This work aimed to better understand the early molecular events occasioned in human thyrocytes following exposure to genotoxic agents than can lead to thyroid mutagenesis and cancer. Similar in vitro data in human physiological models remain scarce.The reported incidence of papillary thyroid carcinoma (PTC), has been increasing worldwide but the pathogenesis of sporadic PTC remains unravelled. H2O2 has long been postulated by the host laboratory and by other groups as a potential thyroid mutagen since it is capable to cause DNA damage and it affects γ radiation-induced DNA damage in thyrocytes. Thyrotropin (TSH) has recently been advocated as a potential risk factor for PTC: it has been associated with advanced stage disease and with progression of microcarcinoma during active surveillance. TSH signalling was found to be essential for BRAF-induced thyroid carcinogenesis in mouse models. Radiation (γ and β) is the only clearly established environmental risk factor for PTC; the cancer relative risk is rising linearly beyond a threshold of 100mGy thyroid absorbed dose. We studied in human thyrocytes in primary cultures the early molecular events following 131I exposure (β radiation) in comparison to γ radiation and H2O2 exposure, and their modulation by TSH. We assessed cell survival, DNA damage, DNA repair, gene expression, cell proliferation and apoptosis. Although we globally observed that the thyrocyte responses following exposure to β, γ radiation or H2O2 showed similarities, TSH, unlike other proliferative agents tested, specifically increased DNA damage both in non-exposed and in 131I-exposed thyrocytes. TSH did not influence γ radiation- or H2O2-induced damage. The effect of TSH on DNA damage in non-exposed thyrocytes decreased after incubation with an antioxidant agent and in 131I-exposed thyrocytes was partially alleviated after inhibition of iodide uptake. Therefore, in our experimental conditions, TSH seemed to predispose thyroid cells to greater DNA damage after exposure to 131I via Gαq-mediated increase in ROS/H2O2 levels, independent of its action on iodide uptake or proliferation status. DNA repair timing was similar between β, γ radiation and H2O2. Both β and γ radiation resulted in an extended thyroid cell cycle arrest but no apoptosis. In contrast, H2O2 did not appear to affect cell cycle at the same extent.This works includes the first, to our best knowledge, RNA sequencing to obtain the gene expression profile of human thyrocytes following 131I exposure. Overall transcriptional responses of thyrocytes exposed to 131I, γ radiation and H2O2 overlapped. At this stage, we were not able to attribute a distinct molecular signature to each agent, pointing that they probably employ similar cellular toxicity mechanisms. Regulated genes were involved in inflammatory response, cytokine signaling, DNA repair, apoptosis and cell cycle. Again, TSH yielded a more prominent transcriptomic response, both in number and in expression level of regulated transcripts, exclusively following 131I exposure. Better understanding of the early stages of thyroid carcinogenesis could lead to a more precise, personalized management of thyroid cancer patients and even in novel therapeutic strategies. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Diese Dissertation untersucht einen möglichen Zusammenhang zwischen dem beschriebenen klinischen Verlauf bei Patienten mit konstitutiv aktivierenden TSH-Rezeptormutationen und der gemessenen in vitro Aktivität. Konstitutiv aktivierende Mutationen finden sich als somatische Mutationen in autonomen Adenomen und als Keimbahnmutationen bei Patienten mit sporadischer bzw. familiärer nicht-autoimmuner Hyperthyreose. Die in vitro Aktivität der zu Grunde liegenden TSH-Rezeptormutationen wird mit Hilfe der Linearen Regressions-Analyse bestimmt. Dies ist ein Verfahren, welches die basale Produktion des second messenger cAMP (Cyclo-Adenosinmonophosphat) misst, unter Berücksichtigung der Expression des TSH-Rezeptors. Die Analyse der Krankheitsverläufe der sporadischen nicht-autoimmunen Hyperthyreose zeigt keinen eindeutigen Bezug zur gemessenen in vitro Aktivität. Es besteht jedoch eine höhere in vitro Aktivität bei Mutationen, die sowohl bei der nicht-autoimmunen sporadischen Hyperthyreose und in autonomen Adenomen zu finden sind, im Vergleich zu ausschließlich familiären Mutationen. Dies entspricht auch dem klinischen Eindruck. Für die wenigen bekannten Fälle der sporadischen nicht-autoimmunen Hyperthyreose wurden dramatische Verläufe mit häufigen Rückfällen unter medikamentöser Therapie und zahlreichen Komplikationen (z.B. mentale Retardierung, Kraniosynostose, zerebrale Ventrikulomegalie, beschleunigte Knochenreifung) beschrieben.