Mutações inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrófico isolado (HHI) normósmico. A partir desse achado, hipotetizamos que mutações ativadoras na NKB e/ou NK3R resultariam na secreção prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutações inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normósmico. Duzentos e trinta e sete pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normósmico. Um grupo de 150 indivíduos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reação em cadeia da polimerase, seguido de purificação enzimática e seqüenciamento automático direto. Análises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) está localizada na proneurocinina B, e análises in silico sugeriram que ela não altera sítios constitutivos de splicing e é benigna para a estrutura da proteína. A análise de segregação familiar mostrou que a mãe da paciente, a qual apresentou um desenvolvimento puberal normal, também apresentava a alteração p.A63P em heterozigose, sugerindo que essa variante não desempenha um papel direto no fenótipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve início puberal aos treze anos de idade. A análise do grau de conservação da alanina na posição 449 mostrou que esse aminoácido não é conservado entre as diferentes espécies, e análises in silico sugeriram que essa variante não altera os sítios constitutivos de splicing, e é benigna para a estrutura do NK3R. Três novas variantes no NK3R foram identificadas, p.G18D, p.L58L (c.172C>T) e p.W275*, em três pacientes do sexo masculino não relacionados com HHI normósmico. As variantes p.G18D e p.L58L foram identificadas em heterozigose, enquanto que a variante p.W275* foi identificada em heterozigose associada a variante silenciosa p.L58L (c.172C>T), e em homozigose em outro paciente. Análises in silico sugeriram que a variante p.G18D poderia afetar a funcionalidade do NK3R. Estudos in vitro dessa nova variante foram realizados, e mostraram que a mesma não altera a função do NK3R, visto que o aumento na produção de fosfatidil inositol não diferiu significativamente entre o receptor mutado e selvagem. Todas as novas variantes descritas nos genes TAC3 e TACR3 não foram identificadas em 300 alelos controles. Em conclusão, nosso trabalho identificou novas variantes nos genes TAC3 e TACR3 em pacientes brasileiros com distúrbios puberais centrais idiopáticos, e confirmou o envolvimento do complexo NKB/NK3R na etiologia do HHI normósmico / Inactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested that this new variant does not alter splice sites or affects the structure of NK3R. Indeed, it was identified three new distinct variants in the TACR3 gene, p.G18D, p.L58L (c.172C>T) and p.W275*, in three unrelated males with normosmic IHH. Both p.G18D and p.L58L (c.172C>T) were identified in heterozygous state, and the p.W275* variant was identified in two of these males, since one in homozygous and in another in heterozygous state in association with the silent variant p.L58L (c.172C>T). In silico analyses suggested that p.G18D might be damaging to the NK3R. In vitro studies of this variant (p.G18D) showed that the amount of inositol phosphate (IP) was not significantly different in cells transfected with the p.G18D mutant receptor than in cells transfected with the wild type receptor, indicating that this variant did not alter the function of the neurokinin B receptor. All new variants identified in the TAC3 and TACR3 genes were absent in 300 control alleles. In conclusion, we identified new variants in the TAC3 and TACR3 genes in Brazilian patients with idiopathic central pubertal disorders. We confirm the key role of the NKB/NK3R complex in the etiology of normosmic IHH

O hipercortisolismo ACTH-dependente (HAD), também chamado de doença de Cushing, é uma das endocrinopatias mais comumente diagnosticadas na espécie canina. A sintomatologia clínica ocorre, secundariamente, aos efeitos gliconeogênicos, catabólicos, antiinflamatórios e imunossupressores dos glicocorticóides sobre vários sistemas orgânicos. Há uma marcante predisposição da doença na raça poodle e casos familiais têm sido diagnosticados sugerindo uma causa genética. As alterações moleculares que levam ao desenvolvimento do HAD em cães permanecem indefinidas. Dentre os genes implicados no desenvolvimento dos corticotrofos e na regulação do eixo corticotrófico, destacam-se o Tbx19 e o Crhr1, respectivamente. O Tbx19 é um fator de transcrição obrigatório para a transcrição do gene da proopiomelanocortina (POMC) e para a diferenciação terminal dos corticotrofos. Como está presente, exclusivamente, em corticotrofos normais e adenomatosos, foi proposto seu envolvimento na secreção excessiva de ACTH na doença de Cushing. A presença de CRHR1 nos corticotrofinomas na espécie humana e canina levantou a hipótese da sua participação na tumorigênese hipofisária, promovendo uma estimulação celular prolongada, mesmo na ausência de hormônios hipotalâmicos. Um aumento da expressão do CRHR1 foi demonstrado nos tumores corticotróficos, apesar da secreção autônoma de ACTH e