There are a number of links between the endocrine system and the nervous system. In this study, the impact of ischemic stroke on the endocrine system was investigated. Elderly volunteers were studied because data regarding the influence of advanced age on endocrine parameters were lacking. Only small differences in pituitary-thyroid and pituitary-adrenal hormone axes were found between two groups of elderly patients, 60 and 80 years of age. The 80-year-old age group had a lower thyrotropin response to thyrotropin releasing hormone (TRH) and a decline in dopamine excretion. Patients with acute ischemic stroke showed a pronounced hypercortisolism studied by the dexamethasone test and urine free cortisol measurements. In multiple regression analyses, postdexamethasone cortisol levels were positively correlated to proximity of the lesion to the frontal pole of the brain and disorientation. Urine cortisol levels were predicted by limb paresis, disorientation and body temperature. High cortisol excretion was associated with a worse functional outcome. Norepinephrine excretion was correlated to urine cortisol levels and to motor impairment. Patients with acute stroke had elevated free thyroxin indices. A paradoxical growth hormone response to TRH was found in the majority of stroke patients. In a multiple regression model disorientation was negatively correlated to thyrotropin response after TRH and positively correlated to prolactin response. Growth hormone response to TRH was associated with extensive paresis. In a cohort study diabetic and non-diabetic patients were prospectively studied after an initial stroke. Diabetes mellitus adversely influenced survival, the risk for a recurrent stroke and myocardial infarction. / <p>S. 1-66: sammanfattning, s. 69-190: 6 uppsatser</p> / digitalisering@umu

Dedicated to Valentine Mott, M.D. / Last page blank. Microform version available in the Readex Early American Imprints series.

A disfunção tireoidiana durante a gestação cursa com maior morbidade materno-fetal. A deficiência de iodo continua sendo uma causa importante de disfunção tireoidiana. O estado de São Paulo é considerado uma região suficiente em iodo. No entanto, as gestantes são consideradas grupo de risco para deficiência iódica devido a maior necessidade de produção dos hormônios tireoidianos ao longo da gestação. Este trabalho objetiva avaliar a suficiência iódica de gestantes residentes em São Paulo e correlacionar as concentrações de iodo com os valores dos hormônios tireoidianos em cada trimestre de gestação. Métodos: Foi realizado estudo transversal com gestantes em pré-natal de baixo risco nos três trimestres de gestação. Foram analisadas 251 mulheres sem doença tireoidiana prévia ou atual, com autoanticorpos antitireoidianos negativos e que não estavam em uso de polivitamínico contendo iodo. A concentração de iodo urinário foi feita em amostra isolada pelo método Sandell- Kolthoff. As dosagens de TSH, T4, T3, T4 livre (T4L), T3 livre (T3L), tireoglobulina (TG), globulina ligadora da tiroxina (TBG) anticorpo antitireoperoxidase (AntiTPO), antitireoglobulina (AntiTG), estradiol e hCG foram feitas por método eletroquimioluminescente. Ultrassonografia da tireoide foi realizada com aparelho Philips IU-22 e transdutor 7,5-12 mHz. Resultados: Os valores de TSH correspondentes aos percentis 2,5 e 97,5 foram 0,38 uIU/mL e 4,23 uIU/mL, respectivamente. Observou-se valores de TSH > 2,5-3,0 uIU/mL em 13,1% gestantes. Valores de TSH > 4 uIU/mL foi observado em 3,6% dos casos. Os valores de T4T e T3T correspondentes aos percentis 2,5 e 97,5 foram 7,8 ug/dL e 16,1 ?g/dL; 122 ng/dL e 249 ng/dL, respectivamente. Os valores de T4l e T3l correspondentes aos percentis 2,5 e 97,5 foram 0,76 ng/dL e 1,42 ng/dL; 0,21 ng/dL e 0,36 ng/dL, respectivamente. Houve aumento significativo na relação T3:T4 ao longo dos trimestres e, em 8% das amostras, essa relação estava acima do valor de referência para gestantes. As dosagens de iodúria não apresentaram diferenças estatísticas entre os três trimestres da gestação. As medianas foram 135ug/L, 153ug/L e 140ug/L, respectivamente. Encontramos iodúria < 150ug/L em 52,2% das gestantes. Comparando o grupo com iodúria < 150ug/L (deficiente em iodo) e o grupo com iodúria entre 150-250ug/L (suficiente em iodo), observou-se diferença significativa nos valores de TSH e de T3 nas gestantes do 2o Trimestre (média de TSH = 2,24 uIU/mL e TSH = 1,78 uIU/mL e média de T3 = 196 ng/dL e T3 = 181 ng/dL entre o grupo deficiente e suficiente, respectivamente). Não houve diferenças significativas entre os valores de TG e o volume tireoidiano ao longo dos trimestres. Conclusão: A frequência de hipotireoidismo subclínico variou de 3,6% a 13,1%, dependendo dos critérios adotados. Não foi possível estabelecer a frequência de hipotiroxinemia materna, devido à ausência de valores de corte de T4l estabelecido na nossa população. Foi encontrada deficiência iódica em 52,2% das gestantes avaliadas. Embora estas gestantes apresentem deficiência leve de iodo, foram necessários mecanismos adaptativos para equilibrar a função tireoidiana materna com possível produção preferencial de T3, aumento da relação T3:T4 e valores mais elevados de TSH. Conclui-se que, embora esta seja uma região suficiente em iodo, as mulheres poderiam se beneficiar com a suplementação deste nutriente durante a gestação / Thyroid dysfunction in pregnancy is associated with increased rate of obstetrical and neonatal adverse outcomes. Iodine deficiency continues to be a major cause of thyroid dysfunction. The state of São Paulo is considered a sufficient iodine region. However, pregnant women are considered a risk group for iodine deficiency due to an increased demand for production of thyroid hormones throughout pregnancy. This study aims to evaluate the iodine sufficiency of pregnant women in São Paulo and to correlate iodine concentrations with thyroid parameters in each trimester of gestation. Methods: A cross-sectional study was carried out with low-risk pregnant women in the three trimesters of gestation. We analyzed 251 women without history of thyroid disease, with negative antithyroid autoantibodies (anti TPO and anti TG) and who were not taking iodine supplementation. The urinary iodine concentration (UIC) was measured by the Sandell-Kolthoff digestion method in casual morning urine samples. Serum TSH, T4, T3, free T4 (FT4), free T3 (FT3), thyroglobulin (TG), TBG, anti-TPO, anti-TG, estradiol and hCG were measured by the electrochemiluminescence method. Thyroid ultrasound was performed with Philips IU-22 and 7.5-12 mHz transducer. RESULTS: The TSH values corresponding to the 2.5 and 97.5 percentiles were 0.38 IU/mL and 4.23 IU/mL, respectively. TSH levels > 2.5-3.0 IU/mL were observed in 13.1% pregnant women. TSH values > 4 IU/mL were observed in 3.6% of the cases. The T4T and T3T values corresponding to the 2.5 and 97.5 percentiles were 7.8 ug/dL and 16.1 ug/dL; 122 ng/dL and 249 ng/dL, respectively. The FT4 and FT3 values corresponding to the 2.5 and 97.5 percentiles were 0.76 ng/dL and 1.42 ng/dL; 0.21 ng/dL and 0.36 ng/dL, respectively. There was a significant increase in the T3:T4 ratio over the trimesters and in 8% of the samples it was above the reference value for pregnant women. The dosages of UIC did not present statistical differences between the three trimesters of gestation. The medians were 135 ug/L, 153 ug/L and 140 ug/L, respectively. We found UIC < 150 ?g/L in 52.2% of pregnant women. Comparing the group with UIC < 150 ?g/L (iodine deficient) and the group with UIC between 150-250 ug/L (sufficient in iodine), a significant difference was observed in TSH and T3 values in the 2nd trimester (TSH = 2.24 IU/mL and TSH = 1.78 IU/mL and mean T3 = 196 ng/dL and T3 = 181 ng/dL between the deficient and sufficient groups, respectively). There were no significant differences between TG values and thyroid volume over the trimesters. Conclusion: The subclinical hypothyroidism frequency ranged from 3.6% to 13.1%, depending on the adopted criteria. It was not possible to establish frequency of maternal hypothyroxinemia, due to the absence of FT4 cutoff established in our population. Iodine deficiency was found in 52.2% of pregnant women evaluated. Although these pregnant women presented mild iodine deficiency, adaptive mechanisms were necessary to balance thyroid function with possible preferential production of T3, increase in T3:T4 ratio and higher values of TSH. It is concluded that although this is an iodine sufficient region, women could benefit from supplementation of this nutrient during pregnancy

Pendrina é um transportador de ânions inserido na membrana apical de células foliculares. Estudos subseqüentes demonstraram que a proteína pode mediar o efluxo apical do iodeto nos tirócitos. Sendo o iodo fundamental para a síntese de hormônios tiroidianos foi objetivo deste estudo avaliar o efeito da administração aguda de iodo na expressão do mRNA da proteína pendrina, em curtos períodos de tempo (30min à 24h).Ratos Wistar foram divididos em: controle e iodo, que receberam injeção de salina ou NaI, sendo decapitados após 30, 1 e 24h dessa administração.O RNA da tiróide foi extraído para a análise da expressão do mRNA da Pendrina por Real Time PCR e Northern Blot. Para o estudo in vitro utilizou-se a linhagem celular de tiróide de rato PCCl3, que foi tratada ou não com 10-3M de NaI. As células permaneceram sob tratamento por 30´, 1 e 24h, quando então o RNA foi extraído para análise da expressão por Real Time PCR.Houve aumento significativo do mRNA da Pendrina em todos os grupos, indicando que mecanismos foram desencadeados visando o efluxo do iodeto da célula. / Pendrin is a chloride/iodide exchange located at the apical membrane of thyrocytes. Mutations in its gene lead to a defect in iodide organification. This suggested that pendrin could function as an apical iodide transporter in this cells. Since iodine is essential for thyroid hormone synthesis, this study attempted to investigate that possibility by evaluating whether the acute iodide administration, from 30min up to 24h, could regulate the Pendrin mRNA expression. Rats received NaI or saline, and were sacrificed 30´, 1 and 24h later.Thyroid total RNA was extracted and Pendrin mRNA content was evaluated by Northern Blotting and Real-Time PCR. For in vitro study,PCCl3 rat thyroid cells were cultured and treated or no with 103M NaI. After 30´, 1 and 24h, the cells were harvested and total RNA was extracted. The mRNA content was evaluated by Real-Time PCR. The mRNA increased in all groups of study, indicating that excess of iodide leads to an activation of Pendrin gene transcription and as consequence increased efflux of this element.

