Dissertations / Theses on the topic 'Adrenergic and thyroid signalling'

Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Skeletal muscles have, due to their large mass, a big impact on the whole body metabolism. There are many signals that can regulate the functions of skeletal muscles and one such signal is activation of α- and β-adrenoceptors (α- and β-ARs) by epinephrine and norepinephrine. This activation leads to several effects which are examined in this thesis.   Stimulation of β-AR on muscle cells induces glucose uptake, an event that both provides the muscle with energy and lowers the blood glucose levels. We discovered two key components in the β-ARs signal to glucose uptake: the transporter protein GLUT4 and the kinase mTOR, a molecule involved in several metabolic processes but not previously known to be activated by β-ARs.   The classical second messenger downstream of β-ARs, cAMP, was surprisingly found to be only partly involved in the β-adrenergic glucose uptake. We also found that a molecule called GRK2 is very important for this glucose uptake.   A novel effect of β-AR stimulation presented in this thesis is the inhibition of myosin II-dependent contractility in skeletal muscle cells. The intracellular pathway regulating this event was different from that regulating glucose uptake and involved both classical and novel molecules in the β-AR pathway.   Another stimulus that we found to activate insulin-independent glucose uptake in skeletal muscle cells was the natural compound Shikonin. Shikonin increased glucose uptake in skeletal muscle cells via a calcium- and GLUT4-dependent mechanism and improved glucose homeostasis in diabetic rats.   Taken together, we have identified new key molecules in the adrenergic signaling pathway as well as novel downstream effects. We conclude that glucose uptake in muscles can be activated by β-adrenergic stimulation or by Shikonin and that both treatments improves glucose homeostasis in diabetic animals. This knowledge can hopefully be used in the search for new drugs to combat type II diabetes.<br><p>At the time of doctoral defence the following papers were unpublished and had a status as follows. Paper 1: Manuscript; Paper 3: Manuscript</p>

Thyroid dysfunction is frequently associated with skin and hair diseases; however, the underlying pathogenic mechanisms are poorly understood. Pathological activation of the thyroid stimulating hormone receptor (TSHR) is the key feature of both hyper- and hypo-thyrodism. Expression of the (TSHR) has been reported in several extra-thyroidal locations including adipose tissue, bone and skin fibroblasts. TSHR expression may explain the association between the thyroid and skin disease. The TSHR can also be activated by a newly discovered glycoprotein hormone, known as thyrostimulin. This hormone is composed of a dimer of unique α 2 and β 5 subunits. Although thyrostimulin has not been detected in the circulation. However, both subunits have been shown to be expressed in different tissues including the skin. The aim of this study is to examine the expression of the TSHR and thyrostimulin in the skin. In addition, to investigate the expression of a variant form of the TSHR in human and mouse skin and, other mouse tissues. RT-PCR using primers specific for the full length receptor and the truncated variant revealed that although the variant was widely expressed in mouse tissues including skin, it was not found in human skin. The full length receptor and thyrostimulin were found to be co-expressed in eye, testis and skin. Immunohistochemistry of frozen skin and thyroid sections using commercially available antibodies against the extracellular (A9) and transmembrane domains (A7) of TSHR demonstrates that TSHR is not expressed in the epidermis but expressed in dermal fibroblasts and in myoepithelium around sweat glands. A new β5 antibody was characterised by western blotting and immunohistochemistry for future investigation of β5 expression in the skin. These data suggest a functional role for TSHR signalling possibly via thyrostimulin in the skin and that the variant form,although potentially present in some tissues, is unlikely to be important in human skin.

G protein coupled receptors, G proteins and their downstream effectors adenylyl cyclase (ACs) were thought to transiently interact at the plasma membrane by random collisions following agonist stimulation. However a growing number of studies have suggested that a major revision of this paradigm was necessary to account for signal transduction specificity and efficiency. The revised model suggests that signalling proteins are pre-assembled as stable macromolecular complexes together with modulators of their activity prior to receptor activation. How and where these signalling complexes form and the mechanisms governing their assembly and maintenance are not completely understood yet. Initially, we addressed this question by exploring AC2 interaction with beta2-adrenergic receptors (beta2ARs) and heterotrimeric G proteins as parts of a pre-assembled signalling complex. Using a combination of biophysical and biochemical techniques, we showed that AC2 interacts with them before it is trafficked to the cell surface in transfected HEK-293 cells. These interactions are constitutive and do not require stimulation by receptor agonists. Furthermore, the use of dominant-negative Rab/Sar monomeric GTPases and dominant-negative heterotrimeric G protein subunits proved that AC2/beta2AR and AC2/Gbetagamma interactions occurred in the ER as measured using both BRET and co-immunoprecipitation experiments, while interaction of the Galpha subunits with the above complexes occurred at a slightly later stage. Both Galpha and Gbetagamma played a role in stabilizing these complexes. Our data also demonstrated that stimulation of AC was still possible when the complex remained on the inside of the cell but was reduced when the GalphaS/AC2 interaction was blocked, suggesting that the addition of the GalphaS subunit was required to render the nascent complexes functional prior to trafficking to proper sites of action. Next, we tackled the issue of higher order assembly of effectors and G proteins, using two different AC isoforms and GalphaS as a model. We demonstrated that AC2 can form heterodimers with AC5 through direct molecular interaction in unstimulated HEK-293 cells. AC2/5 heterodimerization resulted in a reduced total level of AC2 expression, which affected cellular accumulation of cAMP upon forskolin stimulation. The AC2/5 complex was stable in presence of receptor or forskolin stimulation. We provided evidence that co-expression with GalphaS increased the affinity of AC2 for AC5 as monitored by BRET. In particular, the complex formed by AC2/5 lead to synergistic accumulation of cAMP in presence of GalphaS and forskolin, with respect to either of the parent AC isoforms themselves. Finally, we also showed that this complex can be detected in native tissues, as AC2 and AC5 could be co-immunoprecipiated from lysates of mouse heart. Taken together, we provided evidence for stable formation of signalling complexes involving receptor/G proteins/adenylyl cyclase or G proteins/heterodimeric adenylyl cyclases and that G proteins play a crucial role for their assembly and function.

