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This study concerns halogen bonding between small molecules. Except where otherwise stated herein this investigation was performed exclusively using the M06-2X density functional, in conjunction with the 6-31+G* basis set except for iodine and astatine which were treated using the aug-cc-pVDZ-PP basis set with relativistic pseudopotentials. All calculations were performed in the gas phase. The counterpoise procedure was employed for all full geometry optimisations. Statistical analysis of the Cambridge Structural Database, wherein the frequency of structures as a function of halogen bond angle and distance constituted the sole part of this study not to be based on density functional theory. Except in chapter 5, all halogens from fluorine to astatine are investigated. In chapter 3, halogen bonding between halobenzene and a single water molecule is discussed. Competition between R – X•••OH2 halogen bonding and R – X•••H-O-H hydrogen bonding interactions is described. This system is analogous to the more elaborate microsolvated 1- methyl-5-halouracil system described in chapter 4. In this latter system one 1-methyl-5- halouracil molecule interacts with either one or two water molecules. A central feature of the investigation into this system is competition between R – X•••OH2 and R=O•••H-O-H hydrogen bonding. In chapter 5, halogen bonding is discussed in the context of the thyroid system. In particular halogen bonding between a thyroxine iodine atom and the protein backbone as well as crystal water molecules is the subject of this chapter. The effect of substitution of the iodine atom with an astatine atom is presented. Chapter 6 is concerned with halogen bonding in halogenated DNA base pairs. Interaction energies are compared with those of the canonical base pairs, and the effect of halogen bonding on geometry is also discussed. For each system, halogen bonding was found to become stronger and more tolerant of non- linear bond angles going down the halogen group.

Fish are particularly vulnerable to the exposure of anthropogenic pollutants, with a vast array of endocrine disrupting chemicals (EDCs) introduced into the aquatic environment via sewage discharge, waste disposal and land runoff. Brominated flame retardants (BFRs) are halogenated flame retardants that are used to effectively inhibit the flammability of various materials including plastic products, electrical appliances, construction materials and textiles. BFRs are ubiquitous environmental contaminants and are known to disrupt thyroid hormone (TH) homeostasis in several vertebrate species, including fish. Given the vital role of THs in a wide range of developmental processes and physiological functions, assessing and identifying thyroid disrupting chemicals is crucial for safe guarding the long-term health of humans and wildlife. In fish the molecular mechanisms underlying TH disruption by BFRs and the effects on TH-sensitive tissues during early life stages remains unclear. This has been limited by the lack of fundamental knowledge on the TH system of fish and the difficulties associated with examining transcriptional changes in discrete embryonic-larval tissues. Here I have established the expression profiles of a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) during embryonic-larval stages and their regulation by the biologically active TH (3, 5, 3′- tri-iodothyronine; T3). Using molecular tools (whole mount is situ hybridisation and RT-PCR), I demonstrate that a number of genes display spatial and temporal expression profiles during embryo/larval development, and their regulation by T3 was tissue- and developmental stage-specific. I subsequently demonstrated that TBBPA and BDE-47, two important BFR compounds, disrupted TH homeostasis at multiple levels of the HPT axis of zebrafish embryo-larvae after short sub-acute exposures. These compounds altered the expression of genes associated with TH conjugation and clearance, thyroid follicle development and TH transport. In addition, we suggest that TH target genes in the brain, liver, pronephric ducts and craniofacial tissues of zebrafish embryo-larvae may be particularly vulnerable to TBBPA and BDE-47 exposure. It has been proposed that environmental pollutants can disrupt TH signalling in wildlife by disrupting the activity of thyroid receptors (TRs), ligand-binding transcription factors, which mediate the genomic actions of THs. The ability of BFRs to disrupt fish TRs has not yet been examined. Here I developed an in vitro reporter gene transcriptional assay for zebrafish thyroid hormone receptors (zfTRα and zfTRβ) in human embryonic kidney cells and investigated their interactions with several BFR compounds. The assays were optimised and validated using the natural TR agonist T3 in cells transiently transfected with two reporter vector constructs, pGL4.24-PAL and pGL4.24-DR4. None of the six brominated flame retardants tested, namely, tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), 2,2′,4,4′-tetra-bromodiphenyl ether (BDE-47), 2,2′,4,4′,6-penta-bromodiphenyl ether (BDE-100), 2,2′,3,4,4′,5′,6-hepta-bromodiphenyl ether (BDE-183) and deca-bromodiphenyl ether (BDE-209) had an agonistic effect on zfTRα and zfTRβ activity. These results are consistent with our previous finding which suggests that altered TH homeostasis may be a result of increased metabolism and excretion of THs and/or changes in the production of TH by the thyroid follicles. In conclusion, this investigative work aids the understanding of fundamental TH processes in fish, such as gene expression and regulation, and increases our understanding of the mechanisms and potential targets of BFRs in fish.

The hair follicle (HF) is a human mini-organ that autonomously cycles between phases of growth (anagen), regression (catagen) and relative quiescence (telogen). Whilst many molecular controls are now appreciated to influence hair cycle, what ultimately choreographs the switch between each cycle stage is yet to be elucidated. With the increasing link between molecular clock activities in controlling local tissue physiology, we began by studying the hypothesis that the human HF has a functional molecular clock. Utilising human HF organ culture, qRT-PCR and immunofluorescence we found that the HF does indeed have oscillating clock gene expression over 24 and 48 hours in situ, separate from the suprachiasmatic-nucleus. Moreover, core clock proteins BMAL1 and PER1 are expressed in the human HF with PER1 increasing as HFs enter catagen. Next utilising siRNA mediated gene knock-down of either BMAL1 or PER1 in situ, we were able to show that silencing either clock gene leads to anagen prolongation in cultured HFs, demonstrating that the molecular clock modulates the human hair cycle, namely the anagen-catagen switch in situ. As human pigmentation is tightly coupled to the hair cycle and both human HFs and epidermal melanocytes express clock genes/proteins, this led us to investigate the hypothesis that the molecular clock modulates human pigmentation. By silencing BMAL1 or PER1 in HFs an increase in melanin content (Masson-Fontana) was observed in a hair-cycle independent manner. Furthermore, tyrosinase expression/activity as well as TYRP1 and 2 expression, gp100 protein expression, melanocyte dendricity and the number of HF melanocytes were all significantly increased in BMAL1 and/or PER1-silenced HFs. Mechanistically, BMAL1 knockdown reduced PER1 transcription, and PER1 silencing was found to induce phosphorylation of the master regulator of melanogenesis, MITF, thus stimulating human melanogenesis and melanocyte activity. This provides the first evidence that the peripheral molecular clock influences human pigmentation. Finally, the thyroid hormone (T4) has strong links with peripheral clock activity and has been shown to prolong anagen and increase human HF pigmentation. Moreover, T4 is a commonly prescribed treatment for thyroid disorder. As such, we investigated the hypothesis that T4 influences HF clock gene activity. It was observed that transient T4 treatment reduces the amplitude of clock gene oscillations whilst circadian rhythmicity is maintained. Conversely with longer term treatment clock gene activity was significantly increased compared to a scrambled oligo-control. Here we have demonstrated that the human HF has peripheral molecular clock activity which influences the human hair cycle and pigmentation. Finally we were able to uncover a potential novel target, T4, whose pulsatile administration may potentially be used to treat not only hair growth and pigmentation disorders but may be able to modulate circadian activity in peripheral tissues and treat clock-related disease.

