Lee Mei Lan May. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 48-56). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 中文摘要 --- p.III / ACKNOWLEDGEMENTS --- p.IV / PUBLICATION --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATION --- p.VIII / CONTENTS --- p.IX / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- THYROID CANCER AND ITS EPIDEMIOLOGY --- p.1 / Chapter 1.1.1 --- Histology --- p.1 / Chapter 1.1.2 --- Gender comparison --- p.3 / Chapter 1.1.3 --- Female hormone 一 risk factor --- p.6 / Chapter 1.2 --- BIOLOGICAL BACKGROUND OF HORMONE´-´ؤؤ --- p.7 / Chapter 1.2.1 --- Estrogen --- p.8 / Chapter 1.2.2 --- Estrogen antagonist --- p.9 / Chapter 1.2.3 --- Estrogen receptors --- p.10 / Chapter 1.2.4 --- Estrogen receptor and thyroid cancer --- p.12 / Chapter 1.3 --- THE ROLE OF APOPTOSIS --- p.13 / Chapter 1.3.1 --- Bcl-family protein --- p.13 / Chapter 1.3.2 --- Bcl-family protein and cancer --- p.14 / Chapter 1.3.3 --- Estrogen and Bcl-family protein --- p.15 / Chapter 1.4 --- Objectives --- p.16 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS / Chapter 2.1 --- MATERIALS --- p.17 / Chapter 2.1.1 --- Culture media and treatment reagents --- p.17 / Chapter 2.1.2 --- Reagents for Western blot assay --- p.18 / Chapter 2.1.3 --- Antibodies --- p.19 / Chapter 2.1.4 --- Materials for RT-PCR --- p.19 / Chapter 2.1.5 --- Kits --- p.20 / Chapter 2.1.6 --- Instrumentations --- p.20 / Chapter 2.2 --- Methods --- p.21 / Chapter 2.2.1 --- Cell culture and treatment --- p.21 / Chapter 2.2.2 --- MTT assay --- p.22 / Chapter 2.2.3 --- Western blot analysis --- p.23 / Chapter 2.2.3.1 --- Protein extraction --- p.23 / Chapter 2.2.3.2 --- SDS-PAGE and protein transfer --- p.23 / Chapter 2.2.3.3 --- Immunoblotting analysis --- p.24 / Chapter 2.2.4 --- RNA extraction and RT-PCR --- p.25 / Chapter 2.2.4.1 --- RNA extraction --- p.25 / Chapter 2.2.4.2 --- cDNA synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase Chain Reaction (PCR) --- p.27 / Chapter 2.2.5 --- Statistical analysis --- p.28 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Effect of E2 and tamoxifen on proliferation --- p.29 / Chapter 3.2 --- Comparison of effects of E and testosterone on Proliferation --- p.31 / Chapter 3.3 --- Differential Bcl-xL expression in response to E2 and testosterone stimulation --- p.33 / Chapter 3.4 --- Expression of ERα and ERβ in response to E2 stimulation --- p.35 / Chapter 3.5 --- Bcl-xL and Bax protein expression in response to E2 stimulation --- p.36 / Chapter 3.6 --- Expression of Bcl-xL and Bax mRNA in response to E2 stimulation --- p.39 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.41 / Chapter CHAPTER FIVE: --- CONCLUSION --- p.47 / REFERENCES --- p.48

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Ho Wing Man. / On t.p. "gamma" appears as the Greek letter. / Thesis submitted in: December 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 95-106). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGMENTS --- p.V / ABBREVIATIONS --- p.VI / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.X / CONTENTS --- p.XII / Chapter CHAPTER ONÉؤ --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.1.1 --- Thyroid cancer --- p.2 / Chapter 1.1.2 --- Apoptosis and thyroid cancer --- p.4 / Chapter 1.2 --- Estrogen receptors and apoptosis --- p.5 / Chapter 1.2.1 --- Estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) --- p.5 / Chapter 1.2.2 --- Differential roles of estrogen receptor-α(ERα) and estrogen receptor-β (ERβ) in apoptosis --- p.6 / Chapter 1.2.3 --- Bcl-2 family --- p.8 / Chapter 1.3 --- Peroxisome proliferator-activated receptor-γ (PPARγ) --- p.9 / Chapter 1.3.1 --- Molecular aspects of PPAR --- p.9 / Chapter 1.3.2 --- PPAR/RXR complex --- p.13 / Chapter 1.3.3 --- PPARγ ligands --- p.16 / Chapter 1.3.4 --- PPARγ and apoptosis in thyroid cancer --- p.19 / Chapter 1.3.5 --- PPARγ ligands-mediated apoptosis pathway --- p.21 / Chapter 1.4 --- Previous results from our laboratory --- p.25 / Chapter 1.5 --- Summary of previous studies --- p.27 / Chapter 1.6 --- Perspectives --- p.28 / Chapter 1.7 --- Objectives of this project --- p.29 / Chapter CHAPTER TWÓؤ --- GENERAL MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Cell lines --- p.31 / Chapter 2.1.2 --- Plasmid vectors used in this study --- p.31 / Chapter 2.1.3 --- Antibodies --- p.32 / Chapter 2.1.4 --- Culture media and transfection reagents --- p.32 / Chapter 2.1.5 --- Materials for protein manipulation --- p.33 / Chapter 2.1.6 --- Drugs for treatment --- p.34 / Chapter 2.1.7 --- Kits --- p.35 / Chapter 2.1.8 --- Instruments --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Cell culture --- p.36 / Chapter 2.2.2 --- Cell viability analysis --- p.36 / Chapter 2.2.3 --- Preparation of protein extract --- p.37 / Chapter 2.2.4 --- Determination of the concentration of target protein --- p.37 / Chapter 2.2.5 --- Gel electrophoresis and protein transfer --- p.38 / Chapter 2.2.6 --- Immunoblotting --- p.39 / Chapter 2.2.7 --- Apoptosis detected by Cell Death ELISAplus --- p.41 / Chapter 2.2.8 --- PPARγ-ligand Enzyme Immunoassay --- p.45 / Chapter 2.2.8.1 --- 15d-PGJ3 Enzyme Immunoassay --- p.45 / Chapter 2.2.8.2 --- 15(S)-HETE Enzyme Immunoassay --- p.46 / Chapter 2.2.8.3 --- 13(S)-HODE Enzyme Immunoassay --- p.46 / Chapter 2.2.9 --- Transient tranfection and luciferase activity assay --- p.47 / Chapter 2.2.10 --- Statistical Analysis --- p.52 / Chapter CHAPTER THREÉؤ --- ESTROGEN RECEPTORa (ERa) AND ESTROGEN RECEPTORP(ERP) MEDIATE THE PROLIFERATION AND APOPTOSIS OF HUMAN THYROID PAPILLARY CARCINOMA CELLS --- p.53 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Cell culture and treatment --- p.56 / Chapter 3.2.2 --- Western Blot --- p.56 / Chapter 3.2.3 --- Cell proliferation determined by MTT assay --- p.57 / Chapter 3.2.4 --- Apoptosis detected by Cell Death ELISAplus assay --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- "The expression of ERα, ERβ and PPARγ in NPA, FRO, ARO and WRO thyroid cancer cell lines" --- p.59 / Chapter 3.2.2 --- Effects of PPT and DPN on cell viability --- p.61 / Chapter 3.3.3 --- Apoptotic cells quantification by Cell Death ELISAplus assay --- p.64 / Chapter 3.4 --- Discussion --- p.67 / Chapter CHAPTER FOUŔؤ --- THE RELATIONSHIP BETWEEN PPARγ AND ESTROGEN RECEPTOR AND THE REGULATION OF THE APOPTOSIS IN THYROID CANCER CELL LINES --- p.70 / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Material and Methods --- p.74 / Chapter 4.2.1 --- Transient transfection --- p.74 / Chapter 4.2.2 --- Luciferase assay --- p.74 / Chapter 4.2.3 --- 15d-PGJ2 ELISA assay --- p.75 / Chapter 4.2.4 --- 15S-HETE ELISA assay --- p.76 / Chapter 4.2.5 --- 13S-HODE ELISA assay --- p.77 / Chapter 4.3 --- Results --- p.78 / Chapter 4.3.1 --- "PPT, ERα-agonist, increased thyroid cancer cell proliferation and caused the decrease the level of PPARγ ligands" --- p.78 / Chapter 4.3.2 --- "DPN, ERβ-agonist, inhibited thyroid cancer cell proliferation, induced apoptosis and caused the increase the level of PPARγ ligands" --- p.83 / Chapter 4.3.3 --- PPT did not alter the transcriptional activity of PPARγ --- p.88 / Chapter 4.4 --- Discussion --- p.90 / Chapter CHAPTER FIVÉؤ --- CONCLUSIONS AND FUTURE PROSPECT --- p.92 / Chapter 5.1 --- Summary of results --- p.93 / Chapter 5.2 --- Conclusion --- p.94 / Chapter 5.3 --- Future prospects --- p.94 / REFERENCE LIST --- p.95

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