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-30T20:27:31Z No. of bitstreams: 1 Taise Lima de Oliveira Caracterização clínica e molecular de pacientes com hipertireodismo....pdf: 583769 bytes, checksum: 1d1d8e16ece954c3ead88d0648b4eeea (MD5) / Made available in DSpace on 2012-08-30T20:27:31Z (GMT). No. of bitstreams: 1 Taise Lima de Oliveira Caracterização clínica e molecular de pacientes com hipertireodismo....pdf: 583769 bytes, checksum: 1d1d8e16ece954c3ead88d0648b4eeea (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O hipotireoidismo congênito (HC) é uma das doenças metabólicas mais comuns na infância, com incidência de HC é de 1:3.000 a 1:5.000 nascidos-vivos. Quando primário, o HC caracteriza-se por altos níveis de TSH podendo ser originado por disormonogênese, deficiente produção hormonal ou decorre de disgenesia tireoidiana, defeito embriológico que leva a agenesia, hemiagenesia ou ectopia. Na ausência do tratamento hormonal, o HC leva a grave retardo mental, além de outras alterações clínicas. A interação do hormônio TSH com o seu receptor (TSHR) tem importante função biológica estimulando o crescimento, diferenciação e função tireoidiana. Mutações no gene do TSHR têm sido identificadas como causa de HC hereditário ou congênito, mas parecem ser raras. Objetivos: 1) Obter genealogia das famílias afetadas para determinação do padrão de herança, identificação de afetados e possíveis portadores; 2) Descrever as características clínica-demográficas dos pacientes com HC; 3) Determinar a distribuição mutacional no gene do receptor do hormônio estimulante da tireoide (TSHR) nos afetados; 4) Sugerir ações de saúde pública e de otimização do aconselhamento genético. Material e Métodos: Foram estudados 12 pacientes provenientes de Monte Santo-BA, sendo oito diagnosticados pela triagem neonatal e quatro diagnosticados tardiamente durante expedição à cidade. Estes últimos foram identificados durante a coleta dos dados genealógicos das famílias dos afetados. Todos foram investigados para mutações no gene do TSHR. Toda a região codificadora do gene foi amplificada através do DNA genômico, seguido de SSCP e sequenciamento. Resultados: À época da primeira avaliação, os pacientes apresentaram níveis elevados de TSH, confirmando o caráter primário da doença, associados a sinais e sintomas do HC. Observou-se grande heterogeneidade clínica entre os pacientes mesmo aqueles com grau de parentesco muito próximo. Nenhuma mutação patogênica foi encontrada no gene TSHR. Conclusão: a análise das genealogias permitiu observar a heterogeneidade clínica e genética da doença. Não foi encontrada nenhuma mutação no gene TSHR. O estudo de outros genes poderá ajudar no esclarecimento do HC na região. / Congenital hypothyroidism (CH) is one of the most common metabolic disease in childhood, with an incidence of HC is 1:3,000 to 1:5,000 live births. When primary, CH is characterized by high levels of TSH and may be caused by dyshormonogenesis deficient hormone production, or due to thyroid dysgenesis, embryological defect that leads to agenesis, ectopic or hypoplastic. In the absence of hormonal treatment leads to the CH severe mental retardation and other clinical manifestations. The interaction of stimulanting thyroid growth, differentiation and function. Mutations in the TSHR gene have been identified as a cause of hereditary or congenital CH, but appear to be rare. Objectives: 1) Get genealogy of the affected families to detemine the pattern of inheritance, and possible identification of affected patients; 2) Describe the clinical and demographic characteristics of patients witc CH; 3) Determine the mutational distribution in the TSHR gene in the affected individuals; 4) Suggest public health actions and for genetic counseling optimization. Methods: We studied 12 patients from Monte Santo-Bahia, eight were diagnosed by newborn screening and four diagnosed late during an expedition to the city. The latter were identified during the collection of genealogical data of families those affected. All were investigated for mutations in the TSHR. The entire coding region of the gene was amplified using genomic DNA followed by SSCP and sequencing. Results: At first evaluation, all patients had elevated TSH levels, confirming the primary nature of the disease, associated with signs and/or symptoms of CH. As there was a familial recurrence of the disease, we noticed a large clinical heterogeneity among patients, even those with close family relationship. No pathogenic mutation was found in the TSHR gene. Conclusion: The analysis of pedigrees allowed to observe the clinical and genetic heterogeneity of the disease. We not found mutation in the TSHR. The study of other genes may help in clarifying the CH i the region.

Alguns estudos vêm apontando o hipotireoidismo como um fator de risco para depressão; entretanto, esta associação é ainda controversa. Como objetivo o presente estudo estimou a prevalência de sintomas depressivos numa amostra probabilística de mulheres e investigou se existe uma associação entre níveis de TSH e a presença de sintomas depressivos. Conduzimos um estudo transversal de base populacional onde avaliou-se uma amostra de mulheres com 35 anos ou mais anos de idade, residentes no município do Rio de Janeiro (RJ), que foram entrevistadas e tiveram amostras de sangue coletadas. O desenho de amostra proposto para a pesquisa seguiu um modelo de amostragem probabilística por conglomerado, em três estágios de seleção, tendo por base os setores censitários do município. A função tireoidiana foi medida pelo TSH sérico, a partir das amostras coletadas no domicílio. Para avaliação da presença/ausência de sintomas depressivos utilizou-se um instrumento padronizado e validado (PRIME-MD). Foram também coletados dados sócio demográficos e outras informações de saúde da participante. A prevalência de sintomas depressivos, frequências e associações foram calculadas levando-se em conta o desenho de amostra complexo, utilizando procedimentos específicos do pacote SAS (versão 9.1). Da amostra de 1500 mulheres, 1298 foram entrevistadas, sendo o percentual de não-resposta igual a 13,5%. A amostra final foi composta por 1249 participantes, onde foi encontrada uma prevalência de sintomas depressivos igual a 45,9%. Em relação aos níveis de TSH, 4,8% da amostra apresentaram-se acima do ponto de corte adotado (&#8805;6,0 mUl/ml). Destas, 61,9% apresentaram sintomas depressivos, contra 44,9% entre as mulheres com TSH<6,0 (p=0,04). A análise ajustada mostrou que mulheres com nível de TSH&#8805;6,0 tiveram uma chance duas vezes maior de apresentarem sintomas depressivos do que aquelas com TSH normal (OR=2,04). Ao excluir da análise as participantes que auto-referiram diagnóstico prévio de doença tireoidiana, a associação entre níveis de TSH e sintomas depressivos perdeu a significância. Concluindo, foi observada alta prevalência de sintomas depressivos na amostra, especialmente entre o grupo com níveis elevados de TSH. Os resultados chamam atenção para a importância de se investigar em pacientes deprimidos a comorbidade tireoidiana. / Some studies have been indicating that hypothyroidism is a risk factor for depression; however, this association is still controversial. The present study estimated the prevalence of depressive symptoms in a probabilistic sample of women and investigated whether there was any association between thyroid-stimulating hormone (TSH) levels and the presence of depressive symptoms. A cross-sectional population-based study was carried out and sample of women aged 35 years and over living in the municipality of Rio de Janeiro (RJ) was evaluated. They were interviewed and blood samples were taken. The sample design proposed for this study followed a model of probabilistic sampling by clusters, with three selection stages based on the census tracts of municipality. Thyroid functions was measured from serum TSH levels, using samples collected in the subjects homes. To assess the presence/absence of depressive symptoms, a standardized validated instrument (PRIME-MD) was used. Sociodemographic data and other information on participants health were also gathered. The prevalence of depressive symptoms, frequencies and associations were calculated while taking into account the design of this complex sample, using specific procedures from the SAS statistical package (version 9.1). Out of a sample of 1500 women, 1298 were interviewed. The no-response rate was 13.5%. The final sample was composed of 1249 participants, among whom a prevalence of depressive symptoms of 45.9% was found. Regarding TSH levels, 4.8% of the sample was above the cutoff point adopted (&#8805;6.0 mUl/ml). Of these, 61.9% presented depressive symptoms, versus 44.9% among the women with TSH <6.0 (p=0.04). The adjusted analysis showed that the women with TSH levels &#8805; 6.0 had twice as much chance of presenting depressive symptoms as did those with normal TSH levels (OR= 2.04). when the participants who self-reported a previous diagnosis of thyroid disease were excluded from the analysis, the association between TSH levels and depressive symptoms lost its significance. High prevalence of depressive symptoms was observed in the sample, specially among the group with high TSH levels. The results draw attention towards the importance of investigating thyroid comorbidity in depressed patients.

