O corpo lúteo é uma glândula endócrina temporária que regula tanto o ciclo estral quanto a prenhez, apresentando extrema dependência de aporte sanguíneo adequado. O tratamento superovulatório aumenta a concentração sérica de progesterona (P4) e conseqüentemente as taxas de concepção e de prenhez. Esse trabalho objetivou quantificar a vascularização dos corpora lútea (CLL) de animais controle e superovulados, correlacionando-a com a P4 sérica e expressão de VEGF e seus receptores. Foram utilizadas 30 búfalas, cujos CLL foram divididos em cinco grupos: superovulados (receberam 400 mg de FSH divididos em doses diárias decrescentes: 80 mg, 60 mg, 40 mg e 20mg a cada 12 horas durante 4 dias), corpos hemorrágicos (CH), corpo lúteo maduro (CL), CL em regressão (CR) e corpo albicans (CA), que não receberam nenhum tratamento. Três CLL de cada grupo foram fixados em formol tamponado para quantificação da densidade vascular e imunolocalização de VEGF, VEGFR-1 e VEGFR-2. Os três restantes foram injetados com resina Mercox para análise da microvascularização. Amostras de sangue foram coletadas e a P4 mensurada através de RIA convencional. A densidade capilar média encontrada foi de 37,78 ± 8,89; 17,8 ± 3,33; 11,92 ± 3,57; 10,83 ± 2,42 e 3,46 ± 1,66 vasos/ mm2 respectivamente para os cinco grupos, indicando maior vascularização (p<0,001) para o grupo superovulado. A microvasculatura apresentou comportamento semelhante para ambos os grupos, revelando apenas maior densidade da rede capilar dos CLL de animais superovulados, o que se refletiu nos valores séricos de progesterona que foram significativamente maiores (p<0,05) para estes animais, com concentração média de 5,58 ( 0,97ng/ml vs 2,02 ( 0,16 ng/ml para os animais controle na mesma fase. O VEGF, bem como seus receptores (VEGFR-1 e VEGFR-2) foram encontrados imunohistoquimicamente no CLL de búfalas. A imunoreatividade para o VEGF e receptores, pode ser observada nas células endoteliais e luteínicas a partir do 2° dia após a ovulação (p.o.) até a fase de corpo lúteo em regressão (17° dia p.o.), com forte reação nas fase luteínicas inicial e média. A imunoreatividade foi mais intensa nos animais submetidos a superovulação. / Corpus luteum is a temporary organ, which regulates the estrous cycle and pregnancy; it is extremely dependent on vascularization. During corpus luteum life span P4 production is associated with an increase of capillary number known as angiogenesis. The angiogenic process is modulated by many factors, including VEGF (vascular endothelial growth factor), which is considered the most important of them. Hyperstimulation of ovarian function, a largely spread technique employed in cattle raising, is associated with high levels of estradiol and P4, as well as an increase of capillary invasion of the new formed CL. This study intended to quantify the vascularization of normal and superovulated corpora lutea (CLL), trying to correlate this parameter with blood P4 concentrations and expression of VEGF and its receptors. For that purpose thirty buffalo cows were divided into five groups: superovulated (received 400 mg FSH divided in decreasing doses of 80 mg, 60 mg, 40 mg e 20mg 12/12h during 4 days), corpus hemorragicans (CH), mature corpus luteum (CL), regression CL (CR) e corpo albicans (CA), that received no treatment and were slaughtered at days 2, 9, 17 and 26 after ovulation. After slaughter ovaries were collected and corpora lutea prepared as follows: three animals from each group had their CLL fixed in buffered formol solution, cut into 0,5 cm pieces, dehydrated in increasing ethanol concentrations, cleared in xylene and embedded in paraffin using conventional procedures. Five µm slices were prepared for quantification of vascular density and immunolocalization