Atrial fibrillation (AF), the most common sustained arrhythmia, is associated with abnormal intracellular Ca2+ handling. Understanding AF requires comprehensive understanding of ionic currents, Ca2+ handling, phosphorylation regulation and related signalling pathways, but appropriate models are limited. The aim of this thesis is to develop an ionic model of the mouse atrial myocyte to investigate the cellular proarrhythmic mechanisms. We have developed the first mouse atrial myocyte model that incorporates mathematically detailed ion channels, cellular Ca2+ and Na+ handling and their regulation by Ca2+-calmodulin-dependent protein kinase II (CaMKII) and protein kinase A. For the first time, the inositol 1,4,5-trisphosphate (IP3) production system and its effects on excitation-contraction coupling have also been described. The validated model predicted that: 1) hyperactivity of CaMKII and elevated intracellular Na+ concentration are the crucial factors that induce sarcoplasmic reticulum (SR) Ca2+ spontaneous release and delayed afterdepolarisations; 2) β-adrenergic stimulation may have proarrhythmic effects by exacerbating Ca2+ overload; and 3) enhanced activity in ryanodine receptors during IP3-induced Ca2+ release is the major cause of the arrhythmogenesis in IP3 signalling.

Thesis (MScMedSc)--Stellenbosch University, 2015.<br>ENGLISH ABSTRACT: It is well-established that transient activation of the β-adrenergic signalling pathway with ligands such as isoproterenol, formoterol and dobutamine, elicits cardioprotection against subsequent long periods of ischaemia. Initially the focus was on the β1- and β2-adrenergic receptors (β1-AR, β2-AR), but recently the β3-AR also emerged as a potential target in the treatment of heart disease. In heart failure, β1- and β2-AR are typically known to be down-regulated while β3-ARs, on the other hand, are up-regulated (Moniotte et al., 2001). Thus, it has become important to examine the significance of the β3-AR and its downstream signalling under similar states of stress. It has been shown that β3-AR stimulation is resistant to short term agonist-promoted desensitization in vitro and in vivo (Liggett et al., 1993) and after being activated, this receptor is able to convey continual intracellular signals (Lafontan et al., 1994). Thus, it could be an ideal target for therapeutic intervention, also in ischaemic heart disease. We hypothesized that selective β3-AR stimulation during ischaemia / reperfusion may be cardioprotective, whereas selective inhibition of this receptor may prove useful in the end stages of sustained ischaemia and early reperfusion. Methods: The isolated working rat heart, subjected to 35 min of regional ischaemia (RI) and 60 min reperfusion was used as model. The β3-AR agonist (BRL37344) (1 μM) or antagonist (SR59230A) (0.1 μM) were applied as follows: (i) before 35 min RI (PT), (ii) during the last 10 min of RI (PerT) and /or (iii) at the onset of reperfusion (PostT) and (iv) administration of BRL37344 during the last 10 min of RI BRL37344 (PerT) was followed by SR59230A during first 10 min of reperfusion SR59230A (Post). The contribution of nitric oxide synthase (NOS) in β3-AR was assessed, using the non-specific NOS inhibitor, L-NAME (50 μM). Endpoints were functional recovery and infarct size. In another set of experiments BRL37344 and SR59230A were applied according to the same protocols, but the left ventricle was dissected from the heart and freeze clamped at 10 min reperfusion for Western blot analysis of extracellular signal-regulated kinase (ERK p44/p42), protein kinase B (PKB/Akt), glycogen synthase kinase-3β (GSK-3β), and endothelial nitric oxide synthase (eNOS). Data were analyzed with one or two-way analysis of variance (ANOVA). Results: Administration of the selective β3-AR agonist (BRL37344) (1μM) before 35 min RI (BRL37344 (PT), significantly reduced infarct size when compared to the non-pretreatment group (NPT) (21.43±2.52 vs 43.17±1.20, p < 0.001). BRL37344 had similar effects on infarct size when applied during the last 10 min of regional ischaemia BRL37344 (PerT) (14.94±2.34, vs NPT, p < 0.001) or at the onset of reperfusion BRL37344 (PostT) (19.06±1.81, vs NPT, p < 0.001). When BRL37344 was applied as a (PerT+PostT) strategy, infarct size was once again significantly reduced (20.55±2.01 vs 43.17±1.20, p <0.001). In contrast, administration of the β3-antagonist SR59230A according to the same protocol did not reduce infarct size and values similar to those of untreated hearts (NPT) were obtained. Surprisingly, when BRL37344 was applied during the last 10 min of regional ischaemia followed by the administration of the β3-AR antagonist (SR59230A) at the onset of reperfusion, [BRL37344 (PerT) & SR59230A (PostT)], infarct size was significantly reduced to 20.78±3.02 (p <0.001 vs NPT and SR59230A (PerT + PostT). Involvement of nitric oxide (NO) was shown since the reduction in infarct size elicited by BRL37344 was totally abolished by, L-NAME, when administered in combination with BRL37344 for 10 minutes prior to RI or at the onset of reperfusion for 10 minutes (% infarct size: 41.48±3.18 and 35.75±3.54, p <0.001 vs BRL37344 (PT) and BRL37344 (PostT), respectively. Western blot results show that PKB/Akt is activated by BRL37344 regardless of the time of administration. The intervention BRL37344 (PerT+PostT), exhibited the most significant phosphorylation of PKB/Akt (fold increase: 14.2±3.71, p<0.01 vs NPT and p<0.05 vs BRL37344 (PostT). In addition, BRL37344 (PT), (PerT), (PostT) and [BRL37344 (PerT) +SR59230A (PostT)] showed significant activation of this kinase (2.92±0.22, 5.54±0.43, 4.73±0.47, and 6.60±0.78, respectively). ERKp44/p42 however, was not significantly activated by any of the treatments. Phosphorylation of eNOS and GSK-3β was significant only in the BRL37344 (PerT+PostT) and [BRL37344 (PerT) + SR59230A (PostT)] groups. The activation of eNOS-S-1177 in the BRL37344 (PerT+PostT) group was (2.82±0.46, p<0.01 and 0.05 vs NPT and BRL37344 (PostT), respectively) and in the [BRL37344 (PerT) + SR59230A (PostT)] group was (2.26±0.48, p<0.05 vs NPT). A very significant increased phosphorylation of GSK-3β was seen in the same two groups (68.8±7.73, p<0.001 vs NPT and 25.5±5.42 vs NPT, p<0.05, respectively). Conclusion: β3-AR has potent cardioprotective effects when administered either before, during and after ischaemia during early reperfusion as indicated by the reduction in infarct size as well as activation of PKB, GSK-3β and eNOS. These beneficial effects can be linked to NO production through activation of eNOS.