Alguns estudos vêm apontando o hipotireoidismo como um fator de risco para depressão; entretanto, esta associação é ainda controversa. Como objetivo o presente estudo estimou a prevalência de sintomas depressivos numa amostra probabilística de mulheres e investigou se existe uma associação entre níveis de TSH e a presença de sintomas depressivos. Conduzimos um estudo transversal de base populacional onde avaliou-se uma amostra de mulheres com 35 anos ou mais anos de idade, residentes no município do Rio de Janeiro (RJ), que foram entrevistadas e tiveram amostras de sangue coletadas. O desenho de amostra proposto para a pesquisa seguiu um modelo de amostragem probabilística por conglomerado, em três estágios de seleção, tendo por base os setores censitários do município. A função tireoidiana foi medida pelo TSH sérico, a partir das amostras coletadas no domicílio. Para avaliação da presença/ausência de sintomas depressivos utilizou-se um instrumento padronizado e validado (PRIME-MD). Foram também coletados dados sócio demográficos e outras informações de saúde da participante. A prevalência de sintomas depressivos, frequências e associações foram calculadas levando-se em conta o desenho de amostra complexo, utilizando procedimentos específicos do pacote SAS (versão 9.1). Da amostra de 1500 mulheres, 1298 foram entrevistadas, sendo o percentual de não-resposta igual a 13,5%. A amostra final foi composta por 1249 participantes, onde foi encontrada uma prevalência de sintomas depressivos igual a 45,9%. Em relação aos níveis de TSH, 4,8% da amostra apresentaram-se acima do ponto de corte adotado (&#8805;6,0 mUl/ml). Destas, 61,9% apresentaram sintomas depressivos, contra 44,9% entre as mulheres com TSH<6,0 (p=0,04). A análise ajustada mostrou que mulheres com nível de TSH&#8805;6,0 tiveram uma chance duas vezes maior de apresentarem sintomas depressivos do que aquelas com TSH normal (OR=2,04). Ao excluir da análise as participantes que auto-referiram diagnóstico prévio de doença tireoidiana, a associação entre níveis de TSH e sintomas depressivos perdeu a significância. Concluindo, foi observada alta prevalência de sintomas depressivos na amostra, especialmente entre o grupo com níveis elevados de TSH. Os resultados chamam atenção para a importância de se investigar em pacientes deprimidos a comorbidade tireoidiana. / Some studies have been indicating that hypothyroidism is a risk factor for depression; however, this association is still controversial. The present study estimated the prevalence of depressive symptoms in a probabilistic sample of women and investigated whether there was any association between thyroid-stimulating hormone (TSH) levels and the presence of depressive symptoms. A cross-sectional population-based study was carried out and sample of women aged 35 years and over living in the municipality of Rio de Janeiro (RJ) was evaluated. They were interviewed and blood samples were taken. The sample design proposed for this study followed a model of probabilistic sampling by clusters, with three selection stages based on the census tracts of municipality. Thyroid functions was measured from serum TSH levels, using samples collected in the subjects homes. To assess the presence/absence of depressive symptoms, a standardized validated instrument (PRIME-MD) was used. Sociodemographic data and other information on participants health were also gathered. The prevalence of depressive symptoms, frequencies and associations