Dissertationen zum Thema „Adrenergic and thyroid signalling“

Sabe-se que a ativação do Sistema Nervoso Simpático (SNS) induz osteopenia via adrenoceptores b2 (b2-AR). Para investigar se o hormônio tireoideano (HT) interage com o SNS para regular a massa óssea, estudamos o efeito do HT em associação com isoproterenol ou propranolol (agonista e antagonista b-adrenérgicos) e avaliamos o efeito do HT em camundongos com elevado tônus simpático, devido à dupla inativação gênica do a2A-AR e a2C-AR (a2A/a2C-AR-/-), autorreceptores que inibem a liberação de noradrenalina. Vimos que esses animais apresentam um fenótipo de alta massa óssea, apesar do elevado tônus simpático e de intacta sinalização b2-adrenérgica, sugerindo que o a2A-AR e/ou a2C-AR, além do b2-AR, possam mediar ações do SNS no osso. O propranolol limitou e o isoproterenol acentuou os efeitos deletérios do HT no esqueleto, já os animais a2A/a2C-AR-/- apresentaram resistência à osteopenia induzida pela tireotoxicose, o que sugere que há interação entre SNS e o HT para regular a massa óssea, e que esta depende tanto do b2-AR como do a2A- e/ou a2C-AR.
It is known that the sympathetic nervous system (SNS) activation induces ostepenia, via b2-adrenoceptors (b2AR). To investigate if thyroid hormone (TH) interacts with the SNS to regulate bone mass, we studied the effect of TH in association with isoproterenol or propranolol (b-adrenergic agonist and antagonist) and evaluated the effect of TH in mice with a chronic elevated sympathetic tone, due to double disruption of a2A-AR and a2C-AR (a2a/a2c-AR-/-), autoreceptors that inhibit noradrenalin release. We showed that KO mice present a high bone mass phenotype in spite of an elevated sympathetic tone and of intact b2-adrenergic signaling, which suggests that a2A- and/or a2C-AR, besides b2-AR, may also mediate the SNS actions in the bone. Propranolol limited and isoproterenol accentuated the deleterious effects of TH in the skeleton, while a2A/a2C-AR-/- mice presented resistance to the T3-induced osteopenia, which suggest that there is an interaction between the SNS and TH to regulate bone mass, and that it is dependent on b2-AR and a2A-AR and/or a2C-AR signaling.

Despite advances in research and therapy, cardiovascular diseases are still the leading cause of death worldwide. A closer understanding of the endogenous protective mechanism may improve pharmacological interventions for the treatment of heart diseases. Cold acclimation or hardening has strong potential for reducing cardiovascular risk and the literature shows that it stimulates the β-adrenergic and thyroid systems in tissues. At the same time, the adrenergic system in the heart is one of the main regulators of cardiac activity. However, these signaling pathways have surprisingly not been studied at the protein level in the heart yet, and no studies can be found on the subject matter in current literature. Our results show a reduced infarct size induced by ischemic injury in cold-acclimated rats (CA) at 8 ř C for 5 weeks and then returning to normothermic conditions for 2 weeks (CAR). The aim of this dissertation is to determine, the degree of involvement of the adrenergic system in the myocardium during acclimation after 3 days, 10 days, 5 weeks of CA and subsequent CAR at the level of all three β-adrenergic receptor isoforms (β-ARs) and their signaling pathways. The results show unchanged signaling of β1-AR-Gs-adenylyl cyclase-protein kinase A in the cardioprotective regimes CA and CAR, whose...

GPCRs are known to form dimeric structures, and this affects their pharmacological properties. The ?2AR and AT1aR are GPCRs that are involved the regulation of the adrenergic and renin-angiotensin systems. The ?2AR is polymorphic at position 164, affecting its responsiveness to adrenergic ligands. Both receptors have been shown to form dimers, but little is known on how dimerization affects their trafficking and signalling following ligand treatments. Plasma membrane localization, arrestin-2 recruitment, and G-protein interactions were determined between receptor dimers using molecular biological techniques. This study demonstrates that the formation of heterodimers can change the expected response to ligand treatments, along with associated trafficking events. It was determined that ligands bind to dimers, resulting in conformational changes to the dimeric complexes. Both the ?2AR and AT1aR are targeted in cardiovascular disease and this research demonstrates the importance of dimerization when prescribing drug therapies to avoid potential unwanted drug side effects.

博士
長庚大學
基礎醫學研究所
94
Thyroid hormones (T3) regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors or associated pathways. Both thyroid hormone receptor (TR) and testicular orphan receptor 4 (TR4) belong to the nuclear hormone receptor superfamily and are ligand-dependent transcription factors. Because of TR4 and TR sharing similar response element configurations, known as a direct repeat with four nucleotide spacings, this study investigated the possibility of cross-talk between the two ligand-dependent signal transduction pathways. The transcriptional activity of TR mediated via various thyroid hormone response elements (TREs) reporter construct was repressed by TR4 by up to 92%. The glutathione S-transferase (GST) pull-down assay was performed to determine whether the TR4 interacts with TR to account for the TR4 repressed the TR trans-activation. And the results indicate that both the C (DNA binding) domain of TRα1 and the A/B and C domain of TRβ1 interact with the TR4 protein while the TR4 interacts with the TRα1 or TRβ1 proteins through the DNA and ligand binding domains. To further understand the mechanism of TR4 suppresses T3-signalling, electrophoretic mobility shift assay (EMSA) was carried out. TR homodimer or TR/RXR (retinoid X receptor) heterodimer reduced up to 65% by adding increasing amounts of TR4 protein. Therefore, EMSA and GST pull-down assays demonstrated the direct binding of TR proteins to TR4 and revealed that the interaction is important to the TR4-mediated suppression of TR-transactivation. Additionally, furin was elevated approximately 3.4-fold and 2.8-fold in HepG2-TRα1 cells at the protein and mRNA levels, respectively, after 48 hr of 100 nM T3 treatment. Increasing expression of TR4 repressed approximately 20 to 60% of furin promoter activities, which were stimulated roughly 5-fold by 10 nM T3 for 24 hr. Similarly, the up-regulated furin protein and mRNA levels by T3 were gradually suppressed by increasing amounts of TR4. In addition, α-fetoprotein (AFP) is negatively regulated by T3. And the suppression of AFP by T3 was gradually reversed by co-transfection with increasing amounts of TR4 expression plasmid. Taken together, interaction of TR4 with TR proteins may reduce TR binding to its cognate TRE. Subsequently, the interaction between TR4 and TR alters TR target gene stimulation or repression by T3. These findings suggest that TR4 may influence furin and AFP metabolism regulated by T3/TR at the transcriptional level. This study proposes a mechanism for cross-talk and potential antagonism between TRs and TR4.

The effects of adrenergic agonists in the heart were investigated in isolated cardiac tissues and in the whole heart in 8 week diabetic and age-matched control rats by measuring contractile force following administration of PE in presence of timolol, or ISO. Plasma thyroid hormones as well as insulin, glucose and triglyceride was measured. The contractile force of the isolated cardiac tissues was measured in tissue baths under physiological conditions and results expressed by construction of concentration response curves to adrenoceptors agonists, 10⁻⁹ to 10⁻⁴ M for ISO and 10⁻⁷ to 10⁻³ M for PE. The contractile force of the whole heart was measured as left ventricular systolic pressure, without pharmacological intervention and following administration of 10⁻⁶ M PE or 10⁻⁵ M ISO in the perfusate. The hearts were clamp-frozen at 0, 15 and 30 seconds, and 1, 4 minutes following adrenoceptor agonists for measurement of cAMP levels. Cardiac cAMP was measured in the frozen ventricles by employing Amersham commercial SPA kit while protein was measured by using Bio-Rad Dye method. The tissue content of cAMP was expressed as pmol cAMP/mg protein. It was found that the contractile response to PE was increased whereas that to ISO was decreased significantly in diabetic papillary muscle but not the left atria from the same group of animals. The chronotropic effect of PE was increased and that of ISO was decreased significantly in diabetic right atria. The contractile force of the whole heart preparation was increased in diabetic hearts following both adrenoceptor agonists and the increased positive inotropic effect of ISO was accompanied by an increase in cAMP levels. In all of the diabetic animals, the thyroid hormone levels were significantly lower than those of the controls. These findings confirmed that the cardiac alterations and the low thyroid state in ALX-induced diabetic rats are similar to those that occur in STZ-induced diabetic rats although further studies would be needed to reveal the interrelation between the diabetesassociated low thyroid state and diabetic cardiomyopathy.

The RET proto-oncogene encodes a receptor tyrosine kinase (RTK) that is widely expressed in neuroendocrine tissues and is essential for embryonic development of the kidney and enteric nervous system. Mutations leading to constitutive activation of the RET protein underlie various tumours of endocrine tissues. Conversely, loss-of-function mutations of RET lead to Hirschsprung disease, a congenital disorder characterized by a loss of enteric neurons throughout the colon and small intestine. Intracellular trafficking of RTKs through multiple cellular compartments has been shown to impact on downstream signalling. To date, the intracellular trafficking of RET has not been investigated. Here, we show that RET is rapidly internalized after activation and that trafficking to cytoplasmic endosomes plays an important role in downstream signalling. RET is alternatively spliced into multiple isoforms that are co-expressed in cells; therefore, we further investigated RET internalization in an isoform-specific context. This study revealed a number of differences between RET isoforms including differences in sub-cellular localization pre-activation, rate of internalization, and ability to recycle to the plasma membrane. Differential trafficking of RET isoforms alter their downstream signalling properties, providing an additional mechanism to explain the distinct contributions of RET isoforms to cellular processes. Finally, we investigated the impact of altered sub-cellular localization in the context of thyroid carcinoma. Activation of RET has been implicated in a number of thyroid tumours that differ in their inherent oncogenicity. We observed that altered subcellular localization of oncogenic forms of RET, RET/PTCs, enhance their oncogenicity. Interestingly, RET/PTC tumours are indolent and rarely metastasize compared to other RET-mediated forms of cancer. Further investigation revealed that RET/PTC oncogenes are expressed off relatively weak promoters, resulting in quantitatively less RET/PTC oncoprotein expression in these tumours compared to mutant RET expression in more aggressive cancers. Together, our results represent the first in-depth study of the trafficking properties of RET and indicate the importance of proper sub-cellular localization and trafficking in the maintenance of normal cell metabolism.
Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2009-11-19 22:51:47.38

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018
As células cancerígenas são o resultado de um processo gradual e complexo chamado oncogénese. Durante este processo, as células normais transformam-se progressivamente em células cancerígenas através da acumulação de diversas alterações genéticas, que eventualmente culminam numa ou mais características definidas como ‘’hallmarks of cancer’’. Estas características foram definidas como propriedades essenciais ao desenvolvimento cancerígeno por Hanahan and Weinberg e correspondem a: sustentar sinalização proliferativa, escapar aos supressores tumorais, resistir à morte celular, possibilitar imortalidade replicativa, induzir angiogénese e ativar processos de invasão e metastastização. Este conjunto de características foi mais tarde alargado, considerando igualmente a importância da instabilidade genómica e da inflamação, bem como da reprogramação do metabolismo e do escape à vigilância imunitária no desenvolvimento dos processos tumorais. O carcinoma da tiroide é a neoplasia maligna mais frequente do sistema endócrino e a sua incidência tem vindo a aumentar ao longo dos últimos anos. De acordo com as suas características histológicas e morfológicas, o carcinoma da tiroide pode ser subdividido em quatro subtipos principais: carcinoma medular da tiroide (MTC, medullary thyroid cancer), carcinoma papilar da tiroide (PTC, papillary thyroid cancer), carcinoma folicular da tiroide (FTC, follicular thyroid cancer) e carcinoma anaplástico da tiroide (ATC, anaplastic thyroid cancer). Os subtipos PTC, FTC e ATC desenvolvem-se a partir das células epiteliais foliculares da glândula da tiroide, enquanto que o subtipo MTC deriva das células parafoliculares. Para além disso, dentro dos grupos que se desenvolvem a partir das células foliculares, os subtipos papilar e folicular são considerados carcinomas da tiroide bem diferenciados (WDTC, well-differentiated thyroid cancer), enquanto que o subtipo anaplástico corresponde a um tipo de carcinoma da tiroide indiferenciado. O carcinoma papilar da tiroide é o subtipo mais frequentemente diagnosticado, correspondendo a cerca de 80% dos casos dos carcinomas da tiroide. Normalmente, os doentes com estas formas apresentam um prognóstico favorável após remoção total ou parcial da glândula da tiroide e, quando se justifique, terapia com iodo radioativo. No entanto, existe um subconjunto de doentes que apresentam formas agressivas da doença, frequentemente associadas a resistência à radioterapia com iodo e para os quais não existem alternativas terapêuticas eficazes, sendo por isto essencial o desenvolvimento de novas estratégicas terapêuticas. As alterações genéticas mais frequentemente associadas ao carcinoma papilar da tiroide incluem mutações pontuais no gene BRAF ou rearranjos RET/PTC. Uma vez que estas alterações promovem a ativação constitutiva da via de sinalização MAPK (mitogen activated protein kinase), esta por sua vez é considerada essencial ao desenvolvimento do cancro da tiroide. Por outro lado, mutações pontuais no gene RAS também podem ser encontradas em doentes com o subtipo papilar. À semelhança dos rearranjos RET/PTC, as mutações em RAS têm a capacidade de ativar tanto a via de sinalização das MAPK, como a via PI3K/Akt/mTOR. Neste sentido, também a via de sinalização PI3K/Akt/mTOR tem vindo a ser considerada um elemento importante durante o desenvolvimento e progressão do cancro da tiroide. Sendo o carcinoma papilar da tiroide, um cancro que envolve frequentemente a ativação constitutiva da via MAPK, uma terapêutica dirigida à inibição da mesma poderia ser uma opção. No entanto, efeitos secundários indesejados associados ao uso de inibidores desta via, têm vindo a ser reportados em doentes com diferentes formas de carcinoma da tiroide, bem como o escape à terapêutica após longos períodos de tratamento. Desta forma, a compreensão dos mecanismos moleculares subjacentes à oncogénese do subtipo papilar e, em particular, da interação entre diferentes vias de sinalização implicadas, poderá ser uma mais valia no desenvolvimento de novas terapias dirigidas aos doentes com as variantes agressivas. A via de sinalização PI3K/Akt/mTOR é umas das vias mais estudadas no contexto da tumorigénese, devido ao seu papel determinante na proliferação e sobrevivência celular. No carcinoma da tiroide, mutações que afetam esta via costumam ser mais comuns nos tipos foliculares e anaplásticos. No entanto, pensa-se que esta via tem um papel importante na progressão de PTC para formas mais agressivas. Para além disso, como algumas das mutações associadas ao carcinoma papilar da tiroide também têm a capacidade de promover uma ativação da via de sinalização PI3K/Akt/mTOR, também esta via acaba por representar um alvo apelativo ao desenvolvimento de novas terapêuticas dirigidas, visando as formas agressivas. O NF-κB é um fator de transcrição, cuja desregulação pode facilmente promover condições favoráveis ao desenvolvimento cancerígeno, devido ao controlo que exerce sob diversas funções biológicas, tais como na inflamação ou em mecanismos associados à apoptose, crescimento e proliferação celular. No contexto do cancro da tiroide, este fator de transcrição tem sido descrito como um elemento envolvido na resistência à terapêutica, o que leva a suspeitar da presença de algum tipo de interação entre a via de sinalização do NF-κB e as vias de sinalização mais relevantes ao processo oncogénico da tiroide. De