<br>AFRIKAANSE OPSOMMING: Dit is bekend dat verbygaande aktivering van die β-adrenerge seinpad, met ligande soos isoproterenol, formoterol en dobutamien, die hart teen daaropvolgende lang periodes van iskemie beskerm. Aanvanklik was die fokus op die β1- en β2-adrenerge reseptore (β1-AR, β2-AR); maar onlangs is ook die β3-AR as 'n potensiële teiken in die behandeling van hartsiektes ge-eien. In hartversaking, is dit bekend dat β1- en β2-AR afreguleer word, terwyl β3-ARs, aan die ander kant, opreguleer word (Moniotte et al., 2001). Dit het dus belangrik geword om die belang van die β3-AR en sy stroomaf seinpad onder soortgelyke strestoestande te ondersoek. Dit is bewys dat β3-AR stimulasie teen korttermyn agonis geïnduseerde desensitisering in vitro en in vivo bestand is (Liggett et al., 1993) en wanneer geaktiveer, is hierdie reseptor in staat om intrasellulêre seine voortdurend oor te dra (Granneman, 1995). Dit kan dus ‘n ideale teiken vir terapeutiese intervensie wees, ook in iskemiese hartsiekte. Ons hipotetiseer dat selektiewe β3-AR stimulasie tydens iskemie / reperfusie kardiobeskermende mag wees, terwyl selektiewe inhibisie van hierdie reseptor effektief kan wees in die eindstadia van volgehoue iskemie en vroeë herperfusie. Metodes: Die geïsoleerde werkende rothart, onderwerp aan 35 min van streeksiskemie (SI) en 60 min herperfusie, is as model gebruik. Die β3-AR agonis (BRL37344) (1μM) of antagonis (SR59230A) (0.1 μM), is as volg toegedien: (i) voor 35 min SI (PT), (ii) gedurende die laaste 10 min van SI (PerT) en / of (iii) tydens die aanvang van herperfusie (PostT) en (iv) gedurende die laaste 10 min van SI is BRL toediening BRL37344 (PerT) gevolg deur SR59230A tydens die eerste 10 min van herperfusie SR59230A (Post). Die rol van stikstofoksiedsintase (NOS) in β3-AR is met behulp van die nie-spesifieke NOS inhibitor, L-NAME (50 μM) ondersoek. Eindpunte was funksionele herstel tydens herperfusie en infarktgrootte. In 'n ander reeks eksperimente is BRL37344 en SR59230A volgens dieselfde protokolle toegedien, maar die linker ventrikel is uit die hart gedissekteer na 10 min herperfusie en gevriesklamp vir Western klad analise van ekstrasellulêre-sein gereguleerde kinase (ERK p44/p42), proteïen kinase B (PKB/Akt), glikogeen sintase kinase-3β (GSK-3β), en endoteel stikstofoksied- sintase (eNOS). Data is met een of twee-rigting variansie analise (ANOVA) ontleed. Resultate: Administrasie van die selektiewe β3-AR agonis (BRL37344) (1μM) voor 35 min SI BRL37344 (PT), het die infarktgrootte beduidend verminder vergeleke met die nie-behandelde groep (NPT) (21.43±2.52 vs 43.17±1.20, p<0.001). BRL37344 het ‘n soortgelyke effek op infarktgrootte wanneer dit gedurende die laaste 10 min van streeksiskemie BRL37344 (PerT) (14.94±2.34, vs NPT, p<0.001) of by die aanvang van herperfusie (BRL37344 (PostT) (19.06±1.81, vs NPT, p<0.001) toegedien word. Wanneer BRL37344 as 'n (PerT+PostT) strategie toegedien is, was infarktgrootte weereens beduidend verlaag (20.55±2.01 vs 43.17±1.20, p<0.001). In teenstelling hiermee, het administrasie van die β3-antagonis SR59230A volgens dieselfde protokol, nie infarktgrootte verminder nie en waardes soortgelyk aan dié van onbehandelde harte (NPT) is verkry. Interessant, wanneer BRL37344 gedurende die laaste 10 min van streeksiskemie toegedien is, gevolg deur die administrasie van die β3-AR antagonis (SR59230A) by die aanvang van herperfusie, [BRL37344(PerT) & SR59230A(PostT)], was infarktgrootte aansienlik verminder tot 20.78±3.02 (p<0.001 vs NPT en SR59230A (PerT+PostT). Die betrokkenheid van stikstofoksied (NO) is waargeneem deurdat die vermindering in infarktgrootte ontlok deur BRL37344, heeltemal deur L-NAME opgehef is, wanneer dit in kombinasie met BRL37344 vir 10 minute voor SI of by die aanvang van herperfusie vir 10 minute toegedien is (% infarktgrootte: 41.48±3.18 en 35.75±3.54, p<0.001 vs BRL37344 (PT) en BRL37344 (PostT) onderskeidelik). Western kladresultate toon dat PKB/Akt deur BRL37344 geaktiveer word ongeag die tyd van die administrasie. Die intervensie BRL37344 (PerT+PostT), toon die mees beduidende fosforilering van PKB/Akt (voudige toename: 14.2±3.71, p<0.01 vs NPT en p<0.05 vs BRL37344 (PostT). Daarbenewens het BRL37344 (PT), (PerT), (PostT) en [BRL37344 (PerT) + SR59230A (PostT)] ook beduidende aktivering van hierdie kinase tot gevolg gehad (2.92±0.22, 5.54±0.43, 4.73±0.47 en 6.60±0.78, onderskeidelik). ERKp44/p42 is egter nie deur enige van die behandelings geaktiveer nie. Fosforilering van eNOS en GSK-3β was net beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. Die aktivering van eNOS-S-1177 was beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. 'n Baie beduidende toename in fosforilering van GSK-3β is in dieselfde twee groepe (68.8±7.73, p<0.001 en 25.5±5.42, p<0.05 vs NPT onderskeidelik) waargeneem. Gevolgtrekking: β3-AR het kragtige kardiobeskermende effekte wanneer dit, hetsy voor, tydens en na iskemie gedurende vroeë herperfusie toegedien word, soos deur die vermindering in infarktgrootte sowel as die aktivering van PKB, GSK-3β en eNOS aangedui is. Hierdie voordelige effekte kan aan NO produksie deur aktivering van eNOS gekoppel word.