of VEGF, VEGFR-1 and VEGFR-2. The ovaries of the remaining animals were injected with Mercox resin through ovarian artery in order to have the microvascularization evaluated by electron scanning microscopy. Blood samples were collected and P4 measured through conventional RIE. Capillary density during CL life span was 37,78 ± 8,89; 17,8 ± 3,33; 11,9 ± 3,57; 10,83 ± 2,42 and 3,46 ± 1,66 vessels/ mm2 respectively for superovulated (CS), CH, CL, CR and CA, indicated higher vascularization (p<0,001) for superovulated group. The microvasculature showed similar behavior: the density of capillary network was higher in CLL of superovulated animals. Values of serum progesterone were significantly higher (p<0,05) for CS animals: 5,58 ± 0,97 ng/ml vs 2,02 ± 0,16 ng/ml for control animals in the same stage of estrous cycle. The VEGF system and its receptors (VEGFR-1 e VEGFR-2) were immunolocalized in CLL of buffalo cows. The immunoreactivity could be detected in endothelial and luteal cells since day two post ovulation (p.o.) until the stage of regression corpus luteum (Day 17 p.o.), with strong immunostaining at the early and midluteal phase. The immunostaining was more intense in CLL of animals submitted to superovulation.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Concern has increased that widespread adverse effects are occurring in humans and wildlife populations as a result of exposure to environmental chemicals (mostly man-made) that disrupt the normal functioning of the endocrine system. Many pharmaceutical, agricultural and industrial chemicals, acting as endocrine modulating compounds (EDCs), have been detected in an accumulated form in food, in drinking water and in the environment. Although the levels of these chemicals can be measured analytically, it is important to evaluate biological activity. For this, animal models are used and relevant bioassays developed. These assays are based on biological markers, which are specific xenobiotically-induced physiological responses and are usually deviations in cellular or biochemical components, processes or structures. Vitellogenin is a large protein complex, produced in the liver under estrogen control and circulates in the plasma, destined for incorporation into the developing oocyte in female oviparous vertebrates. Since vitellogenin production is low or nonexistent in males, its presence may therefore be interpreted as evidence of exposure to estrogenic environmental contaminants. In this study the African Clawed Frog, Xenopus laevis was used as model to study the effects of estrogens on biochemical and physiological parameters associated with vitellogenesis. As a starting point the seasonal female reproductive cycle in a natural Xenopus laevis population in terms of ovarian state, plasma vitellogenin and plasma estrogen levels was studied. It was shown that plasma vitellogenin and estrogen levels were seasonal and correlated to a seasonal ovarian cycle, which peaked during spring. However, although seasonality existed, there were reproductively active individuals at any time during the year. Increases in plasma estrogen levels predated increases in plasma vitellogenin levels and ovarian maturation. Lipoprotein profiles, as well as plasma cholesterol, triglyceride and phospholipid concentrations were determined and it was shown that estrogen affected these in such a way that these parameters could be used as additional markers in estrogen contamination studies. In order to develop an in vitro bioassay to screen for estrogenic activity, the use of hepatic tissue cultures was investigated. Optimal culture conditions were established and increased sensitivity in the estrogenic response was obtained by using liver slices from male frogs that were pre-treated with