were calculated while taking into account the design of this complex sample, using specific procedures from the SAS statistical package (version 9.1). Out of a sample of 1500 women, 1298 were interviewed. The no-response rate was 13.5%. The final sample was composed of 1249 participants, among whom a prevalence of depressive symptoms of 45.9% was found. Regarding TSH levels, 4.8% of the sample was above the cutoff point adopted (&#8805;6.0 mUl/ml). Of these, 61.9% presented depressive symptoms, versus 44.9% among the women with TSH <6.0 (p=0.04). The adjusted analysis showed that the women with TSH levels &#8805; 6.0 had twice as much chance of presenting depressive symptoms as did those with normal TSH levels (OR= 2.04). when the participants who self-reported a previous diagnosis of thyroid disease were excluded from the analysis, the association between TSH levels and depressive symptoms lost its significance. High prevalence of depressive symptoms was observed in the sample, specially among the group with high TSH levels. The results draw attention towards the importance of investigating thyroid comorbidity in depressed patients.

La progression dans le cycle cellulaire est gouvernée par l’activation séquentielle d’une série de complexes cycline/CDK. La CDK4 initie le passage du point de restriction (point R, à partir duquel l’achèvement du cycle cellulaire devient indépendant des facteurs extracellulaires) en phosphorylant les protéines « antioncogéniques » de la famille pRb. Dans les thyrocytes de chien en culture primaire, l’AMPc (TSH ou forskoline) induit la prolifération et la différenciation alors que la voie mitogénique des facteurs de croissance (l’EGF, par exemple) est associée à une dédifférenciation. Dans ce modèle physiologiquement relevant, la stimulation mitogénique par l’AMPc diffère des cascades des facteurs de croissance puisqu'elle n’induit pas les cyclines D mais au contraire augmente l’accumulation de l’inhibiteur de CDK p27 kip1. Le contrôle positif du cycle cellulaire par l’AMPc requiert néanmoins l’activité de la CDK4. L’AMPc stimule l’assemblage des complexes cycline D3-CDK4 ainsi que leur translocation nucléaire associée à leur liaison à p27. Notre but était d’élucider les différents mécanismes menant au passage du point de restriction dans les cellules épithéliales thyroïdiennes stimulées par l’AMPc ou les facteurs de croissance. Dans ce travail, nous montrons que l’arrêt de la stimulation des thyrocytes de chien par l’AMPc entraîne une diminution rapide de la phosphorylation de pRb et de l’activité de la CDK4 sans affecter la formation des complexes cycline D3-CDK4-p27. Par une approche utilisant le haut pouvoir de résolution de l’électrophorèse bidimensionnelle, nous avons identifié la phosphorylation activatrice de la CDK4 comme cible du contrôle par l’AMPc du passage du point de restriction. Ceci constitue un premier exemple d’une régulation de la phosphorylation et de l’activité de la CDK4 indépendante de son association avec une cycline ou un inhibiteur de CDK. Ces résultats contrastent avec l’absence de modulation d’expression, de localisation subcellulaire et d’assemblage des complexes cycline H-CDK7-Mat1, la CAK considérée comme responsable de la phosphorylation activatrice de la CDK4. Ceci suggère que les CAKs régulées activant la CDK4 n’ont pas encore été identifiées. D’autre part, alors que la TSH induit une accumulation de p27, nous montrons à présent que l’expression de la p21 