Rozprawy doktorskie na temat „Endocrine glands”

Discovering biomarkers and molecular drivers of head and neck endocrine tumors was the inspiration for this thesis. Here, I describe the molecular evaluation of tumors of the thyroid and parathyroid endocrine glands for the purpose of identifying somatic driver alterations in these cancers. While molecular interplay of the germline genomic background of an individual and the somatic genome that emerges throughout the lifetime plays significant roles in increasing the susceptibility to cancer and in driving the malignant phenotype, the major known contributors to cancer remain the acquired somatic mutations. Analysis of a sporadic and recurring parathyroid carcinoma, with incidence of 1 per million population, revealed mutations in mTOR, MLL2, CDKN2C and PIK3CA and comparison of patient-matched primary and recurrent malignant tumors uncovered loss of PIK3CA activating mutation during the evolution of the tumor. Loss of the short arm of chromosome 1 along with somatic missense and truncating mutations in CDKN2C and THRAP3 provided new evidence for the potential role of these as tumor suppressors. Hürthle cell thyroid carcinoma accounts for a small proportion of all thyroid cancers; however, this malignancy often presents at an advanced stage and poses unique challenges. Genomic analysis revealed large regions of copy number variation encompassing nearly the entire genomes accompanied also by near haploidization. Moreover, I identified loss-of-function mutations of the tumor suppressor gene MEN1 in 4% of patients. Repeated alterations of the epigenetic machinery in anaplastic thyroid carcinoma, one of the most fatal of all adult solid malignancies, and novel gene fusions including MKRN1-BRAF, FGFR2-OGDH and SS18-SLC5A11 are reported here. The transcriptomic analysis suggested known drug targets such as FGFRs, VEGFRs, KIT and RET to have low expressions in this cancer; however, through integrative data analysis, I identified the mTOR signaling pathway as a potential therapeutic target for anaplastic thyroid cancer. Molecular analysis of papillary thyroid carcinoma and benign thyroid nodules revealed very low mutation rates in these tumors with CYP1B1, PTPRE, CTSH and RUNX1 emerging as promising diagnostic markers. The key somatic mutations identified in these studies can serve as novel diagnostic markers as well as therapeutic targets.
Science, Faculty of
Graduate

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

S. 1-66: sammanfattning, s. 69-190: 6 uppsatser

digitalisering@umu

Thesis (M.A.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 29, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--Michigan State University. Dept. of Zoology-Environmental Toxicology, 2008.
Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Multiple endocrine neoplasia syndrome type 1 (MEN1) is an autosomal dominantly inherited disease, which is described as an association of tumors mainly in endocrine organs, including pancreas and adrenal glands. Pancreatic neuroendocrine tumors (PNETs) are the most common cause of death in MEN1 patients. More than one third of the MEN1 patients also develop enlargement of the adrenals. MEN1 is caused by a germline mutation of MEN1 gene, a tumor suppressor gene that is located on the human chromosome 11. As noticed, the MEN1 related tumors often develop prior to inactivation of both wild type alleles, indicating MEN1 haploinsufficiency. In this thesis, I utilized a conventional Men1 mouse model that has the phenotype mimicking the human MEN 1 traits, in order to investigate MEN1 tumorigenesis in endocrine pancreas and adrenal glands.   The microvascular aberrations contributing to development and maintenance of PNETs were characterized. The increased vascular density of PNETs developed in the Men1 mice was paralleled by an early and extensive redistribution of pericytes within endocrine tissue. These morphological alterations were supported by fine-tuned variations in expression of several angiogenic regulators  (VEGF, FGF and PDGF) and were further potentiated by hypoxia. Vascular reactivity and blood perfusion of tumor arterioles were significantly altered in response to glucose and L-nitro-arginine methyl ester. Investigation of adrenals from10-month-old Men1 mice showed 681 proteins in mass spectrometry data sets, in which 52 proteins were commonly found in the Men1+/+ and Men1+/- adrenals, and the differential expression between the genotypes reached significant levels. Prdx3, catalyzing the reduction of oxidative stress to cell survival, is one of the overexpressed proteins. Some proteins belonging to the PPARα pathway, e.g. ACLY were also overexpressed. Subsequent microRNA (miRNA) profiling analysis of adrenals from the same age group revealed 31 miRNAs whose expression was significantly altered in comparison between the genotypes. The tumor suppressor miRNAs, miR-486, miR-330 and miR-214, were significantly downregulated in Men1+/- adrenals. The latter, miR-214, is known to inhibit ACLY expression. This finding was in concordance with the proteomic analysis. The oncogene miRNAs, miR-132 and miR-494, were significantly enhanced in the Men1+/- adrenals. Gene ontology analysis demonstrated overrepresentation of the miRNA-targeted genes that are involved in nucleic acid metabolism, vasculature development, angiogenesis, and transcription. Together, these finding after validation in humans may be exploited to improve MEN1 cancer treatment.

The effect of forced molt on pituitary function and other endocrine parameters was investigated in three year old hens subjected to a dietary forced molting procedure. In addition to molting, fasting caused cessation of egg production, body and organ weight loss, alterations in hormone secretion and morphological changes in some endocrine glands. Body and ovary weights decreased but returned to normal with ad libitum feeding. Pituitary, thyroid and adrenal weights were not affected but serum hormone levels measured by RIA revealed a decrease in LH, FSH and PRL and increases in TSH, T₃ and GH all of which returned to higher levels with ad libitum feeding. Serum P₄ levels remained low (and egg-laying stopped) until ad libitum feeding was resumed and then increased and egg-laying returned to a typically productive level. Serum ACTH and T₄ increased with fasting and remained elevated. Gonadotrophs and corticotrophs increased in numbers with fasting and/or food restriction but thyrotrophs, somatotrophs and lactotrophs decreased. Correlations between cell populations and serum hormone levels was quite common. Colloid-filled follicles resembling a hypertrophic thyroid gland occurred throughout the pituitary pars distalis. Granules appear to be discharged into the follicular lumen through exocytotic pores in the apical plasmalemma of follicular cells. Lactotrophs, corticotrophs and somatotrophs are commonly arranged in follicles or clusters. PRL-containing granules are in the center of some follicles and are concentrated near pituitary cysts. Pituitary cysts, lined with ciliated epithelium and sparse mucous cells, are more prevalent in fasted hens and decline with the resumption of feeding. Reduced lactotroph populations and presumptively degenerated lactotrophs in cyst lumens are correlated with reduced serum PRL levels. Necrotic cells occurred in the pituitary parenchyma of fasted birds but dilated RER in the thyrotrophs of fasted hens indicate enhanced activity of these cells. Ultrastructural evidence presented here indicates that pituitary secretion by lactotrophs occurs both intraluminally and perivascularly.

'Endocrine disrupting chemicals (EDCs) are compounds found in the environment that have the potential to disrupt normal endocrine function. Estrogenic EDCs (e-EDCs) is a subclass of EDCs and is defined as substances contaminating the environment that may mimic or inhibit the effect of endogenous estrogen and therefore may influence developmental and reproductive health in humans and animals. The aim of this study was to develop, validate and implement a battery of in vitro and in vivo screening assays for e-EDCs. The study was concluded by implementing this battery of assays to assess the Eerste River, South Africa at three sampling sites, namely Jonkershoek, Stellenbosch sewage treatment works (STW) effluent and Spier for e-EDCs. The control site, Jonkershoek contained very low levels of estrone. Water from this site showed no estrogenic activity when the E-screen and the ER_ induction in MCF-7 cells. Some of the water samples collected at this site tested positive for estrogenicity when analysed with the juvenile tilapia VTG assay, whereas the rest were negative. The estrone levels in the sewage effluent extracts as well as Spier were significantly higher. The assay using ER_ protein induction by the MCF-7 cell line, the MCF-7 proliferation assay and the tilapia in vivo screen for estrogenicity showed that these samples are estrogenic. Results obtained for estrogenicity at the three different sampling sites for each of the assays in the battery were comparable. In this study we developed, validated and also implemented a battery of assays encompassing both in vitro and in vivo assays, based on different biological mechanisms, to detect estrogenic EDCs. To our knowledge, this is the first study that has used a battery of bioassays to specifically assess a South Africa river for estrogenicity...'

