Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hypothyroidism is characterized by a disorder resulting from deficiency of thyroid hormones and is related to lipid metabolism dysfunction and cardiovascular diseases development risk. However, these changes in hypothyroidism need to be understood. Thus, this study aimed to evaluate the association between lipid, inflammatory and oxidative stress markers in patients with hypothyroidism and antioxidant effects of quercetin in these markers, using hypothyroidism experimental model induced by methimazole in rats. The methodology and results are presented in the form of articles. In article 1, were evaluated the oxidative stress biomarkers in 20 patients with subclinical hypothyroidism (SH) (49.12 ± 10.85 years). Thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT) and arylesterase (ARE) were analyzed in SH patients and controls. In addition, were measured plasmatic lipids: total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). TBARS levels and CAT activity were higher in subclinical hypothyroidism patients, such as TC and LDL-C plasmatic levels. Arylesterase activity was lower in the SH group. Correlations were observed between plasmatic lipids and oxidative stress biomarkers and thyroid-stimulating hormone (TSH). TSH was correlated with TBARS, CAT, and SOD. The second study (manuscript 1) aimed to investigate the association between inflammatory biomarkers and overt hypothyroidism (OH). Plasmatic levels of cytokines were determinate: interleukin 1 (IL-1), interleukin 6 (IL- 6), tumor necrosis factor alpha (TNF-α), interferon gamma (INF-ɣ) and the levels of cell free DNA (cf-DNA). Furthermore, we evaluated lipid profile and prothrombotic markers (fibrinogen and D-dimer). OH patients had pro-inflammatory profile, resulted from high levels of cytokines and cf-DNA. Lipids and prothrombotic markers also showed elevated. Significant associations between inflammatory status and lipid profile were observed in hypothyroid patients. Manuscript 2 evaluates the effect of quercetin on oxidative stress biomarkers in methimazole (MMI) - induced hypothyroid rats. Hypothyroidism was induced by administering MMI at 20 mg/100 ml in the drinking water, for 30 days. After this period, rats received orally 10 or 25 mg/kg of quercetin (QT) for 8 weeks. Sixty male wistar rats were randomly divided into six groups (group I, control; group II, QT10; group III, QT25; group IV, hypothyroid; group V, hypothyroid + QT10; group VI, hypothyroid + QT25). Hypothyroid rats showed hepatic, renal and serum TBARS levels increased, along with increased protein carbonyl (PCO) in liver and increased ROS levels in liver and kidney. Quercetin administration (QT10 and 25), was effective in decreasing TBARS levels in serum and kidney, PCO in liver and ROS generation in liver and kidney tissues. Moreover, in hypothyroid group were observed high TBARS levels in cerebral cortex and hippocampus. QT25 treatment decreased the levels in both tissues. Administration of QT25 to hypothyroid rats resulted in decreased SOD activities in liver and whole blood and increased liver CAT activity. Ascorbic acid levels and total oxidative scavenging capacity (TOSC) were increased in liver and kidney rats after QT10 and QT25 treatment. These results suggest association between oxidative stress and hypothyroidism that may potentially modulated by antioxidant supplementation such as quercetin. These findings are of great importance in understanding the biochemical dysfunctions and oxidative status in hypothyroidism, as well as, in research of antioxidants strategies to be used as adjuncts in the treatment of this disorder. / O hipotireoidismo é caracterizado por uma desordem decorrente da deficiência de hormônios tireoideanos, estando relacionado a disfunções no metabolismo lipídico e ao risco de desenvolvimento de doenças cardiovasculares. Entretanto, estas alterações no hipotioreodismo precisam ser melhor compreendidas. Assim, este trabalho teve como objetivo avaliar a associação de marcadores lipídicos, inflamatórios e de estresse oxidativo em pacientes com hipotireoidismo e o efeito antioxidante da quercetina nestes marcadores, utilizando como modelo experimental o hipotireoidismo induzido por metimazol em ratos. A metodologia e resultados são apresentados sob a forma de artigos. No artigo 1, foram avaliados biomarcadores de estresse oxidativo em 20 pacientes com hipotireoidismo subclínico (HSC) (49,12 ± 10,85 anos). Os níveis de substâncias reativas ao ácido tiobarbitúrico (TBARS), e as atividades das enzimas superóxido dismutase (SOD), catalase (CAT) e arilesterase (ARE) foram determinadas em pacientes com HSC e controles. Além disso, foram investigados os níveis de lipídeos plasmáticos: colesterol total (CT), triglicerídeos (TG) e as lipoproteínas de alta (HDL) e baixa densidade (LDL). Os níveis de lipoperoxidação determinado pela medida do TBARS e a atividade da enzima CAT estavam aumentados nos pacientes hipotireóideos, bem como os níveis plasmáticos de CT e colesterol LDL. A enzima ARE mostrou-se diminuída no grupo HSC. Foram evidenciadas correlações entre lipídeos plasmáticos e biomarcadores de estresse oxidativo e com o hormônio de estimulação da tireóide (TSH). O TSH foi correlacionado com TBARS, CAT e SOD. O segundo estudo (manuscrito 1) teve por objetivo investigar a associação entre biomarcadores inflamatórios e o hipotireoidismo clínico (HC). Foram determinados os níveis plasmáticos das citocinas: interleucina 1 (IL-1), interleucina 6 (IL-6), fator de necrose tumoral alfa (TNF- α), interferon gama (INF- ɣ) e os níveis de DNA circulante livre. Além disso, foram avaliados o perfil lipídico e marcadores prótrombóticos (fibrinogênio e D-dímero). Os pacientes com HC apresentaram perfil próinflamatório, resultante dos níveis elevados das citocinas e do DNA livre. Os lipídeos e os marcadores pró-trombóticos também se apresentaram elevados. Associações significativas entre o perfil inflamatório e o perfil lipídico foram observadas nos pacientes hipotireóideos. No manuscrito 2 avaliou-se o efeito da quercetina sobre biomarcadores de estresse oxidativo em um modelo de hipotireoidismo induzido por metimazol (MMI) em ratos. O hipotireoidismo foi induzido pela administração de MMI na concentração de 20mg/100mL na água de beber, por um período de 30 dias. Após este período, os animais receberam oralmente 10 ou 25 mg/kg de quercetina (QT) por um período de 8 semanas. Ratos machos wistar (n=60) foram divididos em seis grupos (grupo I, controle; grupo II, QT10; grupo III, QT25; grupo IV, hipotireóideo; grupo V, hipotireóideo + QT10; grupo VI, hipotireóideo + QT25). Os ratos hipotireóideos apresentaram níveis de TBARS hepático, renal e séricos aumentados, bem como os níveis de proteína carbonil (PCO) no fígado e os níveis de espécies reativas de oxigênio (ERO) no fígado e rins. A administração de quercetina (QT 10 e 25) diminuiu os níveis de TBARS em soro e rins, a PCO no fígado e a geração de ERO nos tecidos hepático e renal. Além disso, no grupo hipotireóideo foram observados altos níveis de TBARS no córtex cerebral e hipocampo. O tratamento com QT25 reduziu os níveis em ambos os tecidos. A administração de QT 25 aos ratos com hipotireoidismo diminuiu a atividade da SOD em fígado e sangue total e aumentou a atividade hepática da CAT. Os níveis de ácido ascórbico e a capacidade antioxidante total aumentaram no fígado e rins dos ratos após tratamento com QT10 e QT25. O conjunto dos resultados sugeriu associação entre estresse oxidativo e hipotireoidismo que pode ser potencialmente modulado por suplementação de antioxidantes como a quercetina. Estes achados são de grande importância no entendimento das disfunções bioquímicas e do status oxidativo no hipotireoidismo como também na busca de estratégias antioxidantes a serem utilizadas como coadjuvantes no tratamento desta disfunção.

L’hypothyroïdie congénitale (HC) est une maladie relativement fréquente, touchant un nouveau-né sur 3000-4000, et dont la majorité des cas sont causés par un défaut dans le développement embryonnaire de la glande. Il existe plusieurs arguments en faveur d’une cause génétique dans une minorité de ces dysgénésies thyroïdiennes mais, à ce jour, seuls quelques cas ont put être reliés à une mutation dans l’un ou l’autre de trois gènes codant pour des facteurs de transcription impliqués dans le développement de la thyroïde (TITF1, PAX8 et FOXE1).<p>Au cours de notre travail de thèse, nous avons caractérisé la 6ème mutation du gène PAX8 à l’état hétérozygote, dans un cas familial d’HC. Nous avons étudié l’impact fonctionnel de cette altération qui entraîne une perte de liaison de la protéine mutée à une séquence cible spécifique, ainsi qu’une diminution drastique de la synergie de transactivation en association avec Titf1. Le phénotype des patients est également intéressant à plus d’un titre. En effet, nous avons pu observer que les mutations de PAX8 sont compatibles avec le développement d’une thyroïde en place et de taille normale à la naissance, pouvant conduire à un diagnostic erroné de dyshormonogenèse. De plus, un des trois patients présente un phénotype rénal qu’il est tentant de relier à la mutation de PAX8 étant donné que ce gène est exprimé durant le développement de cet organe, tout comme dans la thyroïde.<p>Notre travail a également consisté en l’élaboration d’une librairie SAGE du bourgeon thyroïdien en développement, afin de rechercher de nouveaux gènes candidats impliqués dans le développement de la glande thyroïde. Grâce à une technique d’amplification d’ARN, nous avons obtenu une librairie d’environ 94.000 étiquettes à partir de bourgeons thyroïdiens provenant d’embryons de souris au 11ème jour de développement. Cette librairie nous a permis d’identifier une nouvelle isoforme du transcrit de Titf1 modifiant l’extrémité 3’-non codante du messager, ainsi qu’un gène de fonction inconnue mais dont le profil d’expression ainsi que sa grande conservation au cours de l’évolution laissent suggérer un rôle important, tant dans les tissus embryonnaires que dans les tissus adultes.<p>Ces deux découvertes valident le caractère prédictif de notre librairie qui constitue un outil de choix pour l’identification de nouveaux gènes de développement thyroïdien et donc de nouveaux candidats pour l’étude des mécanismes étiopathogéniques à la base des dysgénésies thyroïdiennes.<p>/<p>Congenital hypothyroidism (CH) is a relatively frequent disease affecting 1 every 3000-4000 newborns. The majority of CH cases are caused by a defect in the embryonic development of the gland. There exists several arguments in favor of a genetic cause for a minority of these thyroid dysgeneses but, to this day, only a few cases have been related to a mutation in one of three genes coding for transcription factors implicated in thyroid development (TITF1, PAX8 and FOXE1).<p>In the course of this work, we have caracterized the 6th mutation of the PAX8 gene, in the heterozygous state, in a familial case of CH. We have studied the functional impact of this modification which leads to a loss of the protein’s DNA-binding properties and to a severe reduction in the transactivation synergy in association with Titf1. The phenotype of the patients presents also interesting features. Indeed we have observed that the PAX8 mutations are compatible with the development of an in-place, normal-sized thyroid at birth, which could lead to an erroneous diagnostic of dyshormonogenesis. Moreover, one of the three patients presents with a renal phenotype (unilateral kidney agenesis) which is tempting to relate to the PAX8 mutation, given this gene is expressed during kidney development.<p>Our work also consisted in the generation of an embryonic thyroid bud SAGE library, which we used to search for new candidate genes implicated in thyroid development. With the help of a RNA amplification technique, we obtained a library of roughly 94.000 tags starting from mous thyroid buds at E11. This library allowed us to identify a new Titf1 splicing variant modifying the 3’-UTR of the transcript, and a gene of unknown function. The latter’s expression profile and high conservation throughout evolution suggest a crucial role in embryonic as well as adult tissues.<p>These two findings validate the predictive character of our library which constitutes a powerful tool to identify new thyroid developmental genes and new candidate genes for the study of the etiopathogenic mecanisms responsible for CH.<p><p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Overt hypothyroidism is characterized by decreased of thyroid hormones synthesis, with elevation of thyroid-stimulating hormone (TSH). It s a common disorder in population with highest prevalence in women and with aging. Frequently is associated with lipid metabolism alterations, represented by lipid parameters elevation and consequently with atherosclerosis development. The association between hypercholesterolemia and oxidative stress already well established, however oxidative stress presence in hypothyroidism is controversy. The oxidative stress is characterized by an increase in oxygen reactive species (ROS) generation or antioxidant system deficiency. In the present study were determined biochemical and oxidative stress biomarkers in overt hypothyroidism patients. The biochemical markers, total cholesterol (TC), LDL-cholesterol(C-LDL), cholesterol HDL (C-HDL) and trycerides (TG) were measured in serum samples in these patients. Lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) levels. Antioxidant system evaluation was performed by superoxide dismutase (SOD) and catalase (CAT) actvities and non-enzymatic antioxidants levels were evaluated by reduced glutathione (GSH) and vitamin E (VIT E) levels.The results demonstrated an increase in biochemical markers (TC, C-LDL and TG) in hypothyroid group, when compared to control group. In relation to lipid peroxidation, was observed an increased in TBARS levels in patients with hypothyroidism when compared to control group. The same was observed for antioxidants defenses SOD and CAT. Nonenzymatic antioxidants, such vitamin E, were higher in hypothyroid group in relation to controls, while GSH levels remained unchanged with hypothyroidism. These results suggest the association between overt hypothyroidism and hypercholesterolemia and oxidative stress. The high levels of cholesterol presented by hypothyroidism patients, has a stronger influence under oxygen reactive species (ROS) generation and in consequence under the oxidative stress. The increase in SOD and CAT activities, suggest antioxidant system induction as a mechanism to combat the ROS generation and high lipid peroxidation. In conclusion, overt hypothyroidism has association with hypercholesterolemia and oxidative stress biomarkers increase. / O hipotireoidismo clínico é caracterizado pela diminuição na síntese dos hormônios tireoideanos, com elevação dos níveis do hormônio tireoestimulante (TSH). É uma desordem comum na população com maior incidência no sexo feminino e com a progressão da idade. Freqüentemente está associado a alterações no metabolismo lipídico, representadas pela elevação nos parâmetros lipídicos e consequentemente com o desenvolvimento de aterosclerose. A associação entre hipercolesterolemia e estresse oxidativo já é bem estabelecida, entretanto a presença de estresse oxidativo no hipotireoidismo é controversa. O estresse oxidativo é caracterizado por um aumento na produção de espécies reativas de oxigênio (ERO) ou deficiência do sistema antioxidante. Neste trabalho determinaram-se marcadores bioquímicos e de estresse oxidativo em pacientes com hipotireoidismo clínico. Os marcadores bioquímicos, colesterol total (CT), colesterol-LDL (LDL-C), colesterol HDL (HDL-C) e triglicerídeos (TG) foram medidos em soro dos pacientes. A peroxidação lipídica foi medida através dos níveis de substâncias reativas ao ácido tiobarbitúrico (TBARS). A avaliação do sistema antioxidante enzimático foi realizada através da medida da atividade das enzimas superóxido dismutase (SOD) e catalase (CAT) e os níveis de antioxidantes não-enzimáticos através dos níveis de tióis totais (SH) e vitamina E (VIT E). Os resultados demonstraram um aumento dos marcadores bioquímicos (CT, LDL-C e TG) no grupo hipotireóideo, com relação ao grupo controle. Em relação a peroxidação lipídica, observou-se um aumento dos níveis séricos de TBARS de pacientes com hipotireoidismo quando comparados com o grupo controle. Esse aumento também foi observado para as defesas antioxidantes enzimáticas, SOD e CAT. Com relação aos antioxidantes não-enzimáticos, ocorreu um aumento nos níveis séricos de VIT E no grupo hipotireóideo com relação ao grupo controle, enquanto que para SH não foi observada diferença entre os grupos estudados. Estes resultados sugerem a associação do hipotireoidismo clínico com hipercolesterolemia e estresse oxidativo. Os altos níveis de colesterol apresentados pelos pacientes com hipotireoidismo, exercem forte influência sobre a geração de espécies reativas de oxigênio (ERO) e por conseqüência sobre o estresse oxidativo. Os aumentos das enzimas SOD e CAT, sugerem a indução do sistema antioxidante enzimático, na tentativa de combater a formação de ERO e a elevada peroxidação lipídica. Concluí-se então, que o hipotireoidismo clínico, está associado à hipercolesterolemia e ao aumento dos biomarcadores de estresse oxidativo.

Objetivo: Valorar si existe asociación entre el hipotiroidismo subclínico y la resistencia a la insulina en pacientes no diabéticos de una clínica privada de Lima. Materiales y métodos: Se realizó un estudio observacional analítico de corte transversal. Se incluyeron como participantes a adultos que acudieron a consulta ambulatoria de endocrinología de una clínica privada de Lima en los años 2012 a 2018. Se excluyeron a los pacientes que previamente habían sido diagnosticados de diabetes o patologías tiroideas, que recibían alguna terapia con corticoides sistémicos o hipolipemiantes, o que estaban gestando. Las variables de estudio principales fueron hipotiroidismo subclínico y resistencia a la insulina, las cuales se trabajaron como variables categóricas. Para definir la variable hipotiroidismo subclínico se utilizaron los valores de TSH mayor o igual a 4.5 mU/L, y T4L en valores normales, mientras que la variable resistencia a la insulina se definió utilizando el valor de HOMA-IR mayor a 2.79. Asimismo, también se estudiaron otras variables como IMC, porcentaje de grasa corporal y sexo. Para valorar la asociación entre el hipotiroidismo subclínico y la resistencia a la insulina, se llevó a cabo un modelo lineal generalizado de la familia Poisson con varianza robusta crudo y ajustado. Se presentó como media de asociación a la razón de prevalencia (RP) con su intervalo de confianza al 95% Resultados: Se analizó 1389 pacientes, el promedio de edad fue 39.56 (13.54) años (SD) y el porcentaje de varones fue 25.87%. La prevalencia de HSC fue 13.17% (n=183) y la prevalencia de RI fue 40.4% en el grupo con HSC, y 46.2% en el grupo de eutiroideos. En el modelo de regresión crudo se encontró que los pacientes con HSC tenían un 12% menos probabilidades de tener RI en el análisis crudo (PR 0.876 IC 95%: 0.727-1.055) y 7% menos en el ajustado (PR 0.927, IC 95%: 0.792-1.084); no obstante, ninguno obtuvo una significancia estadística. Secundariamente, se encontró asociaciones entre RI con el IMC, porcentaje de grasa corporal y el sexo masculino. Conclusiones: En nuestra población de estudio no se encontró asociación entre hipotiroidismo subclínico y resistencia a la insulina. / Objective: To assess whether there is an association between subclinical hypothyroidism and insulin resistance in non-diabetic patients at a private clinic in Lima. Materials and methods: An analytical observational cross-sectional study was carried out. Participants were adults who attended the endocrinology outpatient clinic of a private clinic in Lima in the years 2012 to 2018. Patients who were previously diagnosed with diabetes or thyroid diseases, who received systemic corticosteroids, who took lipid-lowering drugs, were excluded. or that they were brewing. The main variables were subclinical hypothyroidism and insulin resistance, which were worked as categorical variables. To define the subclinical hypothyroidism variable, TSH values ​​greater than or equal to 4.5 mU / L and T4L were used in normal values, while the variable insulin resistance was defined using the HOMA-IR value greater than 2.79. Likewise, other variables such as BMI, percentage of body fat and sex were also studied. To assess the association between subclinical hypothyroidism and insulin resistance, a generalized linear model of the Poisson family with crude and adjusted robust variance was carried out. It was presented as an association measure to the prevalence ratio (PR) with its 95% confidence interval. Results: We analyzed 1389 patients, the average age was 39.56 (13.54) years (SD) and the % of men was 27.87%. The prevalence of HSC was 13.17% (n = 183) and the prevalence of IR was 40.4% in the HSC group and 46.2% in the non-HSC group. In the crude regression model, it was found that patients with HSC had a 12% lower probability of having IR in the crude analysis (PR 0.876 IC 95%: 0.727-1.055) and 7% less in the adjusted one (PR 0.927, IC 95%: 0.792-1.084); however, none obtained statistical significance. Secondarily, associations were found in IR with the IMC, percentage of body fat and male sex. Conclusions: In our study population there was no association between subclinical hypothyroidism and insulin resistance. / Tesis

The incidence of thyroid diseases in pregnancy in the Czech Republic reaches 10- 15%. Emphasis on early diagnosis and treatment is laid not only during pregnancy but also in the time preceding conception due to the impact of thyroid diseases on fertility, the course of pregnancy, birth and fetal development. The aim of the dissertation was to assess the effectiveness and economical aspects of screening for thyroid disease in pregnancy and in women with fertility disorders in the conditions of the Czech Republic. The dissertation consists of four published studies. The first study is a prospective cross-sectional study of 200 positively screened pregnant women. In the study we come to conclusion that pregnant women who are at high- and low-risk for thyroid disease have similar clinical and laboratory characteristics and screening, currently focused only on risk groups, is ineffective. The second study of 5 223 pregnant women is a case-control study. We find that the age of women over 30 cannot be regarded as a risk factor for thyroid disease in pregnancy, although addition this age criterion to the case-finding screening strategy improve its efficiency and ATA (American Thyroid Association) include it in their guideline 2011. The third publication is a retrospective cross-sectional study of 188...