apparentée est augmentée par l’EGF + sérum et réprimée par la TSH. En réponse à l’EGF + sérum ou à la TSH, respectivement, la p21 ou la p27 supportent la localisation nucléaire, la phosphorylation et l’activité de la CDK4. Les « inhibiteurs » de CDK p21 et p27 pourraient donc être utilisés différentiellement comme régulateurs positifs de la CDK4 lors des stimulations des cellules épithéliales thyroïdiennes de chien par la TSH (p27) ou par l’EGF + sérum (p21). Nous avons également montré que les complexes cycline D1-CDK4 et cycline D3-CDK4 phosphorylent pRb sur des sites partiellement différents. Cette nouvelle observation a été reproduite pour des complexes cycline D-CDK4 surexprimés en cellules CHO ainsi que pour des complexes exprimés de manière endogène dans différents types cellulaires. Cette différence de spécificité de substrat entre la cycline D1 et la cycline D3 conduit à différents profils de phosphorylation de pRb dans les thyrocytes de chien stimulés par la TSH ou les facteurs de croissance, ce qui est dû à l’utilisation préférentielle de la cycline D3 dans les thyrocytes stimulés par la TSH alors que les facteurs de croissance induisent surtout la cycline D1. Comme différentes fonctions de pRb sont régulées par phosphorylation sur différents résidus, ce résultat indique que les complexes cycline D1-CDK et cycline D3-CDK pourraient affecter de manière partiellement différente la fonction de cette protéine. Enfin, nous avons comparé les stimulations mitogéniques par la TSH ou l’EGF + sérum dans les thyrocytes humains normaux en culture primaire. En accord avec leurs modulations différentes, la cycline D3 et la cycline D1 sont utilisées différentiellement dans les voies mitogéniques stimulées par la TSH ou l’EGF + sérum respectivement. De plus, ce système nous a permis de confirmer la régulation de l’activité de la CDK4 au niveau de sa phosphorylation activatrice comme mécanisme déterminant de la réponse mitogénique.

Bei der vorliegenden Arbeit handelt es sich um eine experimentelle Untersuchung zu Unterschieden in der Signaltransduktion von drei mutierten Thyrotropin-Rezeptoren (mTSHR), die in Rattenschilddrüsenzellen stabil exprimiert werden. Mittels Proteomanalyse konnte die Proteinexpression von mehreren Signalproteinen quantitativ untersucht werden und mit der Proteinexpression in Zellen, die Wildtyp (WT)-Rezeptoren exprimieren, verglichen werden. In den Zellen mit mutierten Rezeptoren konnte eine gesteigerte Expression von Proteinen nachgewiesen werden, die in der Endozytose eine Schlüsselrolle spielen. Weiterhin konnte hier eine verstärkte Internalisierung der mTSHR festgestellt werden. Der Phosphatidyl-Inositol-3-Kinase (PI3K) Signalweg wurde in mTSHR-Schilddrüsenzellen im Vergleich zu WT-Zellen verstärkt aktiviert, und dies geschah Proteinkinase A (PKA) -unabhängig. Zudem konnte eine vorübergehende Aktivierung des Proteins Exchange protein directly activated by cAMP (Epac) durch das zyklische Adenosinmonophosphat (cAMP) nachgewiesen werden, die möglicherweise mit der Aktivierung des PI3K-Signalwegs in Zusammenhang steht. Die Ergebnisse gewähren somit neue Einblicke in die biologische Aktivität von mTSHR. Zum einen bleiben wahrscheinlich die mTSHR nach Internalisierung weiterhin aktiv, zum anderen reduziert sich die weitere Signaltransduktion nicht nur auf cAMP-PKA/Inositoltrisphosphat-Signalwege. Diese Ergebnisse könnten zu einem besseren Verständnis für den Verlauf der Schilddrüsenautonomie beitragen.