Les hormones thyroïdiennes (HT) contrôlent l’homéostasie énergétique, les taux élevés impactant négativement sur le vieillissement. Notre étude compare des souris contrôles (C57BL/6J) avec la lignée WSB/EiJ (WSB) ayant une longévité exceptionnelle, un taux d’HT faible et une résistance à l’obésité. Nous avons analysé l’effet d’un régime gras sur plusieurs mesures physiologiques (poids, HT, glycémie, insulinémie, calorimétrie indirecte), révélant une grande plasticité métabolique des WSB. Les fonctions mitochondriales ont été suivies comme indicateur des réponses cellulaires. Pour étudier les liens entre HT et métabolisme, des études transcriptomiques (RNAseq), furent effectués afin d’identifier les gènes impliqués dans la régulation centrale du métabolisme. Nos résultats suggèrent que la longévité des WSB serait liée à une meilleure plasticité centrale et périphérique métabolique associée à l’HT, impliquant un rôle important de l’intégration des informations au niveau hypothalamique<br>Thyroid hormones (TH) control energy homeostasis, and high levels have a negative impact on aging. Our study compared control mice (C57BL/6J) with the WSB/EiJ (WSB) strain, which demonstrate exceptional longevity, low circulating TH and resistance to obesity. We analysed the effect of a high fat diet (HFD) on several physiological parameters (weight, TH, glycaemia, insulinaemia, indirect calorimetry), the results of which indicated a high capacity for metabolic plasticity in WSB. Mitochondrial function was studied as an indicator of cellular responses. To better understand the links between TH and metabolism, transcriptomic analyses (RNAseq) were carried out in order to indentify genes implicated in the central regulation of metabolism. Our results suggest that the longevity of WSB is linked to a better metabolic plasticity at both central and peripheral levels. This effect can be associated with TH, involving an important role for signal integration by the hypothalamus

Mirtazapine is an atypical antidepressant employed clinically for the treatment of major depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs). serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions at the α2a-AR have been proposed to play a role in its putative earlier onset of action. However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a- ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine, as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR functions. Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H), a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo) were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine, yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a- AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation respectively. The results from the current study show that mirtazapine binds to the α2a-AR with an affinity value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells. Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y cells does not alter muscarinic receptor function (different from fluoxetine and imipramine), but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells (typically associated with chronic antidepressant activity). Although inconclusive, the data also suggests that mirtazapine may reduce α2a-AR function.<br>Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Over 500,000 people in the UK have heart failure (HF). After an initial insult to the heart, sympathetic drive increases which leads to detrimental remodelling of cardiac β-adrenergic receptors (βAR) and further cardiac dysfunction. The main βAR expressed in the heart are the β1AR and β2AR. In heart failure, remodelling is characterised by reduced β1AR density, desensitisation of the remaining β1AR and aberrations of normal βAR signal compartmentalisation. Caveolae, flask-shaped lipid rafts, are present in most cells including cardiac myocytes and are characterised by the presence of caveolin and cavin proteins. Caveolar proteins create distinct micro-domains within the membrane and play a key role in compartmentalisation of signalling from both the β1AR and β2AR. Isolated reports of changes in caveolar structure and proteins in HF have implications for β-AR signalling, however the full array of caveolar protein changes in HF has not previously been assessed. Here we establish how the expression and membrane location of β-AR cascade and caveolar proteins changes in rat models of right ventricular (RV) and left ventricular (LV) failure induced by monocrotaline and aortic banding, respectively. For the RV model, we examined changes in β-AR responsiveness, and tested the potential for reversing functional and caveolar remodelling using a common LV therapy (the β-blocker metoprolol). Quantitative analyses of caveolar protein expression in myocyte and myocardial samples was also carried out using custom-designed calibrating peptides (CavCATs). Both HF models showed a reduction in caveolar protein expression, with protein redistribution also found in the RV model. Decreased expression of β-AR signalling proteins (β1AR, adenylyl cyclase) accompanied by increased expression of inhibitory proteins (Gαi, GRK2) was also observed in both models, with some remodelling of membrane distribution. β-blocker treatment in RV failure partially recovered expression of caveolar and β-AR cascade proteins. Cardiac β1AR responsiveness was reduced in RV failure and again, this was partially recovered by β-blocker treatment. Quantitative work highlights the importance of studying non muscle-specific caveolar protein isoforms in the cardiac myocyte given e.g. similar expression of Cav 3 and Cav 1 in these cells. Caveolae are dynamic membrane compartments which change in HF. This work suggests that caveolar changes affect β-AR signalling protein membrane location, which contributes to aberrations of signalling which (in the case of RV failure) can be reversed by β blockers.

Diabetes (DM) increases mortality after myocardial infarction and deteriorates post-ischaemic cardiac remodelling. This study investigated possible implications of thyroid hormone (TH) signalling in either reducing or preventing this response. TH signalling has a regulatory role in metabolism, cardiac function, growth and ischaemic stress. Acute myocardial infarction (AMI) was induced in age–match healthy control rats (AMI-C) and in streptozotocin (STZ)-induced type I diabetic (DM) animals (DM+AMI) using 35 mg/kg body weight while sham operated animals served as controls (SHAM). The results show that AMI in tissue hypothyroidism caused significant down-regulation of TH receptors, TRα1 and TRβ1, in the diabetic myocardium without changes in T3, T4 levels in plasma. This response was associated with increased expression of β-MHC and distinct changes in cardiac function and geometry. Ejection fractions (EF%) was decreased in DM-AMI as compared to DM+AMI animals. Systolic and diastolic chamber dimensions were increased without concomitant increase in wall thickness and thus, WTI (the ratio of LVIDd/2*Posterior Wall thickness), an index of wall stress, was significantly elevated. The absence of wall thickening in DM+AMI hearts was associated with changes in stretch-induced kinase hypertrophic signalling p38 MAPK. In contrast, ERK, p-ERK and p-p38 MAPK levels were not changed in DM+AMI as compared to non-infarcted hearts (DM+SHAM). TH administration after AMI prevented hypothyroidism and resulted in decreased β-MHC expression, increased wall thickening and normalized wall stress, while stretch-induced p38 MAPK activation was restored. The results show that diabetes can exacerbates post-ischaemic cardiac remodelling and tissue hypothyroidism and TH treatment can prevent this response and improve cardiac haemodynamics.

<p>The alpha-2 adrenergic receptor (α₂-AR) subfamily includes three different subtypes, α_2A-, α_2B- and α_2C-AR, all believed to exert their function through heterotrimeric G_i/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α₂-ARs.</p><p>Evidence is provided for a strong G_s-protein coupling capability of the α_2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α_2A- and α_2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α_2A-AR with the corresponding domain of α_2B-AR enrolled the chimeric α_2A/α_2B receptor with functional α_2B-AR properties. Dual G_i and G_s coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α_2B-AR activation is largely dependent on the ratio of G_i- to G_s-protein amounts in the cell. Also G_i- and G_o-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with G_i or G_o and the α_2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through G_o, whereas activation of G_i potentiated the activity. </p><p>α₂-ARs mobilize Ca²⁺ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca²⁺ response of α₂-ARs.</p><p>In conclusion, α₂-ARs can couple to multiple G-proteins, including G_i, G_o and G_s. The cellular response to α₂-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).</p>

A time-of-day variation in myocardial contraction and its response to sympathetic stimulation of β-adrenoceptors (b-ADR) exists, which is reflected by time-of-day variations in intracellular calcium [Ca2+]i regulation and electrical activity. There are three isoforms of β-ADR (β1/β2/β3) and the functional outcome of each receptor differs. β1/β2-ADRs result in positive inotropism, whereas β3-ADR induces a negative inotropic effect, associated with nitric oxide (NO) signalling. The aims of this thesis were to determine the role of the β3-ADR in the time-of-day variation in the response to sympathetic stimulation previously shown by our group and to investigate the role β1-ADR, β2-ADR and β3-ADR play the control of Ca2+ regulation in isolated ventricular myocytes. Ventricular myocytes were isolated during the rest-period (ZT3) and active-period (ZT15) of male Wistar rats by enzymatic digestion. [Ca2+]i was measured in myocytes loaded with Fura-2. Arrhythmic activity was determined from video imaging, myocytes were electrically field-stimulated at 1Hz. Measurement of action potentials, transient outward (Ito) and L-type calcium (LTCC) currents were made using whole-cell patch-clamp recordings. Investigation of the time-of-day variation in response to sympathetic stimulation revealed a time-of-day variation in basal systolic [Ca2+]i and in the increase in systolic [Ca2+]i following β-ADR activation with ISO, greatest in rest-period (ZT3) myocytes. This did not appear to be governed by the time-of-day variation in action potential duration I observed or in the response of β3-ADR agonist-induced stimulation. Unlike conventional β1-ADR, β2-ADRs are tightly coupled to phosphodiesterase’s (PDE) through the inhibitory G-protein (Gi). Our data suggests this coupling to Gi and PDE activation is responsible for the reduced response of systolic Ca2+ to β2-ADR agonists, reducing the arrhythmic potential of β2-ADR activation. The reduction in systolic [Ca2+]i by β3-ADR activation with BRL37344 shows a time-of-day variation, with a greater negative inotropic response in rest-period (ZT3) myocytes, mediated via NO and PDE, leading to an increase in Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) activity but a reduction in SR Ca2+ content. This action contributes to an anti-arrhythmic action of the β3-ADR agonist BRL37344 against β1-ADR agonist induced arrhythmia. The ability of β3-ADR activation to reduce arrhythmic activity in response to further sympathetic stimulation is likely to exhibit a time-of-day variation. This work highlights the importance of chrono-pharmacology in the use of β3-ADR agonists as a treatment in arrhythmia management.