Uma série de 205 casos de lesões nodulares da tireóide foi revista morfologicamente de acordo com a nova classificação da Organização Mundial da Saúde (OMS, 2004) e analisada quanto à imuno-expressão das citoqueratinas 14 (CK 14) e 19 (CK19) e de citocromo-oxidase. Especial ênfase foi dada à avaliação da oxifilia nos nódulos, valorizando não só a oxifilia clássica (mais de 75% de células oncocíticas), mas também o que aqui chamamos de oxifilia parcial, e ao diagnóstico dos tumores foliculares de potencial incerto de malignidade. Amostras parafinadas dissecadas de carcinomas foram analisados para mutações dos genes BRAF e RAS (H-RAS, KRAS e N-RAS) mediante PCR, SSCP e seqüenciamento e para rearranjo dos genes PAX8-PPARg mediante FISH e RT-PCR. A oxifilia esteve presente, com freqüência variável, em todos os subtipos de nódulos da tireóide, benignos e malignos: 20,7 a 69,7% dos nódulos exibiram oxifilia clássica e 58,6 a 90,6% oxifilia de qualquer intensidade (clássica ou parcial). A imuno-expressão de citocromo-oxidase mostrouse associada ao achado morfológico de oxifilia, principalmente nos nódulos benignos e nos carcinomas foliculares. A CK 19 apresentou reatividade intensa apenas nos carcinoma papilíferos e em um caso de carcinoma folicular com áreas sugestivas de evolução para carcinoma pouco diferenciado. Houve associação significativa entre o diagnóstico de carcinoma papilífero e expressão de CK19 (p<0.001), mas a associação entre oxifilia e expressão de CK 19 não foi significativa. Nos carcinomas papilíferos, a oxifilia ausente ou parcial associou-se a maior freqüência de multicentricidade e a oxifilia clássica à maior idade média dos pacientes. A mutação do gene BRAF (V600E) esteve presente apenas nos carcinomas papilíferos e não teve influência nas variáveis clínico-patológicas. O rearranjo PAX8-PPARg teve incidência nas lesões foliculares oncocíticas (22,2%) bem superior à relatada na literatura. Associou-se a menor idade média dos pacientes e menor diâmetro tumoral nos carcinomas foliculares. A presença de mutações do RAS nas lesões foliculares associou-se significativamente com invasão capsular e oxifilia parcial. Todas os padrões de alterações moleculares, embora presentes, foram menos freqüentes nos tumores oncocíticos. Nossos resultados evidenciam que a oxifilia é muito freqüente em todos os subtipos de nódulos da tireóide, inclusive nos recém-descritos tumores foliculares de potencial incerto de malignidade. Merece destaque o fato de, nas lesões foliculares, a presença de oxifilia, especialmente em sua forma clássica, ter sido significativamente mais freqüente nos carcinomas, seguidos dos tumores foliculares de potencial incerto, sendo menos prevalente nos adenomas foliculares. Embora a oxifilia tenha sido associada com menor freqüência de mutações nos genes BRAF, RAS e rearranjo PAX8-PPARg, a detecção de sua coexistência com estas mutações e a presença freqüente de oxifilia em todos os tipos de nódulos da tireóide favorecem o conceito de que a oxifilia e a tumorigênese possam ser eventos distintos, muitas vezes paralelos. / A series of 205 thyroid nodules was morphologically reviewed according to the new World Health Organization (WHO, 2004) classification and analyzed regarding the immunoexpression of cytokeratins 14 (CK14) and 19 (CK19) and mitochondrial enzyme cytochrome oxidase. Special emphasis was given to the oxyphilia evaluation in the nodules, concerning not only the classical oxyphilia (more than 75% of oncocytic cells) but also the presence of smaller amounts of oncocytic cells, named herein partial oxyphilia, and to the recently purposed diagnosis of follicular tumours of unknown malignancy potential. Dissected paraffin-embedded samples of carcinomas were screened for BRAF and RAS mutations (H-RAS, K-RAS and NRAS) by PCR, SSCP and direct sequencing, whereas FISH and RT-PCR were used for assessment of PAX8-PPARg rearrangement. The oxyphilia was present, with heterogeneous frequence, in all subtypes of thyroid nodules: 20.7 to 69.7% of the nodules showed classical oxyphilia and 58.6 to 90.6% of them, oxyphilia of any intensity (classical or partial). The immunoexpression of cytochrome-oxidase was associated to the morphological finding of oxyphilia, especially in benign nodules and in follicular carcinomas. CK19 showed intense reactivity only in papillary carcinomas and in one case of follicular carcinoma with areas that could be already poorly differentiated. There was a significant association between the diagnosis of papillary carcinoma and CK 19 expression (p<0.001), but the association between oxyphilia and CK 19 expression was not statistically significant. Papillary carcinomas without or with partial oxyphilia were associated to a higher frequency of multicentricity and those with classical oxyphilia to older mean ages of patients. The BRAF gene mutation (V600E) was present only in papillary carcinomas and had no influence on clinical-pathological variables. The PAX8-PPARg rearrangement rates in oncocytic follicular lesions were higher than those previously reported. This rearrangement was associated to a younger mean age of patients and to smaller follicular carcinomas. The presence of RAS mutations in follicular lesions was significantly associated to capsular invasion and partial oxyphilia. All patterns of molecular alterations assessed herein, although present, were less frequent in oncocytic tumours. Our study further demonstrates the prevalence of classical and partial oxyphilia within all subtypes of thyroid nodules, including the recently described follicular tumours of unknown malignancy potential. A remarkable finding is that, among follicular neoplasms, oxyphilia, especially in classical pattern was significantly more prevalent in carcinomas, intermediate in follicular tumours of unknown malignancy potential and less common in adenomas. The absence of a significative relationship between oxyphilia and malignancy in most nodules is noteworthy. However, an association between oxyphilia and malignancy was observed in follicular neoplasms. Although oxyphilia has been associated with a lower mutation frequency in BRAF and RAS genes and PAX8- PPARgrearrangement, it can coexist with these mutations. This finding and the high prevalence of oxyphilia in all subtypes of thyroid nodules support the concept that oxyphilia and tumourigenesis can be distinct, and many times, parallel events.

