Persistent organic pollutants (POPs) are ubiquitous environmental contaminants that are lipophilic, slow to degrade, bioaccumulate in aquatic food webs and threaten the health of both humans and wildlife. Predatory marine mammals, such as harbour seals (Phoca vitulina), are at particularly risk for the accumulation of high POP concentrations and resultant increased risk of toxic effects. EIevated levels of certain POPs have been implicated in the disruption of the endocrine system in marine mammals. The main purpose of this study was to assess whether current levels of POP are affecting thyroid hormone physiology of free-ranging harbour seals (Phoca vitulina) in British Columbia (BC), Canada and Washington State (WA), U.S.A. TH functions mainly by binding to nuclear thyroid hormone receptors (TRs) in target tissues and modulating specific gene expression programs. TR isoforms α (TRα) and β (TREβ) from harbour seals were isolated and quantified in internal and external organs. Harbour seals inhabiting industrialized regions exhibited a contaminant-related increase in blubber TRa and a decrease in circulating total thyroxine (TT4) concentrations. Our TRa expression results provide evidence of contaminant-related disruption of TH action at the Ievel of regulation of gene expression. Our findings of a metabolically active blubber layer. and a contaminant-related disruption of blubber TRa expression, suggest that, in addition to disruption of normal development, contaminant exposure could have important implications for lipid metabolism in seals. Consequently, the disruption of blubber TRa expression could influence such critical life processes as energy storage, thermoregulation, and buoyancy in marine mammals. In this study, the use of gene expression biomarkers in combination with a biopsy-based sampling approach was successfully applied to a small marine mammal (i.e. harbour seal) and demonstrates great promise for investigations in other sentinel species (i.e. cetaceans).

The endocrine system is vulnerable to a range of chemicals in the environment. Endocrine disrupting chemicals (EDCs) are exogenous agents that can induce responses on the endocrine system because of their hormone-like activity and toxicity. Specific to this study are thyroid disrupting chemicals (TDCs), these are EDCs that specifically disrupt the thyroid hormone signalling pathway, and this may result in adverse health effects. Thyroid hormones play a crucial part in metabolism, growth, maintenance of brain function and fertility; hence disruption of the thyroid signalling axis implicates human health. We are exposed to TDCs regularly, and studies have shown an association between TDC exposure and neurobehavioural disorders, reproductive abnormalities and obesity. There is a lack of data associated to thyroid hormone receptor activity in surface and drinking water. Hence, the potential human health risks posed by thyroid disruption may therefore be underestimated. The aim of the study was to optimise and validate the GH3.TRE.Luc reporter gene bioassay that can measure thyroid hormone receptor mediated activity and cytotoxicity in drinking and source water, with relevance to water monitoring. The GH3.TRE.Luc reporter gene bioassay was established, optimized and validated to detect thyroid hormone receptor activity. The luciferace assay was used to test for metabolic activity and the resazurine cell proliferation assay was used to assess cell viability. The assay was applied to compounds with agonistic and antagonistic properties; triidothyronine (T3), thyroxine (T4), triac, tetrac, amiodarone, sodium arsenite, pentachlorophenol (PCP), ethylene thiourea, 2,2,4,4-tetrahydroxybenzophenone (THBP) and methimazole. It was also applied to environmental and drinking water samples from the Global Water Research Coalition (GWRC). Finally, the assay was applied to 48 water samples from a water treatment plant in South Africa, collected over a period of 12 months. Every month, four samples were collected. Two samples were source water samples, with one going into the treatment plant and coming out as 2 distribution pipelines (drinking water). For optimisation and validation, the dose response curves obtained for T3, T4, tetrac and triac (agonists) were comparable to literature. Antagonistic behaviour was seen in sodium arsenite, amiodarone, PCP and methimazole. Spiked water samples from the GWRC showed thyroid hormone receptor activity. Sixteen of the 48 water samples collected from the water treatment plant were positive for thyroid hormone disruptor activity. Highest activity was seen in the winter season, accounting for seasonal variations. High TDCs activity reported in the source water may be due to activities occurring near the dam. The water treatment plant seemed effective for only one of the distribution pipelines, and not the other. This study confirms that GH3.TRE.Luc Reporter Gene Bioassay is a sensitive and effective tool to identify and quantify TDC activity in pure chemicals and in complex environmental mixtures present in water. Further monitoring of water sources for TDCs is recommended to ensure water quality and safety. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