estrogen. Validation studies proved that this bioassay could be employed for routine screening of water and chemical samples. In order to refine the Xenopus laevis vitellogenin ELISA and liver slice bioassay, existing polyclonal anti-vitellogenin antibodies were replaced by culturing monoclonal antibodies. Selected antibodies were characterised and ELISAs developed and validated. This study showed that the newly developed Xenopus laevis vitellogenin ELISA and liver slice bioassay have the potential to be employed in environmental monitoring programmes. / AFRIKAANSE OPSOMMING: Daar is toenemende besorgdheid dat afwykings in mens- en dierbevolkings voorkom as gevolg van blootstelling aan chemikalieë (hoofsaaklik mensgemaak) in die omgewing wat die normale werking van die endokrienstelsel versteur. Verskeie farmaseutiese, landbou- en industriële chemikalieë, wat as endokrienversteurders optree, is in die omgewing gevind. AI kan die vlakke van hierdie stowwe analities bepaal word, is dit belangrik om hulle biologiese aktiwiteit te evalueer. Hiervoor word diermodelle aangewend om toepaslike toetse daarvoor te ontwikkel. Hierdie toetse word baseer op biologiese merkers, spesifieke xenobioties-geïnduseerde fisiologiese reaksies, en is gewoonlik afwykings van sellulêre- of biochemiese komponente, -prosesse of - strukture. Vitellogeen ('n dooiervoorloper) is 'n lipoproteïenkompleks wat, onder estrogeenbeheer, in die lewer vervaardig word en in die plasma sirkuleer vir uiteindelike inkorporasie in ontwikkelende oësiete van vroulike, ovipare werweldiere. Aangesien daar min of geen vitellogeen in manlike diere geproduseer word, is die teenwoordigheid daarvan 'n aanduiding dat die dier aan estrogeniese omgewingsbesoedeling blootgestel is. In hierdie studie is die Platanna, Xenopus laevis, as model gebruik om die gevolge van estrogene op biochemiese en fisiologiese veranderlikes, wat met vitellogenese geassosieer word, te bestudeer. As vertrekpunt is die seisoenale voortplantingsiklus van die wyfie, in terme van vitellogeen en -estrogeen vlakke in die plasma en aktiwiteit van die ovaria bepaal. Daar is aangetoon dat die estrogeen- en vitellogeenvlakke in die plasma met die ovariumsiklus, wat gedurende die lente hoogtepunte bereik, korreleer. Alhoewel daar seisoenaliteit bestaan, was daar dwarsdeur die jaar wyfies wat ovarium dooierneerlegging getoon het. Verhoging in estrogeenvlakke het vitellogeenpieke en rypwording van die ovaria voorafgegaan. Lipoproteïenprofiele, sowel as die cholesterol- , trigliseried- en fosfolipiedkonsentrasies in die plasma is bepaal en daar is aangetoon dat estrogeen hierdie medeveranderlikes in só 'n mate affekteer dat hulle as bykomende biomerkers vir estrogeenblootstelling in besoedelingstudies gebruik kan word. In vitro Xenopus laevis lewersnitte in die weefselkultuur omgewing is ondersoek om 'n biotoets te onwikkel vir die gebruik in vinnige estrogenisiteits-toetsing van watermonsters en chemise stowwe. Die mees gunstige kultuurtoestande is bepaal en die sensitiwiteit van estrogeenreaksies is verhoog deur lewer van mannetjies, wat vooraf met estrogeen behandel is, te gebruik. Hierdie biotoets se geldigheid is gestaaf en kan in roetine eerstevlaktoetsing van watermonsters gebruik word. Die gebruik van poliklonale teenliggaampies in 'n bestaande enzyme-linked immunosorbent assay (ELISA) is vervang deur spesiaal-ontwikkelde monoklonale anti-Xenopus laevis vitellogeen teenliggaampies. Uitgesoekte teenliggaampies, spesifiek teen die estrogeengeïnduseerde proteïene, is gekarakteriseer en ELISAs saamgestel en die geldigheid gestaaf. Hierdie studie het aangetoon dat die nuut-onwikkelde Xenopus laevis vitellogeen-ELISA en lewerkultuurbiotoets die potensiaal het om In omgewingsmoniteringprogramme gebruik te word.