INTRODUÇÃO: O carcinoma papilífero da glândula tireoide é a neoplasia maligna endócrina mais prevalente com incidência em torno de 95%. Apresenta alto índice de disseminação linfática cervical, principalmente para os linfonodos do compartimento central ipsilateral, no nível VI, sendo os paratraqueais e os pré-traqueais os mais acometidos. Porém, existe uma área específica, não descrita nos compêndios anatômicos, que também é sede de metástase e de recidiva tumoral e está localizada entre o nervo laríngeo recorrente direito e as artérias carótida comum direita e tireóidea inferior direita. Uma abordagem cirúrgica nessa área causa maior morbidade devido à anatomia topográfica entre essas estruturas e os linfonodos ali localizados. Dessa forma, essa área pouco explorada é objetivo desse estudo, que pretende comparar dados antropométricos e demográficos com a presença ou não de linfonodos. MÉTODOS: Estudo anatômico transversal, com dissecção de 32 cadáveres não formalizados do Serviço de Verificação de Óbitos da Capital, onde se dissecou a região cervical à direita até a exposição da região localizada entre o nervo laríngeo recorrente direito e as artérias carótida comum direita e tireóidea inferior direita, com subsequente ressecção em bloco do tecido fibrogorduroso dessa região para análise dos linfonodos após preparação específica para esse fim. RESULTADOS: As características demográficas da população estudada foram as seguintes: Idade(anos): 66,75 ± 13,89; Sexo: masculino 20/32 (62,5%) e feminino 12/32 (37,5%); Peso (Kg): 63,1 ± 12,36; Altura (metros): 1,66 ± 0,09; IMC (Kg/m2): abaixo do peso 5/32 (15,6%), normal: 20/32 (62,5%), obeso: 3/32 (9,4%), obesidade mórbida: 4/32 (12,5%); Raça: branca 21/32 (65,62%), negra 10/32 (31,25%), amarela 1/32 (3,12%); Dados antropométricos: brevilineo 8/32 (25%), normolíneo 15/32 (46,88%), longilíneo 9/32 (28,12%). A presença de linfonodos em 22/32 (68,75%), IC 95%, nos casos em que se encontraram linfonodos, a média foi de 1,65 ± 0,29 linfonodos por cadáver. Presença de linfonodos em cadáveres classificados como normolíneos, pelo teste exato de Fischer (p=0,03) e da raça branca, pelo teste exato de Fischer (p=0,04). Não se observou relação entre IMC e a presença de linfonodos, pelo teste de Sperman (p=0,461). CONCLUSÕES: A presença de linfonodos foi confirmada em 22/32 das espécimes estudadas (68,75%); Quanto às características dos linfonodos encontrados, o número variou de zero a 6 e, contando somente os casos em que os linfonodos foram achados, a média foi de 1,65 ± 0,29 linfonodos por cadáver. O tamanho variou de 0,9 a 5,39 mm no diâmetro transverso e de 1,36 a 11,64 mm no diâmetro longitudinal. Houve correlação antropométrica com presença de linfonodos nos cadáveres considerados normolíneos (p=0,03) e da raça branca (p=0,04) / INTRODUCTION: The papillary carcinoma of thyroid gland is the most prevalent endocrin malignant neoplasm, whose incidence is around 95%. This type of cancer causes increased likelihood of lymphatic spread in neck region, mainly to lymph nodes of the ipsilateral central compartiment, in level VI, such as the paratraqueal and the pretraqueal lymph nodes. However, there is a specific area, not described in the anatomic literature, that is receptor of metastasis and of tumor recurrences and is located among the right recurrent laryngeal nerve and the right common carotid and right inferior thyroid arteries. A surgical approach in this area causes more morbidity because of the topographic anatomy among those structures and the lymph nodes located there. Thus, this unexplored area is objective of this scientific study, which pretends to compare anthropometric and demographic data with the lymph nodes presence or absence. METHODS: Cross-sectional anatomic study, with dissection of 32 non-preserved corpses from the Serviço de Verificação de Óbitos da Capital, this study there had happened dissection of the right neck region until the exposition of the region located among the right recurrent laryngeal nerve and the right common carotid and right inferior thyroid arteries. Moreover, there was made resection in block of fibroadipose tissue of this region to analyse the lymph nodes after being prepared with specific material for this goal. RESULTS: Those are the demographic characteristics of this studied population: Age (in years): 66,75 ±13,89; Gender: male 20/32 (62,5%) and female 12/32 ( 37,5%); Weight(Kg): 63,1 ± 12,36; Height(meters): 1,66 ± 0,09; BMI (body mass index) (Kg/m2): underweight 5/32 (15,6%), normal: 20/32 (62,5%); obese: 3/32 (9,4%); morbid obesity: 4/32 (12,5%); Race: caucasian 21/32 (65,62%), afrodescendants 10/32(31,25%), asian 1/32 (3,12%); anthropometric data: brevilineo 8/32 (25%), normolineo 15/32 (46,88%), longilineo 9/32 (28,12%). There are lymph nodes in 22/32 (68,75%); Confidence Interval 95%, in the cases that lymph nodes were found, the average was 1,65 ± 0,29 lymph nodes for each corpse. There are lymph nodes in corpses classified as normolineos by accurate Ficher\'s Test(p= 0,03) and of the white race by accurate Ficher\'s Test(p=0,04). There is no observed correlation between BMI and lymph nodes found, by Spearman\'s correlation test( p=0,461). CONCLUSIONS: lymph nodes were found in 22/32 studied corpses (68,75%). 0 to 6 is the gap of the number of lymph nodes found in the same body. In those bodies which lymph nodes were found, the average is 1,65 ± 0,29 lymph nodes for each body. Their size varies from 0,9 to 5,39 mm in the transversal diameter and from 1,36 to 11,64 mm in the longitudinal diameter. There is a anthropometric correlation with lymph nodes presence in corpses considered as normolineos(p=0.03) and of the white race (p=0,04)