The incidence of thyroid diseases in pregnancy in the Czech Republic reaches 10- 15%. Emphasis on early diagnosis and treatment is laid not only during pregnancy but also in the time preceding conception due to the impact of thyroid diseases on fertility, the course of pregnancy, birth and fetal development. The aim of the dissertation was to assess the effectiveness and economical aspects of screening for thyroid disease in pregnancy and in women with fertility disorders in the conditions of the Czech Republic. The dissertation consists of four published studies. The first study is a prospective cross-sectional study of 200 positively screened pregnant women. In the study we come to conclusion that pregnant women who are at high- and low-risk for thyroid disease have similar clinical and laboratory characteristics and screening, currently focused only on risk groups, is ineffective. The second study of 5 223 pregnant women is a case-control study. We find that the age of women over 30 cannot be regarded as a risk factor for thyroid disease in pregnancy, although addition this age criterion to the case-finding screening strategy improve its efficiency and ATA (American Thyroid Association) include it in their guideline 2011. The third publication is a retrospective cross-sectional study of 188...

Cardiovascular diseases (CVD) have been the leading cause of death worldwide for many years, making it a devasting and increasing concern across the globe. The risk factors of CVD include postnatal factors and prenatal factors. For the prenatal CVD risk factors study, we focused on maternal hypothyroidism (MH), which is a common clinical condition. Studies have shown MH progeny have increased susceptibility to both acquired cardiovascular disease in adulthood and congenital heart disease, but the underlying mechanisms are not well understood. The goal of the present experiments was to test the hypothesis that MH reduces early postnatal cardiac myocyte proliferation in the progeny so that their adult hearts have a smaller complement of cardiac myocytes, which leads to adverse cardiac disease responses. MH model was induced by thyroidectomy (TX) with total thyroxine (TT4) under 1ng/dl after surgery. The progeny from mice that underwent Sham or TX surgery was termed WT (wild type) or MH (maternal hypothyroidism) progeny, respectively. Hearts were collected from WT and MH progeny to determine heart weight (HW), CM size, CM proliferation, and cell culture. RNA-seq was performed on heart tissue at postnatal day 5 (P5) and P60. Transverse Aortic Constriction (TAC) was performed to cause pressure overload-induced cardiac hypertrophy and/or heart failure (HF) in adult WT and MH progeny. ECHO (in-vivo) and histological (ex-vivo) studies were performed at specific times after TAC. Thyroid hormone treatment (levothyroxine, T4) for MH mother was administered. The results showed that the Heart weight (HW) to body weight (BW) ratio at P60 was no difference between groups, but the MH progeny had a larger CM size, consistent with fewer CM numbers. MH progeny had lower EdU+, Ki67+, and PH3+ CMs, and fewer mononucleated CMs, which shows they had a decreased CM proliferation capacity. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH progeny, including Bmp10. Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation in the presence of thyroid hormone. In adult progeny, RNA-seq data showed that MH mice had genes upregulated in the inflammatory response before TAC surgery. Six weeks after TAC, the MH progeny had a greater HW/BW ratio, larger CM size, and more severe LV fibrosis consistent with more severe cardiac pathological remodeling compared with WT progeny. T4 supplemented treatment for MH mothers preserved progeny’s early postnatal CM proliferation capacity and the excessive pathological remodeling after TAC. Concluding, CM proliferation during the early postnatal development stage was significantly attenuated in MH progeny, which results in fewer CMs and CM hypertrophy in adult MH progeny. These changes are associated with worse cardiac disease responses under pressure overload in adult MH progeny. For the postnatal CVD risk factors study, we focused on calcium overload and metabolic disorder, which play a critical role in heart failure with preserved ejection fraction (HFpEF). HFpEF is defined as HF with an EF ≥50% and elevated cardiac diastolic filling pressures. The underlying causes of HFpEF are multifactorial and not well-defined. A transgenic mouse with low levels of cardiomyocyte (CM)-specific inducible Cavβ2a expression (β2a-Tg mice) showed increased cytosolic CM Ca2+, and modest levels of CM hypertrophy and fibrosis. This study aimed to determine if β2a-Tg mice develop an HFpEF phenotype when challenged with two additional stressors, a high-fat diet (HFD) and L-NAME (LN). Four-month-old wild-type (WT) and β2a-Tg mice were given either normal chow (WT-N, β2a-N) or HFD and/or L-NAME (WT-HFD, WT-LN, WT-HFD-LN, β2a-HFD, β2a-LN, and β2a-HFD-LN). Some animals were treated with the HDAC (hypertrophy regulators) inhibitor suberoylanilide hydroxamic acid (SAHA) (β2a-HFD-LN-SAHA). Echocardiography was performed monthly. After four months of treatment, terminal studies were performed, including invasive hemodynamics and organ weight measurements. Cardiac tissue was collected. Our results showed that four months of HFD plus L-NAME treatment did not induce a profound HFpEF phenotype in FVB WT mice. β2a-HFD-LN (3-Hit) mice developed features of HFpEF, including increased natriuretic peptide (ANP) levels, preserved EF, diastolic dysfunction, robust CM hypertrophy, increased M2 macrophage population, and myocardial fibrosis. SAHA reduced the HFpEF phenotype in the 3-Hit mouse model by attenuating these effects. Concluding, the 3-Hit mouse model induced a reliable HFpEF phenotype with CM hypertrophy, cardiac fibrosis, and an increased M2 macrophage population. This model could be used for identifying and preclinical testing of novel therapeutic strategies. / Biomedical Sciences

Thesis (M.Med. (Radiation Oncology)) -- University of Limpopo, 2022 / Background Hypothyroidism in head and neck cancer patients after radiotherapy is known to occur, yet thyroid function tests are not routinely monitored in all patients post radiation therapy. Routine post radiation therapy thyroid function testing is currently not part of the follow-up protocol in these patients at Pietersburg Hospital. The aim of this study is to evaluate post radiation therapy hypothyroidism among head and neck cancer patients treated with radiotherapy at Pietersburg Hospital Methods A prospective (cohort) observational study was carried out among head and neck cancer patients receiving radiotherapy at the radiation oncology department in Pietersburg Hospital. Sample size of n=37 was calculated using Statistica V13.0. Thyroid function tests were performed at the start of radiation therapy and repeated on the first day of follow up, 6 weeks after completing radiation therapy. During follow up, participants were also interviewed for the presence of symptoms of hypothyroidism such as dry skin, dry hair, fatigue, cold intolerance, or weight gain. Data analysis was done with STATA version 16. Descriptive statistics were used to characterise variables, and summarised in tables, graphs and charts. Changes in thyroid function tests and other variables were analysed. A p-value of 0.05 was deemed statistically significant. Results Thirty-seven patients were enrolled in the study, 26 males and 11 females. The mean age of the patients was 53.1 ±12.3 standard deviation [SD]) with a range of 40.8 to 65.4 years. The most common diagnoses were cancer of the larynx and hypopharynx, forming 29.7% and oral cavity cancer, 29.7%. Only three patients (8%) had an early stage cancer (Stages 1 and 2), 11 patients (29.7%) moderately advanced cancer (Stage 3) while the majority (62%; n =23) had locally advanced cancer (Stage 4). Majority of the patients received 70Gy in 35 daily fractions, five fractions per week via 3-D conformal radiotherapy. Only 29 patients who had complete pre- and post radiotherapy thyroid function tests were included in the final analysis. Of these, none had clinical hypothyroidism at 3 months. Two patients (6.8%) had sub-clinical hypothyroidism, with post radiation therapy TSH values greater than 3.5mIU/ml. The mean post radiation therapy TSH values increased by 8.3% and the mean fT4 values decreased by 2.05% compared to the pre-radiation therapy values. Both changes were not statistically significant (p=0.99 and p=0.82 respectively). There was no statistically significant correlation between changes in TSH and fT4 versus age (p=0.88 and p=0.92 respectively), sex (p=0.55 and p=0.15 respectively), cancer stage (p=0.21 and p=0.78 respectively), and cancer site (p=0.17 and p=0.74 respectively). The most common post radiotherapy symptom was fatigue (62%) followed by cold intolerance (54%), weight gain (43%) and dry skin or dry hair (43% each). Conclusion The results of the study suggest that sub-clinical hypothyroidism is detectable early post radiation therapy presenting as clinical symptoms.