Background: Radiotherapy is used in the treatment of over 50% of cancer patients and bar surgery, is the most effective cancer intervention. However, in the clinic secondary malignancies have been observed following radiotherapy and in vitro increased cell migration and invasion have been seen following radiation. The Src/FAK signalling pathway is known to play an important role in the metastatic phenotype through its involvement in cell adhesion, migration and invasion and we have previously demonstrated that radiotherapy can activate this pathway along with the phosphoinositide 3-kinase (PI3K) pathway, also associated with tumour metastases and an aggressive phenotype. Using pharmacological inhibitors, we have investigated combination approaches to evaluate whether Src and PI3K targeting is beneficial in a radiotherapy context, especially focusing on metastatic phenotype. We wished to relate pathway activation to cellular phenotype and increase understanding of the metastatic cascade, the processes involved and the signalling pathways taking the lead. Method: Using thyroid carcinoma cell lines FTC133 and 8505c the effects of Src inhibition using AZD0530, FAK inhibition using FAKi and PI3K inhibition using GDC-0941 were studied. The effects of radiotherapy alone, and in combination with the above inhibitors, were also studied. In vitro MTT, apoptosis and clonogenic assays were used to assess cell proliferation and cell survival and scratch assays, cell adhesion and cell spreading assays were used to assess the effects of the drugs on metastatic characteristics. In vivo tumour growth, survival and ex vivo clonogenics were used to measure the effects of AZD0530 and GDC-0941. Western blotting, immunofluorescence and immunohistochemistry was used to observe the effects on pathway activation and protein localisation. Results: Src and FAK inhibition reduced metastatic characteristics of thyroid carcinoma cell lines in vitro such as cell spreading and migration. FAK inhibition showed a greater effect on cell survival by MTT, clonogenic and apoptosis. In the thyroid carcinoma cell lines radiotherapy enhanced the metastatic phenotype. This was seen by enhanced activation of the Src and PI3K pathways, increased migration and invasion in vitro and enhanced tumour metastasis in vivo. By combining Src inhibition with radiation a reduction in metastatic characteristics was observed and by combining PI3K inhibition with radiotherapy radiosensitivity could be improved. With the triple combination of Src and PI3K inhibition with radiotherapy a significant reduction in cell survival was demonstrated in vitro compared to radiation alone and either inhibitor combined with radiation, with a corresponding significant reduction in tumour growth being observed in vivo. With the combination of Src and PI3K inhibition significant reductions in metastatic characteristics were also observed both in vitro and in vivo seen by a reduction in cell migration and tumour metastasis. Finally combined inhibition of the Src and PI3K pathway reduced the radiation enhanced activation of several pathways in vivo including Src and PI3K.Conclusions: Together these results suggest that the Src and PI3K pathways play a role in radiation enhanced metastatic characteristics in thyroid carcinoma and through combined inhibition of the pathway the negative effects of radiation, enhanced migration and invasion, can be inhibited and the cells can be made more radiosensitive. Full characterisation of the pathways involved in radiation induced motility and radioresistance will provide further rationale for combination therapies and provide potential for application of these therapies in the clinic.

Background: Differentiated thyroid cancer (DTC), although rare, is the commonest endocrine malignancy, with a significant recent increase in incidence. Natural history is well understood: the majority of patients have an extremely good prognosis, but with a tendency for late recurrences, up to twenty years post diagnosis. These features have combined to effect a lack of evidence underlying current practice, the majority of guidelines based around retrospective series and opinion. A number of validated risk stratification systems exist, unique to DTC, some applicable at the point of intervention. Although these systems reliably predict outcome, they are inconsistently used to guide the extent of intervention. As such, the three main facets of treatment, surgery to the thyroid, surgery to the lymph nodes, and radioiodine (RAI), are variably applied to the same extent of disease. This may depend at least partly upon the clinician‟s preference towards use of risk stratification systems. Consequently, low risk patients may be at risk of morbidities, both short and long term from unnecessarily aggressive interventions, despite consistently good prognosis. Rationalisation of the approach is attractive from a patient, provider, and health service perspectives; without controlled or long term data, such change in practice requires justification. Aim The overall aim of this thesis is to describe and compare the clinical effectiveness of risk stratified (RS) management of DTC, compared to non risk stratified (NRS) management, in an adult population. UK context is provided by description of current practice and long term disease specific outcomes. Three strands of work are combined to address this aim. Part One: Systematic Review A comparison of RS/NRS approaches necessitated objective and reproducible definition of practice overall, encompassing the three facets of intervention. Thereafter, rigorous methodology was applied to examine the clinical effectiveness of the two approaches, based on review of 76 datasets identified from the literature, with RS/NRS approach assigned; there were no relevant randomised trials nor prospective comparative series. Patients, disease and practice were described and compared, with outcome data grouped by safety, disease control, and survival. Sensitivity analysis was planned around population and practice items. Study quality vi was objectively assessed. Weighted mean effect sizes were calculated for RS/NRS cohorts and compared across the two intervention groups for each outcome, with findings further tested on sensitivity analysis. Broadly, equivalence was shown across the two treatment groups by outcome, with small differences consistent with disease biology/magnitude of intervention. Risk stratification appeared to be applicable not only to primary intervention, but also follow-up strategies. Variations in outcome definitions were highlighted, and a pragmatic, patient-centred definition favoured as an approach for future work. Lack of time-specific data was a significant limiting factor. Part Two: Description and Analysis of UK Practice With no UK dataset within the systematic review, validity for the findings was required, as well as the need to describe and compare practice and outcomes in a contemporary local context. The ideal dataset was defined and participants for a UK collaborative sought. Five centres contributed datasets of adequate quality, at least in part prospective, with time-specific outcome measures. Overall, UK practice was by definition risk stratified – and appeared increasingly so over time. There was considerable variation in practice across the five centres (2 NRS, 3RS), but all with consistent, equivalent outcomes compared with the review, despite patients of somewhat higher risk. Utilising methodologies from the systematic review, the analysis was repeated and augmented, confirming consistency in effect directions. Time-specific, adjusted outcome measures demonstrated a possible lack of effectiveness of RAI in high risk disease – a new finding worthy of further exploration. The comprehensive datasets further reinforced the need to work towards prospective, long term, time specific data with common agreed definitions, and suggestions are made how this can be achieved. Part Three: National Survey of Practice Preference The current UK preference for RS management, notwithstanding current service provision around thyroid cancer, is little known. Through cross-specialty survey, utilising a number of case scenarios validated by external experts, and gathering data on clinician demographics, this information was sought, in order to give further context to the above findings. A high degree of variation was identified within, and across scenarios; the least RS preference for intervention applied to the lowest risk scenarios, implying risk of over-treatment. RS preference was associated independently with fewer years in practice and high volume practice. Conclusion Based on the best available data, and supplemented by congruent, contemporary UK data and perspectives, this thesis confirms safety and equivalence of effectiveness of a risk stratified approach to the management of differentiated thyroid cancer, demonstrates considerable variations in practice and suggests possible tools towards building a better evidence base for the future.

