Faced with an environment of constantly changing nutrient availability, mammals have adapted complex homeostatic mechanisms to maintain energy balance. Deviations from this balance are largely corrected through a concerted, multi-organ effort that integrates hormonal signals with transcriptional regulatory networks. When these relationships are altered, as with over-nutrition and insulin resistance, metabolic disease ensues. Here, I present data concerning two distinct transcriptional pathways--one for thyroid hormone (TH) and one for insulin--that confer hormone responsiveness on metabolic gene programs that preserve energy homeostasis.

Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are added to polyurethane foams in a variety of industrial and consumer products to prevent flame ignition. The gradual release of these flame retardants (FRs) from such products leads to contamination of various abiotic and biotic media, including wild birds. Recent studies demonstrated endocrine-disrupting effects of TCPP and TDCPP, including alteration of circulating thyroid hormone (TH) levels. The TH-pathway is essential for normal growth and development in birds. There are limited data on the toxicological effects of TCPP and TDCPP in avian species and, prior to this work, no study has examined their effects in avian embryos. This M.Sc. thesis investigates the developmental, molecular and biochemical effects of TCPP and TDCPP in chicken (Gallus gallus domesticus) embryos via egg injection studies. TCPP delayed pipping at doses ≥9.24 μg/g, both TCPP and TDCPP reduced embryo growth at the highest dose (51.6 μg TCPP/g and 45 μg TDCPP/g), and TDCPP decreased free plasma thyroxine and gallbladder size at 7.64 μg/g and 45 μg/g, respectively. Real-time reverse transcription polymerase chain reaction was used to measure changes in mRNA levels of hepatic genes that were responsive to these FRs in a previous in vitro study. TCPP dysregulated the expression of TH-responsive genes and xenobiotic metabolizing enzymes (cytochrome P450s; CYPs), whereas TDCPP only affected CYPs. Less than 1% of the administered TCPP or TDCPP was detected in egg contents following 19 days of incubation, indicating extensive metabolism of the parent compounds. DNA microarrays were used to perform a global transcriptional analysis on liver samples from embryos that exhibited adverse effects following TDCPP injection. 47 differentially expressed genes were identified at the 45 μg/g dose. Functional analysis revealed that immune function and lipid and steroid metabolism were major targets of TDCPP toxicity and indicated a state of cholestatic liver/biliary fibrosis. Since the TH-pathway is a key regulator of metabolic homeostasis, its disruption early in development is a potential cause of the observed adverse effects. This thesis demonstrates, for the first time, developmental and endocrine-disrupting effects of TCPP and TDCPP in an avian species and attempts to link phenotypic changes to molecular-level disruptions in hopes to improve the understanding of their modes of action.

Thyroid hormones (THs) are important hormones involved in developmental processes, including foetal brain maturation. THs are also involved in the maintenance of homeostasis. One in three people in Canada are considered to have some form of thyroid disorder. One reason for the high level of thyroid disorders may be the increasing amount of anthropogenic chemicals released into the environment that affect normal hormone action. Amphibian metamorphosis is completely dependent on TH and provides a model to study such chemicals. This thesis uses the Rana catesbeiana tadpole as a model to study potential TH disrupting chemicals by developing a novel screening assay called the cultured tail fin biopsy assay, or the “C-fin” assay. The C-fin assay uses tail biopsies from premetamorphic tadpoles, Taylor-Kollros stage VI-VIII. The biopsies are cultured in serum-free media along with the test chemical for 48 hours. QPCR is used to measure the mRNA steady-state levels of selected gene transcripts. Two TH-responsive gene transcripts were measured: the up-regulated gene transcript, thyroid hormone receptor β (TRβ) and the down-regulated gene transcript, Rana larval keratin type I (RLKI). Heat-shock protein 30 (HSP30) and catalase (CAT) were used as indicators of cellular stress. Another model system used in this thesis is rat pituitary cells, or GH3 cells. QPCR was used to measure the mRNA steady-state levels of three TH-responsive genes growth hormone (GH), deiodinase I (DIOI), and prolactin (PRL); heat-shock protein 70 (HSP70) was used as an indicator of cellular stress. Nanoparticles, used in various consumer products, were one class of chemicals examined. Using the C-fin assay, nanosilver and quantum dots (QDs) caused perturbations in TH-signalling and also showed signs of cellular stress. There was no overt toxicity observed as was determined by the normalizer, house-keeping gene transcript, ribosomal protein L8. The GH3 cells also detected TH disrupting effects by both nanosilver and QDs; however, nanosilver did not appear to cause cellular stress whereas QDs did. Nitrate and nitrite, major waterway contaminants, were also examined and there were no TH-perturbations observed using the C-fin assay. Finally, two antimicrobials used in many consumer products, triclocarban (TCC), triclosan (TCS) and its metabolite, methyl-TCS (mTCS) were examined using both the C-fin assay and GH3 cells. Both the C-fin assay and the GH3 cells determined mTCS to be more potent than TCS in disrupting TH action. TCC also caused perturbations in TH-signalling as well as causing a significant amount of cellular stress. Overall the C-fin assay and the GH3 cells proved to be excellent models in studying the potential disruptors of the TH axis. The C-fin assay and GH3 cells detected novel TH disruptors and gave further insight into already known disruptors of the TH axis.