facto, uma relação entre a via de sinalização MAPK e a via canónica do NF-κB, foi já descrita por vários autores em diferentes modelos de carcinoma da tiroide, incluindo o subtipo papilar. No entanto, uma interação entre as vias NF-κB e PI3K/Akt/mTOR não se encontra ainda descrita no contexto das neoplasias da tiroide. O principal objetivo deste trabalho foi investigar esta interação em modelos celulares de carcinoma papilar da tiroide. Neste sentido, foram estabelecidas três abordagens experimentais que consistiam na avaliação da atividade do NF-κB: i) na presença de inibidores químicos da via PI3K/Akt/mTOR, ii) na presença de inibidores químicos da via PI3K/Akt/mTOR e com estimulação exógena da via canónica do NF-κB e iii) na presença combinada de inibidores químicos da via PI3K/Akt/mTOR e da via canónica do NF-κB. Os efeitos observados foram ainda comparados entre modelos celulares de PTC com diferentes contextos genéticos. A nível da análise da atividade transcricional do NF-κB, foi verificado um aumento da expressão de um alvo transcricional, em resposta à inibição química da via de sinalização PI3K/Akt/mTOR. Curiosamente, o mesmo não se verifica na presença de estimulação exógena da via canónica do NF-κB, onde a inibição da via PI3K/Akt/mTOR parece não ter impacto na atividade transcricional do NF-κB. Foi no entanto observada uma aparente inconsistência entre a avaliação da ativação de NF-κB com base na sua atividade transcricional e a avaliada através da análise da translocação nuclear da subunidade p65 deste fator de transcrição. Nesta última situação, os resultados indicam um decréscimo da translocação nuclear da subunidade p65 do NF-κB, em resposta à inibição da via de sinalização PI3K/Akt/mTOR. Este fenómeno ocorre tanto na ausência de estímulos exógenos da via canónica do NF-κB, como na presença dos mesmos. No seu conjunto, os resultados deste trabalho sugerem que a via de sinalização PI3K/Akt/mTOR poderá influenciar o estado de ativação do fator de transcrição NF-κB. No entanto, devido à aparente inconsistência entre a atividade transcricional e a translocação nuclear do NF-κB, não foi possível esclarecer se o resultado final do impacto da via de sinalização PI3K/Akt/mTOR no estado de ativação deste fator de transcrição é no sentido de inibir ou estimular a sua atividade. Assim, experiências futuras serão necessárias de forma a compreender e clarificar esta interação, bem como as suas implicações biológicas no contexto do cancro da tiroide. Compreender as possíveis interações entre diferentes vias de sinalização envolvidas na tumorigénese da tiroide será uma mais valia para o desenvolvimento e adequação de terapêuticas dirigidas, particularmente relevante na gestão de doentes com formas agressivas da doença.
Thyroid cancer is the most frequent endocrine malignancy and its incidence has been rising over the past few years. Accounting for more than 80% of the cases, the papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer. In general, PTC patients have a good prognosis after surgery which, in specific cases, is followed by radioiodine therapy. However, a subset of patients present advanced forms of the disease, with lesions that are frequently unresectable or unresponsive to radioiodine therapy. For these patients, no effective alternative treatment exists and new therapeutic options are needed in order to increase patients’ survival rate and lifespan. Throughout cancer development, several genetic changes occur that deregulate different signalling pathways controlling cancer survival, progression and invasion. The most common genetic alterations involved in papillary thyroid cancer include BRAFV600E point mutation and RET/PTC rearrangements, affecting positively the activity of the pro-tumorigenic MAPK pathway. Nonetheless, RET/PTC rearrangements can also activate the PI3K/Akt/mTOR pathway. Besides, RAS activating mutations have been detected in PTC patients and, similar to RET/PTC, can signal through both MAPK and PI3K/Akt/mTOR pathways. Thus, despite MAPK being considered the main signalling pathway involved in thyroid cancer oncogenesis, PI3K/Akt/mTOR can be expected to play an important role during this process. Therefore, targeting the PI3K/Akt/mTOR pathway becomes an attractive therapeutic option, also in the context of thyroid cancer. NF-κB transcription factor has been described as an important anti-apoptotic factor in thyroid carcinomas as well as being involved in acquired resistance to therapy. The interplay of NF-κB with both MAPK and PI3K/Akt/mTOR pathways has been described in several cancers. Considering that in thyroid carcinomas, an interplay between NF-κB and MAPK has been described it may also be relevant to analyse a possible crosstalk between NF-κB and PI3K/Akt/mTOR pathways. Thus, aiming to address this potential crosstalk, the impact of PI3K/Akt/mTOR in NF-κB activation status was analysed in PTC cellular models. NF-κB activity was evaluated in three different conditions: i) upon inhibition of PI3K signalling; ii) upon inhibition of PI3K signalling in the presence of exogenous stimulation of the NF-κB canonical pathway and iii) upon inhibition of both PI3K and NF-κB signalling. Altogether our results suggest the existence of a crosstalk between NF-κB and PI3K/Akt/mTOR signalling. However, whether PI3K/Akt/mTOR pathway exerts a positive or negative impact in the overall NF-κB activation status as well as the molecular mechanisms behind this interplay and its biological significance, require further clarification.

Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing an especially crucial role in postembryonic development. Given its importance, many studies have focused on further elucidating the initial TH signal response and its method of transduction. Although the primary mechanism of TH response is genomic signalling, alternative mechanisms of early TH signal transduction have been relatively poorly studied. The North American bullfrog, Rana catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development, or metamorphosis, can be experimentally induced through exposure to TH. The experimental induction of the TH signalling program leads to similar morphological endpoints as seen in natural metamorphosis in the transition of a tadpole to a juvenile froglet, such as regression of the tail. This TH-induced developmental program can also be manipulated through temperature where, as temperatures lower, developmental rate is delayed and at 5°C metamorphosis is completely stalled. Interestingly, when tadpoles exposed to TH at 5°C are introduced to permissive temperatures (24°C), an accelerated developmental program ensues, even when no more endogenous TH signal remains. Previous research has shown that this phenomenon can also be seen on the molecular level where only a select few transcripts have been shown to be responsive to TH at 5°C. However, the characteristic, if not augmented, TH response program is seen on the transcriptomic level when tadpoles are shifted to 24°C. This indicates that there is a molecular memory where the TH signal is induced in cold temperatures but not executed until more permissive temperatures arise. The extent and regulation of the transcriptomic program involved in this TH-induced molecular memory has yet to be understood. Herein we use the broader probing technique of RNA-seq analysis to identify potential components of the molecular memory. Eighty-one gene transcripts were TH-responsive at 5°C in cultured R. catesbeiana tail fin indicating that the molecular memory is more complex than previously thought. A number of these transcripts encoded regulators of transcription. Closer examination of select transcripts including a novel krüppel-like factor family member, klfX, at 5oC indicated that not all of the candidate molecular memory transcripts are regulated through active transcription and active translation is not required. When moved into 24°C an accelerated transcriptomic response occurred even when no additional TH is added, suggesting that a priming event occurs by TH exposure at 5°C allowing an accelerated metamorphosis at permissive temperatures. The molecular memory may be used as a means to isolate the initiating TH signalling response and the regulation of this program to allow further elucidation of early TH signalling in post-embryonic development.
Graduate

L’hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT) est la condition endocrinienne néonatale la plus fréquemment rencontrée, avec une incidence d’un cas sur 4000 naissances vivantes. L’HCDT comprend toutes les anomalies du développement de la thyroïde. Parmi ces anomalies, le diagnostic le plus fréquent est l’ectopie thyroïdienne (~ 50% des cas). L’HCDT est fréquemment associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdisme) pouvant conduire à un retard mental sévère si non traitée. Le programme de dépistage néonatal assure un diagnostic et un traitement précoce par hormones thyroïdiennes. Cependant, même avec un traitement précoce (en moyenne à 9 jours de vie), un retard de développement est toujours observé, surtout dans les cas les plus sévères (c.-à-d., perte de 10 points de QI). Bien que des cas familiaux soient rapportés (2% des cas), l’HCTD est essentiellement considérée comme une entité sporadique. De plus, plus de 92% des jumeaux monozygotiques sont discordants pour les dysgénésies thyroïdiennes et une prédominance féminine est rapportée (spécialement dans le cas d’ectopies thyroïdiennes), ces deux observations étant clairement incompatible avec un mode de transmission héréditaire mendélien. Il est donc cohérent de constater que des mutations germinales dans les facteurs de transcription thyroïdiens connus (NKX2.1, PAX8, FOXE1, and NKX2.5) ont été identifiées dans seulement 3% des cas sporadiques testés et furent, de plus, exclues lors d’analyse d’association dans certaines familles multiplex. Collectivement, ces données suggèrent que des mécanismes non mendéliens sont à l’origine de la majorité des cas de dysgénésie thyroïdienne. Parmi ces mécanismes, nous devons considérer des modifications épigénétiques, des mutations somatiques précoces (au stade du bourgeon thyroïdien lors des premiers stades de l’embryogenèse) ou des défauts développementaux stochastiques (c.-à-d., accumulation aléatoire de mutations germinales ou somatiques). Voilà pourquoi nous proposons un modèle «2 hits » combinant des mutations (épi)génétiques germinales et somatiques; ce modèle étant compatible avec le manque de transmission familial observé dans la majorité des cas d’HCDT. Dans cette thèse, nous avons déterminé si des variations somatiques (épi)génétiques sont associées à l’HCTD via une approche génomique et une approche gène candidat. Notre approche génomique a révélé que les thyroïdes ectopiques ont un profil d’expression différent des thyroïdes eutopiques (contrôles) et que ce profil d’expression est enrichi en gènes de la voie de signalisation Wnt. La voie des Wnt est cruciale pour la migration cellulaire et pour le développement de plusieurs organes dérivés de l’endoderme (p.ex. le pancréas). De plus, le rôle de la voie des Wnt dans la morphogénèse