1. Sphingolipid-cholesterol domains (lipid rafts) in normal human and dog thyroid follicular cells are not involved in thyrotropin receptor signalling. <p>Thyroid hormone regulates growth and development throughout the animal kingdom. The thyroid which secretes it, is controlled by TSH and its receptor TSHR. TSH and its receptor TSHR act through TSHR-coupled G proteins to control thyroid functions, with a stronger coupling of the TSHR with Gs protein than with Gq protein in human thyrocytes. Gq is not activated by TSH/TSHR in dog, whereas dog TSHR activates it in CHO transfected cells. To better understand TSHR and its downstream effectors G proteins, we attempted to answer the questions by the role of “lipid rafts/caveolae” in TSH action.<p>Lipid rafts/caveolae are sphingolipids-cholesterol-enriched microdomains on plasma membrane that have been proposed to play a role in signal transduction. By concentrating the signal molecules, lipid rafts/caveolae increase the efficiency of the interactions between the molecules and sequestrate them from the bulk membranes. The compartmentation of signal proteins in lipid rafts/caveolae might provide a possible explanation for the relationship between TSHR and G proteins in human and dog thyroctyes.<p>To answer these questions, we first tested the existence of such lipid microdomains in human and dog thyrocytes. By northernblot and RT-PCR of caveolin-1 mRNA, we demonstrated its existence in thyrocytes. The immunohistochemistry of caveolin-1 showed that caveolin/caveolae are present on the apical membrane of thyrocytes, opposite to the TSHR localization on the basolateral membranes. The isolation of lipid rafts/caveolae by Triton X-100/OptiPrep density experiments showed that TSHR and Gq are not in the rafts, even though other proteins such as insulin receptor, flotillin-2 and partially Gs are present in these lipid domains, as expected. Testing the function of the TSH receptor on its main cascade (Gs-Adenylyl cyclase-cAMP) after treating the follicles with Methyl β-cyclodextrin (a cholesterol chelator), we observed no modification of the cAMP levels by this treatment. This is in agreement with our conclusion that the TSHR-Gs-cAMP pathway does not involve the lipid rafts/caveolae domain.<p>TSH-activated signalling does not take place in these membrane domains. Therefore, the differences between species, concerning the TSHR-G proteins coupling cannot be explained by the presence of these membrane domains.<p>2. Species specific thyroid signal transduction: conserved physiology, diverged mechanisms<p>As mentioned above, Gq proteins are activated in human but not in dog thyroid, in response to TSHR. However the dog TSH receptor is able to activate Gq, as demonstrated in transfected CHO cells. Thus, different thyroid signal transduction pathways exist in different species. <p>In this study, we investigated the effects of TSH on its two signal transduction cascades, the cAMP pathway and the phospholipase C – IP3 – DAG pathway, as measured by cAMP levels and inositol phosphate generation. We also measured the effects of TSH and of agents stimulating specifically one of these cascades, forskolin for the cAMP pathway and Ca++ ionophore (ionomycin) and phorbolmyristate ester (TPA) for the phospholipase C pathway, on markers of thyroid hormone synthesis (H2O2 generation and iodide binding to proteins) and on thyroid hormone secretion in vitro in the various thyroids. <p>We demonstrated that in all species investigated, the TSH receptor activates both hormone synthesis and secretion. While in some species, including humans, rats and mice, the TSH receptor activates both the cAMP and phospholipase C– IP3 – DAG cascades, in others (e.g. dog) it only stimulates the first. The cAMP pathway activates the limiting step in thyroid hormone synthesis, the generation of H2O2, in dog, rat and mice but not in human, pig, horse and beef. Thus physiology remains but the pathways to achieve it differ. On a practical point of view, these results allow to choose adequate animal models for investigating different aspects of human thyroid signalling.<p><p>3. Duoxes -TPO association and its regulation in human thyrocytes: the thyroxisome<p>Duox (Dual Oxidase) and TPO (thyroid peroxidase) are the crucial enzymes for the thyroid hormones biosynthesis (T3/T4). TPO uses the hydrogen peroxide (H2O2) produced by Duox1 and Duox2 isoenzymes to covalently link oxidized iodide to tyrosines of thyroglobulin and couple the iodinated tyrosines to form triiodothyronine (T3) and thyroxine (T4). An excess of H2O2 is considered to be toxic for cells although at appropriate concentrations H2O2 may carry out signalling functions. Even though thyrocytes show a better resistance to H2O2 than other cells, it would be beneficial for thyrocytes if Duox and TPO localize closely to increase the working efficiency and avoid an excessive H2O2 spillage. In this study, we explored the association of Duox with TPO, and the possible factors affecting their interaction in the human thyrocyte model. This association was established by co-immunoprecipitation approaches on purified plasma membranes from human thyrocytes and COS-7 transfected cells. <p>Our results show that 1) Duox and TPO localize closely at the plasma membranes of human thyrocytes, 2) this association is up-regulated through the Gq-PLC-Ca2+-PKC pathway and down-regulated through the Gs-cAMP-PKA pathway. 