determined and found that enzyme activity increased by the addition of either detergent at all concentrations (0.1%-1.0%). The apparent Vmax and Km values of bovine liver microsomal FMO for methimazole substrate were found as 1.23 nmol/min/mg and 0.11 mM, respectively. Thermostability of bovine liver microsomal FMO was studied at four different temperatures / 24 &amp / #61616 / C, 37 &amp / #61616 / C, 50 &amp / #61616 / C and 65 &amp / #61616 / C. The incubation time required for the complete loss of enzyme activity was 5 minutes at 65 &amp / #61616 / C, 10 minutes at 50 &amp / #61616 / C and 6.5 hours at 37 &amp / #61616 / C. 68 % of the activity was still detectable at the end of 53 hours at 24 &amp / #61616 / C. Bovine liver microsomal activity towards two drug substrates, imipramine and chlorpromazine, was also determined and found to be 3.73 and 3.75 nmol NADPH oxidized/min/mg, respectively. Effects of two drug substrates, imipramine and chlorpromazine, on bovine liver microsomal FMO-catalyzed methimazole oxidation activity was also studied and found that they inhibit FMO activity at all concentrations studied. Modulation of bovine liver microsomal FMO activity was studied using three different heavy metal ions / Ni+2, Cd+2 and Hg+2. At all other concentrations studied for each heavy metal ion and at all substrate methimazole concentrations (0.1, 0.2, 0.5, 1.0 mM), FMO-catalyzed methimazole oxidation activity decreased compared to control activity. KI values for Ni+2, Cd+2 and Hg+2 were found to be 0.5 mM, 0.085 mM, 4.6 &amp / #61549 / M, respectively. From the Dixon plot, the pattern of inhibition for three heavy metal ions was observed to be noncompetitive.

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)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hypothyroidism is characterized by a disorder resulting from deficiency of thyroid hormones and is related to lipid metabolism dysfunction and cardiovascular diseases development risk. However, these changes in hypothyroidism need to be understood. Thus, this study aimed to evaluate the association between lipid, inflammatory and oxidative stress markers in patients with hypothyroidism and antioxidant effects of quercetin in these markers, using hypothyroidism experimental model induced by methimazole in rats. The methodology and results are presented in the form of articles. In article 1, were evaluated the oxidative stress biomarkers in 20 patients with subclinical hypothyroidism (SH) (49.12 ± 10.85 years). Thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT) and arylesterase (ARE) were analyzed in SH patients and controls. In addition, were measured plasmatic lipids: total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). TBARS levels and CAT activity were higher in subclinical hypothyroidism patients, such as TC and LDL-C plasmatic levels. Arylesterase activity was lower in the SH group. Correlations were observed between plasmatic lipids and oxidative stress biomarkers and thyroid-stimulating hormone (TSH). TSH was correlated with TBARS, CAT, and SOD. The second study (manuscript 1) aimed to investigate the association between inflammatory biomarkers and overt hypothyroidism (OH). Plasmatic levels of cytokines were determinate: interleukin 1 (IL-1), interleukin 6 (IL- 6), tumor necrosis factor alpha (TNF-α), interferon gamma (INF-ɣ) and the levels of cell free DNA (cf-DNA). Furthermore, we evaluated lipid profile and prothrombotic markers (fibrinogen and D-dimer). OH patients had pro-inflammatory profile, resulted from high levels of cytokines and cf-DNA. Lipids and prothrombotic markers also showed elevated. Significant associations between inflammatory status and lipid profile were observed in hypothyroid patients. Manuscript 2 evaluates the effect of quercetin on oxidative stress biomarkers in methimazole (MMI) - induced hypothyroid rats. Hypothyroidism was induced by administering MMI at 20 mg/100 ml in the drinking water, for 30 days. After this period, rats received orally 10 or 25 mg/kg of quercetin (QT) for 8 weeks. Sixty male wistar rats were randomly divided into six groups (group I, control; group II, QT10; group III, QT25; group IV, hypothyroid; group V, hypothyroid + QT10; group VI, hypothyroid + QT25). Hypothyroid rats showed hepatic, renal and serum TBARS levels increased, along with increased protein carbonyl (PCO) in liver and increased ROS levels in liver and kidney. Quercetin administration (QT10 and 25), was effective in decreasing TBARS levels in serum and kidney, PCO in liver and ROS generation in liver and kidney tissues. Moreover, in hypothyroid group were observed high TBARS levels in cerebral cortex and hippocampus. QT25 treatment decreased the levels in both tissues. Administration of QT25 to hypothyroid rats resulted in decreased SOD activities in liver and whole blood and increased liver CAT activity. Ascorbic acid levels and total oxidative scavenging capacity (TOSC) were increased in liver and kidney rats after QT10 and QT25 treatment. These results suggest association between oxidative stress and hypothyroidism that may potentially modulated by antioxidant supplementation such as quercetin. These findings are of great importance in understanding the biochemical dysfunctions and oxidative status in hypothyroidism, as well as, in research of antioxidants strategies to be used as adjuncts in the treatment of this disorder. / O hipotireoidismo é caracterizado por uma desordem decorrente da deficiência de hormônios tireoideanos, estando relacionado a disfunções no metabolismo lipídico e ao risco de desenvolvimento de doenças cardiovasculares. Entretanto, estas alterações no hipotioreodismo precisam ser melhor compreendidas. Assim, este trabalho teve como objetivo avaliar a associação de marcadores lipídicos, inflamatórios e de estresse oxidativo em pacientes com hipotireoidismo e o efeito antioxidante da quercetina nestes marcadores, utilizando como modelo experimental o hipotireoidismo induzido por metimazol em ratos. A metodologia e resultados são apresentados sob a forma de artigos. No artigo 1, foram avaliados biomarcadores de estresse oxidativo em 20 pacientes com hipotireoidismo subclínico (HSC) (49,12 ± 10,85 anos). Os níveis de substâncias reativas ao ácido tiobarbitúrico (TBARS), e as atividades das enzimas superóxido dismutase (SOD), catalase (CAT) e arilesterase (ARE) foram determinadas em pacientes com HSC e controles. Além disso, foram investigados os níveis de lipídeos plasmáticos: colesterol total (CT), triglicerídeos (TG) e as lipoproteínas de alta (HDL) e baixa densidade (LDL). Os níveis de lipoperoxidação determinado pela medida do TBARS e a atividade da enzima CAT estavam aumentados nos pacientes hipotireóideos, bem como os níveis plasmáticos de CT e colesterol LDL. A enzima ARE mostrou-se diminuída no grupo HSC. Foram evidenciadas correlações entre lipídeos plasmáticos e biomarcadores de estresse oxidativo e com o hormônio de estimulação da tireóide (TSH). O TSH foi correlacionado com TBARS, CAT e SOD. O segundo estudo (manuscrito 1) teve por objetivo investigar a associação entre biomarcadores inflamatórios e o hipotireoidismo clínico (HC). Foram determinados os níveis plasmáticos das citocinas: interleucina 1 (IL-1), interleucina 6 (IL-6), fator de necrose tumoral alfa (TNF- α), interferon gama (INF- ɣ) e os níveis de DNA circulante livre. Além disso, foram avaliados o perfil lipídico e marcadores prótrombóticos (fibrinogênio e D-dímero). Os pacientes com HC apresentaram perfil próinflamatório, resultante dos níveis elevados das citocinas e do DNA livre. Os lipídeos e os marcadores pró-trombóticos também se apresentaram elevados. Associações significativas entre o perfil inflamatório e o perfil lipídico foram observadas nos pacientes hipotireóideos. No manuscrito 2 avaliou-se o efeito da quercetina sobre biomarcadores de estresse oxidativo em um modelo de hipotireoidismo induzido por metimazol (MMI) em ratos. O hipotireoidismo foi induzido pela administração de MMI na concentração de 20mg/100mL na água de beber, por um período de 30 dias. Após este período, os animais receberam oralmente 10 ou 25 mg/kg de quercetina (QT) por um período de 8 semanas. Ratos machos wistar (n=60) foram divididos em seis grupos (grupo I, controle; grupo II, QT10; grupo III, QT25; grupo IV, hipotireóideo; grupo V, hipotireóideo + QT10; grupo VI, hipotireóideo + QT25). Os ratos hipotireóideos