thyroïdienne est supporté par de récentes études sur le poisson-zèbre qui montrent des anomalies du développement thyroïdien lors de la perturbation de la voie des Wnt durant différentes étapes de l’organogénèse. Par conséquent, l’implication de la voie des Wnt dans l’étiologie de la dysgénésie thyroïdienne est biologiquement plausible. Une trouvaille inattendue de notre approche génomique fut de constater que la calcitonine était exprimée autant dans les thyroïdes ectopiques que dans les thyroïdes eutopiques (contrôles). Cette trouvaille remet en doute un dogme de l’embryologie de la thyroïde voulant que les cellules sécrétant la calcitonine (cellules C) proviennent exclusivement d’une structure extrathyroïdienne (les corps ultimobranchiaux) fusionnant seulement avec la thyroïde en fin de développement, lorsque la thyroïde a atteint son emplacement anatomique définitif. Notre approche gène candidat ne démontra aucune différence épigénétique (c.-à-d. de profil de méthylation) entre thyroïdes ectopiques et eutopiques, mais elle révéla la présence d’une région différentiellement méthylée (RDM) entre thyroïdes et leucocytes dans le promoteur de FOXE1. Le rôle crucial de FOXE1 dans la migration thyroïdienne lors du développement est connu et démontré dans le modèle murin. Nous avons démontré in vivo et in vitro que le statut de méthylation de cette RDM est corrélé avec l’expression de FOXE1 dans les tissus non tumoraux (c.-à-d., thyroïdes et leucocytes). Fort de ces résultats et sachant que les RDMs sont de potentiels points chauds de variations (épi)génétiques, nous avons lancé une étude cas-contrôles afin de déterminer si des variants génétiques rares localisés dans cette RDM sont associés à la dysgénésie thyroïdienne. Tous ces résultats générés lors de mes études doctorales ont dévoilé de nouveaux mécanismes pouvant expliquer la pathogenèse de la dysgénésie thyroïdienne, condition dont l’étiologie reste toujours une énigme. Ces résultats ouvrent aussi plusieurs champs de recherche prometteurs et vont aider à mieux comprendre tant les causes des dysgénésies thyroïdiennes que le développement embryonnaire normal de la thyroïde chez l’homme.
Congenital hypothyroidism from thyroid dysgenesis (CHTD) is the most common congenital endocrine disorder with an incidence of 1 in 4,000 live births. CHTD includes multiple abnormalities in thyroid gland development. Among them, the most common diagnostic category is thyroid ectopy (~ 50 % of cases). CHTD is frequently associated with a severe deficiency in thyroid hormones (hypothyroidism), which can lead to severe mental retardation if left untreated. The newborn biochemical screening program insures the rapid institution of thyroid hormone replacement therapy. Even with early treatment (on average at 9 d), subtle developmental delay is still be observed in severe cases (i.e., IQ loss of 10 points). Although there have been some reports of familial occurrence (in 2% of the cases), CHTD is mainly considered as a sporadic entity. Furthermore, monozygotic (MZ) twins show a high discordance rate (92%) for thyroid dysgenesis and female predominance is observed in thyroid dysgenesis (especially thyroid ectopy), these two observations being incompatible with simple Mendelian inheritance. In addition, germline mutations in the thyroid related transcription factors NKX2.1, PAX8, FOXE1, and NKX2.5 have been identified in only 3% of sporadic cases and linkage analysis has excluded these genes in some multiplex families with CHTD. Collectively, these data point to the involvement of non-Mendelian mechanisms in the etiology of the majority of cases of thyroid dysgenesis. Among the plausible mechanisms are epigenetic modifications, somatic mutations occurring in the thyroid bud early during embryogenesis, or stochastic developmental events. Hence, we proposed a two-hit model combining germline and somatic (epi)genetic variations that can explain the lack of clear familial transmission of CTHD. In this present thesis, we assessed the role of somatic (epi)genetic variations in the pathogenesis of thyroid dysgenesis via a genome-wide as well as a candidate gene approach. Our genome wide approach revealed that ectopic thyroids show a differential gene expression compared to that of normal thyroids, with enrichment for the Wnt signalling pathway. The Wnt signalling pathway is crucial for cell migration and for the development of several endoderm-derived organs (e.g., pancreas). Moreover, a role of Wnt signalling in thyroid organogenesis was further supported by recent zebrafish studies which showed thyroid abnormalities resulting from the disruption of the Wnt pathway during different steps of organogenesis. Thus, Wnt pathway involvement in the etiology of thyroid ectopy is biologically plausible. An unexpected finding of our genome-wide gene expression analysis of ectopic thyroids was that they express calcitonin similar to normally located (orthotopic) thyroids. Such a finding, although in contradiction with our current knowledge of the embryological development of the thyroid attributes C cell origins to extrathyroidal structures (ultimobrachial bodies) upon fusion with a fully-formed, normally situated gland. Using a candidate gene approach, we were unable to demonstrate any differences in the methylation profile between ectopic and eutopic thyroids, but nevertheless we documented the presence of a differentially methylated region (DMR) between thyroids and leukocytes in the promoter of FOXE1, a gene encoding the only thyroid related transcription factor known to play a crucial role in regulating the migration of the thyroid precursors during development as shown by animal studies. We demonstrated by in vivo and in vitro studies that the methylation status of this DMR is correlated with differential expression of FOXE1 in non-tumoral tissues (thyroids and leukocytes). Knowing that DMRs are hotspots for epi(genetic) variations, its screening among CTHD patients is justifiable in our search for a molecular basis of thyroid dysgenesis, currently underway in a case-control study. The results generated during my graduate studies represent unique and novel mechanisms underlying the pathogenesis of CHTD, the etiology of which is still an enigma. They also paved the way for many future studies that will aid in better understanding both the normal and pathogenic development of the thyroid gland.