dos níveis portais suprimidos de CRH em pacientes humanos e caninos com doença de Cushing. Os objetivos do presente trabalho foram pesquisar a presença de mutações germinativas nas regiões codificadoras dos genes Tbx19 e Crhr1 em cães com HAD. Para tanto, estudamos 50 cães da raça poodle com hipercortisolismo ACTH-dependente (33 fêmeas e 17 machos), com idade média de 8,71 anos e 50 cães controle da mesma raça (32 fêmeas e 18 machos) com idade superior a 6 anos (média de 9,38 anos) e sem endocrinopatias. O DNA genômico foi extraído e amplificado através da reação de polimerização em cadeia (PCR), utilizando-se oligonucleotídeos (primers) específicos para os genes Tbx19 e Crhr1. Foi identificada uma nova variante alélica tanto no Tbx19 como no Crhr1, ambas não descritas na literatura. No gene Tbx19, a variante p. S343G foi encontrada em dois cães não aparentados, mas também em dois controles normais, sugerindo tratar-se de um novo polimorfismo. Já a variante p. V97M do Crhr1 foi encontrada, em heterozigose em um animal com HAD, porém não foi observada em cem alelos normais. O códon 97 está localizado no domínio extracelular aminoterminal do gene Crhr1, de extrema importância para a ligação com alta afinidade ao ligante. O estudo molecular da estrutura quartenária da proteína mutada, seguido da avaliação da energia de ligação da superfície de contato entre o hormônio e o receptor revelou um rearranjo estrutural com alteração da superfície de contato entre o CRH e o seu receptor CRHR1, resultando em uma energia de ligação 17% superior à do receptor selvagem. Em conclusão, esse estudo não identificou alterações no gene Tbx19 associadas ao hipercortisolismo ACTH-dependente canino, mas por outro lado, identificou pela primeira vez, uma mutação ativadora no Crhr1, provavelmente responsável pelo hipercortisolismo ACTH-dependente em um cão da raça poodle. / The ACTH-dependent hypercortisolism (ADH), also called Cushing\'s disease, is one of the most commonly diagnosed endocrine diseases in dogs. The symptoms occur due to glucocorticoids excess leading to gluconeogenic, catabolic, anti-inflammatory and immunosuppressive effects in multiple organs and systems. There is a high incidence of Cushing\'s disease in Poodles and familial disease has been identified suggesting a genetic involvement. The molecular changes that lead to the development of ACTH-dependent hypercortisolism in dogs remain undefined. Among genes implicated in corticotroph development and in corticotropic axis regulation, we would like to point out Tbx19 and Crhr1, respectively. Tbx19 gene is a transcription factor required for transcription of the proopiomelanocortin gene and for terminal differentiation of the corticotroph. Inactivating mutations in that gene are associated with human isolated ACTH deficiency. Since Tbx19 is present exclusively in normal and adenomatous corticotroph cells, its involvement in the secretion of ACTH in Cushing\'s disease was proposed. The presence of CRHR1 in corticotrophinomas in humans and dogs raised the possibility of its involvement in pituitary tumorigenesis, promoting prolonged cell stimulation, even in the absence of hypothalamic hormones. An increased expression of the CRHR1 mRNA was demonstrated in human and canine ACTH-secreting pituitary adenomas, despite the autonomous ACTH secretion and the low portal levels of CRH. The aim of this study was to investigate Tbx19 and Crhr1 coding region mutations in Poodle dogs with ACTH-dependent hypercortisolism. We studied 50 Poodle dogs with ADH (33 females and 17 males) with a mean age of 8.71 years and 50 control dogs of the same breed (32 females and 18 males) older than 6 years (mean 9.38 years) and without endocrinopathies. Genomic DNA was extracted from peripheral blood, amplified by the polymerase chain reaction (PCR) using specific intronic primers and submitted to automatic sequence. We identified a new allelic variant in the Tbx19 and Crhr1 coding regions. The allelic variant p. S343G in the Tbx19 gene was found in two unrelated dogs, but also in two normal controls, suggesting that this is a new polymorphism. The Crhr1 allelic variant p. V97M was found in heterozygosity in one animal with ACTH-dependent hypercortisolism, but was not observed in one hundred normal alleles. The codon 97 is located in the extracellular amino terminal domain of the Crhr1 and is extremely important for high affinity ligand binding. The molecular analysis of the quaternary structure of normal and mutated proteins, followed by evaluation of the binding energy of the contact surface between the hormone and the receptor showed a structural rearrangement of the mutated protein by changing the contact surface between the CRH and its receptor CRHR1, resulting in a binding energy 17% higher than the wild type. In conclusion, this study did not identify Tbx19 mutations associated with canine ACTH-dependent hypercortisolism, but on the other hand, we first identified a Crhr1 gain-of-function mutation probably responsible for ACTH-dependent hypercortisolism in a Poodle dog of our cohort.