MILHOME, Marcus Vinicius Liberato. Interação da frutalina com neoplasias de tireóide. Estudo comparativo com marcadores tumorais em uso. 2008. 112 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2008. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-20T17:58:10Z No. of bitstreams: 1 2008_tese_mvlmilhome.pdf: 9609801 bytes, checksum: c941b15b38e13278f721f7bd633a3802 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-26T20:27:26Z (GMT) No. of bitstreams: 1 2008_tese_mvlmilhome.pdf: 9609801 bytes, checksum: c941b15b38e13278f721f7bd633a3802 (MD5) / Made available in DSpace on 2016-07-26T20:27:26Z (GMT). No. of bitstreams: 1 2008_tese_mvlmilhome.pdf: 9609801 bytes, checksum: c941b15b38e13278f721f7bd633a3802 (MD5) Previous issue date: 2008 / Increased interest has been devoted to cell surface carbohydrates, since they are related to cell differentiation and maturation. Cell transformation in malignant tumors is associated to altered surface membranes, particularly with an abnormal composition of glycoconjugates, determining characteristics of neoplastic cells as invasive behavior and metastasis. Lectins, proteins widely distributed in the plant and animal kingdom, have the distinctive property of binding to carbohydrates of specific structure and configuration. Lectins have been used as tools in many areas of diagnostic investigation especially related to changes in the expression of membrane and cytoplasmatic carbohydrates. The aim of this work was to compare the binding pattern of the biotinilated frutalin, Artocarpus incisa α-galatose binding lectin (FTL-B), with the markers usually available (CK19, HBME-1, GAL3) and the new papillary thyroid carcinoma marker 373-E1 in various thyroid lesions and normal thyroid tissue. FTL-B was obtained by affinity chromatography and biotinilated. CK19, GAL3 and HBME-1 were obtained from Novocastra Laboratories, Ltd., UK and 373-E1 from Labvision, UK. Formalin-fixed, paraffin-embedded thyroid tissues were used to build tissue macro-arrays that were cut at 2 µm and stained using FTL-B, CK19, GAL3, HBME-1 and 373-E1 by strepABC-HRP method. The cases included papillary carcinoma (90), follicular carcinoma (6), Hürthle cell carcinoma (2), anaplastic carcinoma (4), nodal metastasis of papillary carcinoma (6), Hürthle cell adenoma (2), follicular adenoma (3), goiter (4) and Hashimoto’s thyroiditis (3). There was no staining on benign thyroid lesions or normal thyroid tissue using FTL-B or the usual markers. Papillary carcinoma and metastasis from papillary carcinoma showed strong membranar stain using FTL-B. FTL-B and the usual markers showed similar binding pattern suggesting that FTL-B could be used as a thyroid tumoral marker similar to GAL3, CK19, HBME-1 and 373-E1. / O interesse nos carboidratos de superfície celular tem aumentado devido ao fato de estarem relacionados a diferenciação e maturação celular. A transformação celular em neoplasias malignas está associado a alterações na superfície de membranas celulares, particularmente na composição dos glicoconjugados, determinando sua capacidade em relação a invasividade e potencial metastático. Lectinas, proteinas amplamente distribuidas nos reinos animal e vegetal, tem a capacidade de ligarem-se a carboidratos de estrutura e configuração específicas. Tem sido utilizadas como ferramentas em muitas áreas de investigação diagnóstica especialmente naquelas relacionadas a mudanças na expressão de carboidratos no citoplasma e na membrana celulares. O objetivo deste trabalho foi comparar o padrão de ligação da frutalina, lectina α-galactose ligante de Artocarpus incisa, biotinilada (FTL-B) com os marcadores usuais (CK19, GAL3, HBME-1) e o novo marcador para carcinomas papilares da tireóide, 373-E1, em várias lesões malignas e benignas da tireóide e em tecido tireoidiano normal. FTL-B foi obtida por cromatografia de afinidade e biotinilada. CK19, GAL3 e HBME-1 foram obtidos de Novocastra Laboratories, Ltd., UK e 373-E1 de Labvision, UK. Lesões tireoidianas e tecido tireoidiano normal em parafina foram utilizados para a construção de tissue micro-arrays os quais foram cortados a 2 µm e marcados com CK19, GAL3, HBME-1 e 373-E1 pelo método imuno-histoquímico da estreptoavidina-biotina-peroxidase e com FTL-B por método lectino-histoquímico. As lesões incluíam carcinomas papilares (90 casos), carcinomas foliculares (6 casos), carcinoma de células de Hürthle (2 casos), carcinoma anaplásico (4 casos), metástases linfonodais de carcinomas papilares (6 casos), adenoma de células de Hürthle (2 casos), adenomas foliculares (3 casos), bócio (4 casos) e tireoidite de Hashimoto (3 casos). Não houve marcação em tecido tireoidiano normal ou nas lesões benignas usando FTL-B ou os marcadores usuais. Carcinomas papilares e metástases de carcinomas papilares marcaram fortemente a membrana celular com FTL-B. Os marcadores usuais e FTL-B mostraram padrões de marcação semelhantes, sugerindo que FTL-B poderia ser usada como um marcador tumoral da mesma forma que CK19, GAL3, HBME-1 e 373-E1.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Orientador: Gláucia Maria Ferreira da Silva Mazeto / Resumo: Contexto: O carcinoma medular de tireoide (CMT) é um tumor raro, cujo diagnóstico precoce é extremamente relevante uma vez que sua evolução pode ser bastante agressiva. A dosagem sérica de calcitonina pode indicar o diagnóstico do CMT e é utilizada como marcador da presença de doença durante o seguimento dos pacientes. No Hospital das Clínicas da Faculdade de Medicina de Botucatu (HC-FMB), a dosagem do hormônio passou a ser realizada em 2008, sendo sua solicitação de livre acesso. Objetivos: Avaliar as solicitações de dosagem de calcitonina sérica no HC-FMB, independentemente do diagnóstico ou procedimento terapêutico realizados. Pacientes e Métodos: Foram avaliadas, retrospectivamente, todas as dosagens de calcitonina realizadas no Laboratório de Análises Clínicas do HC-FMB, no período de setembro de 2008 a junho de 2017. Foram então comparados os casos com calcitonina detectável ou não, aqueles com e sem CMT e os casos com solicitação de dosagem considerada adequada ou inadequada/não informada. Foi ainda quantificado o custo com as dosagens adequadas e inadequadas/não informadas. Resultados: Foram realizadas 1453 dosagens de calcitonina, em 915 pacientes, com concentrações detectáveis que variaram de 2,00 pg/mL a 527,00 pg/mL (mediana= 4,1 pg/mL). A mais frequente justificativa da coleta foi nódulo tireoidiano (73,9%). Dezenove pacientes (2,1%) apresentaram diagnóstico final de CMT. A maior parte dos casos apresentou calcitonina indetectável (82,7%). A maioria dos pacientes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Context: Medullary thyroid cancer (MTC) is a rare tumor, which early diagnosis is extremely relevant since its evolution can be quite aggressive. Serum calcitonin dosage may indicate the diagnosis of MTC and is used as a marker of the disease presence during the patients’ follow-up. At the Clinics Hospital of the School of Medicine of Botucatu (HC-FMB), the dosage of the hormone was first carried out in 2008, being its request of free access. Objectives: To evaluate the serum calcitonin dosage requirements in the HC-FMB, regardless of the diagnostic or therapeutic procedure carried out. Patients and Methods: All calcitonin dosages carried out at the Clinical Analysis Laboratory of HC-FMB from September 2008 to June 2017 were retrospectively evaluated. The cases with or without detectable calcitonin, as well as the cases with or without MTC, and the cases with adequate or inadequate/not informed dosage request were then compared. Costs with appropriate and inadequate/not informed dosages were also quantified. Results: 1453 calcitonin dosages have been carried out in 915 patients with detectable concentrations ranging from 2.00 pg/mL to 527.00 pg/mL (median = 4.1 pg/mL). The most frequent justification for the collection was thyroid nodule (73.9%). Nineteen patients (2.1%) were eventually diagnosed with MTC. The majority of cases had undetectable calcitonin (82.7%). The majority of MTC patients (73.7%) had detectable calcitonin, and 42.1% of those patients had concentrations ab... (Complete abstract click electronic access below) / Mestre

Alterações genéticas nos genes RET, RAS e BRAF resultam na ativação constitutiva da sinalização MAPK e estão presentes em aproximadamente 70% dos carcinomas papilífero de tiroide, a forma mais prevalente de câncer de tiroide. Múltiplas vias de sinalização podem atuar em conjunto com a via MAPK na oncogênese tiroidiana. Nesse estudo, testamos a hipótese que a via MAPK regula a sinalização Notch e que o crosstalk entre as vias de sinalizações são importantes na regulação da diferenciação e proliferação celular no câncer de tiroide. A ativação condicional dos oncogenes RET/PTC3 e BRAFT1799A em linhagem de célula folicular normal de tiroide aumentou a atividade da via de sinalização Notch. Por outro lado, o bloqueio farmacológico da sinalização MAPK reduziu a sinalização Notch na linhagem celular TPC-1 derivada de carcinoma papilífero de tiroide. Glândulas tiroide de animais transgênicos expressando BRAFT1799A e amostras de carcinoma papilífero de tiroide apresentaram elevados níveis de NOTCH1. A superexpressão de NOTCH1 em célula folicular normal de tiroide aumentou a expressão proteica de NIS. A inibição farmacológica e por RNA de interferência da sinalização Notch apresentou um efeito anti-proliferativo em linhagem de CPT. Além disso, a combinação do inibidor farmacológico de Notch e MAPK diminuiu a proliferação de células de carcinoma papilífero de tiroide. Esses dados sugerem um importante papel da sinalização Notch na oncogênese do carcinoma papilífero de tiroide induzida pela sinalização MAPK e que a via Notch pode ser uma potencial terapia adjuvante no câncer de tiroide. / Genetic alterations in RET, RAS and BRAF result in constitutive activation of the MAPK signaling and are present in approximately 70% of papillary thyroid carcinomas, the most prevalent form of thyroid cancer. Multiple signaling pathways can act with MAPK pathway in thyroid oncogenesis. In this study, we tested the hypothesis that MAPK pathway control Notch signaling and that the crosstalk between these pathways plays an important role in thyroid cancer cell differentiation and proliferation. The conditional activation of RET/PTC3 and BRAFT1799A enhanced Notch signaling pathway in normal follicular thyroid cell. By contrast, pharmacological inhibition of MAPK reduced Notch signaling in TPC-1 cell line derived from papillary thyroid carcinoma. Transgenic mice expressing BRAFT1799A restrict in thyroid gland and human papillary thyroid carcinoma samples showed higher Notch1 expression. NOTCH1 overexpression in normal thyroid follicular cell increased NIS protein expression. Pharmacological inhibition and RNA interference of Notch signaling showed an anti-proliferative effect in papillary thyroid carcinoma cells. Furthermore, the combination of MAPK and Notch signaling inhibitors reduced papillary thyroid carcinoma proliferation. These data suggest an important role of Notch signaling in papillary thyroid carcinoma induced by MAPK-related oncogenes and that Notch signaling pathway could be a potential adjuvant therapy in thyroid cancer.