RESUMO: As concentrações circulantes de cálcio são notavelmente constantes a despeito das variações diárias na absorção intestinal e na eliminação renal deste elemento. A regulação da calcémia é um sistema complexo que compreende vários factores controladores (a calcémia, a fosforémia, as concentrações circulantes de paratormona (PTH) e calcitriol além de muitos outros factores como hormonas esteróides em geral, outros iões como o magnésio e outros factores hormonais) e vários órgãos alvo (glândulas paratiroideias, osso, rim e intestino). As respostas dos órgãos alvo também são muito variadas. No caso mais simples, a cristalização de sais de cálcio corresponde a uma mudança de fase em que participam moléculas orgânicas que a iniciam, aceleram ou inibem. Em geral a combinação de um factor controlador com o respectivo receptor de membrana (para polipeptídeos ou iões) ou intracelular (hormonas esteróides) é apenas o primeiro passo de uma cadeia bioquímica que introduz uma enorme amplificação na resposta. A esta variedade de mecanismos de resposta correspondem grandes diferenças nos tempos de resposta que podem ser de minutos a semanas. É hoje possível “observar” (medir) com apreciável rigor nos líquidos biológicos (sangue, urina, fezes, etc.) os factores mais importantes do sistema de regulação da calcémia (cálcio, fósforo, paratormona e calcitriol) assim como administrar estes factores em experiências agudas. Esta possibilidade reflecte – se na literatura neste campo que tem vindo a crescer. O advento das técnicas da biologia molecular tem permitido a caracterização molecular de algumas das disfunções da homeostase do cálcio e é de esperar um diagnóstico fisiopatológico cada vez mais rigoroso dessas disfunções. Com o avanço dos conhecimentos nesta área que não cessa de aumentar temos cada vez maiores capacidades para fazer diagnósticos e é cada vez mais difícil interpretar com rigor os correspondentes quadros metabólicos. A análise ou síntese de sistemas complexos é a actividade mais nobre dos engenheiros que lhes permite desenhar pontes, diques, barcos, aviões ou automóveis. Com o aparecimento de computadores de médio ou grande porte foi – lhes possível utilizar descrições matemáticas não só para desenhar sistemas como ainda para interpretar eventuais falhas na sua operação. Essas descrições matemáticas consistem numa sequência de operações realizadas num computador segundo um “programa informático” que receberam a designação genérica de modelos, por analogia com as famosas leis (equações) da física que foram deduzidas a partir de um certo número de postulados e que permitem representar matematicamente processos físicos. As famosas leis de Newton são talvez os exemplos mais famosos de “modelos” de sistemas físicos. A introdução de modelos matemáticos em biologia e particularmente em medicina só se deu recentemente.MÉTODOS No trabalho que aqui se apresenta construiu - se um modelo simplificado da homeostase do cálcio destinado ao cálculo de variáveis observáveis (concentrações de cálcio, fósforo, PTH e calcitriol) de modo a poderem comparar-se valores calculados com valores observados. A escolha dos componentes do modelo foi determinada pela nossa experiência clínica e pela informação fisiopatológica e clínica publicada. Houve a preocupação de construir o modelo de forma modular de modo a ser possível a sua expansão sem grandes transformações na descrição matemática (e informática) já existente. Na sua fase actual o modelo não pode ser usado como instrumento de diagnóstico. É antes uma ferramenta destinada a esclarecer “em princípio” mecanismos fisiopatológicos. Usou – se o modelo para simular um certo número de observações publicadas e para exemplificar a sua eventual aplicação clínica na simulação de situações hipotéticas e na análise de possíveis mecanismos fisiopatológicos responsáveis por situações de hipo ou hipercalcémias. Simultaneamente fez – se uma análise dos dados acumulados relativos a doentes vistos no Serviço de Endocrinologia do Instituto Português de Oncologia de Francisco Gentil – Centro Regional Oncológico de Lisboa, S.A. CONCLUSÕES Numa população de 894 doentes com patologias variadas do Instituto Português de Oncologia de Lisboa os valores da calcémia tiveram uma distribuição normal unimodal com uma média de 9.56 mg/dl, e um erro padrão de 0.41 mg/dl. Estas observações sugerem que a calcémia está sujeita a regulação. A partir dos resultados publicados em que o metabolismo do cálcio foi perturbado por infusões de cálcio, calcitriol ou PTH, de estudos bioquímicos e fisiológicos sobre os mecanismos de acção de factores controladores da calcémia e do estudo do comportamento de órgãos alvo (paratiroideias, intestino, osso e rim) foi possível construir um modelo matemático de parâmetros concentrados do sistema de regulação da calcémia. As expressões analíticas usadas foram baseadas na cinética enzimática de modo a que os seus parâmetros tivessem um significado físico ou fisiológico simples. O modelo revelou apreciável robustez e flexibilidade. É estável quando não perturbado e transita entre estados estacionários quando perturbado. Na sua forma actual gera simulações que reproduzem satisfatoriamente um número apreciável de dados experimentais colhidos em doentes. Isto não significa que possa ser usado como instrumento de diagnóstico aplicável a doentes individuais. O desenho do modelo comporta a adição posterior de novas relações quando surgirem situações para as quais se revele insuficiente. A utilização exaustiva do modelo permitiu explicitar aspectos do metabolismo do cálcio que ou não estão contidas na sua formulação actual – o aparecimento de hipertrofia ou de adenomas das paratiroideias e as alterações na estrutura óssea , a participação de outros factores controladores – magnésio, ou estão insuficientemente descritas – alterações do metabolismo do fósforo nos hipoparatiroidismos. A análise dos dados relativos aos doentes do Serviço de Endocrinologia do IPO permitiu o início da caracterização dos tipos de patologia que representam e de possíveis mecanismos fisiopatológicos subjacentes. Estas observações são o ponto de partida para análises futuras. São exemplos das relações encontradas: a distribuição dos doentes por dois grandes grupos conforme a calcémia é determinada pelas concentrações circulantes de PTH ou estas são determinadas pela calcémia; a distribuição sazonal das concentrações de Vit. D25. no sangue; a correlação negativa entre estas e as concentrações de PTH no sangue. Também foi possível extrair a cinética do controlo da PTH sobre a síntese de calcitriol. O estudo dos níveis circulantes de PTH no pós-operatório imediato de doentes paratiroidectomizados permitiu determinar as suas taxas de degradação metabólica. O modelo permitiu simular as relações Ca/PTH no sangue, Ca/Fracção excretada da carga tubular, Ca/P no sangue para valores normais ou altos de Ca. Foram feitas simulações de situações fisiopatológicas (em “doentes virtuais”): infusões crónicas de cálcio, PTH e calcitriol; alterações no comportamento de receptores. Estas simulações correspondem a experiências que não podem ser realizadas em humanos. São exemplos da utilização do modelo na exploração de possíveis mecanismos fisiopatológicos através da observação de resultados quantitativos inacessíveis à intuição. O modelo foi útil em duas fases do trabalho: Primeiro, durante a sua síntese implicou uma escolha criticamente selectiva de informação, sua análise quantitativa e processamento, uma explicitação rigorosa (analítica) das relações funcionais entre os controladores e as variáveis e da sua integração numa estrutura global; Segundo, a simulação de situações experimentais ou clínicas (dados do Serviço de Endocrinologia do IPO) em doentes obrigou a explicitar raciocínios fisiopatológicos habitualmente formulados em bases puramente intuitivas. Esta prática revelou comportamentos óbvios após as simulações – acção reduzida das infusões PTH (simulação de hiperparatiroidismos primários) enquanto não há inibição total da respectiva secreção, necessidade de aumento da massa secretora da paratiroideia nas insuficiências renais avançadas, etc. A síntese e utilização do modelo não implicaram uma preparação matemática avançada e foram possíveis mercê da disponibilidade de “software” interactivo especificamente desenhado para a simulação de sistemas dinâmicos em que os programas se escrevem em inglês usando a simbologia simples da álgebra elementar. A função nobre de modelos desta natureza é semelhante à dos modelos usados pelos físicos desde o século XVII: permitir explicações de carácter geral funcionando como uma ferramenta intelectual para manipulação de conceitos e para a realização de “experiências pensadas” (“thought experiments”) respeitando certos princípios físicos (princípios de conservação) que estabelecem as fronteiras da realidade. -------ABSTRACT: Calcium blood levels are remarkably constant despite great variations in calcium daily intake, intestinal absorption and renal excretion. The regulation of the calcium concentration in the blood is achieved by a complex system that includes several controller factors (mainly the serum levels of calcium, phosphorus, parathyroid hormone (PTH) and calcitriol but also of steroid hormones, ions such as magnesium and other hormonal factors) and several target organs (parathyroid glands, bone, kidney and intestine). The functional response to the controlling factors obeys a variety of kinetics. The precipitation of calcium salts is a simple phase transition in which organic molecules may provide nucleation centres or inhibit the process. The combination of a controller factor with its receptor located in the cell membrane (for peptides or ions) or in the nucleus (for steroid hormones) is only the first step of a biochemical chain that introduces a huge amplification in the response. To this great variability of response we have to add the times of response that vary from minutes to weeks. It is possible to “observe” (measure) with great accuracy in biological fluids (blood, urine, faeces, etc.) the most important factors intervening in the calcium regulation (calcium, phosphorus, PTH and calcitriol). The response of the system to acute infusions of the controlling factors has also been studied. Using molecular biology techniques it has been possible to characterize some calcium homeostasis dysfunctions and better physiopathological diagnosis are expected. With the increasingly new knowledge in this area we have better capacity to diagnose but it is harder to explain correctly the underlying metabolic mechanisms. The analysis or synthesis of complex systems is the noble activity of engineers that enables them to draw bridges, dams, boats, airplanes or cars. With the availability of medium-large frame computers it was possible to use mathematical descriptions not only to draw systems but also to explain flaws in its operations. These mathematical descriptions are generally known as models by analogy with the laws (equations) of physics that allow the mathematical description of physical processes. In practice it is not possible to find general solutions for the mathematical descriptions of complex systems but (numeric) computations for specific situations can be obtained with digital computers. The introduction of mathematical models in biology and particularly in medicine is a recent event. METHODS In this thesis a simplified model of calcium homeostasis was built that enables the computation of observable variables (concentrations of calcium, phosphorus, PTH and calcitriol) and allows the comparison between the simulation values and observed values. The choice of the model’s components was made according to our clinical experience and to the published clinical and physiopathological data. The model has a modular design that allows future expansions with minor alterations in its structure. In its present form the model cannot be used for diagnosis. It is a tool designed to enlighten physiopathological processes. To exemplify its possible clinical application in the simulation of hypothetical situations and in the analysis of possible mechanisms responsible for hypo or hypercalcemias the model was used to simulate a certain number of published observations. An analysis of clinical and laboratory data from the Endocrinology Department of the Portuguese Cancer Institute (I.P.O.F.G.-C.R.O.L.,S.A.) is also presented. CONCLUSIONS In a population of 188 patients without an identifiable disease of the calcium metabolism at the Portuguese Cancer Institute the calcemia levels had a unimodal distribution with an average of 9.56 mg/dL and a S.E.M of 0.41 mg/dL. This observation confirms that serum calcium is regulated. Using published data; in which calcium metabolism was disrupted by calcium, PTH or calcitriol infusions; from biochemical and physiological studies of the action of controller factors on the calcemia; in which the response of target organs (parathyroid glands, intestine, bone, kidney) was studied it was possible to build a mathematical model of concentrated parameters of the calcium homeostasis. Analytical expressions used were based on enzymatic kinetics. The model is flexible and robust. It is stable when not disturbed and changes between steady states when disturbed. In its present form it provides simulations that reproduce closely a number of experimental clinical data. This does not mean that it can be used as a diagnostic tool for individual patients. The exhaustive utilisation of the model revealed the need of future expansions to include aspects of the calcium metabolism not included in its present form –hypertrophy or adenomas of the parathyroid glands, bone structure changes, participation of other controller factors such as magnesium – or insufficiently described – phosphate metabolism in hypoparathyroidism. The analysis of the data collected from the I.P.O.’s Endocrinology Department allowed the initial characterization of the different pathologies represented and of their possible physiopathological mechanisms. These observations are a starting point for future analysis. As examples of the relations found were: the distribution of patients in two groups according to the dependency of calcium by PTH levels or PTH levels by calcium concentration; the seasonal distribution of the serum concentrations of D25; its negative correlation with PTH concentration. It was also possible to extract the kinetics of the control of the synthesis of calcitriol by PTH. The analysis of immediate post-surgical levels of PTH in parathyroidectomized patients allowed the determination of its metabolic clearance. The model also allowed the simulation of the relations between Ca/PTH in blood, serum Ca/Fraction of tubular load excreted and Ca/P in blood for normal and high values of calcium. Simulations were made of pathological situations (in “virtual patients”): chronic infusions of calcium, PTH and calcitriol; changes in the characteristics of receptors. These simulations are not possible in real persons. They are an example of the use of this model in exploring possible mechanisms of disease through the observation of quantitative results not accessible to simple intuition. This model was useful in two phases: Firstly, its construction required a careful choice of data, its quantitative analysis and processing, an analytical description of the relations between controller factors and variables and their integration in a global structure. Secondly, the simulation of experimental or clinical (I.P.O.’s Endocrinology Department) data implied testing physiopathological explanations that previously were based on intuition. The construction and utilisation of the model didn’t demand an advanced mathematical preparation since user-friendly interactive software was used. This software was specifically designed for the simulation of dynamic systems. The programs are written in English using elementary algebra symbols. The essential function of this type of models is identical to that of those used by physicists since the XVII century which describe quantitatively natural processes and are an intellectual tool for the manipulation of concepts and the performance of “thought experiments” based in certain physical principles (conservation principles) that are the frontiers of reality.------------------RESUMÉE: Les concentrations circulantes de calcium sont constantes même pendant des variations de l’absorption intestinale et de l’élimination rénale de cet élément. La régulation de la calcémie est un système complexe qui comprend plusieurs éléments contrôleurs (la calcémie, la phosphorémie, les concentrations circulantes de l’hormone parathyroïdienne (PTH) e du calcitriol et d’autres comme les hormones stéroïdes ou des ions comme le magnésium) et plusieurs organes (glandes parathyroïdiennes, l’os, le rein et l’intestin). Les réponses de ces organes sont variées. Dans le cas plus simple, la cristallisation des sels de calcium correspond à un changement de phase dans lequel y participent des molécules organiques que la débutent, l’accélèrent ou l’inhibent. Généralement la combinaison d’un élément contrôleur avec leur récepteur de membrane (pour les peptides ou les ions) ou intracellulaire (pour les hormones stéroïdes) n’est que le premier pas d’une chaîne biochimique qu’introduit une grande amplification de la réponse. A cette variété de réponses correspondent des grandes différences des temps de réponses qu’y vont des minuits a semaines. Il est possible « observer » (mesurer) dans les fluides biologiques (sang, urine, fèces, etc.) les éléments plus importants du système de régulation de la calcémie (calcium, phosphate, PTH et le calcitriol) et les administrer en expérimentes aigus. Cette possibilité est visible dans la littérature publiée dans ce domaine qui est en croissance permanente. L’avenir des techniques de biologie moléculaire a permis caractériser des nombreuses dysfonctions de la régulation de la calcémie et on attend un diagnostique physiopathologique de ces dysfonctions chaque fois plus rigoureuses. Les connaissances dans ce domaine s’agrandissent et on a de plus de capacités pour faire des diagnostiques et il est chaque fois plus difficile les interpréter. L’analyse ou synthèse de systèmes complexes est l’activité plus noble des ingénieurs qui les permit dessiner des ponts, bateaux, avions ou automobiles. Avec des ordinateurs de médium ou grand port il les est possible utiliser descriptions mathématiques pour dessiner les systèmes et interpréter des éventuelles fautes d’opération. Ces descriptions mathématiques sont une séquence d’opérations réalisées dans un ordinateur selon « un programme informatique » qui ont reçu la désignation générique de modèles, pour analogie avec les équations de la physique qui ont été déduits d’un nombre de postulées et qu’ont permit représenter des processus physiques en équations mathématiques. Les fameuses équations de Newton sont peut-être les exemples plus connus des systèmes physiques. L’introduction des modèles mathématiques en biologie et en particulier en médecine est un évènement récent. Dans ce travaille, on a construit un modèle simplifié de l’homéostasie du calcium pour calculer les variables observables (concentrations de calcium, phosphate, PTH et calcitriol) pour les comparer. Les choix des components a été déterminés par notre expérience clinique et par l’information physiopathologique et clinique publiée. Le modèle a été construit de façon modulaire ce que permit leur postérieur expansion sans des grandes altérations dans la description mathématique et informatique déjà existante. Dans cette forme le modèle ne peut être utilisé comme un instrument de diagnostique. Il est un outil pour éclairer la physiopathologie. Le modèle a été utilisé pour simuler un certain nombre d’observations publiées et pour exemplifier leur possible utilisation clinique dans la simulation des hypothèses et de la physiopathologie des situations d’hypo ou hypercalcémie. On a fait une analyse des éléments des procès cliniques des malades observées dans le Service d’Endocrinologie de l’IPOFG-CROL, SA. Dans une population de 894 malades avec des différentes pathologies les valeurs de calcémie on une distribution uni modale avec une Médie de 9.56 mg/dL et une erreur standard de 0.41 mg/dL. Ces observations suggèrent que la calcémie soit sujette de régulation. En utilisant des résultats de travaux publiés dans lesquels le métabolisme du calcium a été changé par des infusions de calcium, calcitriol ou PTH, des études biochimiques et physiologiques sur des mécanismes d’action des éléments contrôleurs de la calcémie et de l’étude du comportement des organes cible (parathyroïdes, intestin, rein, os), il a été possible de construire un modèle mathématique de paramètres concentrés du système de régulation de la calcémie. Les expressions analytiques utilisées ont été basées sur la cinétique enzymatique de façon à que les paramètres aient eu une signification physique ou biologique. Le modèle est stable quand il n’est pas perturbé et transit entre états stationnaires quand il est sujet a des perturbations. A ce moment il fait des simulations qui reproduisent de façon satisfaisant un nombre d’observations expérimentales. La construction du modèle permit l’addiction de nouvelles relations dans les cas ou il est insuffisant. L’utilisation exhaustive du modèle a permit expliciter des aspects du métabolisme du calcium qui y ne sont pas compris – l’hyperplasie ou la formation des adénomes des parathyroïdes, les altérations de la structure des os, la participation d’outres éléments régulateurs (magnésium), ou sont insuffisamment décrites – les altérations du métabolisme des phosphates dans l’hypoparathyroidism. L’analyse de l’information des malades du Service d’Endocrinologie a permit caractériser les pathologies représentées et leurs possibles mécanismes physiopathologiques. Ces observations sont le point de départ pour les analyses futures. Sont des exemples des relations trouvées: la distribution des malades par deux groupes: ceux dans lequel la calcémie est déterminée par la PTH ou ceux dans lesquels la PTH est déterminée par la calcémie; la distribution sazonale de la concentration de la vitamine D; la corrélation négative entre la vitamine D et la PTH. On a eu la possibilité de déduire la cinétique de control de la PTH sur la synthèse du calcitriol. L’étude des niveaux circulants de PTH sur des sujets parathyroidectomisées a permit déduire leur taux de dégradation métabolique. Le modèle a permit simuler les relations Ca/PTH dans le sang, Ca/fraction éliminée par le rein, Ca/P dans le sang pour des valeurs normales ou hautes de calcium. On a fait des simulations de situations physiopathologiques (dans “malades virtuelles”): Infusions chroniques de calcium, PTH ou calcitriol; altérations des récepteurs. Ces simulations ne peuvent pas être réalisées dans les humains. Sont des exemples d’utilisation du modèle dans l’exploration des possibles mécanismes de la physiopathologie en observant des résultats quantitatifs inaccessibles à l’intuition. Le modèle a été utile pendant deux étapes des travaux: La première, dans sa construction on a choisi l’information disponible, son analyse quantitative, l’explicitation rigoureuse (analytique) des relations fonctionnelles entre les contrôleurs et les variables et sa intégration dans une structure globale. La deuxième, la simulation de situations expérimentales ou cliniques (du Service d’Endocrinologie) a obligé d’expliciter des raisonnements physiopathologiques généralement formulés utilisant l’intuition. Cette pratique a montré des comportements – action réduite des infusions de PTH (jusqu’à l’inhibition totale de leur respective sécrétion), nécessité d’augmenter la masse sécréteuse de la parathyroïde dans les insuffisants rénales, etc. La synthèse et utilisation du modèle n’ont pas besoin d’une formation avancée en mathématique et sont possibles grâce à un programme interactif qui a été conçu pour la simulation des systèmes dynamiques dans lesquels le programme se construit en anglais en utilisant la symbolique élémentaire de l’algèbre. La fonction noble de ces modèles est semblable à celles des physiques du XVII siècle: Permettre établir explications générales en fonctionnant comme un outil intellectuel pour manipuler des concepts et pour la réalisation d’expérimentes pensées en respectant certains principes de la physique (principe de la conservation) qu’établissent les frontières de la réalité.

Thesis (Ph. D.)--Michigan State University. Dept. of Zoology & Environmental Toxicology, 2008.
Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Thesis (MSc)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: The uropygial gland of most birds produces a variety of hydrocarbons, lipids, waxes, fatty acids, alcohols and other organic compounds. These compounds have two widely recognized functions, viz. they are considered essential for the maintenance of a good plumage condition, and may be used for fungicidal, bactericidal or other hygienic purposes. Scimitar-billed woodhoopoes, Rhinopomastus cyanomelas, are groupterritorial birds that live in groups comprising between two and twelve individuals. Individuals enter the roost cavities shortly after sunset and exit the following morning soon after sunrise. During the period that the birds are inside the roost, they are vulnerable to a range of vertebrate predators, including snakes, genets and rats. When disturbed while roosting, woodhoopoes immediately face away from the threat hence presenting their uropygial glands in the direction of the threat. Typically, a drop of brown, highly pungent secretion is then formed at the tip of the papilla to the uropygial gland, and kept in place by a few tuft-like feathers. This response pattern has led some observers to believe that the secretion serves an anti-predatory role. It has been found that the synthetic volatile constituents of the uropygial secretion of the green woodhoopoe, P. purpureus, individually or as a mixture, have potent defensive properties against feline and reptilian predators. In addition, the compounds also showed activity against a range of bacteria. The aim of the present study was to determine the chemical composition of the uropygial secretion of the scimitar-billed woodhoopoe, Rhinopomastus cyanomelas, as a first step towards the evaluation of, inter alia, the semiochemical function of the secretion. Using gas chromatography-mass spectrometry, 179 constituents of the uropygial secretion of the scimitar-billed woodhoopoe have been identified. The majority of the constituents of the secretion are branched and unbranched aldehydes (aliphatic and aromatic), acids (aliphatic and aromatic), sulfides and ketones. This group of volatile compounds is responsible for the obnoxious odour of the secretion and possibly also for its defensive action against predators. The secretion also contains a large number of branched and unbranched alkanes and wax esters. The chemical composition of the secretion was compared with the secretion of P. purpureus as well as with that of the hoopoe, Upupa africana. The uropygial gland secretion of the scimitar-billed woodhoopoe is quite similar to that of the green woodhoopoe, although it is much more complex than that of the green woodhoopoe. In contrast to the uropygial secretions of the green and the scimitar-billed woodhoopoes, the secretion of Upupa africana does not have a strongly obnoxious odour and it also does not contain large quantities of alkanes and wax esters.
AFRIKAANSE OPSOMMING: Die uropigiale klier van die meeste voëls produseer 'n verskeidenheid van koolwaterstowwe, lipiede, was-esters, vetsure, alkohole en ander organiese verbindings. Hierdie verbindings het twee algemeen erkende funksies, naamlik die instandhouding van die goeie kondisie van die vere, en 'n swam- en kiemdodende werking. Swartbekkakelaars (Engels: scimitar-billed woodhoopoes ), Rhinopomastus cyanomelas, is groep-territoriale voëls wat in groepe van tussen twee en twaalf saam woon. Individue gaan hul neste net na sononder binne en verlaat dit weer die volgende oggend net na sonsopkoms. Terwyl die voëls binne die neste is, is hulle kwesbaar ten opsigte van aanval deur verskeie gewerwelde roofdiere, insluitende slange, muskeljaatkatte en rotte. Wanneer hulle in hul neste gesteur word, sal kakelaars onmiddellik wegdraai van die bedreiging sodat die uropigiale klier in die rigting van die bedreiging gekeer is. 'n Druppel bruin, uiters onwelriekende afskeiding vorm dan by die punt van die papil na die uropigiale klier, en word in posisie gehou deur 'n verekwassie. Hierdie gedragspatroon het aanleiding gegee tot die gedagte by sommige waarnemers dat die afskeiding as afweerstof teen roofdiere dien. Daar is gevind dat die sintetiese vlugtige komponente van die uropigiale afskeiding van die groenkakelaar, P. purpureus, individueel of as 'n mengsel, sterk afweer-eienskappe teen katte en reptiele toon. Daarbenewens het die verbindings ook aktiwiteit getoon teen 'n reeks van bakterieë. Die doel van die huidige studie was om die chemiese samestelling van die uropigiale afskeiding van die swartbekkakelaar, Rhinopomastus cyanomelas, te bepaal as 'n eerste stap met die oog op die evaluering van, onder andere, die semiochemiese funksie van die afskeiding. Deur van gaschromatografie-massaspektrometrie gebruik te maak, is 179 komponente van die uropigiale afskeiding van die swartbekkakelaar geïdentifiseer. Die meeste van die komponente is vertakte en onvertakte aldehiede (alifaties en aromaties), sure (alifaties en aromaties), sulfiede en ketone. Hierdie groep vlugtige verbindings is verantwoordelik vir die afstootlike reuk van die afskeiding en waarskynlik ook vir sy afweer-aksie teen roofdiere. Die afskeiding bevat ook 'n groot aantal vertakte en onvertakte alkane en wasesters. Die chemiese samestelling van die afskeiding is vergelyk met die van P. purpureus sowel as dié van die hoepoe, Upupa africana. Die uropigiale klierafskeiding van die swartbekkakelaar stem tot 'n groot mate ooreen met dié van die groenkakelaar, alhoewel dit veel meer kompleks is as dié van die groenkakelaar. In teenstelling met die uropigiale afskeidings van die groen- en die swartbekkakelaars, het die afskeiding van Upupa africana nie 'n afstootlike reuk nie en bevat dit ook nie groot hoeveelhede alkane en was-esters nie.

Total parenteral nutrition (TPN) provides an experimental situation where adequate nutrition is provided intravenously, bypassing the gastrointestinal tract. Under these conditions the importance of orally ingested nutrients in the control of gastric and pancreatic endocrine secretion can be assessed. The objectives of this thesis were two-fold. First, to examine the effects of TPN on the enteroinsular axis component of insulin secretion. Second, to study the importance of orally ingested nutrients in the regulation of gastric hormone secretion using the TPN rat model. In order to carry out these objectives, techniques for TPN and enteral feeding (TEN) of the rat were first developed. A dietary regimen for use in TPN and TEN rats was formulated from commercially-available, human TPN components. Under most circumstances, the TPN/TEN regimen met or exceeded the nutritional requirements for growing rats, as determined by the National Research Council (1978). Hematological analysis revealed few side effects of — intravenous or intragastric feeding. Parenterally and enterally-fed animals demonstrated comparable weight gain to that of a control group (ORAL) fed a rat chow (#5012, Ralston Purina) diet ad libitum. In addition, both TPN and TEN animals appeared healthy after the 7-day infusion period. These studies indicated that the infusion formulation was suitable for chronic intravenous and intragastric feeding. In the first series of experiments, the effects of TPN and TEN on the hormonal component of the enteroinsular axis were studied. TPN animals exhibited hyperinsulinemia and mild hyperglycemia. Conversely, TEN animals exhibited normal plasma glucose and immunoreactive insulin (IRI) concentrations. These data suggested that enterally delivered nutrients were assimilated with greater efficiency than intravenously administered nutrients. It was hypothesized that gut factors normally released by oral food intake facilitated the disposal of nutrients by hepatic and/or peripheral tissues. During the infusion period, TPN animals exhibited chronically depressed circulating IR-gastric inhibitory polypeptide (GIP) levels, in contrast to TEN animals where IR-GIP was elevated. Seven days of TPN or TEN resulted in no change in fasting plasma IRI or IR-GIP levels. However, an exaggerated insulin response to an oral glucose challenge (OGC) occurred after TPN, while the glucose response was reduced. The insulin response from the perfused pancreata of TPN animals to a GIP gradient was 20% and 40% greater than from ORAL and TEN pancreata respectively. Shorter periods of TPN (3 and 5-day periods) indicated that the hypersensitivity of the pancreas to GIP was a progressive condition, increasing with longer periods of infusion. Immunocytochemical and morphometric analysis revealed no differences in the jejunal GLP-cell population after chronic (7-day) intravenous or intragastric feeding. In addition, these routes of feeding had no effect on pancreatic islet area or endocrine cell composition of the islets. Based on these results, it was hypothesized that the increased B-cell sensitivity to GIP may have been causally related to the exposure of the pancreas to chronically low plasma GIP levels during the infusion period. To further test this hypothesis, chronically depressed plasma GIP levels, observed during TPN, were elevated by exogenous GIP infusion to levels seen in TEN rats. Chronic GIP —treatment in TPN animals (TPN-GIP) resulted in normalization of the insulin response to an OGC and in the in vitro insulin response of the isolated pancreas to GIP. These data were taken as further evidence that B-cell sensitivity to GIP was affected by ambient plasma GIP levels, and it was hypothesized that changes in sensitivity may be mediated by alteration at the receptor or post-receptor level. The effect of TPN on nutrient and neuronally mediated insulin release was also investigated. During TPN, metabolites and neuronal elements provided the main stimulus for insulin release, since hormonal components of the enteroinsular axis remained inactive. The present experiments indicated that the B-cell was hypersensitive to glucose, vagal stimulation and the cholinergic agonist methacholine, but normally sensitive to vasoactive intestinal polypeptide (VIP) and the insulinotropic amino acid arginine. These results indicated that TPN was associated with an increased B-cell sensitivity to specific hormonal, nutritive and neuronal stimuli. It was hypothesized that an increased B-cell sensitivity to these specific stimuli contributed to hyperinsulinemia observed in TPN animals during the infusion period, and to the exaggerated insulin response observed after an oral glucose challenge. Total parenteral nutrition also provided an experimental situation in which to study the importance of gastric nutrients in the regulation of Gl-hormone secretion. TPN resulted in a rapid and progressive depletion of circulating gastrin levels. G-cell secretory activity in vivo under basal and stimulatory conditions was also reduced by TPN. This condition persisted in vitro in the isolated stomach. The antral G-cell population was shown to decrease progressively with longer TPN periods, but G-cell hypoplasia and reductions in antral gastrin content were less dramatic than reductions in G-cell secretory activity. It was hypothesized that reductions in G-cell secretory activity were in part causally related to antral G-cell hypoplasia. The present data further suggested, however, that mechanisms which control synthesis and/or secretion within G-cells may have also been impaired, since various stimulants of gastrin release could not reverse gastrin hyposecretion observed during basal periods. Gastrin hyposecretion also could not be reversed by chronic bombesin administration, but was reversed by a 6-day period of oral — refeeding, indicating that the presence of nutrients in the gastric lumen was the primary regulator of tissue gastrin levels and G-cell secretory activity. The gastric D-cell was much less affected by the absence of nutrients in the gastric lumen than was the G-cell, and antral somatostatin hypersecretion may have contributed to G-cell hyposecretion. The experiments presented in this thesis indicated that total parenteral nutrition had marked effects on both B- and G-cell secretory activity. These studies clearly demonstrated the importance of enteral feeding in the maintenance of normal pancreatic and gastrointestinal endocrine secretion.
Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate

Thesis (MSc)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: Bontebok, Damaliscus pygargus pygargus, formerly classified as D. dorcas dorcas, are territorial animals with interdigital glands between the toes of the forelegs. Males regularly defecate on dung heaps, on which they often lie, to communicate with other members of their species. They also communicate by means of visual displays, scent marking and occasionally with scraping or pawing of dung heaps. It is assumed that scent marking with the interdigital secretion serves to define territories frequented by these antelope. These glands secrete a complex mixture of volatile and non-volatile compounds and the volatile compounds in the secretion serve as a chemical signal for other bontebok. It has been suggested that the interdigital secretion is not produced in its final composition by the interdigital gland alone, but that microbial activity is responsible for many of the compounds present in the secretion. In general, many compounds can be attributed to the by-products of microbial hydrolysis of triglycerides, a common characteristic of sebum. It is well documented that micro-organisms inhabit the deep recesses of sebaceous glands and the presence of micro-organisms has been found to be consistent in all antelope exocrine glandular areas. This study involved the chemical characterisation of the volatile metabolites produced in vitro by micro-organisms from the interdigital cavity of the bontebok. Various comparative studies were made, one of which was comparison of the metabolites produced by the individual microbial species as well as the total community of bacteria incubated in different media. A comparison of the compounds identified in the interdigital secretion and the metabolites produced by the micro-organisms in the different media was also made. The volatile metabolite extracts of the individual bacterial species and of the total community were chemically characterised by low-resolution gas chromatography-mass spectroscopy. Classes of compounds identified from the volatile metabolite extracts include: • Acids - Aliphatic (saturated and unsaturated) • Alcohols - Aliphatic (saturated and unsaturated) • Aldehydes - Aliphatic (saturated and unsaturated) • Aromatic compounds • Ketones - Aliphatic (saturated and unsaturated) • Pyrazines • Dimethyldisulphide • Squalene and cholesterol Several qualitative differences were found between the compounds identified in the volatile metabolite extracts of the micro-organisms when incubated in tryptic soy broth (TSB) and minimal salt medium (MSM). In particular, when the microbes were incubated in TSB medium a number of pyrazines were found that were not present when utilising MSM as a medium. Additional qualitative differences were found between the compounds identified in the metabolite extracts of the individual bacterial species and the total community of bacteria, when incubated in both TSB and MSM media. A comparison of the interdigital secretion and the metabolite extracts of the microbial communities incubated in TSB and MSM revealed that many compounds produced in MSM corresponded to the compounds identified in the interdigital secretion. These corresponding compounds were found to be saturated and unsaturated acids, aldehydes and squalene. Furthermore, there was only one corresponding compound in the case of TSB as medium.
AFRIKAANSE OPSOMMING: Die bontebok, Damaliscus pygargus pygargus, voorheen geklassifiseer as D. dorcas dorcas, is 'n territoriale dier met interdigitale kliere tussen die kloutjies van die voorpote. Ramme ontlas gereeld op mishope, waarop hulle dikwels lê, om met ander lede van die spesie te kommunikeer. Hulle kommunikeer ook deur middel van visuele seine, reukmerking en soms deur mishope met die voorpote te kap of te skraap. Reukmerking met die interdigitale afskeiding dien klaarblyklik om gebiede wat deur hierdie diere bewoon word, af te baken. Die interdigitale kliere skei 'n komplekse mengsel van vlugtige en nie-vlugtige verbindings af en die vlugtige verbindings dien as chemiese sein vir ander bontebokke. Die vermoede bestaan dat die interdigitale klier nie alleen verantwoordelik is vir die finale samestelling van die interdigitale afskeiding nie, maar dat mikrobiese aktiwiteit bydra tot die produksie van baie van die verbindings wat in die afskeiding aanwesig is. Sekere verbindings kan in die algemeen toegeskryf word aan die vorming van die neweprodukte van mikrobiese hidrolise van trigliseriede, 'n algemene eienskap van sebum. Dit is bekend dat die diep holtes van vetkliere 'n goeie teelaarde is vir mikroorganismes en daar is gevind dat mikroorganismes feitlik deurgaans voorkom in alle anteloop eksokriene klierareas. Hierdie studie behels die chemiese karakterisering van die vlugtige metaboliete wat in vitro deur mikroorganismes van die interdigitale klierholte van die bontebok geproduseer word. Verskeie vergelykende studies is uitgevoer waarvan een die vergelyking was van die metaboliete wat deur die individuele mikrobiese spesies sowel as die totale gemeenskap van bakterieë geproduseer word tydens inkubasie in verskillende media. Vergelyking van die verbindings wat in die interdigitale afskeiding geïdentifiseer is met die metaboliete wat in verskillende media geproduseer is, het ook deel van die studie uitgemaak. Die vlugtige metaboliet ekstrakte van die individuele bakteriese spesies en van die totale gemeenskap is chemies gekarakteriseer deur middel van laeresolusie gaschromatografie-massaspektrometrie. Die volgende groepe verbindings is onder andere in die vlugtige metaboliet ekstrakte geïdentifiseer: • Sure - Alifaties (versadig en onversadig) • Alkohole - Alifaties (versadig en onversadig) • Aldehiede - Alifaties (versadig en onversadig) • Aromatiese verbindings • Ketone - Alifaties (versadig en onversadig) • Pirasiene • Dimetieldisulfied • Skwaleen en cholesterol Verskeie kwalitatiewe verskille is gevind tussen die verbindings wat geïdentifiseer is in die vlugtige metaboliet ekstrakte van die mikroorganismes onderskeidelik in TSB medium en MSM geïnkubeer. Opvallend was byvoorbeeld die voorkoms van pirasiene in gevalle waar mikroorganismes in TSB medium geïnkubeer is, terwyl hierdie groep verbindings afwesig was wanneer MSM gebruik is. Onderlinge kwalitatiewe verskille is ook gevind tussen die verbindings wat geïdentifiseer is in die metaboliet ekstrakte van die individuele bakteriese spesies en die totale gemeenskap van bakterieë, wanneer in TSB medium sowel as in MSM geïnkubeer is. Vergelyking van die verbindings in die interdigitale afskeiding en in die metaboliet ekstrakte van die mikrobiese gemeenskappe, het getoon dat 'n aantal verbindings wat in MSM geproduseer is, ooreenstem met verbindings wat in die interdigitale afskeiding geïdentifiseer is. Daar is gevind dat hierdie verbindings versadigde en onversadigde sure en aldehiede en skwaleen is. Met TSB as medium was daar slegs een ooreenstemmende verbinding.

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%).

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.

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

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

O presente relatório foi realizado no âmbito do estágio curricular do Mestrado Integrado em Medicina Veterinária da Universidade de Évora, sendo constituído por duas partes. Na primeira parte é realizada uma análise da casuística acompanhada ao longo do estágio curricular no Hospital Veterinário das Laranjeiras, em Lisboa. A segunda parte é composta por uma revisão bibliográfica sobre o hipoadrenocorticismo canino, ou doença de Addison, acompanhado da apresentação e discussão de um caso clínico alusivo ao tema. O hipoadrenocorticismo é uma doença endócrina pouco comum caracterizada pela deficiência em glucocorticóides e mineralocorticóides. O hipoadrenocorticismo é classificado como primário, secundário, típico ou atípico. Não obstante o difícil diagnóstico face à similaridade com outras afeções, o entendimento da fisiopatologia da doença pode impulsionar o médico veterinário a proceder ao teste de estimulação com ACTH que confirma definitivamente o diagnóstico. O tratamento, desde que bem monitorizado, proporciona ao cão uma boa qualidade de vida; Abstract: Surgery and medicine of small animals This report was completed in the scope of Integrated Masters in Veterinary Medicine of the University of Évora internship’s which is divided in two parts. The first part consists of an analysis of the casuistry followed during the traineeship held at Hospital Veterinário das Laranjeiras, in Lisbon. The second part is composed by a literature review about canine hypoadrenocorticism, or Addison's disease, accompanied by the presentation and discussion of a clinical case alluding to the theme. Hypoadrenocorticism is an uncommon endocrine disease characterized by deficiency in glucocorticoids and mineralocorticoids. Hypoadrenocorticism is classified as primary or secondary and typical or atypical. Notwithstanding the difficult diagnosis due to the similarity with other affections, the comprehension of the pathophysiology of the disease may impel the veterinarian to perform an ACTH stimulation test which definitively confirms the diagnosis. The treatment, when well monitored, provides a good quality of life to the dog.

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

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.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Tese (Doutorado em Tecnologia Nuclear)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
FAPESP:11/21708-6

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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
This thesis involved the study of the adrenal and thyroid glands. The study of adrenals was based, primarily, on epidemiological and pathological aspects of the lesions observed in the glands of 300 dogs necropsied in the Laboratório de Patologia Veterinária at the Universidade Federal de Santa Maria (LPV/UFSM). For this purpose, a prospective study was performed from March 2012 to June 2013, totaling 16 months. Of the 300 dogs examined, 82% had one or more adrenal lesions. Only 18% of these dogs did not have adrenal changes. The hyperplastic lesions were the most prevalent, comprising 91.5% of the cases, followed by degenerative (26%), circulatory (20.3%), neoplastic (15%), inflammatory (4.1%) and other lesions (14.2%). Most of the animals with hyperplastic lesions consisted of females, elderly and purebred dogs. Fatty degeneration was the most common degenerative lesion (54.7%). Among the neoplastic changes, primary tumors were the most prevalent (75.7%), with a prevalence of adenomas (57.1%), followed by pheochromocytomas (25%) and cortical carcinomas (17.9%). Metastatic tumors corresponded to 24.3% of all cases of neoplastic changes. The adrenal glands were the site of metastasis of mammary gland carcinoma, osteosarcoma, melanoma, lymphoma, cholangiocarcinoma, histiocytic sarcoma and lung adenocarcinoma. Under "other lesions", adrenal of stress was the most prevalent change (37.1%). It was characterized histologically by a cortex with irregular appearance. For the study of thyroid lesions, it was reviewed the protocols of biopsies and necropsies performed at the LVP/UFSM between 2004 and 2014, and all cases of thyroid tumors were selected. Epidemiological data, clinical significance of tumors, presence of cervical swelling, affected lobes, tumor size, presence and location of metastasis and histological pattern of the neoplasm. It were recorded 26 tumors of the thyroid gland. Of the affected dogs, 53.9% were females and 46.1% were males; 65.4% were purebred. Of these, Boxers were the most affected (15.4%). In 23 protocols the age was informed; 60.9% of the dogs were elderly and 39.1% were adults. The protocols evaluation allowed establishing that 38.5% of the animals had shown cervical swelling, at the time of clinical examination. From the dogs that underwent to necropsy, only 13.6% died or were euthanized due to complications caused by metastases of thyroid tumors. Thus, in 86.4% of the dogs, the neoplasms were considered as incidental necropsy findings. Glandular unilateral involvement occurred in 75% of the dogs. The histological diagnosis of primary tumors showed that 61.5% of the neoplasms were classified as follicular cell carcinomas, 23.1% as follicular cell adenomas, 7.7% as carcinosarcomas and 7.7% as tumors of C cell. Additionally, two cases of thyroid follicular cell carcinoma in dogs that developed myelopathy secondary to cervical spinal epidural metastasis were minutely evaluated, and separately reported.
Esta tese envolveu o estudo das adrenais e da tireoide. O estudo das adrenais baseou-se, principalmente, nos aspectos epidemiológicos e patológicos das lesões observadas nas glândulas de 300 cães necropsiados no Laboratório de Patologia Veterinária da Universidade Federal de Santa Maria (LPV/UFSM). Esse estudo foi realizado de forma prospectiva, de março de 2012 a junho de 2013. Dos 300 cães analisados, 82% tinham uma ou mais alterações nas adrenais e 18% não tinham alterações. As lesões hiperplásicas foram as mais prevalentes, com 91,5% das ocorrências, seguidas de lesões degenerativas (26%), circulatórias (20,3%), neoplásicas (15%), inflamatórias (4,1%) e outras lesões (14,2%). A maioria dos animais que apresentou lesões hiperplásicas era constituída de fêmeas, idosos e cães com raça definida. A degeneração gordurosa foi a lesão degenerativa mais frequente (54.7%). Entre as alterações neoplásicas, tumores primários foram mais prevalentes (75,7%), com predomínio de adenomas (57,1%), seguido de feocromocitomas (25%) e carcinomas corticais (17,9%). Os tumores metastáticos perfizeram 24,3% dos casos de alterações neoplásicas. As adrenais foram sítio de metástase de carcinoma mamário, osteossarcoma, melanoma, linfoma, colangiocarcinoma, sarcoma histiocítico e adenocarcinoma pulmonar. No item outras lesões , adrenal de estresse foi a alteração mais prevalente (37,1%). Essa alteração era caracterizada histologicamente por córtex de aspecto variegado. Para o estudo das lesões de tireoide foram revisados os protocolos de biópsias e necropsias do LPV/UFSM entre os anos de 2004 e 2014 e todos os casos de tumores tireoidianos foram selecionados. A partir disso, foram anotados dados epidemiológicos, significado clínico dos tumores, presença de aumento de volume cervical, lobos afetados, tamanho, presença e localização de metástases e padrão histológico dos neoplasmas. Foram diagnosticados 26 neoplasmas tireoidianos. Dos cães acometidos, 53,9% eram fêmeas e 46,1% eram machos; 65,4% eram cães com raça definida. Desses, Boxers foram os mais afetados (15,4%). Dos 23 protocolos em que a idade foi informada, 39,1% eram cães adultos e 60,9% eram idosos. A avaliação dos protocolos permitiu constatar que 38,5% dos animais haviam apresentado aumento de volume cervical no momento do exame clínico. Apenas 13,6% dos cães necropsiados morreram ou foram submetidos à eutanásia em decorrência de complicações causadas por metástases dos tumores de tireoide. Dessa forma, em 86,4% dos cães os neoplasmas foram considerados achados incidentais de necropsia. O acometimento unilateral da glândula ocorreu em 75% dos cães. Os diagnósticos histológicos dos tumores primários permitiram constatar que 61,5% dos neoplasmas eram carcinomas de células foliculares, 23,1% eram adenomas de células foliculares, 7,7% eram carcinossarcomas e 7,7% eram tumores de células C. Além disso, foram estudados de forma detalhada e relatados separadamente dois casos de carcinoma de células foliculares da tireoide em que os cães desenvolveram mielopatia secundária à metástase epidural cervical espinhal.

Thesis (MSc)--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: Water quality analysis forms the basis in assessing and monitoring catchments. As urban development continuously increase, pollution sources increase in either point source (wastewater treatment works, industrial effluents) and/or non-point source origin (storm water discharge, domestic pollutants), accumulating pollutants in the environment. It was only recently discovered that certain pollutants have subtle disrupting effects on the endocrine system resulting in health related problems associated with the reproductive system and thyroid system (growth and development) of animals and potentially humans. Natural water resource management proves to include limited biological assays measuring endpoints for cytoxicity, inflammatory activity and endocrine disruption. The broad objective of this study was therefore to include several bioassays, not normally used in municipal (City of Cape Town) monitoring programmes, along with water quality data collected by the City of Cape Town. The Eerste/Kuils River catchment system, Western Cape, under the auspices of the City of Cape Town was chosen, and although this catchment does not contribute to drinking water resources, is subjected to a range of anthropogenic influences (industrial effluents, household wastewater, agricultural runoff). Within the short time-frame available for this study (six months) two months, July (following a dry summer and autumn season) and October (following a wet winter and early spring season) were selected for water quality monitoring. Spatial variation (with relevance to specific point and non-point contamination) among sampling sites were also obtained by choosing several (n=10) along the catchment. Specific aim of the study therefore included: Firstly (Chapter 2), the use of in vitro bioassays, lactate-dehydrolises assay (LDH) for cytotoxic activity, pro-inflammatory hormone Interleukin-6 (IL-6) secretion by human blood cells and a specific Salmonella ELISA for faecal contamination, in conjunction with routine chemical and biological (mostly microbiological) monitoring activities. The study indicated significant variation among sites in all microbiological measures as well in IL-6 secretion and Salmonella presence. Between months, variations were also evident in certain variables. Secondly (Chapter 3), two bioassays using the yolk precursor protein, vitellogenin (Vtg) as endpoint was implemented in a) an in vitro Xenopus laevis liver slice assay (five day exposure) and b) an in vivo Zebrafish (Danio rerio) bioassay (seven day exposure) assessing estrogenic activity in the Eerste/Kuils River catchment. Although estrogen spiked positive control water samples stimulated Vtg production in vitro as well as in vivo, no dramatic estrogenic activity was measured at any of the selected sites. Thirdly (Chapter 4), a bioassay using the thyroid controlled metamorphosis in Xenopus laevis tadpoles to assess effects on the thyroid hormonal system was implemented. Thyroid stimulatory activity, compared with a negative control sample, was measured at two sites along the catchment. Although the practical implementation of the tadpole semi-static exposure protocol (water replacement) proved to be labour intensive, all the added bioassays proved to be valuable tools to add valuable information regarding water quality. It is clear that more research related to anthropogenic influences along the Eerste/Kuils River catchment system are needed, specifically in monitoring monthly variations to better understand annual variation in several of the endpoints studied.
AFRIKAANSE OPSOMMING: Waterkwaliteit vorm die basis vir die evaluering en monitering van opvangsgebiede. Voortdurende stedelike ontwikkeling gee aanleiding tot ‘n toename in die voorkoms van besoedelstowwe in die natuurlike omgewing deur gelokaliseerde (punt) bronne (rioolwerke/industriële uitvloeisel) en/of nie gelokaliseerde (nie punt) bronne (vloed uitlaat/huishoudelike uitvloeisels) van besoedeling. Dit het onlangs aan die lig gekom dat van hierdie chemise besoedelstowwe subtiel die endokriene sisteem versteur en so aanleiding gee tot gesondheidsprobleme in terme van die voortplantingsisteem en tiroied sisteem (groei en ontwikkeling) by diere en moontlik ook die mens. Daar is beperkte gebruik van biologiese toetse wat inligting verskaf oor sitotoksiteit, inflammatoriese aktiwiteit en endokriene versteuring. Die doel van hierdie studie was dus om van hierdie biologiese toetse, wat normaalweg nie deel uitmaak van die roetine munisipale (Stad van Kaapstad) opvangsgebied monitering nie, gebruik te maak. Die Eerste-, Kuilsrivier, Wes Kaap, onder beheer van Stad Kaapstad is gekies en alhoewel die opvangsgebied nie water bydra tot drinkwaterbronne nie, word die opvangsgebied beïnvloed deur verskeie mensgemaakte bronne van besoedeling (afloop vanuit omliggende lanbougebiede). Binne die kort tydsraamwerk van die projek (ses maande) is besluit om twee maande, Julie (volg ‘n droë somer en herfs seisoen) en Oktober (volg ‘n nat winter en vroeë lente seisoen) vir water kwaliteit monitering te kies. Ruimtelike variasie langs die loop van die opvangsgebied is ingesluit deur moniteringspunte (n=10), met in ag name van die potensiële besoedelingsbronne. Spesifieke doelwitte van die projek sluit in: Eerstens (Hoofstuk 2), om die in vitro biotoetse, laktaat hidrolise (LDH) vir sitotoksisiteit, proinflammatoriese hormoon Interleuikin-6 (IL-6) vir inflammatoriese aktiwiteit, vrygestel deur menslike bloedselle en ‘n Salmonella ELISA vir ontlasting besoedeling saam met bestaande chemise en biologiese (hoofsaaklik migrobiologiese) veranderlikes te gebruik. Die studie het getoon dat beduidende variasie in alle mikrobiologiese toetse asook IL-6 vrystelling en Salmonella voorkoms bestaan het tussen versamelpunte. Maandelikse variasie in sekere van die veranderlikes het ook voorgekom. Tweedens (Hoofstuk 3), is twee biotoetse wat die dooiervoorloperproteïen, vitellogeen (Vtg) as eindpunt gebruik geimplimenteer in a) ‘n in vitro Xenopus laevis lewersnit biotoets (vyf dag blootstelling) en b) ‘n in vivo Zebravis (Danio rerio) biotoets (sewe dag blootstelling) om estrogenisiteit in die Eerste-, Kuilsrivier opvangsgebied te evalueer. Alhoewel, die estrogeen behandelde positiewe kontrole water monsters Vtg produksie veroorsaak het in beide die in vitro lewer-kulture en in vivo vistoets, is geen dramatiess estrogeniese aktiwiteit by enige van die moniteringspunte gevind nie. Derdens (Hoofstuk 4), is ‘n biotoets wat die tiroïedbeheerde metamorfose in Xenopus laevis paddavisse gebruik om effekte op die tiroïedsisteem te evalueer. Die differensiële stimulering (versnelling), in vergelyking met ‘n negatiewe kontrole watermonster, van die tiroied sisteem is by twee moniteringspunte in die opvangsgebied waargeneem. Alhoewel die praktiese implementering van die paddavis semi-statiese (water word gereeld vervang) biotoets arbeidsintensief is, het alle bykomstige biotoetse waardevolle bykomstige inligting oor water kwaliteit verskaf wat ‘n belangrike bydrae tot ingeligte bestuursbesluite kan verleen. Dit is duidelik dat meer navorsing int verband met die menslike invloed langs die Eerste-, Kuilsrivier opvangsgebied nodig is, veral met maandlikse monitering vir seisoenale veranderinge.

[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.

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.

La Néoplasie Endocrinienne Multiple de type 1 (NEM1) est un syndrome héréditaire caractérisé par l'association de lésions des glandes endocrines. La détection de mutation du gène MEN1, prédisposant à la NEM1, est importante pour le suivi médical des patients. Les techniques de diagnostic actuellement utilisées permettent d'identifier une mutation chez 95 % d'entre eux. Afin de contribuer à l'amélioration de ces analyses, nous avons développé de nouveaux outils diagnostics grâce auxquels nous avons identifié une large délétion du gène MEN1. L'étude de l'ARNm a également permis d'identifier les conséquences fonctionnelles de certaines anomalies atypiques du gène sur le processus d'épissage, prouvant ainsi leur pathogénicité. Enfin, nous avons développé un système d'étude de la fonction de la protéine ménine, codée par le gène MEN1, normale ou mutée dans des cellules ß pancréatiques afin d'analyser les conséquences fonctionnelles des mutations faux-sens.

L'homéostasie buccale conditionne fortement les perceptions gustatives ; elle repose sur un épithélium buccal sain et le bon fonctionnement des glandes salivaires, qui sont finement régulés par hormones sexuelles. Le but de cette thèse était de savoir si une exposition orale en bisphénol A, un migrant d'emballage alimentaire et de composites dentaires, et une alimentation riche en phyto-œstrogènes (soja) pouvaient modifier l'homéostasie buccale. Deux expérimentations ont été conduites : une étude dose-effet du BPA (5µg à 12,5 mg/kj/j) chez le rat adulte, et une étude d'interaction d'un régime riche en soja sur les effets du BPA. Sur la base de tests gustatifs et d'une approche histologique et moléculaire (qPCR-TR), la première étude identifie pour la première fois une action du BPA sur la sècheresse buccale. Nous avons constaté que le BPA était responsable d'une moindre consommation d'eau (p<0.01), d'une préférence augmenté au sel (p<0.05) et diminué au sucre (p<0.05), et d'une altération des sécrétions salivaires. Une réversibilité partielle des effets à l'arrêt du traitement. A contrario un régime riche en phyto-œstrogène augmente la prise d'eau (p<10-6) et diminue la préférence au sel (p<0.05) par rapport à un régime semi-synthétique, et peut s'opposer aux effets du BPA. Ces études montrent que BPA et phyto-œstrogènes exercent des effets œstrogéniques agonistes ou antagonistes en fonction de la cible biologique considérée, et qu'un régime à base de soja peut gommer la plupart des effets observés

Les tumeurs endocriniennes sont des tumeurs rares, présentant des caractéristiques métaboliques communes qui déterminent tant leur prise en charge que les modalités d'imagerie. Notre étude a porté sur l'apport des traceurs radioisotopiques ciblant des systèmes métaboliques spécifiques de ces tumeurs. La mIBG, structurellement proche de la noradrénaline, est recaptée par les terminaisons adrénergiques via un transporteur spécifique (NAT). La scintigraphie à la mIBG radioiodée visualise donc les vésicules de stockage des catécholamines et constitue un outil pour la mise en évidence des tumeurs dérivées de la crête neurale. Les études préliminaires ont montré son intérêt dans la recherche du neuroblastome avec une spécificité et une sensibilité très élevées. L'octréotide est un analogue de la somatostatine qui se fixe de façon spécifique sur les récepteurs de type SSTR 2. Ainsi, la scintigraphie à l'octréotide marqué 111In permet l'évaluation des tumeurs endocrines dérivées de l'endoderme qui expriment ces récepteurs membranaires. Le 18FDG est un traceur du métabolisme glucidique dont l'utilisation repose sur l'augmentation de l'activité glycolytique au niveau des tumeurs. Son intérêt n'est plus à démontrer en oncologie. Dans le cancer thyroi͏̈dien, plusieurs études documentent son apport potentiel et suggèrent une supériorité du TEP-FDG sur les méthodes conventionnelles de diagnostic des métastases. Nous avons cherché à optimiser les paramètres d'utilisation de ces méthodes afin de permettre une détection complète des localisations tumorales chez un individu donné, notamment dans l'exploration de tumeurs peu différenciées, d'homogénéiser les pratiques scintigraphiques, dans un souci de reproductibilité, et de poser les bases d'un traitement de type radiométabolique. En particulier, nous avons démontré que le recours à des activités élevé de mIBG n'augmentait pas de façon significative la sensibilité de l'examen scintigraphique. Nous avons également montré que la scintigraphie à l'octréoscan a une place limitée dans la stratégie diagnostique du cancer médullaire de la thyroi͏̈de. La scintigraphie au 18FDG, par contre, est d'après notre étude, un examen performant, complémentaire de l'imagerie conventionnelle pour le diagnostic des localisations secondaires dans les cancers thyroi͏̈diens ne fixant pas l'iode 131. Afin d'améliorer les performances de ces méthodes, il serait intéressant de pouvoir développer d'autres voies métaboliques. Ainsi différents traceurs sont à l'étude, particulièrement ceux basés sur les techniques de tomographie par émission de positrons. A ce propos, nous avons rapporté notre expérience sur l'utilisation d'un nouveau traceur dans l'exploration des tumeurs endocriniennes : le 11C-acetate

Maturation du foie au cours du développement. L'ornithine transcarbamylase hépatique est localisée dans la mitochondrie et intervient dans l'uréogenèse, son activité augmente lentement chez le foetus au cours des derniers jours de la gestation et croît fortement chez le nouveau-né. Rôle de régulation de l'enzyme pour les variations de l'insulinémie