Abstract not available

生物科技為目前世界各國極力投入之新興產業，此產業無需大量天然資源及低污染的特性，同時又能解決人類的醫療問題並提昇生活水準的未來發展空間，更使得此一新興產業吸引了眾人的眼光。 　　本論文提出一個評估的模式，讓此產業之技術、產品或專利之價值能夠經系統化的分析後完整呈現，並可基於此分析結果作出策略上之判斷，並以GnRH vaccine技術為例。因為GnRH分子之序列早已發表，經本研究分析得兩種常用的專利保護策略，第一種是將GnRH與不同的載體進行結合，而新生成一個嵌合化合物，並申請此新化合物之專利；第二種是進行GnRH分子結構上之改變以取得新序列的專利保護，取得效果優於自然設計之分子，或藉以增加與免疫載體之結合率。經分析後發現，GnRH疫苗應用理論已經相當成熟，未來幾年應該會有成熟產品通過上市之審核過程；再隨著新科技的進步，可預期將會有更多的新產品會上市，而進一步地有效提昇產品功效並降低製造成本。 　　本研究最後針對GnRH未來在人用藥品及動物用藥之未來市場加以分析，並對於以重複免疫原在重組型疫苗製備法之開發提出行銷、財務、通路、價格、推廣、臨床實驗之建議，以期能經由策略規劃的角度將生物技術的價值作最大化的管理。 / Biotechnology attracts high attentions among many countries recently. New biotechnology will provide a key solution to cure diseases and potentially will be able to increase human life quality without damaging the environment and consuming massy natural resources. 　　In this thesis, we presented a possible process, which involved in academic, marketing and patent information analysis, to effectively evaluate the feasibility and potential of any novel biological technologies, so as to provide the managerial and strategic suggestions based on the analysis. GnRH repeat immunogens vaccine development was taken as an example to demonstrate if the proposed process can be executed adequately. After discretly cross examining the collected information, we concluded that many independent GnRH vaccine patents are available but will not be effective enough to stop to new competitors to get in this sector because GnRH sequence was disclosed in very early research and nobody held the patent for it and most of GnRH patents were granted to either different GnRH-Carrier chimeras or the GnRH analog sequences. Analysis results show that there is only one company closely approaching the final product approval at this moment. However, any improved technology can easily employed to provide a better product with higher effectiveness and lower cost following the first product's upcoming marketing clearance and then make the future market very complicated. 　　Based on those findings, we further analyze the Taiwanese current environment and provide a few managerial suggestions to maximize the value of a novel GnRH vaccine preparation technology.