O teleósteo Astyanax altiparanae, popularmente conhecido como lambari importância econômica e ecológica. Poucos são os trabalhos que visam demonstrar a morfologia dos testículos de brasileiros. O objetivo deste estudo foi classificar e demonstrar as alterações morfológicas do testículo de Astyanax altiparanae ao longo do ciclo gonadal e quantificar tais alterações. Foram encontrados os estádios: regredido, maturação inicial, maturação média, maturação final e regressão. O estudo ultraestrutural revelou que as células secretoras que revestem os ductos possuem organelas relacionadas à síntese de macromoléculas e gotículas de lipídio. O estudo imuno-histoquímico revelou detalhes da distribuição de receptor de andrógeno, claudina-1, citoqueratina, actina, colágeno do tipo I, laminina e fibronectina neste órgão. O presente trabalho revelou que os machos de Astyanax altiparanae são capazes de desenvolver o intersexo, já que foram encontrados oócitos no interior dos testículos de alguns indivíduos. / The Astyanax altiparanae (Characiformes, Characidae), popularly known as lambari, is a fish that has ecologic and economic importance. Morphological studies on testis of South American teleost species are not frequently found in the appropriated literature. The aim of this work was to show the changes in the morphology of the testis of Astyanax altiparanae occurred through the gonadal cycle. It was found five stages of the gonadal cycle: regressed, early maturation, mid maturation, late maturation and regression. Ultrastructural study, reveals that secretory cells showed a great amount of organelles responsible for macromolecules synthesis, and lipids droplets. Immunohistochemical studies reveal details of the distribution of androgen receptor, claudin-1, actin, cytokeratin, type I collagen fibers, laminin and fibronectin in this organ. This work demonstrated that males of Astyanax altiparanae were able to develop the intersex, once oocytes were found in the testis of some individuals.

[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.

Lancée par une communication du célèbre professeur Brown-Séquard en 1889 sur les effets de l’auto injection d’un suc testiculaire, l’opothérapie – technique de soin par le suc de glandes – s’inscrit dans la ligne d’une longue tradition de médication animale. Les publications de médecins et de pharmaciens nous ont permis d’établir comment cette nouvelle thérapeutique s’inscrit dans le paysage d’une médecine qui se scientifise au tournant du XIXe-XXe siècles. L’opothérapie, dont le développement est tributaire de l’évolution des connaissances sur les glandes endocrines, se développera grâce aux succès thérapeutiques enregistrés dans les affections thyroïdiennes et gynécologiques et grâce à la mise à la disposition du public de spécialités issues d’une pharmacie qui s’industrialise et qui fournit une médication sous une forme qui permet de s’affranchir d’un geste médical. L’opothérapie, qui se démarque de l’hormonothérapie par l’usage d’objets thérapeutiques naturels mal identifiés qui ont suscité de nombreux débats sur leur composition et leur mode d’action, connaitra son plus grand développement aux alentours de la Première guerre mondiale et persistera, malgré le développement de l’hormonothérapie s’appuyant sur des molécules de synthèse, jusque dans les années 1990. / Launched by a communication from the famous Professor Brown-Sequard in 1889 on the effects of self-injection of testicular juice, organotherapy – a technique of care using the juice of glands – falls within a long tradition of animal medication. Publications of doctors and pharmacists have allowed us to establish how the new treatment is part of the landscape of medicine that became more scientific at the turn of the nineteenth and twentieth centuries. Opotherapy/Organotherapy, whose development depends on the development of knowledge on the endocrine glands, develops through therapeutic successes in thyroid and gynecological diseases and by making pharmaceuticals produced by industrializing pharmacists which provided medication in a form that eliminates a medical procedure, available to the public. Organotherapy, which stands out from hormone therapy by the use of natural misidentified drugs that have generated a great number of debates on their composition and mode of action, will know its greatest development around the First World War and will persist despite the development of hormone therapy based on synthetic molecules until the 1990s.

Lancée par une communication du célèbre professeur Brown-Séquard en 1889 sur les effets de l’auto injection d’un suc testiculaire, l’opothérapie – technique de soin par le suc de glandes – s’inscrit dans la ligne d’une longue tradition de médication animale. Les publications de médecins et de pharmaciens nous ont permis d’établir comment cette nouvelle thérapeutique s’inscrit dans le paysage d’une médecine qui se scientifise au tournant du XIXe-XXe siècles. L’opothérapie, dont le développement est tributaire de l’évolution des connaissances sur les glandes endocrines, se développera grâce aux succès thérapeutiques enregistrés dans les affections thyroïdiennes et gynécologiques et grâce à la mise à la disposition du public de spécialités issues d’une pharmacie qui s’industrialise et qui fournit une médication sous une forme qui permet de s’affranchir d’un geste médical. L’opothérapie, qui se démarque de l’hormonothérapie par l’usage d’objets thérapeutiques naturels mal identifiés qui ont suscité de nombreux débats sur leur composition et leur mode d’action, connaitra son plus grand développement aux alentours de la Première guerre mondiale et persistera, malgré le développement de l’hormonothérapie s’appuyant sur des molécules de synthèse, jusque dans les années 1990. / Launched by a communication from the famous Professor Brown-Sequard in 1889 on the effects of self-injection of testicular juice, organotherapy – a technique of care using the juice of glands – falls within a long tradition of animal medication. Publications of doctors and pharmacists have allowed us to establish how the new treatment is part of the landscape of medicine that became more scientific at the turn of the nineteenth and twentieth centuries. Opotherapy/Organotherapy, whose development depends on the development of knowledge on the endocrine glands, develops through therapeutic successes in thyroid and gynecological diseases and by making pharmaceuticals produced by industrializing pharmacists which provided medication in a form that eliminates a medical procedure, available to the public. Organotherapy, which stands out from hormone therapy by the use of natural misidentified drugs that have generated a great number of debates on their composition and mode of action, will know its greatest development around the First World War and will persist despite the development of hormone therapy based on synthetic molecules until the 1990s.

Sickle cell anaemia (SCA), the most prevalent hereditary disease in the world, presents, among many complications, endocrine-metabolic changes, especially growth retardation and puberty in children and adolescents. The final height of adults with SCA is, however, still controversial and there are few studies about it in literature. This dissertation consists of two original articles. In the first article, "Growth and puberty in a prospective cohort of patients with sickle cell anemia: assessment in ten years", it was observed that patients with SCA showed growth impairment and pubertal delay when compared to healthy controls, however, albeit belatedly, they reach normal sexual maturation. Moreover, they reached normal height in adulthood, unlike what occurred with weight and BMI, finding even more evident in males. In the second article, "Endocrine-metabolic profile of adults with sickle cell anemia", it was observed that men with SCA had higher levels of gonadotropins than controls. Levels of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol were lower in the SCA group. The high prevalence of low HDL (96%) in the SCA group and its positive correlation with the hemoglobin concentration and its negative correlation with reticulocytes count and leukocytes count the possibility of HDL be considered a marker to assist in predicting adverse clinical events in this population. Although it has not been observed difference in the levels of 25-hydroxyvitamin between the groups, there was a higher prevalence of its deficiency and insufficiency in SCA group, which enhances the risk, already existing in this population, of osteoporosis. The findings of both studies characterize patients with SCA about the evolution of their growth and development into adulthood and show endocrinemetabolic changes that deserve to be investigated during follow-up, in order to prevent other complications that can compromise their quality life. / A anemia falciforme (AF), doença hereditária mais prevalente no mundo, apresenta, dentre diversas complicações, alterações endócrino-metabólicas, destacando-se retardo de crescimento e puberdade em crianças e adolescentes. A estatura final dos adultos com AF, entretanto, ainda é controversa e pouco estudada na literatura. Esta dissertação é composta por dois artigos originais. No primeiro artigo, “Crescimento e puberdade em uma coorte prospectiva de pacientes com anemia falciforme: avaliação em dez anos”, observou-se que os pacientes com AF apresentaram comprometimento de crescimento e atraso puberal quando comparados a controles saudáveis, todavia, ainda que tardiamente, atingiram maturação sexual normal. Além disso, alcançaram estatura normal na idade adulta, diferentemente do que ocorreu com peso e IMC, achado ainda mais evidente no sexo masculino. No segundo artigo, “Perfil endócrino-metabólico de adultos portadores de anemia falciforme”, observou-se que os homens com AF apresentaram maiores níveis de gonadotrofinas. Níveis das lipoproteínas de alta densidade (HDL) e baixa densidade (LDL) também foram menores no grupo AF. A elevada prevalência de HDL reduzido (96%) no grupo AF e sua correlação positiva com a concentração de hemoglobina e negativa com contagem de reticulócitos, contagem de leucócitos apontam para a possibilidade de o HDL poder se configurar um marcador que auxilie na predição de eventos clínicos adversos nesta população. Apesar de não ter havido diferença dos níveis de 25-hidroxivitamina entre os grupos, constatou-se maior prevalência de sua deficiência e insuficiência no grupo AF, o que potencializa o risco, já existente nesta população, de osteoporose. Os achados de ambos os estudos caracterizam os pacientes com AF em relação à evolução de seu crescimento e desenvolvimento até a fase adulta e mostram alterações endócrinometabólicas que merecem ser investigadas durante seu seguimento, a fim de prevenir outras complicações que possam comprometer sua qualidade de vida.

O teleósteo Astyanax altiparanae, popularmente conhecido como lambari importância econômica e ecológica. Poucos são os trabalhos que visam demonstrar a morfologia dos testículos de brasileiros. O objetivo deste estudo foi classificar e demonstrar as alterações morfológicas do testículo de Astyanax altiparanae ao longo do ciclo gonadal e quantificar tais alterações. Foram encontrados os estádios: regredido, maturação inicial, maturação média, maturação final e regressão. O estudo ultraestrutural revelou que as células secretoras que revestem os ductos possuem organelas relacionadas à síntese de macromoléculas e gotículas de lipídio. O estudo imuno-histoquímico revelou detalhes da distribuição de receptor de andrógeno, claudina-1, citoqueratina, actina, colágeno do tipo I, laminina e fibronectina neste órgão. O presente trabalho revelou que os machos de Astyanax altiparanae são capazes de desenvolver o intersexo, já que foram encontrados oócitos no interior dos testículos de alguns indivíduos. / The Astyanax altiparanae (Characiformes, Characidae), popularly known as lambari, is a fish that has ecologic and economic importance. Morphological studies on testis of South American teleost species are not frequently found in the appropriated literature. The aim of this work was to show the changes in the morphology of the testis of Astyanax altiparanae occurred through the gonadal cycle. It was found five stages of the gonadal cycle: regressed, early maturation, mid maturation, late maturation and regression. Ultrastructural study, reveals that secretory cells showed a great amount of organelles responsible for macromolecules synthesis, and lipids droplets. Immunohistochemical studies reveal details of the distribution of androgen receptor, claudin-1, actin, cytokeratin, type I collagen fibers, laminin and fibronectin in this organ. This work demonstrated that males of Astyanax altiparanae were able to develop the intersex, once oocytes were found in the testis of some individuals.

Congenital hypothyroidism (CH) is the most frequent of the rare endocrine diseases (e.g. Addison's disease, Cushing's syndrome, Congenital adrenal hyperplasia.), which affects 1:2000 – 4000 newborns. If not immediately diagnosed after birth, thyroid hormones deficiency causes severe defects in brain and skeletal development leading to a complex clinical scenario called cretinism. CH can be due to a defective synthesis of thyroid hormones (dyshormonogenesis) or an abnormal embryonic development of the gland. Data obtained using knockout mouse models have shown the pivotal role of four specific transcription factors (NKX2.1, PAX8, FOXE1 and HHEX) for the correct organogenesis or function of the gland. Although mutations in those genes have been identified in few cases of CH patients, the pathogenetic mechanisms remain still elusive in the vast majority of CH cases (95%).<p>For the identification of new genes and molecular events controlling thyroid organogenesis it would be useful to develop an in vitro cellular model to recapitulate thyroid embryogenesis in a dish. Embryonic Stem Cells (ESCs) have recently emerged as system model to recapitulate the embryogenesis of several tissues in vitro.<p>Induced overexpression of defined transcription factors has been shown to have a directing effect on the differentiation of pluripotent stem cells into specific cell types. In this thesis I show that a transient overexpression of the transcription factors NKX2.1 and PAX8 is sufficient to direct the differentiation of murine ESCs into thyroid follicular cells (TFC) and promotes in vitro self- assembly of TFC into three-dimensional follicular structures, when associated to a subsequent thyrotropin (TSH) treatment. Cells differentiated by this protocol showed significant iodide organification activity, a hallmark of thyroid tissue function. Importantly, athyroid mice grafted with mESC-derived thyroid follicles show normalization of plasma T4 levels with concomitant decrease of plasma TSH. In addition, a full normalization of body temperature at 4 weeks after transplantation was observed. Together, these data clearly demonstrate that grafting of our mESC-derived thyroid cells rescues the hypothyroid state and triggers symptomatic recovery along with the normalization of plasma hormone concentrations. The high efficiency of TFC differentiation and follicle morphogenesis in our system will provide an unprecedented opportunity for future studies to decipher regulatory mechanisms involved in embryonic thyroid development, a major research need towards an improved understanding of the molecular mechanisms underlying congenital hypothyroidism, the most common congenital endocrine disorder in humans. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Ο φαρμακευτικός κλάδος μπορεί να επωφεληθεί, με την βοήθεια της σημερινής τεχνολογίας των ηλεκτρονικών υπολογιστών και συγκεκριμένα των βάσεων δεδομένων. Σκοπός της διπλωματικής εργασίας είναι η κάλυψη των αναγκών της Ελληνικής συνταγογραφίας των φαρμάκων. Συγκεκριμένα οι φαρμακευτικές βάσεις δεδομένων, που ήδη υπάρχουν, δεν χρησιμοποιούν την τεχνολογία αναζήτησης των αλληλεπιδράσεων των φαρμάκων. Για το σκοπό αυτό σχεδιάστηκε και υλοποιήθηκε ένα μοντέλο βάσεων δεδομένων, που υποστηρίζει πλήρως τις αλληλεπιδράσεις των φαρμάκων, σύμφωνα με τις οδηγίες του εθνικού συνταγολογίου φαρμάκων (Ε.Ο.Φ). Έπειτα έγινε η εισαγωγή των φαρμάκων και δοκιμάστηκε η ακεραιότητα του συστήματος για την επιτυχή λειτουργία των αλληλεπιδράσεων μεταξύ φαρμάκων, καθώς μια εσφαλμένη αλληλεπίδραση μπορεί να αποβεί μοιραία για κάποιους ασθενείς. Τέλος αυτή η διπλωματική εργασία μπορεί να χρησιμοποιηθεί από φαρμακοποιούς και ιατρούς για την ασφαλέστερη και αποτελεσματικότερη συνταγογράφηση. / The pharmaceutical branch can profit, with the help of current technology of computers and concretely with the use of data bases. The purpose of this diplomatic work is to cover the needs of the Greek prescription on medicines. Particularly the pharmaceutical data bases, that already exist, do not use the search technology of interactions in medicines. For this aim it was drawn and implemented a model of data bases, that completely supports the interactions of medicines, according to the directives of national organization for medicines (N.O.M). The next step was the insertion of medicines and test the integrity of system for the successful operation of interactions between medicines, while a mistaken in interaction it can be turns out fatal for certain patients. Finally this diplomatic work can be used from pharmacists and doctors for secure and more effective prescription.