Les récepteurs couplés aux protéines G (RCPGs) représentent la plus grande famille de cibles thérapeutiques pour le traitement d’une panoplie de pathologies humaines. Bien que plusieurs décennies de recherche aient permis de façonner nos connaissances sur ces protéines membranaires, notre compréhension des déterminants moléculaires de leur activité signalétique reste encore limitée. De ces domaines de recherche, une avancée récente a mis à jour un nouveau phénomène, appelé sélectivité fonctionnelle des ligands, qui a bouleversé les paradigmes décrivant leu fonctionnement de ces récepteurs. Ce concept émane d’observations montrant que l’activité pharmacologique de certains ligands n’est pas nécessairement conservée sur tout le répertoire signalétiques connu du récepteur et peu se restreindre à l'activation sélective d’un sous-groupe de voies de signalisation.Ce nouveau modèle pharmacologique de l'activation des RCPG ouvre de nouvelles possibilités pour la découverte de médicaments plus efficace et sûr, ciblant les RCPGs. En effet, il permet la conception de molécules modulant spécifiquement les voies signalétiques d’intérêt thérapeutique, sans engager les autres voies qui pourraient mener à des effets secondaires indésirables ou de la tolérance. Cette thèse décrit l'utilisation d'une nouvelle approche sans marquage, basée sur la mesure du changement l'impédance cellulaire. Par la mesure des changements cellulaires, comme la morphologie, l’adhésion et/ou la redistribution des macromolécules, cette approche permet de mesurer de façon simultanée l'activité de plusieurs voies de signalisation impliqués dans ces réponses. Utilisant le récepteur β2-adrénergique (β2AR) comme modèle, nous avons démontré que les variations dans l’impédance cellulaire étaient directement liées à l’activation de multiples voies de signalisation suite à la stimulation du récepteur par son ligand. L’agoniste type du β2AR, l’isoprotérénol, s’est avéré induire une réponse d’impédance dose-dépendante constituée, dans le temps, de plusieurs caractéristiques distinctes pouvant être bloquées de façon compétitive par l’antagoniste ICI118,551 Par l’utilisation d’inhibiteurs sélectifs, nous avons été en mesure de déterminer la contribution de plusieurs voies signalétiques canoniques, comme les voies dépendantes de Gs et Gi, la production d’AMPc et l’activation de ERK1/2, sur ces changements. De plus, la dissection de la réponse d’impédance a permis d’identifier une nouvelle voie de mobilisation du Ca2+ contribuant à la réponse globale des changements initiés par la stimulation du β2AR. Dans une autre étude, nous avons rapporté que la réponse calcique induite par le β2AR serait attribuable à une transactivation Gs-dépendant du récepteur purinergique P2Y11, lui-même couplé à la protéine Gq. La mesure d’impédance permettant de distinguer et de décrire une pléiade d’activités signalétiques, nous avons émis l’hypothèse que des ligands arborant des profils signalétiques différents généreraient des réponses d’impédance distinctes. Le criblage d’une librairie de ligands spécifiques au β2AR a révélé une grande variété de signatures d’impédance. Grâce au développement d’une approche computationnelle innovatrice, nous avons été en mesure de regrouper ces signatures en cinq classes de composés, un regroupement qui s’est avéré hautement corrélé avec le profil signalétique des différents ligands. Nous avons ensuite combiné le criblage de composés par impédance avec l’utilisation d’inhibiteurs sélectifs de voies signalétiques afin d’augmenter la résolution du regroupement. En évaluant l’impact d’une voie signalétique donnée sur la signature d’impédance, nous avons été en mesure de révéler une plus grande variété de textures parmi les ligands. De plus, cette méthode s’est avérée efficace pour prédire le profil signalétique d’une librairie de composés non caractérisés, ciblant le β2AR. Ces travaux ont mené à l’élaboration d’une méthode permettant d’exprimer visuellement la sélectivité fonctionnelle de ligands et ont révélé de nouvelles classes de composés pour ce récepteur. Ces nouvelles classes de composés ont ensuite été testées sur des cardiomyocytes humains, confirmant que les composés regroupés dans différentes classes produisent des effets distincts sur la contractilité de ces cellules. Globalement, ces travaux démontrent la pertinence de l’utilisation de l’impédance cellulaire pour une évaluation précise des différences fonctionnelles parmi les composés ciblant les RCPGs. En fournissant une représentation pluridimensionnelle de la signalisation émanant des RCPGs à l’aide d’un seul essai ne requérant pas de marquage, les signatures d’impédance représentent une stratégie simple et innovante pour l’évaluation de la fonctionnalité sélective des ligands. Cette méthode pourrait être d’une grande utilité dans le processus de découverte de nouveaux médicaments.