The clone pR2 was isolated from a chick cDNA library and its insert was sequenced. The predicted amino acid sequence, encoded by the insert, was analysed and the protein was found to be a new member of the steroid/thyroid receptor superfamily. Further analysis revealed that it was a member of a newly discovered class of retinoid receptors, the retinoid X receptors (RXRs). By comparing the amino acid identities with other RXRs isolated from the mouse it was confirmed that our clone encoded chicken RXR-γ (cRXR-γ). At this time the ligand for the RXRs was unknown. All-trans retinoic acid had been shown to activate members of the RXR family but this was considered to represent a spurious cross-reaction. In order to identify the nature of the ligand for RXRs, co-transfections were carried out in Baby Hamster Kidney cells with a construct encoding cRXR-γ and a reporter construct consisting of the chloramphenicol acetyl transferase (CAT) gene under the control of a promoter containing a response element which will bind to the RXRs. Using this system a number of retinoids were tested for their ability to activate the reporter. Of all the retinoids tested, none were found to bring about reporter activation at physiological levels. It was later reported that an isomer of retinoic acid, 9-cis retinoic acid, was the ligand for the RXRs. Structural similarities between 9-cis retinoic acid and the eicosanoids raised the possibility that eicosanoids may also activate RXRs in vivo. Using a similar transfection system it was demonstrated that lipoxygenase metabolites of arachidonic acid (the precursor of eicosanoids) are capable of activating cRXR-γ at submicromolar concentrations. Consistent with this, molecular modelling predicts conformational similarities between some lipoxygenase products and 9-cis retinoic acid. This may explain why certain eicosanoids (the hydroxyeicosatetraenoic acids) mimic some of the actions of retinoids in cell-based assays. As a preliminary step in the analysis of the function of cRXR-γ, a system was set up to block the effect of the receptor by co-expression of a dominant negative mutant. QT6 cells were co-transfected with an expression construct for cRXR-γ and for the ligand binding domain of cRXR-γ. It was shown that by co-expressing this dominant negative mutant, cRXR-γ mediated transactivation was inhibited. A genomic clone was isolated for cRXR-γ and partially sequenced. This allowed the identification of an exon/intron boundary within the cDNA. From this genomic clone a region of 5'-sequence was subcloned into a CAT construct and transfected into QT6 cells. This showed that the sequence contained promoter activity. In summary, these results have lead to the identification of a novel clone as encoding a member of the RXRs. The activation of the receptor by a number of retinoid and non-retinoid compounds has been determined and a genomic clone isolated leading to the discovery of a region containing promoter activity.

Thank You, Neck is a UNO thesis film that was filmed in November of 2022 and delivered in May of 2023. It was written and directed by Marion Forbes who will presume first-person singular (ie. “I”, “Me”, “My”) in the following reflection paper charting the development, pre-production, production, and post-production of the aforementioned short film. The thesis paper discusses the process of creating the film from concept, pre-production, production, post-production, and completion.

Proteins are the primary mediators of cellular activity and play a critical role in disease etiology and treatment. The conformation of proteins, either intrinsic or induced by other interacting partners, can inform their localization, their capacity to transmit signaling cascades, and, ultimately, their function. Immunoglobulins, or antibodies, are glycoproteins generated by the immune system with the primary function of protecting the host by blocking or neutralizing the spread of pathogens. The blocking activity of antibodies can proceed via simple mechanisms such as occlusion of interacting interfaces or through more complex allosteric pathways for blocking pathogenic protein function. Paradoxically, immune system dysregulation can also produce self-reactive, pathogenic antibodies causative for disease. Amongst these disparate examples is a shared feature: the capacity for antibodies to effect protein function through conformational control of their interacting partner. This dissertation uses a combination of cryo-electron microscopy, pharmacological signaling studies, and biochemical reconstitutions to understand how natural, patient-derived, self-reactive antibodies can select for active state conformations of the thyrotropin G protein-coupled receptor, and how synthetic single-domain antibodies can restrict SARS-CoV-2 S protein conformation and subsequent viral entry. In the first chapter, I and others structurally describe the activation mechanism of the thyrotropin receptor both in the context of normal, hormone-mediated signaling and in pathogenic, autoantibody-induced activation. These results describe the hormone mimicry and ensuing conformational selection employed by autoantibodies in the pathogenesis of Graves’ disease in their action at the thyrotropin receptor. This work is also the first to structurally describe how autoantibodies can activate G protein-coupled receptors and further refines the model for glycoprotein hormone receptor activation by the eponymous glycoprotein hormones.In the second chapter, I and others discover and biophysically characterize single-domain antibodies directed against the SARS-CoV-2 S protein. The single-domain antibodies we initially describe only inhibit viral entry through steric occlusion of the angiotensin-converting enzyme II binding interface on the viral S protein. In a structure-guided manner, we further engineer one single-domain antibody into a multivalent construct that restricts the viral S protein into a closed conformation and show enhanced potency in viral entry inhibition after additional affinity maturation. We demonstrate that this construct is stable to common routes of aerosolized delivery and propose its use as a viral prophylactic. This work also describes single-domain antibodies with allosteric, yet incompletely described mechanisms of S protein entry inhibition.

The prospect of spinal cord regeneration in humans is an exciting medical advance, but one that remains elusive from the complicated cellular and molecular mechanisms that prevent regeneration from happening. Various model organisms that do possess regenerative ability have been studied in hopes of understanding how spinal cord regeneration can be facilitated in humans. Recent studies in non-regenerative mammalian organisms however have uncovered the role of T3 signaling pathways in inhibiting regenerative capacity. These previous studies have shown inhibition of T3 in-vitro and in-vivo in various model organisms has increased the capacity for regeneration even in organisms that typically do not have such an ability. My dissertation provides a broad examination of previous literature exploring the barriers to regeneration in a wide range of model organisms, as well as potential therapeutic targets for inducing regeneration. Here, I also show how inhibition of T3 in X. laevis tadpoles allows for increased functional recovery from spinal cord transection.

Current evidence suggests that thyroid hormones (THs) may impact development of the immune system. However, studies that explore the role of THs in immune development are limited, and the mechanisms leading to alterations in immune function are poorly understood. It is important to elucidate the role of THs in immune development given that many environmental contaminants have been shown to disrupt TH homeostasis and may also have negative impacts on the immune system. As such, the main goal of this study was to determine the long-term consequences of early life stage (ELS) hypothyroidism on immune function. To achieve this goal, it was first necessary to further characterize basic immune function in the selected model species, the fathead minnow (FHM, Pimephales promelas). Preliminary studies were conducted to describe the transcriptomic response to Yersinia ruckeri and adapt assays for the assessment of respiratory burst and phagocytic cell activity. To determine the long-term effects of ELS hypothyroidism, FHMs were exposed to the model thyroid suppressant propylthiouracil (PTU) from

Thyroid cancer is the most prevalent endocrine cancer (1). The prevalence of palpable thyroid nodules in the general adult population is 4% to 7% (2). Ultrasound imaging detects thyroid nodules in 19%-68% of randomly selected individuals (3). The rate of thyroid cancer in nodules found on US is 4% to 15% (4). In order to evaluate thyroid nodules patients undergo thyroid ultrasonography and, if needed, a fine-needle aspiration biopsy. Of all fine-needle aspiration biopsies, 15-30% are indeterminate on cytology (5). While only 3% of these nodules are malignant on average, a much higher percentage of nodules are surgically removed in order to rule out malignancy after indeterminate FNA results. Our goal is to identify clinical and ultrasound predictors of benign results in indeterminate nodules, to assist physicians in selecting nodules for surgical removal versus monitoring with ultrasound imaging. Between October 2010 and November 2017 there were 129 patients with 134 thyroid nodules from Temple University Hospital, Jeanes Hospital, and Fox Chase Cancer Center who had a total or partial thyroidectomy after a cytology report of at least one AUS or FLUS thyroid nodule. These patients were evaluated for age, sex, BMI, TSH, fT4, tT3, nodule size, and ultrasonography features to determine if any features were predictive of a benign or malignant thyroid nodule. Additionally, we looked at whether any of these features were more likely to occur in an AUS nodule or a FLUS nodule. We found that none of the demographic factors, thyroid function tests, or ultrasound features were good predictors of malignancy in AUS or FLUS thyroid nodules. We found that AUS nodules are more likely to be malignant than FLUS nodules, and this held true when we accounted for age, sex, smoking history, and BMI. We concluded that demographic factors and thyroid function tests are unable to predict increased risk of malignancy in Bethesda category III nodules, AUS nodules are more likely to be malignant that FLUS nodules, and nodules with at least one suspicious ultrasound feature are more likely to be AUS nodules than FLUS nodules due to AUS nodules having nuclear atypia and FLUS nodules having architectural atypia.

Understanding the physiological response of marine mammals to anthropogenic stressors can inform marine ecosystem conservation strategies. Stress stimulates release of glucocorticoid (GC) hormones, which increase energy substrate availability while suppressing energy-intensive processes. Exposure to repeated stressors can potentially affect an animal’s ability to respond to and recover from subsequent challenges. To assess the endocrine response of a marine mammal to repeated stressors, we administered adrenocorticotropic hormone (ACTH) to free-ranging juvenile northern elephant seals (Mirounga angustirostris; n=7) once daily for four days. ACTH administration induced significant, but transient (

This work has focused on improving our knowledge of the thyroid specification process. Thyroid cells are derived from mouse embryonic stem cells (mESCs) by directed differentiation through multiple intermediate developmental stages, including anterior foregut endoderm (AFE), prior to NKX2-1+ thyroid progenitor specification. To investigate if transient Nkx2-1 expression can increase the efficiency of thyroid specification, we utilized a mESC line double knock-in GFP-T/hCD4-Foxa2 with a doxycycline inducible (Tet-On) Nkx2-1 transgene. Transient activation of the Nkx2-1 transgene at the AFE stage leads to stable induction of high levels of endogenous Nkx2-1 as well as early and mature thyroid-specific markers including Pax8, Foxe1, Tg, Nis, and Tshr. Lung and neuronal NKX2-1+ lineages were not derived in this system. The thyroid progeny mature and organize into follicle-like structures in 3D culture. These follicles express adherens and tight junction proteins indicative of an epithelial character and produce the thyroid hormone thyroxine (T4) in the presence of iodide. Critical determinants of thyroid lineage specification have been revealed by variations in developmental stage timing, signaling pathways, and sorting of AFE-stage subpopulations. To provide further insights into the mechanisms of thyroid specification, RNA-Seq data acquired from relevant stages has identified potential genes involved in early thyroid development. The results demonstrate that Nkx2-1 can act as a stage-specific inductive signal during directed differentiation of mESCs to thyroid follicular cells. We have also developed a mouse model to recapitulate these results in an in vivo context. This work has provided novel insights into thyroid specification and provides an efficient system for deriving and studying thyroid cells, which can be used for in vitro modeling of development and disease.

Hyperthyroidism is the most common feline endocrinopathy. 131I is the treatment of choice, and over 50,000 cats have been treated using an empirical fixed dose. Better treatment responses could be achieved by tailoring the dose based on the severity of disease. Scintigraphy is the best method to quantify the severity of the disease. Previously established scintigraphic quantitative methods, thyroid to salivary ratio (T:S ratio) and % dose uptake, are the most widely recognized measurements. Recently, the thyroid to background ratio (T:B ratio) has been proposed as an alternate method to assess function and predict 131I treatment response. The purpose of this study was to determine the best location of a background ROI, which should be reflective of blood pool activity. We also hypothesized that the T:B ratio using the determined background ROI would provide improved correlation to T4 when compared to T:S ratio and % dose uptake in hyperthyroid cats. Fifty-six hyperthyroid cats were enrolled. T4 was used as the standard measure of thyroid function and was obtained prior to thyroid scintigraphy and 131I therapy. Blood samples were collected at the time of scintigraphy and radioactivity within the sample was measured. The plasma radioactivity was compared to the background ROI count densities in 8 anatomic regions using linear regression analysis for 55 cats. One cat was excluded from the study because of an injection error during scintigraphy. T:B and T:S ratios, and % dose uptake on scintigraphy were then compared to serum T4 by linear regression analysis for 39 cats. Sixteen cats were excluded because of recent methimazole or Y/D diet use, or incomplete data. The heart ROI correlated best to plasma pertechnetate activity (r = 0.70). % dose uptake correlated best to serum T4 (r = 0.74), followed by T:S ratio (r = 0.66), followed by the T:B ratio using the heart ROI (r = 0.59). Placing an ROI over the heart is the best method of quantifying plasma radioactivity. T:B ratio using the heart ROI as the background is a good predictor T4 but percent dose uptake and T:S ratio proved to be better predictors of T4 than any of the T:B ratios. Therefore, our hypothesis was not supported. The T:B ratio may not provide the best scintigraphic measurement of thyroid function. Hence it is unlikely to accurately predict treatment response to 131I therapy.

Micronutrient deficiencies affect billions of people worldwide and often coexist in developing countries due to consumption of diets lacking nutrient diversity. Thus, it is important to consider how micronutrients such as copper (Cu), iron (Fe), and iodine interact physiologically. Cu, Fe, and iodine/thyroid hormone (TH) deficiencies lead to similar brain development deficits, suggesting these micronutrient deficiencies share a common mechanism contributing to the observed derangements. Previous studies in rodents and humans indicate that Cu and Fe deficiencies during adolescence or adulthood lead to impaired TH status. However, prior to this thesis research, relationships between Fe or Cu deficiencies and thyroidal status had not been assessed in the most vulnerable population, the developing fetus/neonate. My first two studies showed that Fe deficiency lowers newborn rat circulating and brain TH concentrations and alters TH-regulated brain gene expression. In a third study, Fe deficiency exacerbated the effect of mild TH insufficiency on neonatal thyroidal status and brain TH-responsive gene expression. Together, these novel findings suggest that impaired neonatal thyroidal status may contribute to some of the brain developmental abnormalities associated with fetal/neonatal Fe deficiency. Fe deficiency also has significant impacts on the developing brain independent of effects on thyroid function. In humans, Fe deficiency often results in anemia, reduced blood oxygen carrying capacity. Decreased oxygen delivery to the brain can induce a compensatory increase in blood vessel outgrowth. My final study demonstrated, for the first time, that Fe deficiency anemia increases blood vessel growth in the neonatal rat brain. The functional contribution of increased vasculature to the developing Fe-deficient brain is unknown but could be adaptive, maladaptive, or both. In summary, my thesis research exploring micronutrient interactions during brain development has identified two novel potential contributors to the brain developmental derangements associated with Fe deficiency: impaired neonatal thyroid function and increased neonatal brain vasculature.

Biological responses to toceranib phosphate have been reported in both canine apocrine gland anal sac adenocarcinomas (AGASACA) and thyroid carcinomas (TC). However, little is known about the molecular biology of either of these tumor types and the mechanism of the biological activity of toceranib. In the present study, we investigated the presence and expression of known targets of toceranib and interrogated AGASACA and TC samples for the expression of VEGFR2, PDGFRα/β, KIT and RET at both the message and protein level to begin to dissect the molecular basis for this observed activity.Samples from 24 primary AGASACA, 11 with paired metastatic lymph nodes, and 15 TC were evaluated using reverse-transcriptase polymerase chain reaction for detection of mRNA, phosphoprotein arrays to screen for evidence of protein phosphorylation, and tissue microarrays for immunohistochemical evaluation of protein expression. Messenger RNA from VEGFR2, PDGFRα/β, KIT and RET was detected in all AGASACA samples. Messenger RNA for VEGFR2, PDGFRα/β, and Kit was detected in all TC samples, while mRNA for Ret was amplified in 10/15 TC samples. Evidence of phosphorylation of VEGFR2, PDGFRα/β, and KIT was not detected on phosphoprotein arrays in either tumor. Evidence of phosphorylation of RET was detected in 54% of the primary AGASACA and 20% of TC. Positive immunoreactivity for VEGFR2 was noted in 19/24 primary and 6/10 metastatic AGASACA and 6/15 TC samples. Positive immunoreactivity for KIT was noted in 8/24 primary and 3/10 metastatic AGASACA and 9/15 TC samples. PDGFRα was noted in all primary and metastatic AGASACA and all of the TC samples. PDGFRβ expression was noted in 4/24 primary and 1/10 metastatic AGASACA and 4/15 TC tumor cells, while intense stromal staining was noted in all tumor samples. Known targets of toceranib are present in both AGASAC and TC at both the level of message and protein expression. Our findings of VEGFR2 and PDGFR protein expression and phosphorylation of RET merit further investigation as to their roles in the biology of AGSACA and TC as well as to their contribution to the observed response to toceranib.

Microchimerism is the presence of cells from one individual in another genetically distinct individual. Pregnancy is the main cause of natural microchimerism through transplacental bidirectional cell trafficking between mother and fetus. The consequences of pregnancy-related microchimerism are under active investigation. However, many authors have suggested a close relationship linking fetal microchimerism and the development of autoimmune diseases. It has been more than ten years now since the demonstration of the presence of a significant high number of fetal microchimeric cells residing in thyroid glands from operated patients with Graves' disease. This intrathyroidal fetal microchimerism is an attractive candidate mechanism for the modulation of Graves' disease in pregnancy and the postpartum period.

OBJECTIVE: Thyroid cancer is the most common endocrine malignancy and patients presenting with thyroid nodules often undergo surgery solely for diagnostic purposes. The goal of our study was to examine the accuracy of Elastic Scattering Spectroscopy (ESS) in distinguishing between benign and malignant thyroid nodules in fresh ex vivo specimens and to design an in vivo ESS probe and device, manufacture it and conduct a clinical trial. METHODS: Patients already undergoing thyroidectomy surgery were consented for the ex vivo study. ESS data was obtained from ex vivo specimens by recording 5 readings per nodule with five repetitive readings per each site. Final pathology reports were used to confirm the diagnosis. The spectra were analyzed using principal component analysis, linear discriminant analysis and leave one out technique. The in vivo ESS study was conceptually designed and IRB approval from Boston Medical Campus was obtained. RESULTS: The ex vivo study showed that ESS could predict the difference between benign and malignant tumors with a sensitivity of 74%, specificity of 90%, positive predictive value of 82% and negative predictive value of 85%. 193 spectra were analyzed from 64 patients, 120 spectra were from benign nodules and 73 from malignant nodules. Subanalysis examined only indeterminate nodules showed sensitivity of 65%, specificity of 79%, PPV 77% and NPV 67%. The in vivo ESS probe was designed and 12 identical instruments were manufactured. Initial experimental readings were taken and parameters were adjusted for the in vivo tissue environment. The clinical trial is underway. CONCLUSIONS: ESS is a practical tool that can accurately identify malignancy in ex vivo thyroid specimens with high specificity and sensitivity. Initial in vivo experimental trials have been conducted and show promise for similar results.

The purpose of this study was to report the scintigraphic and sonographic appearance of the thyroid gland in clinically normal horses so these modalities could be used to assess the thyroid gland in this species. Horses were divided into two age groups. Group A consisted of 8 horses between 3 and 10 years of age and Group B of 7 horses between 11 and 20 years of age. Total T4 concentrations were within the laboratory reference interval. Thyroid to salivary (T/S) ratio, percent dose uptake of pertechnetate and thyroid lobe volume were calculated. Echogenicity of thyroid lobes and presence of nodules were documented. The two groups were compared using appropriate parametric and nonparametic tests. Total T4 concentrations were significantly lower in the older group. Sixty minute mean ± standard deviation (SD) T/S ratios for older versus younger horses were 5.8 ± 3.0 and 5.3 ± 2.2, respectively. Sixty minute median and interquartile ranges for percent dose uptake of pertechnetate for older versus younger horses were 3.64% (1.5 to 3.98%) and 2.55% (2.33 to 2.90%), respectively. Mean ± SD thyroid lobe volumes for older versus younger horses were 18.93 ± 5.16 cm3 and 13.55 ± 3.56 cm3, respectively. Most thyroid lobes were hyper or isoechoic to the sternocephalicus muscle. Prevalence of thyroid nodules did not differ between groups. Older horses had trends for greater T/S ratios, percent dose uptakes and thyroid lobe volumes but had lower total T4 concentrations. Further studies using scintigraphy and ultrasound in horses with thyroid disease are planned.

Background: Abnormal maternal thyroid function during pregnancy is associated with adverse affect to the maternal health and child development. Elevated serum thyroid stimulating hormone (TSH) in pregnant women is associated with impaired neuropsychiatric development of the child. Presence of thyroid peroxidase antibodies (TPO-Ab) is a significant risk factor for maternal postpartum thyroid disease. Objective: This study was to determine prevalences of abnormal TSH and TPO-Ab in Thai pregnancies. Method: Serum sample were obtained from 480 pregnant women as a part of their routine antenatal screening at Ramathibodi Hospital during August and October 2008. Measurement of TSH and TPO-Ab was done using Chemiluminescent Microparticle Immunoassay. Results: The prevalences of abnormal TSH and TPO-Ab in Thai pregnancies were 9.79% and 13.96%, respectively. The highest frequency was found in 30.34 year-old age group. TSH abnormality was significantly associated with gestational age (P = 0.037). Abnormal TSH levels were mostly in first trimester of pregnancies. Conclusion: Since abnormal thyroid function is common in Thai pregnancies, maternal screening of TSH and TPO-Ab should be considered.

This paper is by no means an exhaustive review or ‘stand-in’ encyclopedia for all there is to know about thyroid disorders. Instead, as all science is a compilation and synthesis derived from a collaboration among disciplines, this paper will reflect this trend by analyzing thyroid disorders from biological, histological, medical, and pharmaceutical sources.

The mandible is a rare site for the occurrence of primary bone lymphoma (PBL), a non-Hodgkin lymphoma. We report herein a case of an incidentally diagnosed thyroid incidentaloma by 18Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography in a patient with a previous diagnosis of PBL. Therapeutic options are reviewed and discussed.

Objective: To determine the efficacy and safety of iopanoic acid for the treatment of experimentally-induced hyperthyroidism in cats. Animals: 15 healthy adult domestic short hair cats Procedures: Hyperthyroidism was induced by daily subcutaneous administration of levothyroxine for 42 days. On day 28, cats were randomized to a control group receiving a placebo PO every 12 hours, a low dose group receiving 50 mg iopanoic acid PO every 12 hours, and a high dose group receiving 100 mg iopanoic acid PO every 12 hours. Cats were treated for 14 days. Weight and heart rates were obtained on days -8, 0, 28, 35, and 42. Blood was collected for CBC and biochemical analysis and for T4, T3, and rT3 measurement on days -8, 28, 35, and 42. Results: Two cats were removed prior to day 28 due to prolonged anorexia and another on day 36 because of heart failure. The low dose and high dose groups had significantly lower T3 concentrations on days 35 and 42 compared to the control group and to their own T3 concentrations on day 28. The T3 concentrations in cats administered iopanoic acid were not different from those obtained prior to induction of hyperthyroidism. Body weight and food consumption were not altered by iopanoic acid treatment, while heart rate was decreased in the low dose group on day 35 when compared to day 28. Conclusions and clinical relevance: Iopanoic acid was effective in decreasing T3 concentrations, but its effect on clinical signs of hyperthyroidism was less apparent. Studies evaluating the long-term efficacy in cats with naturally-occurring hyperthyroidism are warranted.

The objective of these studies was to contribute to the understanding and assessment of thyroid function in horses. The first study evaluated methods of assessing thyroid function in horses, including validation of an enzyme immunoassay (EIA) for measuring equine thyroid hormones and development and assessment of a thyrotropin releasing hormone (TRH) response test. Our data indicated that EIA is an acceptable method for the measurement of total (T) and free (F) thyroxine (T4) and triiodothyronine (T3) in equine plasma. Its measurements are not equivalent to values obtained by radioimmunoassay (RIA), but they can be calibrated to predict corresponding RIA values. A protocol was developed for TRH response tests involving administration of 1 mg TRH intravenously, with blood sample collection immediately before, 2.5, 5.0, and 24 h after administration. Analysis of plasma TT4, FT4, TT3, and FT3 revealed that the magnitude of hormone response was best approximated by the area under the curve of hormone plotted against time and by the absolute change in thyroid hormone concentration. Baseline concentrations, peak concentrations, and percent of baseline values were not as well able to predict the magnitude of hormone response. The second study assessed the effects of exercise and feed composition on thyroid status. Thirteen mature Arabian geldings, adapted to either a high sugar and starch (SS) or high fat and fiber (FF) feed, underwent 15 wk of exercise training followed by a treadmill exercise test. The TRH response tests performed before training, after training, and the morning after the exercise test revealed that the exercise test decreased the TT4 and FT4 response, whereas feeding of high levels of sugars and starches increased the response of TT3 and FT3. During the first four weeks of training, increased TT4 and FT4 concentrations occurred simultaneously with increased nonesterified fatty acid concentrations, decreased triglyceride concentrations, and increased insulin sensitivity. The increase in TT4 and FT4 may have provided the cellular signaling necessary for increased lipolysis and insulin sensitivity. These metabolic changes facilitate the increases in lipid and carbohydrate metabolism that are needed to fulfill the additional energy requirements of regular exercise. The third study assessed thyroid status in ponies with different laminitic histories. Total T4, FT4, TT3, and FT3 were measured during March and May 2004 in 126 ponies that were categorized as either previously laminitic (PL; n = 54) or never laminitic (NL; n = 72) and evaluated for current laminitis in May (CL; n = 13). Decreased concentrations of TT4 and FT4 were found in PL ponies when compared to NL ponies in March (P = 0.018, 0.020) and May (P = 0.018, 0.001). However, TT4 and FT4 concentrations in CL ponies were not different than concentrations in NL ponies in May (P = 0.82, 0.72), and when retrospectively separated out in March, were not different than NL ponies (P = 0.90, 0.84). Therefore, basal thyroid hormone concentrations are not useful as a predictor or hormonal characteristic of pasture-associated laminitis. The decreased TT4 and FT4 in PL ponies may be an indication of a response or compensation to laminitis and may facilitate the metabolic changes necessary to cope with the disease.

This study tested the hypothesis that levothyroxine supplementation increases plasma von Willebrand factor (vWf) concentration and enhances vWf function. The effects of levothyroxine administration were evaluated in 8 euthyroid Doberman Pinschers with plasma vWf concentration 70%). All dogs had FVIII:C activity within reference range. The response to placebo versus active levothyroxine treatment revealed no significant differences between groups at any time for BMBT, vWf:Ag, vWf:CBA, and FVIII:C. Serum total thyroxine, fT4, and T3 were significantly higher in the levothyroxine-treated group compared to the placebo group at days 2 and 30. Thyroid stimulating hormone was significantly lower in the levothyroxine-treated group compared to the placebo group at days 2 and 30. Levothyroxine (0.04mg/kg) caused laboratory evidence of hyperthyroidism but did not affect plasma FVIII:C and vWf:Ag concentration or the vWf-dependent functional parameters of collagen binding and BMBT. The results of this study do not reveal a direct action of levothyroxine supplementation on plasma vWf concentration or activity in euthyroid Doberman Pinschers.

The RET/PTC family of oncogenes are detected in human papillary thyroid carcinomas. The overall aim of this study is to investigate the phosphotyrosine signaling pathways downstream of RET/PTC1 oncoprotein that mediate dedifferentiation in PC Cl 3 immortalized rat thyroid cells. Site directed mutagenesis was performed to generate single tyrosine 404 or 451 to phenylalanine (F) mutants. In this study, PC Cl 3 cell lines stably expressing either RET/PTC1 or RET/PTC1 Y/F were generated to 1) investigate the mechanism of RET/PTC1 mediated NIS reduction, 2) investigate the role of pY404 or pY451 on NIS reduction and TSH-independent proliferation and 3) compare global gene expression profiles expression by microarray analysis and identify novel genes that are dysregulated by RET/PTC1 or RET/PTC1 Y/F expression. RET/PTC1 expression was demonstrated to decrease NIS expression and function in PC Cl 3 cells. Stimulation of the cAMP-PKA pathway by forskolin, 8-Br-cAMP or catalytic PKA expression in the nucleus were able to reverse the effect of RET/PTC1 on NIS expression and function. In chapter 3, the effect of single Y/F mutagenesis on RET/PTC1 subcellular localization, catalytic PKA nuclear localization and reduction of NIS protein expression and function were investigated. Phosphotyrosine 451 was important for mediating RET/PTC1 plasma membrane localization while pY Y404 mediated signaling was found to be important for RET/PTC1 mediated NIS reduction. Microarray analysis was performed to compare gene expression profiles between PC Cl 3 parental, RET/PTC1, RET/PTC1 Y/F expressing cell lines. Osteonectin, HIF-1α, aquaporin 5 and alpha crystallin B were identified as novel genes upregulated by RET/PTC1. In order to investigate the role of onset and expression level of RET/PTC1 on thyroid tumorigenesis, PC Cl 3 CMV-Tet-On TRE-RET/PTC1 cell lines which have doxycycline (dox) inducible RET/PTC1 expression were generated and characterized. TRE-RET/PTC1 transgenic mice were also generated and characterization of the mice for dox-inducible RET/PTC1 expression are currently underway. A bi-directional TRE construct expressing RET/PTC1 and luciferase was generated as a first step towards generating bi-transgenic mice with thyroid targeted expression and dox-inducible RET/PTC1 as well as luciferase expression which will serve as a reporter gene for RET/PTC1 expression.

Tricyclic antidepressants have been shown to alter thyroid function in man and laboratory animals, but have not been evaluated in the dog. The effect of administration of clomipramine on canine thyroid function was studied in a prospective protocol in which 14 mature, healthy dogs were administered clomipramine (3 mg/kg PO q12h) for 112 days. Thyroid-stimulating hormone (TSH), total thyroxine (T4), total 3,5,3' triiodothyronine (T3), free thyroxine (fT4), and 3,3',5' triiodothyronine (reverse T3; rT3) concentrations were measured on selected days. Thyrotropin-releasing hormone (TRH) response tests were performed concurrently. Repeated measures analysis of variance was applied to test for effects of day of treatment; when significance (p < 0.05) was noted, it was further investigated using orthogonal polynomial trends. Significant decreases were found in serum T4 (26 ± 1.2 to 17 ± 0.5 nmol/L, p < 0.001), fT4, (29 ± 2.4 to 19 ± 1.3 pmol/L, p < 0.0002), and rT3 (1.2 ± 0.1 to 0.83 ± 0.08 nmol/L, p < 0.0001) concentrations. The effect of time on serum T3 concentration was also significant (p < 0.0001), but no consistent trend could be identified. No significant effect of time was noted in either pre- or post-TRH TSH concentrations. The results of this study indicate that significant and substantial decreases in T4 (35%), fT4 (38%), and rT3 can occur during clomipramine administration. Long-term administration of clomipramine may result in a misdiagnosis of hypothyroidism if a dog is tested while taking this medication and, since decreased serum fT4 occurs, hypothyroidism may result.

This thesis investigated the role of thyroid function in the regulation of seasonal changes in live weight, reproductive activity, pelage and antler development in red deer stags. Experiment 1 examined the effects of removal of thyroid hormones in the regulation of seasonal changes by use of 3 THX stags with 5 euthyroid stags as controls. Thyroidectomy blocked the seasonal decline in testicular diameter, neck girth, plasma testosterone concentration and responsiveness of testosterone and LH to a stimulus of exogenous GnRH, which occurred in controls in spring and early summer. THX stags also failed to undergo the spring growth spurt, antler and pelage changes. Furthermore a seasonal pattern of plasma T3 concentrations was recorded with low values in winter and high values in summer. Experiments 2 and 3 examined the importance of the seasonal patterns of plasma thyroid hormone concentration in regulation of seasonal changes. In Experiment 2 injections of T3 during autumn and winter elevated plasma T3 concentrations in 4 treated stags compared with 4 controls, and in Experiment 3 injections of T4 from winter to spring raised both plasma T4 and T3 concentrations in 4 stags. In spite of the elevation of plasma thyroid hormones there were no changes in seasonal patterns of live weight, reproductive activity or pelage development. These results suggest that the seasonal changes in secretion of thyroid hormones are not instrumental in regulating seasonality of stags. Experiment 4 utilised 4 THX stags treated with T4 and another 4 THX stags as controls to test the hypothesis that thyroid hormones are required for the expression of seasonal changes. Treatment with T4 commencing in spring stimulated gain of live weight induced reproductive regression, antler development and the spring moult, thus confirming the hypothesis. Twenty-four hour melatonin profiles between THX (n=3) and euthyroid stags (n=3) were similar (Experiment 5), which indicated that the blockade of seasonal changes following thyroidectomy was not due to inappropriate secretion of melatonin. Also in Experiment 5, treatment of THX stags with MPA was utilised to check whether development of antlers could be altered by thyroidectomy. The treated stags cast hard antlers and grew a set of new antlers. This indicated that blockade of antler development in THX stags was due to the maintained secretion of testosterone and not to hypothyroidism, and that antler bone growth could proceed normally in the absence of thyroid hormones. In conclusion, results of this thesis demonstrate that in red deer stags thyroid hormones are required for the normal expression of seasonal changes in spring and early summer. Thyroid function affects development of antlers indirectly through its influence on seasonal testicular activity.

A competitive protein binding assay was developed for the measurement of serum total thyroxine (T4) concentrations in horses. This method corrected for the presence of ethanol extractable substances, present in sera, shown to spuriously interfere in such assays. Total T4 and total triiodothyronine (T3) concentrations in 13 cycling and early pregnant mares (determined every two to three days for four weeks during spring) were 2.97 ± 0.7 µg/100 ml, and 73.3 ± 12.7 ng/100 ml respectively. Total and free T4 concentrations were measured in horses in various physiological states, with the concentrations in untrained mares in spring being 3.19 ± 1.3 µg/100 ml and 5.68 ± 2.4 ng/100 ml respectively. Results suggested that in horses in training, total T4 decreases with intensity of training and there is a concomitant alteration in the T4 binding proteins so that free T4 remains at a comparable level to that in the untrained horse. Total and free T4 concentrations are significantly higher in untrained horses during the autumn/winter period, than in the spring. Total and free T4, and total and free T3 concentrations were measured in foals from birth to three months of age. Both total and free T4 concentrations were high at birth (12.1 and 3.1 times the adult concentration), and decreased to be at adult levels by approximately 15 and 10 days of age respectively. Total T3 and free T3 concentrations in cord blood were 6.8 times and 2.3 times the adult level, and began increasing immediately to reach peak concentrations of 15 and 8.7 times the adult level at 2 to 3 days, thereafter declining, but still 2.7 and 2.6 times the adult concentration in 32 day old foals. These very high levels in the newborn foal are related to the maturity of its neuromuscular system and thermogenic capacity. A radioimmunoassay (RIA) method for the measurement of serum FSH in horses was developed. The assay met the requirements for a valid RIA, and is the first reported for the measurement of this hormone. RIA methods for measurement of serum LH in horses were also developed, and modifications of methods for measurement of serum progesterone and oestradiol-17β described. Serum concentrations of FSH, LH, progesterone and oestradiol-17β were measured in cycling mares. Two surges of FSH occur at approximately 10 day intervals during the oestrous cycle, one in the late oestrous/early dioestrous period, and another during mid-dioestrus. Such surges are thought to prepare and prime follicles to respond to the increased LH concentrations during oestrus which result in final maturation processes, ovulation and corpus luteum establishment. In pregnant mares, continued surges of FSH appear responsible for the follicular proliferation which occurs, with PMSG having an "LH-like" action on these FSH prepared and primed follicles and inducing the peri-ovulatory changes and secondary corpus luteum establishment. FSH surges occur during the two to three weeks prior to parturition, with a marked surge within four days of foaling, and a further surge one to five days after foaling. These FSH surges probably prepare and prime follicles for the ovulation 9 to 15 days post-partum. Mares in various depths of acyclicity were treated during a 22 day period with exogenous gonadotrophin releasing hormone (GnRH) and progesterone on a regime to reproduce the gonadotrophin surges, and luteal phase progesterone concentrations occurring in the normal ovulatory cycle. Typical pre-ovulatory follicles were induced by this treatment, but ovulation resulted in corpus luteum maintenance only in mares in shallow acyclicity in which a peri-ovulatory LH surge typical of the normal cycle was also induced. The results suggested that additional FSH surges might be necessary to adequately prepare and prime follicles before the final follicular maturation processes, including steroidogenesis, can occur.

Yearling red deer (Cervus elaphus) stags where either treated with 1 mg triiodothyronine (T₃) weekly from 18 January to 28 March then 5 mg (T₃) weekly from 5 April to 24 May 1990 (n=4), or served as controls (n=4). From 18 January to 14 June weekly blood samples and measurements of live weight, testicular diameter and heck girth were taken. Plasma testosterone and LH responses to a GnRH challenge were determined each month and plasma T₃ concentration was measured in the weekly blood samples. The T₃ treatment produced highly significant elevations of T₃ in plasma. However no effect of increased plasma T₃ was seen in any of the other parameters. These results indicate that the thyroid hormones are not involved in regulation of autumnal transition to the breeding season in red deer stags.