L'hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT, ectopie dans plus de 80 %) a une prévalence de 1 cas sur 4000 naissances vivantes. L’HCDT est la conséquence d'une défaillance de la thyroïde embryonnaire à se différencier, à se maintenir ou à migrer vers sa localisation anatomique (partie antérieure du cou), qui aboutit à une absence totale de la thyroïde (athyréose) ou à une ectopie thyroïdienne (linguale ou sublinguale). Les HCDT sont principalement non-syndromiques (soit 98% des cas sont non-familiale), ont un taux de discordance de 92% chez les jumeaux monozygotes, et ont une prédominance féminine et ethnique (i.e., Caucasienne). La majorité des cas d’HCDT n’a pas de cause connue, mais est associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdie). Des mutations germinales dans les facteurs de transcription liés à la thyroïde (NKX2.1, FOXE1, PAX8, NKX2.5) ont été identifiées dans seulement 3% des patients atteints d’HCDT sporadiques et l’analyse de liaisons exclue ces gènes dans les rares familles multiplex avec HCDT. Nous supposons que le manque de transmission familiale claire d’HCDT peut résulter de la nécessité d’au moins deux « hits » génétiques différents dans des gènes importants pour le développement thyroïdien. Pour répondre au mieux nos questions de recherche, nous avons utilisé deux approches différentes: 1) une approche gène candidat, FOXE1, seul gène impliqué dans l’ectopie dans le modèle murin et 2) une approche en utilisant les techniques de séquençage de nouvelle génération (NGS) afin de trouver des variants génétiques pouvant expliquer cette pathologie au sein d’une cohorte de patients avec HCDT. Pour la première approche, une étude cas-contrôles a été réalisée sur le promoteur de FOXE1. Il a récemment été découvert qu’une région du promoteur de FOXE1 est différentiellement méthylée au niveau de deux dinucléotides CpG consécutifs, définissant une zone cruciale de contrôle de l’expression de FOXE1. L’analyse d’association basée sur les haplotypes a révélé qu’un haplotype (Hap1: ACCCCCCdel1C) est associé avec le HCDT chez les Caucasiens (p = 5x10-03). Une réduction significative de l’activité luciférase est observée pour Hap1 (réduction de 68%, p<0.001) comparé au promoteur WT de FOXE1. Une réduction de 50% de l’expression de FOXE1 dans une lignée de cellules thyroïdienne humaine est suffisante pour réduire significativement la migration cellulaire (réduction de 55%, p<0.05). Un autre haplotype (Hap2: ACCCCCCC) est observé moins fréquemment chez les Afro-Américain comparés aux Caucasiens (p = 1.7x10-03) et Hap2 diminue l’activité luciférase (réduction de 26%, p<0.001). Deux haplotypes distincts sont trouvés fréquemment dans les contrôles Africains (Black-African descents). Le premier haplotype (Hap3: GTCCCAAC) est fréquent (30.2%) chez les contrôles Afro-Américains comparés aux contrôles Caucasiens (6.3%; p = 2.59 x 10-9) tandis que le second haplotype (Hap4: GTCCGCAC) est trouvé exclusivement chez les contrôles Afro-Américains (9.4%) et est absent chez les contrôles Caucasiens (P = 2.59 x 10-6). Pour la deuxième approche, le séquençage de l’exome de l’ADN leucocytaire entre les jumeaux MZ discordants n’a révélé aucune différence. D'où l'intérêt du projet de séquençage de l’ADN et l’ARN de thyroïdes ectopiques et orthotopiques dans lesquelles de l'expression monoallélique aléatoire dans a été observée, ce qui pourrait expliquer comment une mutation monoallélique peut avoir des conséquences pathogéniques. Finalement, le séquençage de l’exome d’une cohorte de 36 cas atteints d’HCDT a permis d’identifier de nouveaux variants probablement pathogéniques dans les gènes récurrents RYR3, SSPO, IKBKE et TNXB. Ces quatre gènes sont impliqués dans l’adhésion focale (jouant un rôle dans la migration cellulaire), suggérant un rôle direct dans les défauts de migration de la thyroïde. Les essais de migration montrent une forte diminution (au moins 60% à 5h) de la migration des cellules thyroïdiennes infectées par shRNA comparés au shCtrl dans 2 de ces gènes. Des zebrafish KO (-/- et +/-) pour ces nouveaux gènes seront réalisés afin d’évaluer leur impact sur l’embryologie de la thyroïde. / Congenital hypothyroidism by thyroid dysgenesis (CHTD) is a common disorder with prevalence at birth of 1 in 4000 live births. CHTD is the consequence of a failure of embryonic thyroid to differentiate or to migrate to its anatomical location (front of the neck), which leads to a total lack of thyroid (athyreosis) or an ectopic thyroid (lingual or sublingual). The most common category is ectopic thyroid diagnosis (up 85%). Most cases of CHTD have no known cause, but are associated with severe deficiency of thyroid hormones (hypothyroidism). The clinical diagnosis of hypothyroidism is usually possible only when permanent brain damage is already present. On the other hand, biochemical screening on the second day of life allows initiating replacement therapy from the second week of life, pre-empting severe intellectual deficit associated with the congenital hypothyroidism. Even with early treatment (an average of 9 days), loss of IQ, which is not exclusively due to the severity of hypothyroidism, can still be observed. Molecular markers may identify patients at risk for intellectual deficit (by e.g., genes involved in neuronal migration and the thyroid during development). These patients might benefit from early intervention to stimulate their neurocognitive development. Cases of CHTD are mainly non-syndromic and sporadic (in 98% of cases, there is no other affected in the family), have a discordant rate of 92% in monozygotic twins, and a female and ethnic (Caucasian) dominance. Germline mutations in thyroid-related transcription factors have been identified in only 3% of patients with sporadic CHTD, and linkage analysis has excluded these genes in rare multiplex families with CHTD. In addition, non-penetrating mutations among close relatives (for Nkx2.5 gene) suggest that modifying genes as germline variants de novo copy number (CNV) and / or somatic mutations are associated with CHTD. To respond to this research questions, we used two different approaches: 1) a candidate gene approach studying FOXE1, the only gene involved in ectopic thyroid in the mouse model and, 2) an approach using next generation sequencing techniques (NGS) to find genetic variants that could explain this pathology using a cohort of mostly sporadic CHTD. Variants and genes discovered by these two different approaches have been validated and their functional impact on the thyroid gland was evaluated by several experiments.