O hormônio tireoideano (T3) é um potente regulador das funções cardíacas e musculares esqueléticas. Desse modo, o presente trabalho identificou o efeito de tal hormônio nas proteínas da banda M sarcomérica, as quais fazem parte das proteínas estruturais cardíacas e musculares esqueléticas. O T3 diminuiu a expressão da proteína M no coração, uma das proteínas da banda M, e agiu diretamente no gene dessa proteína M. No músculo esquelético, T3 aumentou a expressão de EH-miomesina no músculo sóleo e reduziu a expressão de proteína M no músculo extensor digital longo (EDL). Portanto, pode-se concluir que o T3 possui uma importante função na regulação da expressão de proteínas estruturais musculares e a ausência dessas proteínas pode ocasionar lesões das estruturas musculares cardíacas e esqueléticas. / Thyroid hormone (T3) regulates many functions of the heart and skeletal muscle. In this way, the present work identified the effect of T3 on the sarcomeric M band proteins, which are structural proteins from the heart and skeletal muscle. T3 down regulated M protein expression in the heart, one of the M band proteins, and, moreover, T3 could regulate M protein gene directly. In the skeletal muscle, T3 up regulated EH-myomesin expression in soleus muscle and T3 down regulated M protein expression in the extensor digitorum longus muscle (EDL). Therefore, we could conclude that T3 has an essential function in the regulation of muscle structural protein and the absence of these proteins could cause lesions at cardiac and skeletal muscle structures.

Pregnancy is a condition leading to an important strain on thyroid morphology and function.A normal functioning of the thyroid gland in the mother is essential for the early fetal development, since the fetal thyroid does not produce thyroid hormones until the end of the first trimester (approximately 12 to 14 weeks).The impact of thyroid dysfunction (and especially hypothyroidism) during pregnancy is well documented and has been associated with a number of obstetrical complications, such as premature delivery, low birth weight and even fetal death. In view of all changes in thyroid physiology during pregnancy the ATA (American Thyroid Association) guidelines recommend using trimester- and population-specific normality ranges, to define thyroid dysfunction. It is proposed to determine them in pregnant women without thyroid antibodies (TPO) and without severe iodine deficiency. Due to the few numbers of randomized clinical trials, there is still no consensus whether all pregnant women should be screened or only women at risk for the development of thyroid dysfunction during pregnancy.Thyroid dysfunction during pregnancy is caused in most cases by the presence of thyroid autoimmunity (TAI) and also the altered pregnancy outcomes in most studies are associated with the presence of TAI.Besides the presence of TAI, other factors might also change, influence and/or modify thyroid function. When we started our research, there were only few studies that investigated the impact of other variables, such as iron, BMI, smoking habit and/or the background of the pregnant women on the prevalence of thyroid dysfunction during the first trimester of pregnancy.The aims of the thesis were therefore, to investigate: • the association between the iron reserve status (ferritin levels), thyroid (dys)function and autoimmunity, corrected for confounders such as age, BMI, smoking habit and the time of blood sampling;• the impact of the ethnic background of the pregnant woman on thyroid function and autoimmunity, corrected for confounders such as age, BMI, smoking habit, and the time of blood sampling. Furthermore, to determine ethnic-specific reference ranges and investigate their impact on the diagnosis of thyroid dysfunction;• the impact of changes in thyroid function within the normal reference range in women free of thyroid autoimmunity on pregnancy outcomes, corrected for established covariates (age, BMI, smoking) and iron reserve as candidate new variable.• whether targeted high-risk screening for thyroid dysfunction during pregnancy could be improved with the inclusion of iron status and ethnicity to the actual risk factors defined in the ATA-GL.The results can be summarized as follows:Thyroid function during pregnancy can be influenced by variables others than thyroid antibodies such as the iron status and the ethnical background of the women. However, their impact on thyroid function is less important compared to that of thyroid antibodies. No significant impact of well-known variables (BMI, age, smoking) and others such as iron has been shown on clinical pregnancy outcomes when thyroid function remained within the normal range and no thyroid antibodies were present.We have shown that adding variables such as iron deficiency, ethnic background and obesity to the currently provided list of factors leading to a high-risk for the development of thyroid dysfunction during pregnancy, might improve the detection rate of subclinical hypothyroidism to comparable rates obtained in case of universal screening. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

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Around the time of birth, the lung switches from a secretory- to a liquid absorptive organ to enable the fetus to transit from an intra-uterine to an air-breathing environment. This study concerns hormonal control of the liquid reabsorptive mechanism in the fetal lung which allows this transition to take place. Thyroidectomy in the fetal sheep at 118 days gestation (term = 147 days) prevented the development of adrenaline- or cyclic AMP-sensitivity which, in euthyroid fetuses, resulted in the capacity to absorb lung liquid from 130 days onwards. Studies in which T₃ and T₄ were infused to thyroidectornized fetal sheep showed that T₃ was required for the normal evolution of the reabsorptive response. However, infusion of this hormone to immature fetuses (110 days) did not advance the gestation at which adrenaline-sensitive absorption is first seen. Co-infusion of T₃ and hydrocortisone showed that these 2 hormones have a powerful synergistic effect on the absorption mechanism. Within a few hours of infusion of these 2 hormones to immature fetuses, a reabsorptive response to adrenaline similar to that normally seen in mature fetuses was observed. This response was fully reversible on withdrawal of T₃ and hydrocortisone infusion, and the hormonal effect was blocked by the protein synthesis inhibitor, cyclohexirnide. These findings suggest that the normal rise in T₃ and cortisol seen in the fetus in late gestation is responsible for maturation of the liquid absorption mechanism which allows the fetus to make a transition to an independent air-breathing existence. These observations may be of significance in the clinical management of infants born prematurely, who may have had insufficient pre-natal exposure to T₃ and cortisol.

Type II 5'-deiodinase (D2) catalyzes the conversion of T4 to the transcriptionally active T3. When T4 levels are high, D2 activity levels are low. Conversely when T4 levels are low, D2 catalytic activity is high. Immunocytochemistry and biochemical data from cultured rat astrocytes revealed that physiological concentration of T4 and the non-transcriptionally active metabolite rT3, but not T3, initiates the budding of D2 containing endosomes and their subsequent translocation to the perinuclear space. Further analysis showed that this process required a polymerized actin cytoskeleton but not cellular transcription or translation; however the precise mechanism remained unknown. In this present investigation, we characterized the requirement of an unconventional myosin motor protein, myosin 5a, in the actin-based endocytosis of D2 containing vesicles. We developed an in vitro actin binding assay that exploited the T4 dependent binding of D2 containing vesicles to F-actin, and showed that D2p29:F-actin interactions are calcium, magnesium and ATP-dependent suggesting that a calmodulin (CaM) regulated myosin ATPase is required. Introduction of in vitro transcribed and translated vesicle-binding tail, which lacked the actin binding head, of myosin 5a to the in vitro actin binding assay created a dominant negative inhibitor of D2 binding to the actin cytoskeleton by competing with the native myosin 5a. A replication deficient adenoviral vector expressing the fusion protein of the 29 kDa substrate binding subunit of D2 with a green fluorescent protein reporter molecule enabled us to directly examine T4 dependent regulation of D2 in vitro as well as in living cells. Using immunoprecipitation we showed a T4 dependent association between the vesicle binding tail of myosin 5a and D2 containing vesicles. Biochemical analysis of the interaction of the myosin 5a tail with D2 containing vesicles revealed that the last 21 amino acids of myosin 5a were both necessary and sufficient for the attachment of D2 containing vesicles to the F-actin cytoskeleton. Using rapid acquisition time-lapse digital microscopy in p29GFP expressing rat astrocytes, we showed directed T4 dependent p29GFP movement from the plasma membrane to the perinuclear region. This hormone dependent vesicle movement was not observed in cells treated with T3 or no hormone. Time lapse motion studies allowed for the calculation of the velocity and of the distance traveled for individual fusion protein containing vesicles. The velocity for cells treated with T4 or rT3 was identical to that reported for vesicle-laden myosin 5a in mouse melanophores. In contrast cells treated with T3 or those receiving no hormone treatment had velocities similar to diffusion of proteins within the plasma membrane. Astrocytes constitutively expressing both p29GFP and dominant negative myosin 5a inhibitors failed to show hormone induced centripetal movement. These data demonstrate that myosin 5a is the molecular motor responsible for thyroid hormone dependent actin based endocytosis in astrocytes.

Several methods were used to detect changes in thyroid activity and in degradation of thyroxine (T4) in exercising and sedentary control rats. Rats were run in a motor-driven exercise wheel over a three-week period at progressively increasing work loads and then were run 1.5-2 hours per day for 10-14 days before sacrifice. Disappearance of injected T4-125I from plasma was considerably enhanced by exercise. As determined by isotopic equilibration and paper chromatographic methods, T4 concentration was depressed in plasma and elevated in liver of exercised rats. No significant changes in T4 concentration were observed in heart, kidney, or gastrocnemius muscle. Thyroidal iodotyrosine levels were elevated immediately following but not 24 hours after exercise. Serum free T4 was higher in exercised rats. In vitro deiodinating activity in liver, muscle or kidney homogenates was not affected by exercise. The elevated T4 degradation rate accompanying exercise is thus likely due to the elevated free T4. The liver is apparently the tissue site responsible for the increased T4 uptake and degradation.