Nanoparticles (NPs) are engineered in the nanoscale (<100nm) to have unique physico-chemical properties from their bulk counterparts. Nanosilver (nAg) is the most prevalent nanoparticle in consumer products due to its strong antimicrobial action and can be released to the environment during product manufacture, usage and disposal. The predicted environmental concentrations are within the North American guidelines for the protection of aquatic life and in drinking water. While nAg toxicity at high concentrations has been well described, the sublethal effects at environmentally-relevant concentrations are relatively unknown. Initial screening in our lab showed nAg was a potential endocrine disrupting chemical (EDC). Amphibian metamorphosis is mediated by thyroid hormone (TH), and nAg perturbed TH-dependent transcriptional responses in the tailfin of bullfrog (Rana catesbeiana) tadpoles. The primary objective of this thesis was to further investigate and characterize the effects of low, environmentally relevant concentrations of nAg on TH-dependent metamorphosis in R. catesbeiana and Xenopus laevis. Two chronic, 28 day in vivo exposures at 0.06 and 6µg/L nAg were conducted with premetamorphic R. catesbeiana tadpoles using TH to induce precocious metamorphosis. Ionic silver (iAg) was also examined to control for the complete dissolution of Ag. Analysis of metamorphic stage progression demonstrated nAg-induced acceleration of hindlimb growth and development. After 6 days of nAg exposure, analysis with quantitative real-time polymerase chain reaction (QPCR) demonstrated nAg-induced disruption of TH-responsive transcripts in a tissue-specific manner. Furthermore, the nAg effects could not be fully explained by iAg, indicating NP-specific disruption. Two chronic, 28 day exposures to 0.018-1.8 µg/L nAg were conducted on X. laevis premetamorphic and prometamorphic tadpoles. nAg was found to significantly bioaccumulate in tadpole tissue after 28 days. Furthermore, nAg increased the hindlimb length during early premetamorphosis and in post-metamorphic juvenile tadpoles. Using an in-house MAGEX microarray and QPCR transcriptional analysis, 7 biomarkers of nAg exposure were validated. Five of these targets showed disruption to their TH-response. Furthermore, the increased mRNA abundance of two peroxidases indicated that nAg generated reactive oxygen species (ROS) even at low, environmental concentrations. This thesis demonstrates that nAg has consistent EDC actions across two distinct amphibian species, and the data suggest that regulatory guidelines for silver may need revision. A X. laevis derived fibroblast-like TH-responsive cell line, XTC-2, was used in conjunction with the 7 biomarkers of nAg exposure to gain mechanistic insight into the role of ROS in TH signaling disruption. Monocultures were created and validated to increase the specificity of TH-response. While the monocultures were successfully created, the biomarkers were not responsive to nAg in this cell line. Additional investigations were made into the relationship between genetic sex and responsiveness to TH. Genetic sexing methods were used to investigate transcriptional differences between males and females during natural and TH-induced metamorphosis. The sexing protocol was optimized and validated successfully. The genetic sex was determined for premetamorphic and prometamorphic X. laevis tadpoles exposed to TH for 48 h. QPCR and microarray analysis were used to identify three markers that demonstrated transcriptional sex-bias during early gonadal differentiation stages. / Graduate / 0307 / 0383 / 0487 / amanda_carew14@yahoo.ca