Crohns sjukdom är en kronisk inflammatorisk tarmsjukdom av okänd orsak. Det tidigaste tecknet på Crohns sjukdom är mikroskopiska sår i det s.k. follikelassocierade epitelet (FAE) som täcker ansamlingar av immunceller i tarmen. FAE är specialiserat för att fånga innehåll från tarmen och transportera det till underliggande immunvävnad. Denna funktion är viktig för att inducera skyddande immunsvar, men den utgör också en ingångsväg för sjukdomsalstrande bakterier. Crohns sjukdom är associerat med ett kraftigt ökat immunsvar mot bakterier, och sjukdomsförloppet kan ändras av stress. Det övergripande syftet med avhandlingen var att studera effekterna av stress på FAE samt att undersöka rollen av FAE vid utvecklingen av tarminflammation, särskilt vid Crohns sjukdom. Inledningsvis studerades effekterna av psykologisk stress på FAE. Stressade råttor uppvisade ökad genomsläpplighet av bakterier efter stress, och passagen var högre i FAE än i vanligt epitel. Efterföljande experiment visade att stressförändringarna i slemhinnan regleras via kortikotropinfrisättande hormon och mastceller. Vidare visade det sig att vasoaktiv intestinal peptid kunde efterlikna stressens effekter på genomsläppligheten, och att detta kunde förhindras genom att blockera mastcellerna. Studier av tunntarmsslemhinna från patienter med icke-inflammatorisk tarmsjukdom och friska kontroller visade en högre passage av bakterier i FAE än i vanligt epitel. Hos patienter med Crohns sjukdom var bakteriepassagen genom FAE betydligt ökad jämfört med kontroller. Resultaten från detta avhandlingsarbete visar att stress kan förändra upptaget av bakterier från tarmen via FAE, med mekanismer som innefattar kortikotropinfrisättande hormon och mastceller. Detta har gett nya kunskaper kring regleringen av slemhinnebarriären. Vidare presenterar denna avhandling nya insikter i sjukdomsuppkomsten vid Crohns sjukdom genom att påvisa en tidigare okänd defekt i barriärfunktionen i FAE. / The earliest observable signs of Crohn’s disease are microscopic erosions in the follicle-associated epithelium (FAE) covering the Peyer’s patches. The FAE, which contains M cells, is specialised in sampling of luminal content and delivery to underlying immune cells. This sampling is crucial for induction of protective immune responses, but it also provides a route of entry for microorganisms into the mucosa. Crohn’s disease is associated with an increased immune response to bacteria, and the disease course can be altered by stress. The overall aim of this thesis was to study the effects of stress on the FAE and elucidate the role of FAE in the development of intestinal inflammation, specifically Crohn’s disease. Initially, rats were submitted to acute and chronic water avoidance stress to study the effects of psychological stress on the FAE. Stressed rats showed enhanced antigen and bacterial passage, and the passage was higher in FAE than in regular villus epithelium (VE). Further, stress gave rise to ultrastructural changes. Subsequent experiments revealed the stress-induced increase in permeability to be regulated by corticotropin-releasing hormone and mast cells. Furthermore, vasoactive intestinal peptide (VIP) mimicked the stress effects on permeability, and the VIP effects were inhibited by a mast cell stabiliser. Human studies of ileal mucosa from patients with non-inflammatory disease and healthy controls showed a higher antigen and bacterial passage in FAE than in VE. In patients with Crohn’s disease, the bacterial passage across the FAE was significantly increased compared to non-inflammatory and inflammatory controls (ulcerative colitis). Furthermore, there was an enhanced uptake of bacteria into dendritic cells, and augmented TNF-α release in Crohn’s disease mucosa. Taken together this thesis shows that stress can modulate the uptake of luminal antigens and bacteria via the FAE, through mechanisms involving CRH and mast cells. It further shows that human ileal FAE is functionally distinct from VE, and that Crohn’s disease patients exhibit enhanced FAE permeability compared to inflammatory and non-inflammatory controls. This thesis presents novel insights into regulation of the FAE barrier, as well as into the pathophysiology of Crohn’s disease by demonstrating a previously unrecognised defect of the FAE barrier function in ileal Crohn’s disease.

The process of respiration is a pattern of spontaneity and automatic motor control that originate in the brainstem. The mechanism by which the brainstem detects CO2 is termed central CO2 chemoreception (CCR). Since the early 1960’s there have been tremendous efforts placed on identification of central CO2 chemoreceptors (molecules that detect CO2). Even with these efforts, what a central CO2 chemoreceptor looks like remain unknown. To test the hypothesis that inward rectifier K+ (Kir) channels are CO2 sensing molecules in CCR, a series of experiments were carried out. 1) The first question asked was whether the Kir4.1-Kir5.1 channel is expressed in brainstem chemosensitive nuclei. Immunocytochemistry was performed on transverse medullary and pontine sections using antibodies raised against Kir4.1 and Kir5.1. Positive immunoassays for both Kir4.1 and Kir5.1 subunits were found in CO2 chemosensitive neurons. In the LC the Kir4.1 and Kir5.1 were co-expressed with the neurokinin-1 receptor that is the natural receptor for substance P. 2) The second question asked was whether the Kir4.1-Kir5.1 channel is subject to modulation by neurotransmitters critical for respiratory control. My studies demonstrated that indeed the Kir4.1-Kir5.1 channel is subject to modulation by substance P, serotonin and thyrotropin releasing hormone. 3) I performed studies to demonstrate the intracellular signaling system underlying the Kir4.1-Kir5.1 channel modulation by these neurotransmitters. The modulation by all three neurotransmitters was dependent upon the activation of protein kinase C (PKC). The Kir4.1-Kir5.1 but not the Kir4.1 channel was modulated by PKC. Both the Kir4.1 and Kir5.1 subunits can be phosphorylated by PKC in vitro. However, systematic mutational analysis failed to reveal the phosphorylation site. 4) The fourth question asked was whether Kir channels share a common pH gating mechanism that can be identified. Experiments were performed to understand the gating of the Kir6.2+SUR1 channel as specific sites for ligand binding and gating have been demonstrated. I identified a functional gate that was shared by multiple ligands that is Phe168 in the Kir6.2. Other Kir channels appear to share a similar gating mechanism. Taken together, these studies demonstrate the modulation of Kir channels in central CO2 chemoreception.

Dans les cellules somatotropes, la liaison du facteur de libération de l’hormone de croissance (GHRH) à son récepteur (GHRH-R) stimule la synthèse et la sécrétion de l’hormone de croissance (GH) ainsi que la prolifération cellulaire. Chez les mammifères, le vieillissement est caractérisé par une diminution de la sécrétion de GH, liée à une perte de sensibilité des somatotropes au GHRH. Chez le rat âgé, des modifications de niveaux d'ARNm du GHRH-R et une diminution d'affinité et de capacité de liaison du GHRH sont rapportés. Au cours du vieillissement, une augmentation des niveaux de glucose et d’acides gras libres sérique suggère qu’une gluco- ou lipotoxicité puisse contribuer au dysfonctionnement de la fonction somatotrope. À ce jour, la restriction calorique modérée de longue durée (RCMLD) constitue l’intervention la plus efficace pour prévenir ou retarder les détériorations liées à l’âge. Des études ont montré des effets bénéfiques de la RCMLD sur l’axe somatotrope au cours du vieillissement via un maintien des paramètres de liaison du GHRH-R. Compte tenu de l’importance de cet axe, la compréhension des mécanismes menant à la somatopause ainsi que ceux associés aux effets bénéfiques de la RCMLD s’avère importante. Les objectifs principaux de la présente thèse étaient : 1) de déterminer les effets de la RCMLD chez le rat, sur le GHRH-R hypophysaire et la sensibilité des somatotropes au GHRH, 2) d’identifier les mécanismes associés à la somatopause et aux effets bénéfiques de la RCMLD, et 3) de préciser les effets d’une gluco-ou lipotoxicité sur l’axe somatotrope de rats et leur implication dans la somatopause. Des rats de 8 mois ont été soumis à une restriction calorique de 40% jusqu’à l’âge de 18-20 mois et ont été comparés à des rats jeunes et âgés nourris ad libitum. Cette étude a permis de mettre en évidence des effets bénéfiques de la RCMLD sur la régulation et la fonctionnalité du GHRH-R et de proposer que le glucose et les acides gras libres (AGL) circulants soient impliqués dans le vieillissement de la somatotrope. Une étude de micro-puce à ADN à permis d’identifier des gènes associés à des mécanismes de protection et de réparation des dommages cellulaires mis en place dans l’hypophyse antérieure au cours du vieillissement et par la RCMLD. Finalement, les effets d’un stress gluco- ou lipotoxique sur la fonction somatotrope ont été étudiés chez des rats de 2 et 6 mois, infusés 72 h avec une solution de glucose ou d’Intralipides, mimant les niveaux circulants de glucose et d’AGL retrouvés chez le rat âgé. Les résultats obtenus montrent que la glucotoxicité affecte la régulation de certains gènes de la somatotrope, dont le GHRH-R, et suggèrent que la capacité de réponse à ce type de stress est altérée. Les mécanismes par lesquels la glucotoxicité exerce ces effets pourraient inclure la génération de stress oxydant. L’ensemble de ces résultats proposent de nouvelles pistes mécanistiques qui pourraient contribuer au retardement de la somatopause et, ultimement, à l’élaboration de nouvelles stratégies d’intervention nutritionnelles ou pharmacologiques ciblant les mêmes voies que la RCMLD, avec une efficacité similaire ou supérieure. / In somatotroph cells, growth hormone-releasing hormone (GHRH) binding to its receptor (GHRH-R) stimulates growth hormone (GH) secretion and cell proliferation. In mammals, aging is characterized by a decrease of GH, associated with a decline of GHRH somatotroph sensitivity. In the aged rat, changes in GHRH-R mRNA levels and decrease of GHRH affinity and capacity were reported. In the course of aging, significant increase of glucose and free fatty acid (FFA) serum levels are observed, suggesting that gluco- or lipotoxicity could contribute to somatotroph dysfunction. Up to now, long-term moderate caloric restriction (LTMCR) has been the most efficient intervention to prevent or delay age-related deteriorations. Studies have shown beneficial effects of LTMCR on somatotroph axis during aging, through the maintenance of GHRH-R binding parameters. Knowing the importance of this axis, an understanding of the mechanisms associated with the effects of somatopause and benefits of LTMCR is important. Therefore, the main objectives of this thesis were: 1) to determine the effects of LTMCR on rat pituitary GHRH-R and somatotroph sensitivity to GHRH, 2) to identify the mechanisms associated to somatopause and the beneficial effects of LTMCR and 3) to specify the effect of gluco- or lipotoxicity on the rat somatotroph axis and their implications in somatopause. Eight-month-old rats were submitted to a 40% LTMCR until the age of 18 to 20-months and where compared to young and old rats fed ad libitum (AL). This study highlighted beneficial effects of LTMCR on GHRH-R regulation and functionality and suggested that high circulating levels of glucose and FFA could be involved in somatotroph aging. A microarray study was also performed, allowing the identification of genes associated to cellular protection and damage repair mechanisms regulated by aging and LTMCR in the anterior pituitary. Finally, effects of a gluco- or lipotoxic stress on the somatotroph function was assessed in 2- and 6-month-old rats submitted to a 72-h glucose and/or Intralipid infusion, to mimic the levels of glucose and FFA found in aged rats. The results showed that glucotoxicity affects the regulation of specific genes in the somatotroph, such as the GHRH-R gene, and suggest that the response capacity against this type of stress is altered with age very early on. Mechanisms by which glucotoxicity exerts these effects might include oxidative stress production. Altogether, these results proposed novel mechanisms that could contribute to delay somatopause and, ultimately, to the development of new nutritional or pharmacologic interventions targeting the same pathways as LTMCR, with a similar or greater efficiency.

Background: Lithium augmentation is a first-line strategy for depressed patients resistant to antidepressive therapy, but little is known about patients’ subsequent long-term course or outcome predictors. We investigated long-term outcomes of unipolar depressed patients who had participated in a study on the effects of lithium augmentation on the hypothalamic-pituitary-adrenocortical system using the combined dexamethasone/corticotrophin-releasing hormone (DEX/CRH) test. Methods: Twelve to 28 months (mean 18.6 ± 4.6 months) after lithium augmentation, 23 patients were assessed with a standardized interview, of which 18 patients had complete DEX/CRH test results. Relapse was diagnosed by DSM-IV criteria (Structured Clinical Interview for DSM-IV; SCID I). Results: Only 11 patients (48%) had a favorable follow-up, defined as absence of major depressive episodes during the observation period. Patients with a favorable and an unfavorable course did not differ in clinical or sociodemographic parameters, endocrinological results or continuation of lithium. However, fewer previous depressive episodes tended to correlate (p = 0.09) with a favorable course. Conclusion: Results from studies using the DEX/CRH test to predict relapse in depressed patients treated with antidepressants were not replicated for lithium augmentation. Our finding could reflect the elevation of DEX/CRH results by lithium, independent of clinical course. Limitations of the study are its small sample size, the heterogeneous clinical baseline conditions and the lack of lithium serum levels. The fact that lithium continuation did not predict the course might be related to the difference between the efficacy of lithium in controlled studies and its effectiveness in naturalistic settings. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Περιγράψαμε την Διαταραχή Μεταγωγής Σήματος Αυξητικής Ορμόνης (GHTD), σε παιδιά με σοβαρή καθυστέρηση ανάπτυξης, φυσιολογική έκκριση αυξητικής ορμόνης (GH), χαμηλό IGF-I αλλά φυσιολογική ανταπόκριση IGF-I σε χορήγηση hGH, που παρουσιάζουν ελαττωματική φωσφορυλίωση του STAT3. Η διαταραχή θεραπεύεται με hGH. Οι CIS πρωτεΐνες είναι αρνητικοί ρυθμιστές του σηματοδοτικου μονοπατιού της GH που ανταγωνίζονται STATs για θέση πρόσδεσης με τον υποδοχέα GHR ή συμμετέχουν στην αποδόμηση του JAK2/GHR μέσω ουβυκουιτίνης/προτεασώματος. Η αδυναμία φωσφορυλίωσης του STAT3 και JAK2 φαινεται να προσπερνάται με χρήση εναλακτικής οδου. Με ‘διασυνομιλία’ της GH με το μονοπάτι του EGF. Συγκεκριμένα η GH φωσφωρυλιώνει το EGFR μέσω φωσφορυλίωσης JAK2. Τέλος, η φωσφορυλίωση STAT3 προκαλείται και από την 17β-οιστραδιόλη. Υπάρχει μία κλινική οντότητα της “καθυστέρησης της ήβης και ανάπτυξης” όπου γίνεται σημαντική επιτάχυνση στην ανάπτυξη μετά την εφηβεία όπου υπάρχει φυσιολογικό τελικό ανάστημα που ταιριάζει με την πορεία ενός ασθενή. Σκοπός: Μελετήθηκε η αρνητική ρύθμιση της GH σε ινοβλάστες παιδιών με GHTD και φυσιολογικών. Στη συνέχεια, η πιθανή συσχέτιση του σηματοδοτικού μονοπατιού της GH και EGF στα παιδιά και τέλος η πιθανή συσχέτιση των στερεοειδών του φύλου με το GH άξονα σε ένα GHTDπαιδί . Yλικά/μέθοδοι: Σε πρωτογενείς καλλιέργειες ινοβλαστών ούλων μαρτύρων και GHTD παιδιών μελετήσαμε την έκφραση και ενεργοποίηση της CIS και JAK2, με επαγωγή με hGH, με Western blot. Στη συνέχεια μελετήθηκε η έκφραση του EGF και pEGF στα κύτταρα των παιδιών και μαρτύρων με επαγωγή των κυττάρων με GH και EGF έλεγχος με Western blot και coimmunoprecipitation. Τέλος μελετήθηκε η φωσφoρυλίωση STAT3 και JAK2 σε ινοβλαστες (προεφηβικούς και εφηβικούς) του ασθενή με GH και 17-β οιστραδιόλη με Western immunoblotting. Αποτελέσματα: Η έκφραση του CIS με 200 ng/ml hGH έδειξε μόνο στους ασθενείς αύξηση της συνολικής CIS και της ουβικουτινυλιομενής μορφής της. Η έκφραση και ενεργοποίηση τoυ JAK2 μόνο στους ασθενείς με επαγωγή με hGΗ δείχνει καθυστερημένη ενεργοποίηση του. Το STAT3 φωσφωρυλιώνεται φυσιολογικά με επαγωγή των κυττάρων των ασθενών με EGF όχι με GH. Οι pEGFRs φωσφωρυλιώνουν φυσιολογικά το JAK2 στους ασθενείς, ενώ όχι στους μάρτυρες. Ο ένας ασθενής προεφηβικά δε φωσφωρυλιώνει το STΑT3 με GH. Εμφανίζει την ουβικουτινιλιωμένη μορφή του CIS. Δεν φωσφωρυλιώνει το STAT3 με 17β-οιστραδιόλη προ-εφηβικά, και εμφανίζει την ουβικουτινιλιωμένη μορφή του CIS. Ενώ εφηβικά φωσφορυλιώνει το STAT3 με 17β-οιστραδιόλη και δεν εμφανίζει την ουβικουτινιλιωμένη μορφή του CIS. Συμπεράσματα: Βρέθηκε καθυστερημένη ενεργοποίηση του GH άξονα σε 2 ασθενής με GHTD μαζί με υπερέκφραση της CIS και ουβικουτινυλιομενής CIS. Η σηματοδότηση της GH γίνεται φυσιολογικά στους μάρτυρες άρα δεν υπάρχει λόγος εναλλακτικής οδού, σε αντίθεση με ασθενείς που χρειάζεται να χρησιμοποιήσουν το μονοπάτι του EGF. Ο ένας ασθενής ξεπέρασε την αδυναμία να φωσφορυλιώσει το STAT3 χωρίς hGH αλλά με την έναρξη της εφηβείας. Ένας λόγος που το παιδί αυτό έδειξε σημαντική επιτάχυνση στην ανάπτυξη είναι ότι μετά την εφηβεία δεν υπήρχε υπερλειτουργία του ανασταλτικού μηχανισμού της GH, διαμέσου του Ub CIS. / We have previously described a new disorder (GHTD) in 4 children with growth delay, normal provoked and spontaneous GH secretion, and low IGF-I concentrations but normal IGF-I generation test results who have a defect in the phosphorylation of the signal transducer and activator of transcription STAT-3. These children respond with a significant increase in their growth velocity after administration of hGH. CIS proteins are inhibitors of the GH signal transduction pathway, by distinct mechanisms: by competition with STATs for common tyrosine-binding sites on the cytoplasmic tail of GHR or by a proteasome-dependent mechanism. Monoubiquitinated form of CIS protein was observed in 2 GHTD patients.Also STAT3 phosphorylation defect could be overcomed by using an alternative pathway the one of Epidermal Growth factor (EGF). Also in one patient its STAT3 defect was overcomed when he entered puberty, sex steroids may enhanced his growth. Objective: The purpose of the study was the characterization of the molecular mechanisms involved in GH signal transduction pathway in GHTD patients, as a possible cause of an increased expression of its inhibitors. Also to search if there is a possible crosstalk between GH and EGF growth transduction pathways. And finally the role of sex steroids in GH signalling in one GHTD patient. Patients/Material and Methods: In primary fibroblast cell cultures from gingival biopsies of the GHTD patients and age-matched normal children we studied: expression analysis, in cells inducted with GH, of CIS and JAK2 phosphorylationby western immunoblotting and RT-PCR. Also the cells of the same children and controls were inducted with EGF and STAT3 phosphorylation was studied. Finally the cells of one of the patients were inducted with GH and 17β-estradiole before and after puberty and its STAT3 phosphorylation and CIS expression were studied. Results: Expression analysis in the childrens’ fibroblasts showed an overexpression of CIS in 2 patients as compared to normal children. STAT3 defect was not present in the patients fibroblasts that were inducted with EGF. Also one GHTD patient that he couldn't phosphorylate in his inducted fibroblast with GH and 17-b estardiole STAT3 and ubiquitinated CIS was present to his cells before puberty this defect was overcomed after he entered puberty. Conclusions: The overexpression of CIS may inhibit the activation of STAT3 and may be involved in the pathogenesis of the severe short stature of the GHTD children. Also GH signalling pathway has no defect in control patients so there is no need of using an alternative pathway such as the one of EGF that occurs in GHTD patients. Also one GHTD patient that showed a STAT3 defect before puberty was overcomed after he entered puberty and without GH treatment. There is a clinical status that is called 'growth dealy' that matches this patients profile, that shows rapid growth after puberty. A probable cause could be that ubiquitinated form of CIS was not present after he entered puberty.

No description available.