3) H2O2 directly increases the association of Duox and TPO. 4) Partial NH2- or COOH-terminal Duox1 and Duox2 proteins show different binding abilities with TPO in COS-7 transfected cells.<p>The association of the two proteins Duox and TPO thus supports our previous hypothesis of the thyroxisome, a pluriprotein plasma membrane complex in which elements of the iodination apparatus localize closely, thus optimizing working efficiency and minimizing H2O2 spillage. Defect in this association, independently of the catalytic efficiency of the enzyme, could therefore impair thyroid hormone synthesis and be harmful to thyroid cells, leading to thyroid insufficiency.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Os microRNAs são pequenos RNAs importantes na modulação da expressão gênica. O carcinoma papilífero de tireoide (CPT) é responsável pela maior parte dos casos de câncer de tireoide. Mutações somáticas ativam as vias de MAPK e PI3K/AKT e exercem papel importante no desenvolvimento do CPT. Acredita-se que miRNAs regulem genes associados a essas vias. A expressão de miRNAs foi avaliada em amostras parafinadas de CPT caracterizadas quanto à presença da mutação BRAFV600E. A expressão de miRNAs variou devido à heterogeneidade dos tecidos emblocados e entre pacientes, dados importantes quando microRNAs são avaliados como biomarcadores. Microarranjos de DNA mostraram diferenças na expressão de miR-16, miR-19a, miR-21, miR146a, miR-146b, miR-221 e miR-222 entre amostras positivas e negativas para BRAFV600E, indicando um possível papel na modulação de vias influenciadas por esta mutação. Quando amostras adicionais foram analisadas, apenas miR-146b apresentou expressão diferencial entre os dois grupos, ressaltando a variabilidade entre pacientes quanto à expressão de miRNAs. / MicroRNAs are small non-coding RNAs that regulate protein-coding genes. Papillary thyroid cancer (PTC) accounts for most of the thyroid cancer cases. Somatic mutations activate MAPK and PI3K/AKT pathways and play a leading role in PTC. MiRNAs may contribute to the regulation of these pathways. We studied the expression of miRNAs in formalin-fixed, paraffin-embedded (FFPE) PTC samples submitted to mutation characterization. Our results show that miRNA expression varies due to embedded sample heterogeneity and that there is great variability in miRNA expression among patients, both important issues when miRNA expression is evaluated as a biomarker. DNA microarray experiments compared BRAFV600E positive and negative samples. MiR-16, miR-19a miR-21, miR146a, MiR-146b, miR-221 and miR-222 were deregulated, indicating a possible implication in BRAF-related pathways. When additional samples were evaluated, only miR-146b presented consistent variation in expression, highlighting variability among patients

A l’issue d’un accident nucléaire, les produits de fission de l’uranium tel les iodes radioactifs sont dispersés dans l’environnement. L’homme est susceptible d’être exposé à ces éléments majoritairement via l’inhalation d’air et/ ou l’ingestion d’aliments contaminés. L’iode 131 est connu pour être responsable de l’augmentation de l’incidence du cancer de la thyroïde. Une des contremesures pour prévenir cette pathologie est l’ingestion de dose unique de comprimés d’iodure de potassium (KI) à fin de saturer la glande thyroïde par de l’iode stable et d’éviter ainsi l’accumulation de l’iode radioactif. Les scénarios de rejets réitérés d’iodes radioactifs lors des deux accidents majeurs Tchernobyl et Fukushima ont mis en évidence les limites de cette mesure, des prises répétées de KI pour protéger dans le temps les populations s’avèrent nécessaires. Dans la littérature on dispose de peu de données clinique et préclinique sur la prise répétée d’iode stable, quant à son usage ça n’a pas été décrit. La doctrine iode ainsi que l’autorisation de mise sur le marché (AMM) du KI envisage seulement la prise unique à renouveler exceptionnellement chez la population adulte. L’iode est connu pour être un élément clé de la fonction thyroïdienne, on jouant un double rôle à la fois de substrat de régulateur de la thyroïde. Si sa présence est indispensable à la formation des hormones thyroïdienne, son excès exerce un effet inhibiteur transitoire de cette synthèse connu sous le nom de l’effet Wolff-Chaikoff. Les hormones thyroïdiennes jouent un rôle majeur dans le développement et la fonction de presque tous les organes du corps (cerveau, cœur, os...), la moindre variation de leurs niveau peut impacter l’homéostasie du corps. Ainsi, il est difficile d’appliquer la prise répétée du KI en absence connaissances biologiques et toxicologiques. Pour combler ses lacunes et proposer une solution de prophylaxie répétée en cas d’exposition réitérée, le programme de recherche français PRIODAC : PRophylaxie