G protein-coupled receptors (GPCRs) represent the largest family of therapeutic targets for the treatment of a wide variety of human pathologies. Decades of research have provided an extensive base of knowledge about these fascinating membrane proteins, yet significant advancements in the understanding of the structural and functional details of these important drug targets continue to accumulate to this day. One such area of research in particular that has caused a paradigm shift in the way we conceptualize receptor function is a recently identified phenomenon known as ligand functional selectivity. This concept refers to the numerous observations that the pharmacological activity of a ligand at a given receptor is not always conserved over all possible signalling events engaged by the receptor, often resulting in the selectivity of a ligand to modulate only a subset of the receptor’s signalling repertoire. This model of receptor activity reveals exciting new possibilities for the discovery of safer and more efficacious drugs targeting GPCRs; through the design of drugs specifically targeting the pathway of therapeutic interest without modulating other, uninvolved pathways which could lead to tolerance or adverse effects. This thesis will describe the use of a novel, label-free technique based on cellular impedance to further characterize ligand functional selectivity at GPCRs. By measuring changes in higher-order cellular responses, such as changes in morphology, adhesion and redistribution of macromolecules, this approach provides a means to simultaneously measure the activity of multiple signalling pathways converging on these responses. Using the β2-adrenergic receptor (β2AR) as a model system, we have demonstrated that changes in cellular impedance reflect the activity of multiple signalling events elicited following ligand stimulation of the receptor. Isoproterenol, the prototypical agonist of the β2AR, was found to elicit a dose-dependent impedance response consisting of multiple, discrete features over time, which could be blocked in a competitive manner by the antagonist ICI118,551. Using pathway-selective inhibitors, we were able to dissect the contribution of many of the canonical pathways activated by the β2AR, including Gs- and Gi-dependent signalling, as well as cAMP production and ERK1/2 activation. Furthermore, through the pharmacological dissection of this impedance response, we identified a novel Ca2+ mobilization pathway that contributes to the overall cellular response to β2AR stimulation. In a separate study of the mechanism generating this β2AR-promoted Ca2+ response, we revealed a Gs-dependent transactivation mechanism of the Gq-coupled P2Y11 purinergic receptor. Given the ability of impedance measurements to capture this pleiotropic signalling activity, we then reasoned that ligands exhibiting different signalling profiles should generate distinct impedance signatures. In screening a library of functionally selective compounds targeting the β2AR, we obtained a wide variety of impedance signatures. Through the development of a novel computational approach, we were able to cluster these signatures into five distinct compounds classes, which were highly correlated with signalling profiles of the ligands. In an extension of this approach, we then combined impedance screening with the use of pathway-selective inhibitors to determine if this would provide greater resolution in distinguishing among functionally distinct compounds. By assessing if and how a given signalling pathway contributes to a ligand’s impedance signature, we were able to reveal even more texture among ligands targeting the β2AR. Furthermore, this approach was found to be predictive of the signalling profiles of a library of uncharacterized compounds for the β2AR. This work led to the development of a visualization method to express ligand functional selectivity and revealed potentially novel classes of compounds for the receptor. These compound classes were then validated in human cardiomyocytes, confirming that compounds clustering into different classes produced distinct effects on cardiomyocyte contractility. Altogether, this work demonstrates the ability of cellular impedance to accurately measure functional differences among compounds targeting GPCRs. In providing a representation of the pluridimensionality of GPCR signalling using a single, label-free assay, impedance profiling represents an innovative strategy to assess ligand functional selectivity and may be a valuable addition to future drug discovery campaigns.