Sabe-se que o hormônio tireoideano (HT) regula o desenvolvimento e crescimento dos ossos. No estudo, investigamos se o HT interage com o sistema nervoso simpático (SNS) controlando o crescimento longitudinal ósseo (CLO), e se essa possível interação depende do &#945;2A-AR. Para tanto, avaliamos o efeito de 30 dias de hipotireoidismo (HIPO) e hipertireoidismo (HIPER) e &#945;2A-AR-/- de 21 dias de idade. Vimos que os animais &#945;2A-AR-/- apresentam menor comprimento no fêmur, tíbia, rádio, úmero e L4 quando comparados aos animais Selv. Como esperado, o HIPO e HIPER prejudicaram o CLO desses ossos nos camundongos Selv, entretanto, o efeito do HIPO foi mais deletério. A morfologia da LE distal do fêmur mostrou que os animais &#945;2A-AR-/- eutireóideos (EUT) apresentam desorganização de zonas e alterações no número de condrócitos sendo o Hipotireoidismo o tratamento mais deletérios. Dados desse estudos sugerem que as vias PTHrP/Ihh e IGF-1/IGF-1R possam ser vias de convergência do SNS e HT na regulação da morfofisiologia da LE, envolvendo o &#945;2A-AR. Observou-se que os animais &#945;2A-AR-/- apresentam alterações no osso trabecular com potencialização com o hipotireoismo. Além disso, os animais &#945;2A-AR-/- apresentam alterações a conectividade trabecular. Esses achados sugerem que o SNS e interage o HT dependente do &#945;2A-AR.<br>It is known that the thyroid hormone (TH) regulates the development and growth of bones. In the study, we investigated whether the HT interacts with the sympathetic nervous system (SNS) controlling bone longitudinal growth (CLO), and if this possible interaction depends on &#945;2A-AR -/-. Therefore, we evaluated the effect of 30 days of hypothyroidism (HYPO) and hyperthyroidism (HYPER) and &#945;2A-AR -/- 21 days old. We have seen that &#945;2A-AR -/- animals have shorter femur, tibia, radius, humerus and L4 compared to Selv animals. As expected, the HYPO and HYPER damaged the CLO of these bones in Selv mice, however, the effect of HYPO was more deleterious. The morphology of the distal femur LE showed that &#945;2A-AR -/- animals euthyroid (EUT) have clutter zones and changes in the number of chondrocytes and Hypothyroidism most harmful treatment. Data from this study suggest that PTHrP pathways / Ihh and IGF-1 / IGF-1R can be SNS convergence and HT pathways in the regulation of the CO morphophysiology involving the &#945;2A-AR. It was observed that &#945;2A-AR -/- animals show changes in trabecular bone with augmentation with the hipotireoismo. Furthermore, &#945;2A-AR-/- animals show changes trabecular connectivity. These findings suggest that the SNS and interacts the HT dependent &#945;2A-AR.

Dados mostram que a estimulação do Sistema Nervoso Simpático (SNS) induz osteopenia via receptor &beta;2-adrenérgico, que é expresso no osso. Porém, um estudo recente demonstrou que camundongos knockouts (KO) para os receptores adrenérgicos &alpha;2A e &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) apresentam alta massa óssea, mesmo com hiperatividade simpática. Além disso, esses camundongos são resistentes à osteopenia induzida pelo excesso de hormônio tireoidiano. Esses achados sugerem que o &alpha;2A e/ou &alpha;2C-AR também possam mediar as ações do SNS no esqueleto e que esses receptores estão envolvidos na interação do HT com o SNS para regular o metabolismo ósseo. Neste estudo, tivemos como objetivo: avaliar se o &alpha;2CAR interfere na fisiologia óssea e se a ação do HT no tecido ósseo depende do &alpha;2CAR, avaliando o efeito do HT na fisiologia óssea dos camundongos &alpha;2CAR-/- tratados com dose suprafisiológica de T3. A microtomografia computadorizada mostrou que o volume de osso trabecular foi menor e maior nos animais &alpha;2CAR-/-, no fêmur e na vértebra respectivamente. Os animais KO também apresentaram diminuição da resistência óssea quando comparados com os selvagens. Além disso, vimos que os animais KOs foram resistentes aos efeitos deletérios da tirotoxicose no osso, tanto no fêmur quanto na vértebra. Esses achados sugerem que o &alpha;2C-AR apresenta um papel na mediação dos efeitos da ativação do SNS no osso e que o HT interage via &alpha;2C-AR, para regular massa e resistência óssea.<br>Data demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via &beta;2-adrenoceptor signaling. A recent study showed that mice with gene inactivation of the adrenergic receptor &alpha;2A and &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) have a high bone mass phenotype, even presenting SNS hyperactivity. Also, these knockout (KO) mice are resistant to the thyroid hormone-induced osteopenia. These findings suggest that SNS interacts with thyroid hormone (TH) to regulate bone mass and &alpha;2A and/or &alpha;2C adrenoceptors may have an important role in mediating the actions of the SNS in the skeleton. In this study, we had the following objectives: (i) to evaluate whether the isolated inactivation of &alpha;2CAR interferes with the bone metabolism and to evaluate whether the action of HT on bone tissue depends on &alpha;2CAR, treated with 20 times the physiological dose of T3. The microtomography analysis showed that the trabecular bone volume of the femur and of the sixth lumbar vertebra (L6) were, respectively, lower and higher in &alpha;2C-AR-/- mice, when compared with WT animals. Furthermore, we showed a resistance of KO animals on the deleterious effects of TH on bone. These findings suggest: (i) &alpha;2C-AR is involved with bone longitudinal growth; (ii) &alpha;2C-AR may mediates the effects of the SNS in the bone in a skeletal site specific manner, (iii) the actions of thyroid hormone on bone metabolism involves interactions with the SNS via &alpha;2C adrenergic receptors.