répétée par l’IODe stable en situation ACcidentelle (ANR/RSNR), dont fait partie cette thèse vise à exploiter les modalités d’administration répétées du KI chez toutes les tranches d’âge (in utero, adulte et âgé), et d’évaluer la toxicologie de la prise répétée de KI sur les grandes fonctions physiologiques de l’organisme. Trois modèles de rats Wistar ont fait l’objet de ce travail de thèse : le modèle de référence rat adulte (âgé de 3 mois), le modèle à risque, organisme en développement (exposé durant la gestation) et un autre modèle à risque le rat âgé (âgé de 12 mois), ces trois modèles ont reçus 8 prise consécutive de KI 1mg/kg/24h. Les effets biologiques de ce traitement pendant 8 jours ont été évalués à long-terme (30 jours post-prophylaxie). Concernant, le modèle de référence ont n’a pas observé d’impact néfaste à long-terme de la prise répétée du KI (Lebsir, Cohen et al. 2018; Lebsir, Manens et al. 2018) par contre sur les modèles à risque plusieurs effets à long-terme ont été mis en évidence. Chez la progéniture exposée in utero, la coordination motrice ainsi que l’expression de quelques gènes clés du cerveau ont été négativement modifiées par le traitement. Chez le rat âgé la biochimie clinique, l’expression de quelques gènes clés de la fonction cardiovasculaire ainsi que le système rénine-angiotensine-aldostérone ont été significativement impacté par le traitement. En conclusion, les résultats obtenus montrent l’innocuité sur le plan toxicologique du KI administré à 1mg/kg toutes les 24h pendant 8 jours chez le modèle adulte et la nocuité de ce schéma prophylactique chez les modèles à risque in utero et âgé. Ces résultats ont été communiquées à la pharmacie centrale des armées (producteur et détentrice de l’AMM) afin de servir de données d’entrée pour des études de bonne pratique de laboratoire notamment pour le modèle adulte et également, afin de contribuées à l’évolution de la doctrine de l’iode en terme de radioprotection. / Following nuclear accidents, uranium fission products such as radioactive iodines are released into the environment. Humans are likely to be exposed to these elements mainly through inhalation of air and / or ingestion of contaminated food. Iodine 131 is known to be responsible for increasing the incidence of thyroid cancer. One of the available countermeasures is the ingestion of a single dose of potassium iodide (KI) tablets to saturate the thyroid gland with stable iodine and thus prevent the uptake of the radioactive isotope.Repeated releases of radioactive iodine during the two major accidents Chernobyl and Fukushima have highlighted the weaknesses of this measure, repeated intake of KI maybe necessary. In the literature there is little clinical and preclinical data on the repeated intake of stable iodine, regarding its use it has not been described. The iodine doctrine as well as the Marketing Authorization (MA) of the KI considers only the single taking, to renew exceptionally in the adult population. Iodine is known to be a key component of thyroid function, playing a dual role as both a substrate and a regulator of the thyroid. If its presence is essential for the synthesis of thyroid hormones, its excess exerts a transient inhibitory effect on this synthesis known as the Wolff-Chaikoff effect. Thyroid hormones affect the development and function of almost all organs of the body (brain, heart, bone ...), the slightest variation in their level can impact the homeostasis of the body. Hence, the toughness of applying repeated KI intake in the absence of biological and toxicological data. To fill this gap and find a solution in the event of repeated exposure, the French research program PRIODAC: repeated potassium iodide prophylaxis in accidental situation (ANR / RSNR), of which this thesis is part, aims to define the modalities of repeated administration of KI in all age groups (in utero, adult and elderly), and evaluate the biological consequences on the the body’s major physiological functions. Three models of Wistar rats were used in this work: the reference model adult rat (3 months), the first sensitive model offspring (exposed during gestation) and another sensitive model the older rat (12 months ), these three models received 8 consecutive intake of KI 1mg / kg / 24h. And the effects were assessed at long-term (30 days post-prophylaxis). On the reference model, there was no long-term adverse impact of repeated KI intake (Lebsir, Cohen et al. 2018; Lebsir, Manens et al. 2018). On the other hand, sensitive models reveal several long-term effects; the offspring exhibited impaired motor coordination and variation of the expression of some key brain genes. And in the elderly rat urinary biochemistry, expression of some key genes of the cardiovascular function, as well as the renin-angiotensin-aldosterone system were significantly impacted by the treatment. In conclusion, the results obtained show the toxicological safety of KI administered at 1 mg / kg every 24 hours for 8 days in the adult model, and the harm of this prophylactic scheme in sensitive models in utero and elderly. These results were sent to the French central pharmacy of armies and will serve as input data for good laboratory practice studies that eventually will contribute to the evolution of the iodine doctrine and KI MA.