apresentaram níveis de TBARS hepático, renal e séricos aumentados, bem como os níveis de proteína carbonil (PCO) no fígado e os níveis de espécies reativas de oxigênio (ERO) no fígado e rins. A administração de quercetina (QT 10 e 25) diminuiu os níveis de TBARS em soro e rins, a PCO no fígado e a geração de ERO nos tecidos hepático e renal. Além disso, no grupo hipotireóideo foram observados altos níveis de TBARS no córtex cerebral e hipocampo. O tratamento com QT25 reduziu os níveis em ambos os tecidos. A administração de QT 25 aos ratos com hipotireoidismo diminuiu a atividade da SOD em fígado e sangue total e aumentou a atividade hepática da CAT. Os níveis de ácido ascórbico e a capacidade antioxidante total aumentaram no fígado e rins dos ratos após tratamento com QT10 e QT25. O conjunto dos resultados sugeriu associação entre estresse oxidativo e hipotireoidismo que pode ser potencialmente modulado por suplementação de antioxidantes como a quercetina. Estes achados são de grande importância no entendimento das disfunções bioquímicas e do status oxidativo no hipotireoidismo como também na busca de estratégias antioxidantes a serem utilizadas como coadjuvantes no tratamento desta disfunção.

<p>Thyroid hormone is essential for regulating adult metabolism and proper fetal development. Under normal conditions, maternal and fetal thyroid hormones are subject to metabolism at the placenta and within fetal tissues through deiodination and sulfation to regulate fetal exposure to the bioactive hormone. Disruptions of the thyroid hormone system can result in non-thyroidal illness syndrome (NTIS), which is classified as the dysregulation of thyroid hormone homeostasis. The exact cause of the alterations in circulating thyroid hormone levels during NTIS is not well- known. In comparison, hypothyroidism results from the absence of thyroid hormone production and presents as low thyroid hormone levels.</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) crosses the late gestation placenta and causes suppression of circulating maternal and fetal thyroid hormone. Chapter 2 investigates the potential role of thyroid hormone metabolism in this disruption. Pregnant gilts were challenged with PRRSV2 (n=22) or sham inoculated (n=5) at gestation day 85. Samples were collected on day 106, and viral load was assessed in fetal serum and thymus. From the entire fetal population, three distinct subsets of fetuses representing biological extremes were identified, including uninfected with no detectable viral load (UNIF), high viral load viable (HV-VIA), or high viral load with severe meconium staining (HV-MEC). In addition, control fetuses from sham inoculated gilts (CON) were used as a reference group. Samples of fetal liver, kidney, and the corresponding fetal placenta and maternal endometrium for n=10 fetuses per group were then used to evaluate gene expression. A total of 11 genes associated with thyroid hormone metabolism including deiodinases (DIO1,2,3), sulfotransferases (SULT1A3,1B1,1C2,1E1,2A1), sulfatase (STS), and solute carriers (SLC16A2,16A10) were quantified using absolute quantification qPCR. Evidence of fetal decompensation was observed within the high viral fetuses in the form of decreased DIO1 expression within the fetal liver and increased DIO3 expression in both components of the placenta. Circulating levels of T4 and inactive thyroid hormone metabolites, reverse-triiodothyronine (rT3) and two diiodothyronines (3,5-T2 and 3,3’-T2), were measured in fetal serum. While T4 was depressed, no change was observed in circulating rT3 levels, and neither T2 metabolite reached the lower detection limit. This may suggest that alterations in thyroid hormone metabolism generate a localized effect on hormone metabolites in the respective tissues.</p> <p>Alternatively, the low levels of available T3 and T4 limit the production of downstream metabolites to be found in serum.</p> <p>The cause-and-effect relationship between PRRSV infection, fetal thyroid disruption, and the effects on fetal thyroid hormone metabolism are unclear. Therefore, Chapter 3 developed a non-pathogenic model using methimazole (MMI) to induce hypothyroidism in the late gestation fetus and evaluate the impact on fetal development and thyroid hormone metabolism. Pregnant gilts were either treated with oral methimazole or equivalent sham from gestation day 85-106 (n=4/group), followed by classification of all fetuses as live, live but meconium stained, or dead. Fetuses exposed to MMI in-utero were notably hypothyroid with significantly suppressed serum T3 and T4 and histological evidence of goiter. Surprisingly, fetuses from MMI-treated dams were substantially larger but appeared to exhibit non-allometric growth with an increase in girth but not length. The liver, kidney, and the corresponding fetal placenta and maternal endometrium were collected from a subset of 16 fetuses per group to evaluate the relative expression of five genes associated with thyroid hormone metabolism, including three deiodinases and two solute carriers known to transport thyroid hormone. Compensatory transcription of DIO3 was observed in all tissues evaluated, suggesting increased vertical transfer of maternal thyroid hormone at the placenta and decreased breakdown of thyroid hormone within fetal organs.</p> <p>The evaluation of thyroid hormone metabolism within the fetus and within the placenta has allowed us to differentiate suppressed thyroid hormone levels of the pig fetus under pathogenic and non-pathogenic conditions. In the context of PRRSV infection, the observed decompensation of thyroid hormone metabolism would further exacerbate the hypothyroid state and is therefore consistent with NTIS. In contrast, fetuses with induced thyroid hormone suppression following maternal exposure to MMI showed compensatory thyroid hormone metabolism in the same tissues. This indicates true hypothyroidism and clearly demonstrates a fetal capacity to respond to such endocrine disruption.</p>

<p dir="ltr">Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating virus that is endemic to the swine industry. This virus has little direct effect on the dam but results in abortions, stillborn, and delivery of viremic piglets. PRRSV is unable to cross the swine placenta in early gestation but as gestation progresses, the placenta becomes permissible during late gestation. The mechanisms that allow the virus to cross the late gestation placenta are not well understood, but several theories have been presented regarding changes in placental morphology or enzymatic changes. Additionally, piglets who experience IUGR due to uterine crowding have been found to be more resistant to PRRSV infection, having lower viral levels than their normal litter mates. When vertical transmission from the dam to fetus occurs not only can the effects previously mentioned occur, but PRRSV is also known to cause suppression of maternal and fetal thyroid hormone. Thyroid hormone plays numerous roles in fetal development such as accretion of fetal mass, appetite regulation, and coincidently follows a similar increase trajectory to that of fetal growth during mid- to late-gestation. Consequently, any dysregulation of thyroid hormone has the potential to cause severe side-effects and may alter fetal growth. The relationship between thyroid hormone and fetal growth and development is not well understood. Chapter 2 investigates the potential cause-and-effect relationship between fetal growth and thyroid hormone through the induction of fetal hypothyroidism. Pregnant gilts (n=24) were given a sham treatment (CON; n=12) or treated with methimazole (MMI; n=12), a goitrogen capable of crossing the placenta. These gilts were then further subdivided across four gestational timepoints spanning mid- to late-gestation, these being days 55, 66, 76, and 86. Treatment started 21 days prior to these desired dates. Upon completion of treatment the gilts were humanely euthanized, and fetuses were extracted (resulting populations of n=174 MMI and n=166 CON) and fetal body and organ weights were recorded. Collected tissues included heart, liver, lung, kidneys, spleen, brain and thyroid. Fetuses were imaged in the left and right lateral recumbency for phenotypic analysis, including novel head measurements. Placental sample were also taken. Additionally fore- and hind limbs were taken from the centermost male and female from each litter so that radiographs could be taken to analyze bone growth. Statistical analysis of all phenotypic differences was carried out using a linear mixed effect model including gestational age and treatment as fixed effects and gilt as a random effect. The data revealed that the left and right phenotypic parameters are highly correlated (R2>0.9). Upon extraction, goiters were present in the MMI fetuses and there was a significant increase in both absolute and relative thyroid weights. Thus, the use of MMI during this period of gestation was successful in inducing hypothyroidism. Additionally, the MMI treated thyroids had a significant treatment by time interaction with 0.014g and 0.21g increase at day 55 and 66 respectively indicating reduced compensatory action within the fetal hypothalamic-pituitary-thyroid axis during this earliest period. Liver weight as a percentage of body weight decreased from 6.06% to 2.56% between days 55 and 86 in the CON group but, was significantly increased at all time points in response to MMI induced hypothyroidism (P<0.01). Thus, the in brain to liver weight ratio decreases over time, in MMI fetuses (P<0.05). While all other phenotypic parameters were significantly altered by gestation age, there was no significant impact of fetal hypothyroidism. This indicates that fetal thyroid hormone is not the driving factor for the exponential fetal growth seen in mid- to late-gestation. PRRSV virus is a complex and devastating virus to the swine industry, especially when it infects pregnant gilts and sows. PRRSV is unable to cross the swine placenta during mid-gestation but as gestation progresses the virus readily crosses the placenta and is able to infect piglets during this late gestation period. The mechanisms by which PRRSV crosses the highly restrictive porcine placenta are not clear. Additionally, piglets who experience intrauterine growth retardation experience lower virus levels than their normal counterparts. Chapter 3 investigates the changes in three genes of interest that we hypothesized, had the potential to fluctuate throughout gestation and facilitate PRRSV transfer, as well as the morphological changes that occur in the maternal-fetal interface through mid- to late-gestation and how these aspects may vary between IUGR and normal piglets. Placental samples were collected from pregnant gilts (n=12) equally divided across days 55, 66, 76, and 86 of gestation. Samples were taken of each fetus’s placenta adjacent to the umbilical cord. A portion of the sample was cut into 1 cm2 and placed into a mold with optimal cutting temperature media (OCT) for later cryo-sectioning and histology. The remaining portion had the fetal placenta peeled from the endometrium and flash frozen in liquid nitrogen for RNA extraction. A subset of samples was chosen based on fetus’s brain to liver weight ratios (n=96). From each litter two males and two females with the most extreme case of IUGR, based on z-scores, were chosen and the same was done for the two males and females with lowest brain to liver weight ratios, the later were classified as large for gestational age (LGA). 56 of the original 64 had acceptable levels of placental RNA for analysis. A total of 3 genes were chosen for analysis based on their function and previous literature. These included CD163, SIGLEC1 and IL-10. No significant up or down regulation was seen in any of the selected genes and there was no variation between IUGR and LGA fetuses. Additionally, placenta histology was conducted to evaluate populations of CD163 positive macrophages throughout the maternal fetal interface across mid- to late-gestation. Populations of CD163 positive macrophages were found on both the maternal and fetal sides of the maternal fetal interface at all timepoints. Collectively these results show there is no fluctuation in CD163, SIGLEC1, or IL-10 among timepoints or between IUGR and LGA fetuses. Additionally, the histology samples confirm the presence of resident populations of CD163 positive macrophages on maternal and fetal sides of the MFI. Collectively these results indicate that more research needs to be done to determine the underlying mechanisms of PRRSV transmission during late gestation.</p>