O hormônio tireoidiano (HT) é essencial para o desenvolvimento, maturação e metabolismo ósseos, enquanto que o sistema nervoso simpático (SNS) é, também, um potente regulador do remodelamento ósseo. Demonstrou-se que SNS regula negativamente a massa óssea, agindo via receptores ?2-adrenérgicos (?2-AR), expressos em osteoblastos. O nosso grupo demonstrou que os receptores ?2 adrenérgicos (?2-AR) também medeiam ações do SNS no esqueleto e que são expressos em osteoblastos, osteócitos, condrócitos e osteoclastos. Considerando-se que o HT interage com o SNS para regular uma série de processos fisiológicos, e que o excesso de HT e a ativação do SNS causam perda de massa óssea, levantamos a hipótese de que há interação entre o HT com o SNS para regular a massa óssea. Estudos do nosso grupo vêm sustentando essa hipótese, uma vez que camundongos com inativação gênica dos receptores beta2-AR apresentam resistência à osteopenia induzida por doses tóxicas de HT. Considerando-se, ainda, que a interação do HT com o SNS em vários tecidos e/ou órgãos depende da sinalização beta2 adrenérgica, o presente estudo teve como objetivo avaliar se a interação do HT com o SNS para regular a morfofisiologia óssea envolve o beta2-AR. Para tanto, estudamos o efeito de 10x e 20x a dose fisiológica de triiodotironina (3,5ug ou 7.0ug de T3/100g de massa corporal/dia, respectivamente), por 90 dias, na microaquitetura óssea e em parâmetros biomecânicos do fêmur de camundongos com inativação gênica do beta2-AR (beta2-AR-/-), e nos seus respectivos Selvagens (Selv), os camundongos da linhagem FVB. Como esperado, o tratamento com T3 promoveu efeitos deletérios na microarquitetura trabecular das fêmeas Selv, enquanto alguns desses efeitos foram mais brandos ou inexistentes nos animais beta2-AR-/-, revelando resistência do osso trabecular dos animais knockout (KO) aos efeitos deletérios da tireotoxicose. Em contraste, a microarquitetura femoral dos camundongos machos beta2-AR-/- se mostrou mais sensível aos efeitos deletérios da tireotoxicose, em relação aos respectivos Selv. Quanto ao osso cortical femoral, vimos que o tratamento com T3 aumentou o perímetro endosteal e a área medular nos animais Selv machos e fêmeas, mas não nos animais beta2-AR-/-, o que sugere que o T3 promove reabsorção óssea endosteal no osso cortical, em um mecanismo que depende da via de sinalização do beta2-AR. Vimos, ainda, que o tratamento com T3 causou reduções significativas na carga máxima, tenacidade, rigidez e resiliência do fêmur dos camundongos fêmeas Selv. Em contraste, nenhum desses parâmetros biomecânicos foi afetado pelo tratamento com T3 no fêmur das fêmeas KO, evidenciando, mais uma vez, uma resistência desses animais aos efeitos deletérios da tireotoxicose no tecido ósseo. Por outro lado, os camundongos machos Selv e KO se mostraram resistentes aos efeitos deletérios do tratamento com T3 sobre os parâmetros biomecânicos do fêmur, sugerindo a participação de fatores sexuais na interação do HT com o SNS para regular a morfofisiologia óssea. Em conjunto, os achados do presente estudo corroboram a hipótese de que o HT interage com o SNS através da via dos receptores beta2 adrenérgicos para regular a morfofisiologia óssea, especialmente em fêmeas e no osso cortical<br>Thyroid hormone (TH) is essential for bone development, maturation and metabolism, while the sympathetic nervous system (SNS) is also a potent regulator of bone remodeling. SNS has been shown to negatively regulate bone mass, acting via beta2-adrenergic (beta2-AR) receptors expressed in osteoblasts. Our group demonstrated that alpha2-adrenergic (alpha2-AR) receptors also mediate SNS actions in the skeleton and are expressed in osteoblasts, osteocytes, chondrocytes and osteoclasts. Considering that TH interacts with the SNS to regulate a series of physiological processes, and that the excess of TH and the activation of the SNS cause loss of bone mass, we hypothesize that there is interaction between TH and the SNS to regulate the bone mass. Studies of our group have supported this hypothesis, since mice with gene inactivation of alpha2-AR present resistance to the osteopenia induced by toxic doses of TH. Considering that the TH-SNS interaction in various tissues and/or organs depends on beta2-adrenergic signaling, the present study aimed to evaluate whether the interaction of TH with the SNS to regulate the bone morphophysiology involves beta2- AR. Therefore, we studied the effect of 10x and 20x the physiological dose of triiodothyronine (3.5ug or 7.0ug of T3/100g body mass/day, respectively), for 90 days, in the bone microarchitecture and biomechanical parameters of the femur mice with beta2-AR gene inactivation (beta2-AR-/-), and of their respective Wild-type (WT) controls, the FVB lineage mice. As expected, T3 treatment promoted deleterious effects on the trabecular microarchitecture of the WT females, while some of these effects were milder or nonexistent in beta2-AR-/- animals, revealing trabecular bone resistance of knockout (KO) animals to the deleterious effects of thyrotoxicosis. In contrast, the femoral microarchitecture of the male beta2-AR-/- mice was more sensitive to the deleterious effects of thyrotoxicosis, in relation to the respective WT animals. Regarding to the femoral cortical bone, we saw that T3 treatment increased the endosteal perimeter and the medullary area both male and female WT animals, but not in the beta2-AR-/- mice, suggesting that T3 promotes endosteal bone resorption in the cortical bone, in a mechanism that depends on the alpha2-AR signaling pathway. We also found that treatment with T3 caused significant reductions in the maximum load, tenacity, stiffness and resilience of femurs of the WT female mice. In contrast, none of these biomechanical parameters was affected by T3 treatment in the KO females, demonstrating again resistance of these animals to the deleterious effects of thyrotoxicosis on bone tissue. On the other hand, WT and KO male mice were resistant to the deleterious effects of T3 treatment on the biomechanical parameters of the femur, suggesting the participation of sexual factors in the interaction of HT with the SNS to regulate bone morphophysiology. Taken together, the findings of the present study corroborate the hypothesis that TH interacts with the SNS through the beta2 adrenergic receptor pathway to regulate bone morphophysiology, especially in females and cortical bone

