Includes bibliographical references (leaves 191-207) / xiv, 207 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the interactions between thyroid hormones and male reproductive function in mature, prepubertal and post pubertal Merino rams. Hypothyroidism or hyperthyroidism was induced in these rams for 8-10 weeks and their reprodroductive endocrine axis and testis functions were assessed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1986

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 10

1. Sphingolipid-cholesterol domains (lipid rafts) in normal human and dog thyroid follicular cells are not involved in thyrotropin receptor signalling. Thyroid hormone regulates growth and development throughout the animal kingdom. The thyroid which secretes it, is controlled by TSH and its receptor TSHR. TSH and its receptor TSHR act through TSHR-coupled G proteins to control thyroid functions, with a stronger coupling of the TSHR with Gs protein than with Gq protein in human thyrocytes. Gq is not activated by TSH/TSHR in dog, whereas dog TSHR activates it in CHO transfected cells. To better understand TSHR and its downstream effectors G proteins, we attempted to answer the questions by the role of “lipid rafts/caveolae” in TSH action. Lipid rafts/caveolae are sphingolipids-cholesterol-enriched microdomains on plasma membrane that have been proposed to play a role in signal transduction. By concentrating the signal molecules, lipid rafts/caveolae increase the efficiency of the interactions between the molecules and sequestrate them from the bulk membranes. The compartmentation of signal proteins in lipid rafts/caveolae might provide a possible explanation for the relationship between TSHR and G proteins in human and dog thyroctyes. To answer these questions, we first tested the existence of such lipid microdomains in human and dog thyrocytes. By northernblot and RT-PCR of caveolin-1 mRNA, we demonstrated its existence in thyrocytes. The immunohistochemistry of caveolin-1 showed that caveolin/caveolae are present on the apical membrane of thyrocytes, opposite to the TSHR localization on the basolateral membranes. The isolation of lipid rafts/caveolae by Triton X-100/OptiPrep density experiments showed that TSHR and Gq are not in the rafts, even though other proteins such as insulin receptor, flotillin-2 and partially Gs are present in these lipid domains, as expected. Testing the function of the TSH receptor on its main cascade (Gs-Adenylyl cyclase-cAMP) after treating the follicles with Methyl β-cyclodextrin (a cholesterol chelator), we observed no modification of the cAMP levels by this treatment. This is in agreement with our conclusion that the TSHR-Gs-cAMP pathway does not involve the lipid rafts/caveolae domain. TSH-activated signalling does not take place in these membrane domains. Therefore, the differences between species, concerning the TSHR-G proteins coupling cannot be explained by the presence of these membrane domains. 2. Species specific thyroid signal transduction: conserved physiology, diverged mechanisms As mentioned above, Gq proteins are activated in human but not in dog thyroid, in response to TSHR. However the dog TSH receptor is able to activate Gq, as demonstrated in transfected CHO cells. Thus, different thyroid signal transduction pathways exist in different species. In this study, we investigated the effects of TSH on its two signal transduction cascades, the cAMP pathway and the phospholipase C – IP3 – DAG pathway, as measured by cAMP levels and inositol phosphate generation. We also measured the effects of TSH and of agents stimulating specifically one of these cascades, forskolin for the cAMP pathway and Ca++ ionophore (ionomycin) and phorbolmyristate ester (TPA) for the phospholipase C pathway, on markers of thyroid hormone synthesis (H2O2 generation and iodide binding to proteins) and on thyroid hormone secretion in vitro in the various thyroids. We demonstrated that in all species investigated, the TSH receptor activates both hormone synthesis and secretion. While in some species, including humans, rats and mice, the TSH receptor activates both the cAMP and phospholipase C– IP3 – DAG cascades, in others (e.g. dog) it only stimulates the first. The cAMP pathway activates the limiting step in thyroid hormone synthesis, the generation of H2O2, in dog, rat and mice but not in human, pig, horse and beef. Thus physiology remains but the pathways to achieve it differ. On a practical point of view, these results allow to choose adequate animal models for investigating different aspects of human thyroid signalling. 3. Duoxes -TPO association and its regulation in human thyrocytes: the thyroxisome Duox (Dual Oxidase) and TPO (thyroid peroxidase) are the crucial enzymes for the thyroid hormones biosynthesis (T3/T4). TPO uses the hydrogen peroxide (H2O2) produced by Duox1 and Duox2 isoenzymes to covalently link oxidized iodide to tyrosines of thyroglobulin and couple the iodinated tyrosines to form triiodothyronine (T3) and thyroxine (T4). An excess of H2O2 is considered to be toxic for cells although at appropriate concentrations H2O2 may carry out signalling functions. Even though thyrocytes show a better resistance to H2O2 than other cells, it would be beneficial for thyrocytes if Duox and TPO localize closely to increase the working efficiency and avoid an excessive H2O2 spillage. In this study, we explored the association of Duox with TPO, and the possible factors affecting their interaction in the human thyrocyte model. This association was established by co-immunoprecipitation approaches on purified plasma membranes from human thyrocytes and COS-7 transfected cells. Our results show that 1) Duox and TPO localize closely at the plasma membranes of human thyrocytes, 2) this association is up-regulated through the Gq-PLC-Ca2+-PKC pathway and down-regulated through the Gs-cAMP-PKA pathway. 3) H2O2 directly increases the association of Duox and TPO. 4) Partial NH2- or COOH-terminal Duox1 and Duox2 proteins show different binding abilities with TPO in COS-7 transfected cells. The association of the two proteins Duox and TPO thus supports our previous hypothesis of the thyroxisome, a pluriprotein plasma membrane complex in which elements of the iodination apparatus localize closely, thus optimizing working efficiency and minimizing H2O2 spillage. Defect in this association, independently of the catalytic efficiency of the enzyme, could therefore impair thyroid hormone synthesis and be harmful to thyroid cells, leading to thyroid insufficiency.

Activating mutations of genes coding for two different tyrosine kinase receptor, either RET or NTRK1 (also named TRKA), as well as of RAS or BRAF gene are associated with human thyroid papillary carcinoma (PTC). RET or NTRK1 protooncogene encodes a cell-surface transmembrane tyrosine kinase receptor with nerve growth factor as its lignand. Oncogenic potential of these two genes in thyrocytes results from replacement of their 5' portion by regulatory parts of other genes, leading to constitutive activation of their tyrosine kinase activity. The four reported oncogenic rearrangements of NTRK1 (TRK) are the consequences of fusion of its tyrosine kinase domain with one of the three genes (TPM3 gene, TPR gene, TFG gene). In our previous study, a PTC sample was found to express the NTRK1 tyrosine kinase domain without harboring NTRK1 rearrangement. We, therefore, assumed that there might have a novel NTRK1 rearrangement in this sample. 5¡¦RACE strategy was employed to clone the unknown 5¡¦end. Sequence of the cloned DNA fragment demonstrated that it is an aberrant transcription product containing an unspliced intron 9. In addition, the variant of NTRK1 wild type termed TRKA¢¹, which lacks exon 9, was also detected in this particular specimen. We conclude that amplification of TK domain of NTRK1 may serve as a rapid screening method for the presence of NTRK1-related transcript in PTCs. Mutations of the BRAF protein serine/threonine kinase gene have recently been identified in a variety of human cancers, especially in melanoma and papillary thyroid carcinomas. Among benign and malignant thyroid tumors, BRAF V599E mutations were reported to be restricted to papillary carcinomas. In this study, we analyzed mutations of BRAF in conjunction with our previous studies on RAS, RET rearrangement and NTRK1 rearrangement in PTCs to investigate genetic alterations in the RAS/RAF/MEK/MAPK kinase pathway. BRAF V599E mutations were detected in 49 of 105 (47%) PTCs but not in other type of thyroid tumor. There was no overlap between papillary carcinomas harboring RET rearrangement, NTRK1 rearrangement and BRAF mutations. Correlation between BRAF mutations and various clinicopathological parameters in 101 papillary carcinomas did not reveal any association with age, sex, tumor size, cervical lymph node metastasis, extrathyroidal extension, distant metastases and clinical stage. We conclude that BRAF mutations are restricted to papillary carcinomas in thyroid tumor. The overall frequencies in our study are in line with data previously reported. In Taiwan, BRAF mutation is the most prevalent oncogene in papillary thyroid carcinomas so far identified.

No description available.

Abstrate: Ras proto-oncogenes are members of the superfamily of GTP-binding proteins. Many tyrosine kinase receptors, including those for epidermal growth factor, insulin, and nerve growth factor, signal through RAS proteins. The product of members of this oncogene family (H-, K-, N-ras) is a 21 kD protein with nucleotide binding activity, involved in the transduction of information from the cell surface to the nucleus. The three RAS proteins exit in two states: a resting state in which they are bound to GDP and an active state in which they bind GTP. The most common form of mutational activation of Ras oncogenes in human tumors is through single base substations affecting either the GTP-binding of main (codons 12 and 13) or the GTPase domain (codon 61) of the protein. Thus, mutant RAS proteins result in constitutive activation of the downstream signaling cascade because their affinity for GTP is increased or their GTPase activity is decreased, so that the protein cannot return to the resting state. To investigate we have screened 89 thyroid tumor specimens, which include 8 follicular carcinomas (FC), 42 papillary carcinoma (PTC), 2 anaplastic carcinoma (AC),5 Hurthle cell adenoma (HA), 12 follicular adenoma (FA) and 20 nodular goiter (NG), for mutation in three Ras genes using PCR and automatic sequencing. Four tumors contained Ras gene mutation. Of these, three were identified among FC (37.5%), which mutation were in the codon 61 of each Ras genes. One mutation were at codon 61 of N-ras in FA specimens (8.3%). In addition, 33.7% (30/89) of specimens contain H-ras codon 27 polymorphism. In conclusion, our data indicated that the prevalence rates of Ras gene mutation were 5.8% and 2.7% in thyroid carcinoma and thyroid benign adenoma, respectively. Other environmental or genetic factors might also involved in the thyroid tumorigenesis and worth further investigation. The data were further confirmed using the combination of the PCR and denaturing gradient gel electrophoresis (DGGE). Four more cases of possible Ras mutation were detected which did not revealed by automatic sequencing , indicating that DGGE is a more sensitive method in detecting single nucleotide mutation. DGGE analysis should increase the detection rate of Ras gene mutation in our analysis.

The purpose of this study was to design a dosing regimen and evaluate the safety of zonisamide (ZNS) following multiple dosing and to determine appropriate monitoring methods. Clinical pharmacokinetics were studied in 8 adult dogs (4 male and 4 female) ranging from 3 to 4 years of age using a randomized crossover design following single intravenous (IV) and oral administration, 6.85 and 10.25 mg/kg, respectively. Samples were collected intermittently for 48 hours. Dogs were then dosed orally (10.17 mg/kg) twice daily for 8 weeks. Blood samples were collected weekly and at discontinuation of the drug. Additionally, urine was collected to determine 24 hour urine ZNS clearance following IV administration. Safety was based on clinical pathology, thyroid and urine testing during both studies. ZNS was measured using high performance liquid chromatography in serum, plasma, erythrocytes (RBC) and whole blood. Data were subjected to standard non-compartmental pharmacokinetic analysis using computer assisted linear regression (WinNonLin?). Comparisons were made in different compartments using one-way ANOVA to identify any differences. Safety parameters at study beginning and end were compared using a Student t-test. ZNS concentrations differed among blood compartments after single dosing, with oral maximum concentration (Cmax) being greatest in RBC (28.73?g/ml) and least (14.36?g/ml) in plasma. Volume of distribution also differed, being greater (1096.05ml/kg) in plasma and least in (379.23ml/kg) RBC. Clearance of ZNS was 57.55ml/hr/kg from plasma and 5.06ml/hr/kg from RBC. Elimination half- life in plasma was 16.4 hr in serum and 57.4 hr in RBC. Bioavailability was 126.8% for RBC and 189.6% for plasma. Following multiple dosing, at steady-state, Cmax averaged 65.8?g/ml with fluctuations of 17.2% between dosings. Accumulation of ZNS was 3.5 (plasma) and 4.3 (RBC). Concentrations did not differ among blood compartments at the end of multiple dosing. Although differences did occur across time in clinical pathology tests, all were within normal limits at study end except for T4. In conclusion, ZNS dosed at 10 mg/kg twice daily for dogs would maintain therapeutic levels (10 to 70?g/ml) recommended in human epileptic patients. Therapeutic monitoring would be best measured in serum or plasma accompanied with thyroid and urine testing.

Due to the seasonal nature of E. coli O157:H7 shedding and of hormone production by the thyroid gland, two studies were initiated to determine whether chemical inhibition of the thyroid gland influences fecal shedding of Escherichia coli O157:H7. Twenty-four crossbred sheep (68.6 kg BW) were randomly assigned to pen and either 0.0 mg/kg BW PTU or 20 mg/kg BW PTU for 5, 11, or 14 days. Sheep were experimentally infected (d 0) with E. coli O157:H7 11 days prior to PTU treatment. Fecal and serum samples were collected for bacterial enumeration and for analysis of T3 and T4, respectively. Sheep were humanely euthanized and tissue and content samples were collected from the rumen, ileum, colon and rectum. Detection of E. coli O157:H7 increased toward the terminal end of the GI tract. In the treatment group, serum T3 levels decreased to an overall lower level than the control group. A correlation was seen between T3 levels and daily O157:H7 bacterial shedding (P=0.003; r=0.37). In experiment 2, 12 growing lambs (41.04 kg BW) were exposed to either 0.0 mg/kg BW PTU or 40 mg/kg BW PTU for 21 days. Fecal samples were collected for analysis of generic E. coli and body weights were recorded on days 0, 7, 14, and 21. Feed intake was recorded throughout the experiment. Animals were experimentally infected with E. coli O157:H7 on day 15. Sheep were humanely euthanized on day 21 and GI tract tissue and content was collected from the rumen, ilium, colon and rectum. A date by treatment interaction was observed for T4 (P=0.0016) and hormone levels decreased in treated animals. Thyroxine and E. coli O157:H7 display a multivariate treatment (P=0.0005) and date effect (P=0.0174) but no significant interaction. Triiodothyronine and E. coli O157:H7 shedding have a slight date trend (P=0.065) but no significant treatment or treatment by date interaction. Generally, the treatment group shed genreric E. coli at higher levels throughout the study period with slightly more than a log count difference between groups at the last collection point (control = 3.8 CFU/gram of feces (log10); treatment = 4.9 CFU/gram of feces (log10)). Results from these experiments suggest that correlations exist between both E. coli O157:H7 and generic E. coli shedding in sheep.

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)

Ph.D.