Sköldkörteln (tyroidea) är ett endokrint organ som påverkar ämnesomsättningen. Tyreoidea stimulerande hormon (TSH) frisätts från hypofysen och stimulerar syntes och frisättning av tyreoideahormonerna trijodtyronin (T3) och tyroxin (T4). Tyreoidea hormonerna har i sin tur en negativ påverkan på frisättningen av TSH. TSH, T3 fritt och T4 fritt analyseras på serum med immunokemiska metoder. Becton Dickinson (BD) har tagit fram ett snabbkoagulerande provrör för analys av serum. Detta provrör heter BD Vacutainer® RST™ (rapid serum tube). Akutmottagningen på Centrallasarettet i Växjö använder detta provrör för vissa analyser och har önskemål om att kunna utföra tillbeställningar på S-TSH efter provtagning. Syftet med studien var att undersöka om det går att analysera S-TSH, S-T3 fritt och S-T4 fritt på BD Vacutainer® RST™ med instrumentet ADVIA Centaur XP. För studien togs venprover på 30 personer (20 kvinnor och 10 män), ett RST™ och ett SST™ (serum separation tube). SST™ användes som referensrör. Analysen av S-TSH, S-T3 fritt och S-T4 fritt utfördes med ADVIA Centaur XP och analysresultaten jämfördes genom regressionsanalys, Pearsons korrelationskoefficient och bias. Regressionsanalysen gav för alla tre analyserna linjer som låg nära varandra och k = 1, samt Pearsons korrelationskoefficient i närheten av 1. Bias visade en spridning på främst ±10 % med två avvikande resultat ett på 28 % och ett annat på -13 %. Dessa avvikande resultat förklarades genom att båda resultaten var låga värden och en liten skillnad mellan resultaten ger en större spridning. Slutsatsen blev att går det att analysera S-TSH, S-T3 fritt och S-T4 fritt på RST™ med ADVIA Centaur XP vilket kommer att minska antalet provtagningar på akuten och ge ett snabbare analysresultat på fler analyser.

The role of thyroid-stimulating hormone (TSH) in the regulation of peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T₄), the principal hormone released from the thyroid gland in response to TSH stimulation, circulates with a robust daily rhythm in the sciaenid fish, red drum. Previous research has suggested that the red drum T₄ cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T₄ feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with driving this T₄ cycle, I developed quantitative real time RT-PCR (qPCR) techniques to quantify the daily cycle of expression of the pituitary TSH subunits GSU[alpha], and TSH[beta]. I found that pituitary TSH expression cycled inversely to, and 6-12 hours out of phase with, the T₄ cycle, consistent with the hypothesis that TSH secretion drives the T₄ cycle. To examine the potential role of deiodinases in negative feedback regulation of this TSH cycle, I also utilized qPCR to assess the pituitary expression patterns of the TH activating enzyme outer-ring deiodinase (Dio2) and the TH deactivating enzyme inner ring deiodinase (Dio3). Whereas Dio2 was not expressed with an obvious daily cycle, Dio3 was expressed in the pituitary mirroring the TSH cycle. These results are consistent with T₄ negative feedback on TSH and suggest that TH inactivation by pituitary cells is an important component of the negative feedback system. To further examine the TH regulation of this Dio3 cycle, I developed an immersion technique to administer physiological doses of T₃ and T₄ in vivo. Both hormones persist in static tank water for at least 40 hours. Immersion in 200ng/ml T₄ significantly increased both plasma T₄ and T₃ within physiological ranges above control at 4.5 hours. Immersion in 100ng/ml T₃ increased plasma T₃ within physiological ranges over control by 22 hours while significantly decreasing plasma T₄ below control, presumably through inhibition of TSH secretion. T₄ also significantly inhibited the expression of the TSH [alpha] and [beta] subunits at 4.5 and 22 hours of immersion whereas T₃ immersion significantly inhibited the expression of the [alpha] and [beta] subunits of TSH by 22 hours. Both Dio2 and Dio3 expression were significantly diminished by T3 and T₄ at 22 hours. Inhibition of circulating THs with the goitrogen methimazole significantly increased the expression of TSH. These results indicate that both T₄ and T₃ are capable of negative feedback regulation of TSH expression in red drum on a time scale consistent with the T₄ daily cycle, and further support Dio3 destruction of THs in the pituitary, potentially regulated by circulating T₄, as a critical component of negative feedback on TSH. This study supports the importance of central mechanisms acting through pituitary TSH secretion in regulating thyroid function in red drum.

Made available in DSpace on 2014-10-09T12:50:32Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:42Z (GMT). No. of bitstreams: 1 00315.pdf: 1408017 bytes, checksum: fa7119b275b1da64d1ec8115e3701037 (MD5) / Tese (Doutoramento) / IEA/T / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP