Orientador: Vania dos Santos Nunes Nogueira / Abstract: Although being controversial, some services use Whole Body Imaging (WBI) with 131I prior to the ablative dose with this radiopharmaceutical in the treatment of differentiated thyroid cancers (DTC). Proponents of this approach argue for a better optimization of the ablative dose, and opponents argue that this WBI could lead to the "stunning effect" characterized by reduced tissue uptake of 131I in ablative dose treatment, thereby compromising treatment and prognosis of these individuals. Objective: To evaluate whether WBI with 131I, ablative pre-dose, interferes with the efficacy of the therapeutic dose of iodine as remission of the disease after total or near total thyroidectomy in individuals with DTC. Methodology: A systematic review of the literature was conducted in which randomized, non-randomized and observational studies were included in which the patients were in the late postoperative period of total or subtotal thyroidectomy due to one of the DCT and were assigned the diagnostic WBI with I131 before the ablative dose (intervention) or they performed WBI with 123I before the ablative dose, or non-performance of the diagnostic WBI (directly ablative dose with 131I) (control). The primary outcome was disease remission assessed by the ablative success rate at least six months after follow-up. Results: After performing the searches in the following electronic databases Embase (1980-25/04/2017), Pubmed (1966-25/04/2017), CENTRAL (Cochrane Controlled Trials Register) (04/2... (Complete abstract click electronic access below) / Resumo: Apesar de existirem controvérsias, alguns serviços utilizam a pesquisa de corpo inteiro (PCI) com 131I previamente a dose terapêutica com esse radionuclídeo no tratamento dos cânceres diferenciados da tiroide (CDT). Os defensores dessa conduta argumentam uma melhor otimização da dose ablativa, e os contrários afirmam que essa PCI poderia ocasionar o “efeito stunning”, caracterizado pela redução da captação tecidual do 131I no tratamento com a dose ablativa, comprometendo com isso o tratamento e o prognóstico desses indivíduos. Objetivo: avaliar se a PCI com 131I, pré-dose ablativa, interfere na eficácia da dose terapêutica de iodo quanto a remissão da doença após a tireoidectomia total ou quase total em indivíduos com CDT. Metodologia: foi realizada uma revisão sistemática da literatura na qual foram incluídos estudos controlados randomizados, não randomizados e observacionais, nos quais os pacientes estavam em pós-operatório de tireoidectomia total ou subtotal devido um dos CDT, e foram alocados a um dos dois grupos: PCI diagnóstica com 131I realizada antes da dose terapêutica com I131(intervenção), ou a PCI realizada com 123I antes da dose ablativa, ou não realização da PCI diagnóstica antes da dose terapêutica (ambos controle). O desfecho principal foi a remissão da doença avaliada pela taxa de sucesso ablativo em pelo menos seis meses de seguimento. Resultados: depois de realizadas as pesquisas nas bases eletrônicas Embase (1980–25/04/2017), Pubmed (1966–25/04/2017), CENT... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

O ácido araquidônico (AA) é precursor na formação dos eicosanoides, que são mediadores lipídicos com uma série de funções na fisiologia e patologia humana. A maioria dos eicosanoides atuam como mediadores pró-inflamatórios e contribuem para o desenvolvimento e proliferação de tumores. Nesta tese foram avaliados dois mediadores: a 15-deoxi-Δ12,14-PGJ2 (15d- PGJ2) e os ácidos Epoxieicosatrienoicos (EETs), ambos apresentam uma atividade oposta a da maioria dos eicosanoides, ou seja, com uma ação anti-inflamatória e antitumoral. Esses dois mediadores distintos da via do AA foram utilizados nesta tese em dois projetos distintos. Primeiro: A 15d- PGJ2 possui uma atividade antiproliferativa e induziu apoptose para diversos tipos de células tumorais, entretanto, o efeito da15d- PGJ2 em células de cancer da tireoide ainda estava desconhecido. Neste sentido, foram cultivadas in vitro células tumorais da tireoide, da linhagem TPC1, e tratadas com diferentes concentrações de 15d- PGJ2 (0 ate 20 μM), as células tratadas demonstraram uma diminuição na proliferação, e aumento na apoptose, e uma diminuição na liberação e expressão relativa de IL-6. Estes resultados em conjunto sugerem que a 15d- PGJ2 pode ser utilizada como uma nova terapia para o cancer da tireoide. Segundo: Os EETs são metabolizados em seus diois pela epóxi hidrolase solúvel (sEH), para manter a estabilidade dos EETs e a sua atividade antiiflamatoria, foi utilizado um inibidor (TPPU) para sEH em um modelo de periodontite induzida por Aggregatibacter actinomycetemcomitans. O tratamento oral com TPPU, assim como o uso de animais sEH Knockout, levou a uma redução na perda óssea acompanhada da diminuição de moléculas osteoclastogenicas como RANK, RANKL e OPG, demonstrando que a inibição farmacológica da sEH pode ter um valor terapêutico na periodontite e doenças inflamatórias que envolvem a reabsorção óssea. / Arachidonic acid (AA) a precursor in the formation of eicosanoids which are lipid mediators with a number of functions in human physiology and pathology. The most of the eicosanoids act as proinflammatory mediators and contribute to the development and proliferation of tumors. In this thesis we evaluated two mediators: 15-deoxy-Δ12,14-PGJ2 (15d- PGJ2) and epoxieicosatrienoic acids (EETs) both act with an opposite activity of most eicosanoids, with an anti-inflammatory and and anti-tumoral action these two distinct mediators from AA pathway were used in this thesis in two different projects. First: 15d- PGJ2, was described that to have an antiproliferative activity and to induce apoptosis in several types of tumor cells however, the effect of 15d- PGJ2 in thyroid cancer cells was unknown in this sense, we tested in vitro cultured thyroid tumor cells, here in TPC1 cells, and treated with different concentrations of 15d- PGJ2 (0 to 20 uM) the treated cells showed a decrease in proliferation and an increase in apoptosis and a decrease in IL-6 release and relative expression. These key results together demonstrate that 15d- PGJ2 can be used as a new therapy for thyroid cancer. Second: The EETs are converted to their diols by soluble epoxy hydrolase (sEH) to maintain the stability of EETs and their anti-inflammatory activity, an inhibitor (TPPU) against was used to sEH in a periodontitis model induced with Aggregatibacter actinomycetemcomitans. The oral treatment in mice with TPPU and sEH Knockout animals showed bone loss reduction accompanied by a decrease in the osteoclastogenic molecules, like RANK, RANKL and OPG, demonstrating that pharmacological inhibition of sEH may have therapeutic value in periodontitis and inflammatory diseases that involve bone resorption. / Tese (Doutorado)

The aim of this work is to introduce a couple of new exposure scenariosof interest when personnel, family members and general public are externally irradiated from patients treated with Iodine-131. A series of modied ORNL matematical phantoms were used in Monte-Carlo simulations in PENELOPE-2011 to determine dose rates to: a cab driver where thepatient sits in the back seat at opposite side, a newborn child held by an adult patient, a doctor giving treatment to a patient while leaning over the hospital bed and collective effective dose to bus passengers if the patient travels home by bus. The cost to order taxi instead of a public transport ticket for the patient is put in relation to the lowered collective effective dose to general public and compared with other companies price per lowered manSv. Dose rate measurements and the effective half-life of I-131 are used to determine the retained whole body activity at different times post administration. This in combination with the simulations are used to estimate effective doses to the cab driver, bus passengers, newborn and personnel. Whole body-, abdominal- and thyroid source distributions were considered in the patients during simulations. Dose rates within 99.7 % condence intervals were up to: 23.7 ± 0.4, 62.7 ± 0.3, 530 ± 71, 96.8 ± 0.5 µSv/h/GBq, for the cab driver, closest bus passenger, newborn and physician respectively. Collective effective dose to bus passengers is estimated to 291 ± 2 µmanSv/h/GBq. A patient specic tool for estimating more reasonable restriction times on how long to avoid small children is developed, taking into account retained body activity and the home situation for the patient. Effective dose estimations suggest that dose constraints are very unlikely exceeded for the taxi driver, and public transport should be a considered option for thyrotoxicosis patients of lower administered activities and some cancer patients. It is also estimated that hospitals in Sweden are paying 7.6 - 31 MSEK per lowered collective effective dose of 1 manSv by sending patients by taxi instead of bus.

Une des propriétés majeures de la thyroïde est de capter l’iode de la circulation sanguine grâce à la présence d’un transporteur d’iodure (NIS pour Natrium Iodide Symporter). Cette capacité d’accumulation d’iode par les thyrocytes joue un rôle clé dans la synthèse des hormones thyroïdiennes ainsi dans le diagnostic et le traitement des cancers de la thyroïde. Cependant, en raison d’une diminution ou de l’absence de l’expression du NIS dans certaines tumeurs et métastases, des patients deviennent réfractaires à la radiothérapie métabolique et présentent une radiorésistance, causant ainsi un problème de santé publique.L’oncogène BRAFV600E, un puissant activateur de La voie MAP kinase, est détecté dans 40 - 60% des cancers thyroïdiens de type papillaires (CPT) qui représentent 80% de la totalité des cancers thyroïdiens. La mutation BRAFV600E est associée aux tumeurs thyroïdiennes les plus agressives. Cependant l’inhibition pharmacologique de la voie MAP kinase induite constitutivement par l’oncogène BRAFV600E ne permet pas, à elle seule, de rétablir l’expression du NIS chez des patients atteints d’un cancer de la thyroïde muté BRAFV600E. Ceci suggère que d’autres mécanismes compensatoires peuvent contribuer à la radiorésistance. Une étude récente menée sur un modèle murin a montré que la régulation négative du NIS par l’oncogène BRAFV600E est médiée par la voie du TGF beta. Une autre a montré que l’expression du NIS serait dépendante de l’état redox de la cellule, suggérant un rôle des espèces réactives de l’oxygène (ROS). Dans les cellules les ROS peuvent être produites par les NADPH oxydases (NOX/DUOX). La thyroïde en exprime trois : DUOX2 nécessaire à la synthèse des hormones thyroïdiennes ainsi que DUOX1 et NOX4 dont le rôle physiologique reste inconnu. NOX4, surexprimé dans les CPTs, a été montré être un nouvel effecteur clé de la voie du TGF beta dans d’autres cancers.Dans mon projet de thèse, je me suis intéressée à l’étude du rôle de NOX4 dans la régulation négative du NIS dans les CPT mutés BRAFV600E. L’étude du mécanisme, réalisée à partir de deux lignées humaines issues de cancers papillaires mutés pour BRAF (BCPAP et 8505C), a permis d’établir que l’oncogène BRAFV600E contrôle l’expression de NOX4 au niveau transcriptionnel via la voie TGF-beta/Smad3. Ces résultats ont été validés sur une lignée de rat exprimant de manière conditionnelle BRAFV600E ainsi que sur des thyrocytes humains en culture primaire. De manière importante, l’utilisation d’antioxydants tels que le N-acetyl cystéine (NAC) ou l’inhibition spécifique de l’expression de NOX4 par ARN interférence permet de réinduire l’expression du NIS. Ces résultats qui montrent que les ROS produites par NOX4 inhibent l’expression du transporteur de l’iode (NIS) établissent un lien entre l’oncogène BRAFV600E et NOX4. Une analyse comparative de l'expression de NOX4 effectuée à partir de 500 cancers papillaires de la thyroïdes mutés ou non pour BRAF (données TCGA) confirme que NOX4 est significativement augmenté dans les cancers porteurs de la mutation BRAF et que ceci est corrélé à une diminution de l’ARNm du NIS. Par ailleurs, le niveau de NOX4 est inversement corrélé au score de différenciation thyroïdien, suggérant que NOX4 pourrait être impliqué dans le processus de dédifférenciation. Cette étude ouvre une nouvelle opportunité pour l’optimisation de l’utilisation de la radiothérapie métabolique dans le traitement des cancers thyroïdiens réfractaires à l’iode I131 et présente NOX4 comme une cible thérapeutique potentielle. / One of the major properties of the thyroid is iodine uptake from the bloodstream through an iodide transporter (NIS for Natrium Iodide Symporter). This capacity plays a key role in the thyroid hormones synthesis, but also in both diagnosis and treatment of thyroid cancer. However, due to a decrease or absence of the NIS expression in some tumors and metastases, patients become refractory to the metabolic radiotherapy and present a radioresistance, which cause a public health problem.The BRAFV600E oncogene, a potent activator of the MAP kinase pathway, is detected in 40-60% of papillary thyroid cancer (PTC), which represent 80% of total thyroid cancers. The BRAFV600E mutation is associated with the more aggressive thyroid tumors. However, the pharmacological inhibition of the MAP kinase pathway, constitutively induced by the BRAFV600E oncogene, is not able to restore alone the expression of NIS in patients with BRAFV600E mutated thyroid cancer. This suggests that other compensatory mechanisms may contribute to the radioresistance. A recent study in a mouse model demonstrated that downregulation of NIS by BRAFV600E oncogene is mediated through the TGF beta activation. An other showed that the expression of NIS is dependent on the redox status of the cell, suggesting a role for the reactive oxygen species (ROS). In cells, ROS can be produced by the NADPH oxidases (NOX/DUOX). The Thyroid gland expresses three of them: DUOX2, which is necessary for the thyroid hormones synthesis, but also DUOX1 and NOX4 whose the physiological role remains unknown. NOX4, which is overexpressed in the PTCs, has been shown to be a new key effector of the TGF beta pathway.In my thesis project, I was interested in studying the role of NOX4 in the negative regulation of NIS in BRAFV600E mutated CPT. The study of the mechanism, made from two human cell lines derived from BRAF-mutated papillary thyroid cancers (BCPAP and 8505C), has revealed that the oncogene BRAFV600E controls the expression of NOX4 at the transcriptional level via the TGF-beta/Smad3 pathway. These results were validated on both a rat thyroid cell line conditionnaly expressing BRAFV600E and on human thyrocytes in primary culture. Importantly, the use of antioxidants such as N-acetyl cysteine (NAC) or specific inhibition of NOX4 expression by RNA interference allow reinduction of NIS expression. These results, which show that ROS produced by NOX4 inhibit the expression of iodine transporter (NIS), establish a link between the oncogene BRAFV600E and NOX4. A comparative analysis of the NOX4 expression, made from 500 papillary thyroid cancers mutated or not for BRAF (TCGA data), confirms that NOX4 is significantly increased in BRAF-mutated cancers and that this is correlated with a decrease of NIS mRNA. Furthermore, the level of NOX4 is inversely related to thyroid differentiation score, suggesting that NOX4 might be involved in the dedifferentiation process. This study opens a new opportunity for optimizing the use of metabolic radiotherapy in the treatment of thyroid cancers refractory to radioiodine I131and makes NOX4 as a potential therapeutic target.