O iodo é transportado para a célula tireoideana através do simportador sódio-iodo (NIS). Estudo anterior de nosso grupo mostrou que a expressão do RNAm de NIS estava reduzida nos tecidos tumorais (T) tireoideanos quando comparada à dos tecidos não tumorais (NT) adjacentes. A metilação de ilhas CpG localizadas em promotores é um dos mecanismos epigenéticos que regula a expressão gênica. Já foi demonstrado que a metilação aberrante desempenha papel importante na tumorigênese humana, porém, estudos qualitativos não conseguiram definir um padrão de metilação do promotor de NIS no câncer diferenciado de tireóide. Assim, o presente estudo visou (1) investigar o padrão de metilação do promotor do gene NIS em amostras T (benignos e malignos) e NT adjacentes, e correlacionar o grau de metilação com os valores de RNAm, e (2) identificar novas sequencias regulatórias, alvos de metilação do DNA, que pudessem também modular a expressão de NIS. Foram estudados 30 pares de amostras de tecido tireoideano T e NT adjacente, sendo 10 benignos e 20 malignos. Também foi avaliado um par de amostras de paciente com nódulo maligno com alta captação de iodo (hipercaptante). A metilação da ilha1 CpG foi avaliada por PCR Metilação Específica (MSP) semiquantitativa e a metilação da ilha2 CpG foi analisada por bissulfito seqüenciamento; ambas com DNA bissulfito convertido. Novas ferramentas de bioinformática foram utilizadas na análise in silico para identificar ilhas CpG funcionais na região 5\' upstream da seqüência promotora de NIS. Plasmídios foram construídos contendo o fragmento de DNA da Ilha2 CpG na frente de gene reporter (luciferase) e usados em transfecções de células HEK293 para avaliar a atividade regulatória desta ilha. Celulas tumorais com baixa expressão de NIS foram tratadas com agente desmetilante 5Aza e a expressão de RNAm de NIS foi avaliada por PCR em tempo real. Na ilha1 CpG, todas as amostras (T e NT) do grupo benigno e maligno apresentaram metilação, não observando-se diferença no grau de metilação entre os grupos. No entanto, maiores níveis de metilação foram detectados na região 5 desta ilha, que decresceram na direção da extremidade 3. Observou-se maior grau de metilação no tecido T em relação ao tecido NT adjacente (T>NT) em 60% das amostras do grupo benigno e em 35% do grupo maligno. Não foi identificada correlação entre o grau de metilação da ilha1 CpG e a expressão de RNAm de NIS. Nas amostras (T e NT) do paciente com tumor hipercaptante não foi detectada metilação nesta ilha. Assim foi realizada nova análise in silico, a qual identificou por primeira vez uma segunda ilha CpG, ilha2 CpG, com 256pb, localizada entre as posições -2152 e -1887 (em relação ao sítio ATG), contendo 14 sítios CpG. As analises da metilação da ilha2 CpG indicaram que essa região também é alvo de metilação, sendo observada hipermetilação (66.07%) em todas as amostras T quando comparada às amostras NT(23,21%), nos grupos benigno e maligno. A análise individual de cada par de amostras detectou um padrão de metilação do gene NIS em tecidos tireoideanos; sendo que 100% das amostras do grupo benigno e 95% das do grupo maligno apresentavam a ilha2 CpG mais metilada no tecido T do que no tecido NT (T>NT). Além disso, foi possível identificar correlação inversa significativa entre o grau de metilação da ilha2 CpG e a expressão gênica de NIS. Estudos in vitro para caracterização funcional da ilha CpG2 mostraram que esta nova ilha pode ser considerada como um enhancer do gene NIS na presença de fatores de transcrição da tireóide. Também foi possível detectar, em linhagem de carcinoma folicular da tireoide, a reexpressão do RNAm de NIS paralelamente à redução do grau de metilação da ilha2 CpG, após o tratamento com agente desmetilante. Sendo assim, confirmamos que a metilação do promotor NIS é um evento freqüente em amostras T (benignas e malignas) e NT. Este é o o primeiro trabalho que descreve a existência da ilha2 CpG, na região 5 do gene NIS, com atividade de enhancer, que regularia a expressão gênica por metilação do DNA. Pela primeira vez identificou-se a existência de um padrão de metilação no gene NIS em tecido tireoideanos (T>NT), assim como correlação inversa entre o grau de metilação da ilha2 CpG e a expressão de RNAm de NIS. Estes resultados poderiam explicar a reduzida expressão do NIS observada em amostras