Um dos mais importantes achados dos últimos anos foi o de que o remodelamento ósseo está sujeito ao controle do SNC, com o SNS agindo como efetor periférico. Um estudo do nosso grupo demonstrou que camundongos <font face=\"Symbol\">a2A/<font face=\"Symbol\">a2C-AR-/- apresentam um fenótipo de alta massa óssea, como também são resistentes à osteopenia induzida pelo excesso de hormônio HT. Com o intuito de verificar a participação do <font face=\"Symbol\">a2A-AR-/- nestes processos, tivemos como objetivos: caracterizar o fenótipo ósseo de camundongos <font face=\"Symbol\">a2A-AR-/- e avaliar o efeito do HT na estrutura óssea desses camundongos tratados. Pudemos observar que o comprimento longitudinal dos ossos dos animais <font face=\"Symbol\">a2A-AR-/- são menores do que dos animais selvagens e a análise por <font face=\"Symbol\">mCT do fêmur mostrou uma diminuição da porosidade da cortical. Com relação ao tratamento com hormônio tireoideano, os animais <font face=\"Symbol\">a2A-AR-/- tratados com T3 foram resistentes à diminuição do comprimento dos ossos causado pelo excesso de HT e vimos, ainda, que o osso trabecular dos animais <font face=\"Symbol\">a2A-AR-/- foi mais sensível aos efeitos deletérios da tirotoxicose, entretanto o osso cortical e parâmetros biomecânicos ósseos dos animais KOs foram menos sensíveis. Em conclusão, o presente estudo sugere que o <font face=\"Symbol\">a2A-AR está envolvido no processo de crescimento ósseo e que esse receptor possa mediar, pelo menos parcialmente, ações negativas do T3 nesse processo como também do HT no osso cortical.<br>One of the most important finds of the recent years is that bone remodeling is subject to the control of the CNS, with SNS acting as the peripheral effector. However, a recent study of our group showed that mice <font face=\"Symbol\">a2A/<font face=\"Symbol\">a2C-AR-/- have a high bone mass phenotype, even though are resistant to the thyroid hormone-induced osteopenia. In order to verify the role of <font face=\"Symbol\">a2A-AR-/- in these cases, we had as objectives to evaluate whether the isolated inactivation of <font face=\"Symbol\">a2A-AR interferes with the bone structure, and to evaluate the action of HT on these animals. We have observed that the longitudinal length of the bones of <font face=\"Symbol\">a2A-AR-/- animals are lower than those of wild type animals and the analysis of the femur by <font face=\"Symbol\">mCT showed a lower cortical porosity. With regard to treatment with thyroid hormone, we observed that <font face=\"Symbol\">a2A-AR-/- animals were resistant to the bone length decrease caused by thyroid hormone excess. We also noticed that the trabecular bone of <font face=\"Symbol\">a2A-AR-/- animals was more sensitive to the deleterious effects of thyrotoxicosis. Moreover, the cortical bone and bone biomechanical parameters KO animals were less sensitive. In conclusion, the findings of this study suggest that <font face=\"Symbol\">a2A-AR is involved in the process of bone growth and that this receptor may mediate at least partly, negative actions of T3 in this process as well as the HT in the cortical bone.

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.<br>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<br>長庚大學<br>基礎醫學研究所<br>94<br>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.<br>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<br>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.<br>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.<br>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.<br>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.<br>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.