Background: Although radioactive iodine 131 (RAI) has been successfully used in differentiated thyroid cancer (DTC) patients therapy for more than 70 years, thus far published data regarding RAI- induced salivary glands functional impairment have been inconsistent. Due to the trend towards using lower activities or even RRA omission, the possible RAI untoward effects have been widely discussed. Aim: To evaluate and quantify salivary gland function in thyroidectomised DTC patients before and after RRA using activity of 3.7 GBq and to compare salivary gland functional changes in DTC patients after single or repeated RAI treatment (using activities  5.5 GBq) with their age- and sex-matched RAI-naive counterparts using salivary gland scintigraphy with 99m Tc- pertechnetate. In addition, we performed subjective symptoms evaluation. Patients and Methods: Salivary gland scintigraphy (SGS) was performed in 31 RAI-naive patients (6 men, 25 women, median age 52 years) before and 4.6 months after RRA. Salivary gland function was measured also in 23 patients (7 men, 16 women, median age 60 years) with RAI administration history and compared with their age- and sex-matched counterparts. Non-parametric Wilcoxon and Mann-Whitney tests were used (due to non-normal data distribution) for statistical analysis. A p...

Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.:Background; Methods; Results; Discussion; Conclusions

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Pendant les trente dernières années, le taux d'incidence du cancer de la thyroïde chez l'homme et la femme a considérablement augmenté partout dans le monde. Cependant, on estime que d'ici à 2019 le cancer de la thyroïde deviendra le troisième cancer le plus répandu chez les femmes dans tous les groupes d'âge en raison de la tendance d’augmentation plus dramatique chez elles. En général, il n'y a aucune raison claire qui explique l'augmentation mondiale de l'incidence du cancer de la thyroïde et il est émis l'hypothèse que cette recrudescence de l'incidence a une étiologie multifactorielle. Bien qu'il soit clair que le progrès technique des modalités de l’imagerie diagnostique telle que l'échographie peut amener à une augmentation du taux de détection du cancer de la thyroïde secondaire au sur-diagnostic des maladies sous-cliniques, il existe des preuves fortes indiquant une vraie augmentation du cancer de la thyroïde. La cartographie cervicale échographique préopératoire est un outil important dans l'algorithme diagnostic du cancer de la thyroïde. Elle aide à identifier l’étendue des métastases ganglionnaires cervicales afin de guider la dissection chirurgicale anticipée. La dissection chirurgicale du cou orientée selon les compartiments anatomiques et guidée par la cartographie cervicale échographique peut amener à une réduction des risques des complications postopératoires et des récidives tumorales locorégionales. Nous avons effectué une analyse qualitative et quantitative de la cartographie cervicale échographique afin d'évaluer la fiabilité diagnostique de ce test. Nos résultats ont démontré une valeur prédictive positive assez élevée de cette modalité diagnostique ainsi q’une association quantitative forte entre les données de la cartographie échographique et les résultats de l’histopathologie. Nous suggérons que l’utilisation de la cartographie cervicale échographique cible les patients présentant un risque plus important d’une maladie persistante / récidivante. / Over the last 30 years, the incidence rate of thyroid cancer has drastically increased in both genders all over the world. However, due to a more dramatic pattern in females, it is estimated that by 2019 it will become the third most prevalent cancer in women of all ages. Overall, there are no clear reasons behind the worldwide increase in thyroid cancer incidence and it is hypothesized that this upsurge has a multifactorial etiology. Despite the fact that recent advances in imaging modalities such as ultrasound can lead to thyroid cancer overdiagnosis by improving the detection rate for subclinical disease, there is strong evidence indicating a true increase in the occurrence of thyroid cancer as well. Preoperative ultrasound cervical mapping, an important tool in the diagnostic algorithm of thyroid cancer, helps to identify metastatic spread in cervical lymph nodes and guides the surgeon for subsequent surgical dissection. Compartment oriented neck dissection directed by ultrasound mapping decreases locoregional tumor recurrence and lowers the risk of postsurgical complications. We conducted a qualitative and quantitative analysis of ultrasound mapping to evaluate this test’s diagnostic reliability. Our results demonstrated that the positive predictive value of this diagnostic modality was sufficiently high and that there was a strong quantitative association between ultrasound mapping and histopathology results. We therefore recommend that ultrasound mapping be used to target patients with a higher risk of persistent or recurrent thyroid cancer.

Ces travaux de thèse portent sur l'analyse des mélanges réels et synthétiques complexes composés de petites molécules à l'aide de la RMN HRMAS. Dans une première partie, une approche RMN HRMAS basée sur l'analyse métabolomique en combinaison avec des techniques de reconnaissance des formes (PCA et O-PLS-DA) a été appliquée pour le diagnostic des lésions thyroïdiennes indéterminées et étudier également les effets biologiques négatifs des nanoparticules d'aluminium sur pseudomonas brassicacearum. Dans une seconde partie, nous avons étudié la RMN chromatographique en utilisant la silice comme matrice de support qui pourrait fournir une alternative rapide et complète de la LC pour la caractérisation de mélanges complexes. En outre, l'exigence de la suppression du signal dans l'extrait de plantes naturelles et d'hydrocarbures aromatiques conduit à l'élaboration d'une méthode rapide et précise en utilisant des polymères à empreintes moléculaires avec une excellente sélectivité. La sélectivité des polymères à empreintes moléculaires à travers la capture d'une cible moléculaire spécifique est exploitée ici pour éliminer efficacement les signaux RMN. / This thesis work deals with the analysis of natural and synthetic complex mixtures composed of small molecules using HRMAS NMR. In a first part, an integrated HRMAS-NMR based metabolomic analysis in combination with pattern recognition techniques (PCA and O-PLS-DA) has been applied for the diagnosis of indeterminate thyroid lesions and also studied the potential adverse biological effects of aluminium nanoparticles on pseudomonas brassicacearum. In a second part we investigated that chromatographic NMR using silica as the matrix support could provide a quick alternative and complement to LC for the characterization of complex mixtures. In addition, requirement for signal suppression in natural plant extract and aromatic hydrocarbons led to the development of a rapid and accurate method using molecularly imprinted polymers with excellent selectivity. The selectivity of Molecularly Imprinted polymers towards capturing a specific molecular target is exploited here to efficiently remove NMR signals.

Neste estudo, investigamos o envolvimento de galectina-3 na fisiologia e no câncer de tiróide usando vários modelos biológicos e metodologias. Observamos que o gene LGALS3 apresenta um SNP no códon 98, mas não observamos correlação entre os genótipos deste SNP e fenótipo de câncer de tiróide. Na linhagem de tiróide de rato PCCl3, mostramos que a indução da expressão do oncogene RET/PTC promove o aumento da expressão de galectina-3, no entanto, a expressão de galectina-3, por si só, não confere vantagem de proliferação à célula. Por outro lado, na linhagem de carcinoma papilífero de tiróide TPC-1, a galectina-3 contribui para a sobrevivência da célula tumoral e progressão do ciclo celular, aumentando a expressão de c-Myc, diminuindo a expressão de p21 e caspase-3, e favorecendo a ativação de importantes vias envolvidas no controle do ciclo celular. Além disto, em modelos in vivo e in vitro, a galectina-3 interferiu na função e diferenciação da célula folicular tiroidiana, exercendo um papel indireto na regulação da expressão da tireoglobulina e atividade de TTF-1. / In this study, we investigate the involvement of galectin-3 in thyroid physiology and cancer using several biological models and methodologies. We observed that LGALS3 gene presents a SNP in codon 98, but no correlation between the genotype and the phenotype of benign or malignant thyroid tumor was observed. In the rat thyroid cell line PCCl3, we showed that the conditional induction of RET/PTC oncogene expression promotes the increase of galectin-3 expression, however, galectin-3 expression itself did not confer a proliferative advantage to cell. On the other hand, in papillary thyroid carcinoma cell line TPC-1 the galectin-3 contributes to tumor cell survival and cell cycle progression, increasing c-Myc expression, decreasing p21 and caspase-3 expression and cooperating to activation of important signaling pathways which are involved in the cell cycle control. In addition, in vitro and in vivo models the galectin-3 interferes in the differentiation and function of thyroid follicular cell, playing an indirect role in the regulation of thyroglobulin expression and TTF-1 activity.