INTRODUÇÃO: O Hipotireoidismo congênito (HC) é uma das endocrinopatias mais frequentes em pediatria e pode causar retardo mental e do crescimento, se não for tratado precocemente. A determinação do nível do hormônio estimulador da tireoide em sangue total após o nascimento (TSHneo) constitui uma estratégia efetiva para o rastreamento de HC, embora não exista consenso em relação aos níveis considerados seguros para essa detecção. Muitos serviços utilizam os valores de corte do TSH neonatal de 10,0 e 15,0 ?UI/mL, por ensaios imunofluorimétricos. OBJETIVO: Analisar a capacidade de detecção dos casos de hipotireoidismo congênito por diferentes níveis de corte do TSH neonatal e os efeitos destes sobre o sistema de triagem neonatal para essa doença, em nascidos vivos avaliados pelo Programa de Triagem Neonatal (PTN) da rede pública do Estado de Mato Grosso (MT), de 01 de janeiro de 2010 a 31 de dezembro de 2012. MÉTODOS: Estudo de coorte, de corte transversal, com coleta retrospectiva de dados obtidos a partir do banco de dados do Serviço de Referência em Triagem Neonatal do Estado de Mato Grosso, de nascidos vivos no período 01/01/2010 a 31/12/2012 e avaliados pelo PTN-MT. Estes foram divididos em dois grupos: I. Controle: Crianças com exame de triagem neonatal normal; II. Estudo: Crianças com HC. Análise estatística incluiu o uso do teste qui-quadrado ou exato de Fischer para análise das características dos recém-nascidos entre os grupos e o teste t de Student ou não paramétrico de Mann-Whitney para análise dos níveis de TSH em sangue total de ambos os grupos e, avaliação das concentrações de TSH e T4 livre no soro, em crianças com HC. Construiu-se uma curva ROC (Receiver Operating Characteristic), para a avaliação dos pontos de corte do TSHneo. O nível de significância foi p<0,05. RESULTADOS: Entre as 111.705 crianças triadas pelo Programa, 50 tiveram o diagnóstico de HC, sob o ponto de corte do TSHneo de 5,0 ?UI/mL. A prevalência da doença foi de 1:2.234 nascidos vivos. A cobertura do Programa estadual foi de 73,9%. Para o Grupo II, os níveis do TSHneo foram superiores a 20,0 ?UI/mL em 61,4% das crianças e, os níveis de TSH no soro excederam este valor em 83,7%. A curva ROC identificou o ponto de corte do TSHneo de 5,03 ?UI/mL, como o correspondente à sensibilidade de 100% e a maior especificidade associada (93,7%). A área observada sob a curva foi de 0,9898 (p<0,0001). CONCLUSÕES: Observou-se uma cobertura inadequada do PTN-MT. O ponto de corte do TSH neonatal de 5,0 ?UI/mL, adotado pelo PTN-MT, foi confirmado pela curva ROC como o mais seguro para detectar HC e determinou a elevada prevalência da doença no Estado de Mato Grosso / INTRODUCTION: Congenital hypothyroidism (CH) is a very common pediatric endocrine disorder and can cause mental and growth retardation without early treatment. Measuring the total blood thyroid-stimulating hormone level after birth (TSHneo) is an effective screening strategy for CH, although there is not yet a consensus on the appropriate diagnostic levels. Many services use the neonatal TSH cut-off points of 10.0 and 15.0 uIU/mL per imunofluorimetric assay. OBJECTIVE: The aim of the present study was to analyze the ability of various TSHneo cutoff values to detect CH and their effects on the Newborn Screening Program (NSP) of the State of Mato Grosso (MT) from January 1, 2010, to december 31, 2012. METHODS: Cohort study, cross-sectional, based on retrospective data collection obtained from the database of the Reference Service for Neonatal Screening of the State of Mato Grosso, for all live births from January 1, 2010, to December 31, 2012, reviewed by NSP-MT. The infants were divided into two groups: I-Control: infants with normal newborn screening tests and II-Study: infants with CH. Statistical analysis included the chi-square or Fisher\'s exact test to compare the characteristics of the newborns from both groups and Student\'s t-test or the non-parametric Mann-Whitney test to analyse the total blood TSH level from both groups of infants and evaluate the serum TSH and free thyroxine (T4) concentrations in infants with CH. A Receiver Operating Characteristic (ROC) curve was constructed to assess the TSHneo cutoff values. The significance level was p < 0.05. RESULTS: Using a TSHneo cutoff value 5.0 uIU/mL, 50 out of 111,705 screened infants were diagnosed with CH. The prevalence of CH was 1:2,234 live births. The state program coverage was 73.9%. For Group II, the TSHneo levels were higher than 20.0 uIU/mL in 61.4% of infants, and the serum TSH levels exceeded that level in 83.7%. The ROC curve showed that a TSHneo cutoff value of 5.03 uIU/mL had 100% sensitivity and the greatest associated specificity (93.7%). The area under the curve was 0.9898 (p < 0.0001). CONCLUSIONS: An inadequate coverage of the NSP-MT was observed. The ROC curve confirmed that the TSHneo cutoff value of 5.0 ?IU/mL adopted by the NSP-MT was the safest for detecting CH and determined the high prevalence of disease that was found in the State of Mato Grosso