tumorais benignas e malignas e a baixa captação de iodo dos tumores tireoideanos / Iodide is transported from the blood into thyroid cell through the sodium iodide symporter (NIS). We have previously detected reduced NIS mRNA expression in thyroid tumoral tissue (T) when compared with the non tumoral tissue (NT). Methylation of DNAs CpG islands, very common in promoters, is an epigenetic alteration that regulates gene expression. Aberrant gene methylation plays an important role in human tumorigenesis. Previous qualitative studies have failed to define a NIS promoter methylation pattern in thyroid cancer. This study aimed (1) to investigate the methylation pattern of the NIS gene promoter in thyroid tumors (benign and malignant), and to correlate the methylation status with cellular levels of NIS mRNA, in T and adjacent NT samples; (2) to identify new regulatory DNA sequences that could modulate NIS gene expression by methylation of the DNA. Thirty pairs of thyroid samples (tumoral and non-tumoral), 10 benign and 20 malignant tumors were included. A pair of samples of a malignant nodule with high iodide uptake was also included. Methylation of CpG Island1 was evaluated by semiquantitative Methylation Specific PCR (MSP) and methylation of CpG island2 was analyzed by bisulfite sequencing; both using bisulfite converted DNA. New bioinformatic tools were used in in silico analysis to identify functional CpG islands in the 5 upstream region of the NIS promoter. Plasmids were constructed containing the CpG island2 DNA fragment in front of a reporter gene (lucyferase) and used in transfections assays of HEK293 cells to assess the regulatory activity of this island. Tumor thyroid cell cultures with low NIS expression were treated with the demethylating agent 5Aza and NIS mRNA expression was quantified by real time PCR. Methylation of CpG island1 was detected in all the samples (NT and T) of benign and malignant group, no differences were observed in the methylation degree between the groups. However, highest methylation levels were detected in the 5 region of CpG island1, which decreased to the 3 end. The analysis of each pair of samples revealed higher methylation degree in T tissue with respect to NT adjacent tissue (T> NT) in 60% of the benign group and in 35% of the malignant group. No quantitative correlation was found between CpG island1 methylation and NIS mRNA levels in both groups. Methylation was not detected in the NT and T samples of the patient with high iodine uptake. Furthermore, new in silico analysis allowed to identify a second CpG island, CpG island2, with 256pb, located between positions - 2152 and -1887 (relative to ATG), containing 14 CpG sites. The analysis of the CpG island2 indicated that this region is also a target of DNA methylation. Hypermethylation was observed in all T samples (66.07%) when compared with NT samples (23.21%), in benign and malignant groups. The analysis of each pair of samples allowed to detect a NIS gene methylation pattern in thyroid tissues. In 100% of the benign group and in 95% of the malignant group methylation degree of the CpG island2 was higher in the T tissue as compared to NT tissue (T>NT). Moreover, it was possible to identify significant negative correlation between the methylation degree of CpG island2 and NIS mRNA gene expression. In vitro functional characterization of the CpG island2 showed that this new island may be considered an enhancer of NIS gene in the presence of thyroid transcription factors. The treatment of follicular thyroid carcinoma cells with demethylating agent restored the NIS mRNA expression, in parallel with the reduction of the CpG island2 methylation degree. In conclusion, we confirm that the NIS promoter methylation is a frequent event in thyroid T (benign and malignant) and NT samples. This is the first description of a new second CpG island in the 5\'region of the NIS gene, with enhancer activity, which may regulate gene expression by DNA methylation. This is the first report that described the existence of a NIS gene methylation pattern of in thyroid tissue (T>NT) as well as a negative correlation between the methylation degree of CpG island2 and NIS mRNA expression. These results may explain the reduced NIS expression observed in benign and malignant tumor samples as well as the low iodine uptake of thyroid tumors