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

xvi, 112 p. : ill. (some col.) / In seasonal environments, the ability to take advantage of the favorable seasons and avoid or mitigate the effects of the unfavorable ones is essential for organismal fitness. Many polar and temperate organisms use photoperiod (length of day) to time seasonal life history events because photoperiod's regular annual cycle makes it a very reliable indicator of seasonality. This reliability allows organisms to anticipate and properly prepare for seasonal change. Although photoperiodism is widespread in polar and temperate vertebrates, little is known relative to invertebrates regarding how its use varies with environment and this method's underlying genetic and physiological basis. This dissertation is focused on demonstrating the proper methodology for the study of photoperiodism and establishing the threespine stickleback as a model of vertebrate photoperiodism. Chapter I is an introduction to photoperiodism, how it is influenced by environment, the physiological basis of its output, and a summary of the chapters that follow. Chapter II explains an analytical method to test for causality and applies this method to data that have been interpreted as evidence that the circadian clock is causally involved in photoperiodism. Chapter III describes the photoperiodic response of threespine stickleback Gasterosteus aculeatus populations from two latitudes. These results are used to inform an empirical examination of the previously described assertion that the circadian clock is causally involved in photoperiodism. Chapter IV examines the physiological basis of early photoperiodic response using the threespine stickleback as a model teleost fish. Chapter V summarizes the previous chapters, describes their significance, and suggests future research directions. This dissertation includes both previously published and co-authored material. Supplementary Excel files demonstrating the analyses used in Chapter III are also included in this dissertation. / Committee in charge: Eric Johnson, Chairperson; William Cresko, Advisor; William Bradshaw, Member; Judith Eisen, Member; Patricia McDowell, Outside Member

Dados recentes mostram que o remodelamento ósseo está sujeito ao controle do SNC, com o SNS. Para tanto, avaliamos o (CLO) e a morfologia da (LE) do fêmur de camundongos de 21 dias de idade selvagens (WT) e Knockout (KO) para a2CAR, tratados, para mimetizar o hipertiroidismo (Hiper) ou tratados para indução do hipotiroidismo (Hipo). Os animais KO eutiroideos (Eut) apresentaram uma desorganização da ZP e aumento do número de CHM da LE. Nos animais WT, o Hipo promoveu redução do CLO, desorganização da ZP e diminuição do número de CHM. O Hipo promoveu efeitos contrários na LE dos animais KO em relação aos animais WT, sendo capaz de reverter, parcialmente, a desorganização da ZP dos camundongos KO Eut, além de resultar em maior número de CHM, em relação aos animais WT Hipo. Já o Hiper levou a aumento do número de CH nos animais WT e redução nos animais KO. Os animais KO Hiper apresentaram diminuição de CHM, quando comparados aos animais WT Hiper. Esses achados reforçam as hipóteses de que o SNS regula o CLO via a2CAR e que o HT e SNS interagem para regular o CLO. / Recent data show that bone remodeling is under control of the CNS, with the SNS. Thus, we evaluated the bone growth and the morphology of the femoral EGP of 21- day old female wild-type (WT) and a2CAR-/- mice (KO), treated to mimic hyperthyroidism (Hyper), or treated for hypothyroidism (Hypo). The euthyroid KO mice had a disorganization of PZ and increase in the number of MHC of the EGP. In WT animals, Hypo promoted a significant reduction in the BLG, PZ disorganization and a decreased number of MHC. Hypo promoted opposite effects in EGP of KO compared to WT mice. Hypo was able to partially revert the PZ disorganization observed in euthyroid KO, and resulted in a greater number of MHC compared to WT Hypo. On the other hand, Hyper caused an increase in the number of HC in WT mice and a reduction in KO mice. Furthermore, Hyper KO animals showed a reduction in the number of MHC, when compared to the Hyper WT mice. These findings support the hypothesis that the SNS regulates the BLG via a2CAR and that the interaction between SNS and TH can regulate the BLG.

O objetivo deste estudo foi investigar os efeitos do T3 e do seu análogo GC-24, agonista TR<font face=\"symbol\">b seletivo, na proteólise muscular mediada pela via ubiquitina-proteassoma. Avaliamos o efeito do T3 e GC-24 no trofismo radial de fibras musculares, no nível de ubiquitinação e na expressão de genes envolvidos na via ubiquitina-proteassoma. Para tanto foram utilizados, ratos Wistar divididos em 4 grupos (Controle, 12 horas, 1 e 7 dias) e tratados com T3 e GC-24. Determinou-se a área de secção transversa dos cortes histológicos através do programa \"Image Pro-Plus\". O nível de ubiquitinação foi determinado através de Western Blot para proteína ubiquitinada e a expressão gênica por PCR em Tempo real. T3 e GC-24 promoveram redução do diâmetro das fibras musculares e aumentaram o nível de proteínas ubiquitinadas em ambos os músculos. Com relação à expressão gênica, T3 e GC-24 modularam a expressão dos genes analisados de maneira diferenciada, demonstrando que GC-24 é capaz de modular genes pouco ou não responsivos ao T3. / Triiodothyronine (T3) is known to play a key role in the function of several tissues/organs via the thyroid hormone receptor isoforms a/pha (TRa) and beta (TRI3). Abnormalities in skeletal muscle function have been associated with increased leveis of T3, which is a major sarcopenia (Ioss of sarcomeres). Although the phenomenon of sarcopenia induced by T3 has been widely reported, little is known about the molecular mechanisms invo/ved in proteolysis induced by T3. In this study we have investigated the effects of T3 and GC-24, a novel synthetic TRI3¬selective compound, on the ubiquitin proteasome pathway. We analyzed the effect of T3 and GC-24 on the radial trophism, ubiquitination leveis and gene expression of the ubiquitin-proteasome pathway, which are important regulators of muscle proteolysis in the skeletal muscle. We have addressed the ubiquitin ligases (Atrogin¬1, MuRF-1 and E3a) and the deubiquitinating enzymes (UBP45, UBP69 and USP28). Wistar male rats (170-200g) were divided in 4 groups (Control, 12, 1 and 7 days). Rats received T3 (30l-\'g/100g) and GC-24 (16 I-\'g/1 OOg). After decapitation, EDL and soleus muscles were removed for histological ana/ysis, protein expression and gene expression. Cross sectional area was determined in histological sections through the software \"Image-Pro Plus. The ubiquitination leveis was determined by Western Blot and gene expression determined by Real Time PCR analysis. T3 and GC-24 reduced the diameter of the muscle fibers vs control group. Both T3 and GC-24 incresed the ubiquitination leveis, in the soleus and EDL. Regarding gene expression analysis, T3 and GC-24 modulate the gene expression in a differential manner. In the soleus, T3 increased Atrogin-1 and E3 alpha gene expression, while did not alter Murf-1 gene expression. On the other hand, in EDL Atrogin-1 gene expression is not altered, while E3 alpha and Murf-1 are elevated by T3. In the soleus and EDL deubiquitinating gene expression is mostly not altered, exception made for UBP 45, which is reduced by T3 in soleus muscle. GC-24, increased gene expression of E3a and MuRF-1 in the soleus, while did not alter Atrogin-1 gene expression. However, in EDL muscle, GC-24 increased Atrogin-1 and E3a mRNA, while did not alter MuRF-1. Finally, GC-24 decreased UBP 45 gene expression in EDL muscle and USP 28 gene expression was robustly elevated by GC-24 in both muscles analyzed. This data shows that GC-24 is able to strongly modulate genes that are less responsive or even unresponsive to T3, pointing that the GC-24-TRb complex might trans-activate differently target genes. However, both T3 and GC-24 are able to modulate the muscle proteolysis.