Les espèces réactives de l’oxygène ont initialement été identifiées comme des produits délétères dérivés du métabolisme aérobie. Il est maintenant accepté que ces espèces sont produites de manière régulée par des enzymes et interviennent dans des fonctions cellulaires telles que la défense immunitaire, la signalisation intracellulaire, la biosynthèse des hormones et la modification de matrice extracellulaire. Les NADPH (Nicotinamide Adénine Dinucléotide Phosphate) oxydases (NOX) forment une famille d’enzymes transmembranaires capables de former de l’anion superoxyde (O2.-) par transfert d’électrons du NADPH à l’oxygène moléculaire (O2). DUOX1 et DUOX2 sont deux des sept membres composant cette famille qui génèrent directement de l’H2O2 comme produit de réduction de l’O2.<p>Initialement clonés à partir de la thyroïde dans notre laboratoire, les ADNc codant pour les protéines DUOX ont été identifiées dans d’autres tissus, comme par exemple la prostate ou l’épithélium respiratoire où DUOX1 est majoritaire. DUOX2 se retrouve également dans les glandes salivaires, dans la muqueuse rectale et tout le long du tractus digestif. D’autre part, un orthologue de DUOX, appelé Udx1, a été identifié en 2004 au niveau de la membrane ovocytaire chez l’oursin. Dans chacun de ces tissus, l’H2O2 produit par les protéines joue un rôle clef. <p>Le mécanisme d’activation de DUOX dans tous ces tissus n’a été identifié que récemment. En effet, pour être exprimé sous forme active à la surface cellulaire, les protéines DUOX nécessitent un facteur de maturation spécifique. Ces facteurs, appelés DUOXA1 et 2 pour « DUOX activator », suivent l’expression tissulaire de leur DUOX respectif. Nous avons montré que la région COOH-terminale de DUOXA1 est responsable de l’activité génératrice d’H2O2 de DUOX1. DUOX2 peut produire de l’H2O2 ou de l’O2.-. L’extrémité NH2-terminale de DUOXA2 est critique dans cette activité et détermine le type de dérivé oxygéné produit. Dans notre système, DUOXA2 n’est pas détecté à la surface cellulaire, sauf en cas de modification de son extrémité amino-terminale par l’addition d’un épitope. DUOXA1 peut être exprimé à la membrane plasmique mais sa présence n’est pas nécessaire au sein du complexe formé avec DUOX pour que ce dernier soit actif. Les facteurs de maturation jouent donc un rôle de protéine chaperonne, induisant la maturation et la translocation d’une protéine DUOX active à la surface cellulaire. <p>Dans la thyroïde, l’H2O2 produit par les protéines DUOX constitue le cofacteur de la thyroperoxydase catalysant l’oxydation de l’iode et le couplage de sa forme oxydée sur des résidus tyrosines de la thyroglobuline menant in fine à la synthèse des hormones thyroïdiennes T3 et T4 et leur relarguage dans la circulation sanguine. Plusieurs mutations dans le gène DUOX2 ont déjà été décrites chez des patients atteints de dyshormonogenèse transitoire ou permanente. Nous avons mis en évidence qu’une inactivation totale de la protéine DUOX2 était compatible avec un état hypothyroïdien peu sévère et transitoire, indiquant l’intervention probable de DUOX1 dans la synthèse des hormones thyroïdiennes. Le défaut génétique identifié est composé d’une délétion génomique partielle d’un allèle associée à une mutation faux-sens (G1518S) sur l’autre allèle du patient hypothyroïdien. Cette mutation, située dans le site catalytique de l’enzyme, mène à une abolition de l’activité de l’enzyme qui est néanmoins exprimée partiellement à la surface cellulaire. <p>Les messagers des DUOX ont été identifiés récemment dans les tractus digestif et respiratoire. Le rôle joué par l’H2O2 dans ces tissus semble avant tout être un rôle de défense contre les micro-organismes en mettant en jeu la lactoperoxydase oxydant le thiocyanate en composé bactéricide actif. Nous avons montré que l’H2O2 produit par DUOX exerce un effet répulsif sur les bactéries. En effet, l’invasion de cellules CHO exprimant de manière stable DUOX2 et DUOXA2 par Salmonella Typhimurium est diminuée lorsque la production d’H2O2 de ces cellules est stimulée. Cet effet répulsif constituerait un rôle primordial pour DUOX au niveau des muqueuses respiratoire et digestive.<p>Lors de la fertilisation, une explosion respiratoire a lieu et de l’H2O2 est produit. Cet H2O2 fourni à l’ovoperoxydase permettrait la formation d’une enveloppe rigide autour de l’ovocyte, bloquant ainsi l’entrée de spermatozoïdes surnuméraires. Ce phénomène a été largement étudié dans l’ovocyte d’oursin, dans lequel la NADPH oxydase responsable de la production d’H2O2 a été caractérisée: il s’agit de Udx1, l’orthologue de DUOX. Chez les mammifères, le phénomène existe mais le mécanisme est en grande partie inconnu. Nous avons montré que les ARNm des DUOX sont exprimés dans l’ovocyte humain ;ceci nous permet d’émettre l’hypothèse que l’inhibition de la polyspermie chez l’homme pourrait être similaire à celle de l’oursin. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished