Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Made available in DSpace on 2016-08-19T18:16:07Z (GMT). No. of bitstreams: 1 Dissertacao Kenya Mara.pdf: 799958 bytes, checksum: cb09cc18255c1c97c36d29edcb749fd9 (MD5) Previous issue date: 2012-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The pituitary adenomas are the most common cause of a sellar mass of the third decade onwards, accounting for more than 10% of intracranial tumors. Recent studies indicate a prevalence of up to 77.6 per 100,000 inhabitants. Postoperative complications of hypophyseal surgery include: lesion of the carotid artery, cerebrospinal fluid leak, nasal septum perforation, Diabetes Insipidus, Syndrome of Inappropriate Antidiuretic Hormone Secretion, adrenal insufficiency, epistaxis, hyponatremia, sinusitis, visual loss, transient paralysis of the cranial nerve, sphenoidal abscess, meningitis, hemorrhage in the tumor bed and hypothalamic injury. Being the last three the ones of the highest mortality. This study evaluated 28 patients undergoing surgery of Pituitary tumor by transsphenoidal way and 2 by transcranial and watched the most common complications such as: cerebrospinal fluid leak (30 %), Diabetes Insipidus (26.6 %) and adrenal insufficiency (13.3 %) were found more frequently , followed by bleeding in the tumor bed (6.7%), pneumonia (6.7%) and sinusitis (3.3%). The prevalence of mortality was 6.7%. Endocrine complications can lead to death if not treated timely. A thorough clinical and laboratory evaluation by a multidisciplinary team is necessary for good performance in the management of postoperative complications of patients undergoing resection of pituitary tumor, since anticipate complications and minimize its risks. / Os adenomas hipofisários são a causa mais comum de massa selar da terceira década em diante, sendo responsável por mais de 10% das neoplasias intracranianas. Estudos recentes apontam para uma prevalência de até 77,6 por 100.000 habitantes. A ressecção cirúrgica pode ser necessária e as complicações pós-operatórias de cirurgia hipofisária incluem: lesão de artéria carótida, fistula liquorica, perfuração de septo nasal, diabetes insipidus, SIADH, insuficiência adrenal, epistaxe, hiponatremia, sinusite, déficit visual, paralisia transitória de nervo craniano, abscesso esfenoidal, meningite, hemorragia no leito do tumor e lesão hipotalâmica. Sendo as três últimas as de maior mortalidade. Neste estudo avaliamos 28 pacientes submetidos à cirurgia de tumor hipofisário via transesfenoidal e 2 por via transcraniana, onde foram observadas as seguintes complicações: fístula liquórica (30%), diabetes insipidus (26,6%) e insuficiência adrenal (13,3%), hemorragia no leito do tumor (6,7%), pneumonia (6,7%) e sinusopatia (3,3%). A taxa de mortalidade foi de 6,7%. Complicações endócrinas podem levar a óbito se não tratadas em tempo hábil. Uma rigorosa avaliação clínica e laboratorial por equipe multidisciplinar é necessária para o bom desempenho no manejo das complicações pós-operatórias dos pacientes submetidos a ressecções de tumor hipofisário, posto que pode antecipar complicações e minimizar seus riscos.

As mutações no gene PROP1 são a causa genética mais comum da deficiência combinada de hormônios hipofisários. Até o momento, diversas mutações missense e pequenas deleções foram descritas sendo a mutação 301-302 delAG a mais freqüente. Nosso objetivo foi estudar as mutações em DNA de pacientes com hipopituitarismo e padronizar a extração de DNA de células de swab oral, usando um método com NaCl e comparar com um kit comercial (Purigene, Minneapolis, EUA). Amplificamos os 3 exons do gene PROP1 do DNA obtido de células orais e de sangue periférico. Identificamos a mutação 301-302delAG em 6 pacientes, 4 em homozigose (33%) e 2 em heterozigose (16%) e a mutação G51A em heterozigose em um único paciente. Em dois irmãos, filhos de pais consangüíneos, não foi possível amplificar os 3 exons do gene PROP1 enquanto que os os genes LHX3 e LHX4 foram amplificados com sucesso. Para confirmar a hipótese de deleção do PROP1, o Southern blotting foi realizado usando como sonda o produto de PCR do exon 2 do gene PROP1 e um fragmento do gene CYP21A2 como sonda controle. A banda referente ao CYP21A2 estava presente nos pacientes e nos controles enquanto a banda referente ao PROP1 estava ausente nos irmãos e presente na mãe e nos controles. Para definir a extensão da deleção usamos um mapa de STS próximos ao gene e o STS GDB:314805 localizado a 6,0 kb a montante do PROP1 não foi amplificado nos pacientes. Entretanto, o gene Q8N6H0 a 18 kb a juzante e o STS WI-16216 a 59 kb a montante do PROP1 foram amplificados com sucesso nos pacientes e controles indicando que a deleção está localizada dentro de 81 Kb. Para determinar os limites da deleção, várias reações de PCR foram realizadas com primers desenhados progressivamente distantes de gene PROP1, cobrindo toda a região. Isto nos permitiu determinar a região deletada de 9,6 kb a juzante e 11 kb a montante do gene PROP1, com o tamanho máximo deletado de 18,4 kb. Por ambos os métodos de extração obtivemos um DNA de boa qualidade, permitindo o amplificação dos 3 exons do gene PROP1. A extração com NaCl foi mais rápida e mais barata resultando em maior quantidade de DNA quando comparada com o kit comercial. Em conclusão, descrevemos a deleção completa do gene PROP1 em dois irmãos com o fenótipo clássico de hipopituitarismo associado à hipófise hipoplásica ou aumentada e padronizamos a extração de DNA de células de mucosa oral com NaCl, que apresentou custo mais baixo e resultado mais rápido quando comparado a extração por um kit comercial, indicando que o swab oral é uma fonte prática de obtenção de DNA para estudos genéticos. / PROP1 gene mutations are the most common cause of genetic combined pituitary hormone deficiency. To date, several missense mutations and small deletions have been described and the 301-302 del AG is the most frequent. Our objective was to study PROP1 mutations in patients with hypopituitarism and standardize DNA extraction from an oral swab, using the NaCl method, comparing it with a commercial kit (Purigene, Minneapolis, USA). We amplified the 3 exons of PROP1 gene in DNA obtained from oral cells and peripheral blood cells. We identified the delAG301-302 mutation in 6 patients, 4 in homozygous (33%) and 2 in heterozygous (16%) state and G51A mutation in heterozygous state in a single patient. In two siblings, a boy and a girl, born to consanguineous parents we failed to amplify PROP1 gene by PCR whereas LHX3 and LHX4 genes were successfully amplified. To confirm the hypothesis of PROP1 gene deletion, Southern blotting was performed using PROP1 exon 2 gene PCR product as a probe and a fragment of CYP21A2 gene as a control probe. The CYP21A2 band was present in patients and controls whereas PROP1 band was absent in both siblings and present in their mother and in controls. To define the extension of this deletion we used STS mapping approach and no amplification for a STS GDB:314805 6.0 kb downstream of PROP1 was found. However, Q8N6H0 gene located 18 kb upstream and the STS WI-16216 located 59 kb downstream of PROP1 were successfully amplified indicating that the deletion is placed within 81 Kb. To determine the limits of the deletion a number of PCR covering this region were then carried out with primers located progressively distant from PROP1. This allowed us determine the deleted region from 9.6 kb upstream to 11 kb downstream of PROP with a maximum deletion size of 18.4 kb. Both methods yielded good quality DNA, allowing the amplification of 3 exons of PROP1 gene. The NaCl method showed to be faster and less expensive, resulting in a larger amount of DNA when compared with the commercial kit. In conclusion, we describe a complete deletion of PROP1 gene in two siblings with classical hypopituitarism phenotype associated with hypoplastic or enlarged pituitary gland and standardized the DNA extraction of oral cells with NaCl, which presented lower costs and faster results, when compared with the extraction by a commercial kit indicating that oral swabs are a reliable DNA source for genetic studies.

Object: The aim of the study was to evaluate the early changes in pituitary hormone levels after severe traumatic brain injury (sTBI) and compare hormone levels to basic neuro-intensive care data, a systematic scoring of the CT-findings and to evaluate whether hormone changes are related to outcome. Methods: Prospective study, including consecutive patients, 15-70 years, with sTBI, Glasgow Coma Scale (GCS) score <= 8, initial cerebral perfusion pressure > 10 mm Hg, and arrival to our level one trauma university hospital within 24 hours after head trauma (n = 48). Serum samples were collected in the morning (08-10 am) day 1 and day 4 after sTBI for analysis of cortisol, growth hormone (GH), prolactin, insulin-like growth factor 1 (IGF-1), thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), follicular stimulating hormone (FSH), luteinizing hormone (LH), testosterone and sex hormone-binding globulin (SHBG) (men). Serum for cortisol and GH was also obtained in the evening (17-19 pm) at day 1 and day 4. The first CT of the brain was classified according to Marshall. Independent staff evaluated outcome at 3 months using GOS-E. Results: Profound changes were found for most pituitary-dependent hormones in the acute phase after sTBI, i.e. low levels of thyroid hormones, strong suppression of the pituitary-gonadal axis and increased levels of prolactin. The main findings of this study were: 1) A large proportion (54% day 1 and 70% day 4) of the patients showed morning s-cortisol levels below the proposed cut-off levels for critical illness related corticosteroid insufficiency (CIRCI), i.e. < 276 nmol/L (= 10 ug/dL), 2) Low s-cortisol was not associated with higher mortality or worse outcome at 3 months, 3) There was a significant association between early (day 1) and strong suppression of the pituitary-gonadal axis and improved survival and favorable functional outcome 3 months after sTBI, 4) Significantly lower levels of fT3 and TSH at day 4 in patients with a poor outcome at 3 months. 5) A higher Marshall CT score was associated with higher day 1 LH/FSH-and lower day 4 TSH levels 6) In general no significant correlation between GCS, ICP or CPP and hormone levels were detected. Only ICPmax and LH day 1 in men was significantly correlated. Conclusion: Profound dynamic changes in hormone levels are found in the acute phase of sTBI. This is consistent with previous findings in different groups of critically ill patients, most of which are likely to be attributed to physiological adaptation to acute illness. Low cortisol levels were a common finding, and not associated with unfavorable outcome. A retained ability to a dynamic hormonal response, i.e. fast and strong suppression of the pituitary-gonadal axis (day 1) and ability to restore activity in the pituitary-thyroid axis (day 4) was associated with less severe injury according to CT-findings and favorable outcome.

As mutações no gene PROP1 são a causa genética mais comum da deficiência combinada de hormônios hipofisários. Até o momento, diversas mutações missense e pequenas deleções foram descritas sendo a mutação 301-302 delAG a mais freqüente. Nosso objetivo foi estudar as mutações em DNA de pacientes com hipopituitarismo e padronizar a extração de DNA de células de swab oral, usando um método com NaCl e comparar com um kit comercial (Purigene, Minneapolis, EUA). Amplificamos os 3 exons do gene PROP1 do DNA obtido de células orais e de sangue periférico. Identificamos a mutação 301-302delAG em 6 pacientes, 4 em homozigose (33%) e 2 em heterozigose (16%) e a mutação G51A em heterozigose em um único paciente. Em dois irmãos, filhos de pais consangüíneos, não foi possível amplificar os 3 exons do gene PROP1 enquanto que os os genes LHX3 e LHX4 foram amplificados com sucesso. Para confirmar a hipótese de deleção do PROP1, o Southern blotting foi realizado usando como sonda o produto de PCR do exon 2 do gene PROP1 e um fragmento do gene CYP21A2 como sonda controle. A banda referente ao CYP21A2 estava presente nos pacientes e nos controles enquanto a banda referente ao PROP1 estava ausente nos irmãos e presente na mãe e nos controles. Para definir a extensão da deleção usamos um mapa de STS próximos ao gene e o STS GDB:314805 localizado a 6,0 kb a montante do PROP1 não foi amplificado nos pacientes. Entretanto, o gene Q8N6H0 a 18 kb a juzante e o STS WI-16216 a 59 kb a montante do PROP1 foram amplificados com sucesso nos pacientes e controles indicando que a deleção está localizada dentro de 81 Kb. Para determinar os limites da deleção, várias reações de PCR foram realizadas com primers desenhados progressivamente distantes de gene PROP1, cobrindo toda a região. Isto nos permitiu determinar a região deletada de 9,6 kb a juzante e 11 kb a montante do gene PROP1, com o tamanho máximo deletado de 18,4 kb. Por ambos os métodos de extração obtivemos um DNA de boa qualidade, permitindo o amplificação dos 3 exons do gene PROP1. A extração com NaCl foi mais rápida e mais barata resultando em maior quantidade de DNA quando comparada com o kit comercial. Em conclusão, descrevemos a deleção completa do gene PROP1 em dois irmãos com o fenótipo clássico de hipopituitarismo associado à hipófise hipoplásica ou aumentada e padronizamos a extração de DNA de células de mucosa oral com NaCl, que apresentou custo mais baixo e resultado mais rápido quando comparado a extração por um kit comercial, indicando que o swab oral é uma fonte prática de obtenção de DNA para estudos genéticos. / PROP1 gene mutations are the most common cause of genetic combined pituitary hormone deficiency. To date, several missense mutations and small deletions have been described and the 301-302 del AG is the most frequent. Our objective was to study PROP1 mutations in patients with hypopituitarism and standardize DNA extraction from an oral swab, using the NaCl method, comparing it with a commercial kit (Purigene, Minneapolis, USA). We amplified the 3 exons of PROP1 gene in DNA obtained from oral cells and peripheral blood cells. We identified the delAG301-302 mutation in 6 patients, 4 in homozygous (33%) and 2 in heterozygous (16%) state and G51A mutation in heterozygous state in a single patient. In two siblings, a boy and a girl, born to consanguineous parents we failed to amplify PROP1 gene by PCR whereas LHX3 and LHX4 genes were successfully amplified. To confirm the hypothesis of PROP1 gene deletion, Southern blotting was performed using PROP1 exon 2 gene PCR product as a probe and a fragment of CYP21A2 gene as a control probe. The CYP21A2 band was present in patients and controls whereas PROP1 band was absent in both siblings and present in their mother and in controls. To define the extension of this deletion we used STS mapping approach and no amplification for a STS GDB:314805 6.0 kb downstream of PROP1 was found. However, Q8N6H0 gene located 18 kb upstream and the STS WI-16216 located 59 kb downstream of PROP1 were successfully amplified indicating that the deletion is placed within 81 Kb. To determine the limits of the deletion a number of PCR covering this region were then carried out with primers located progressively distant from PROP1. This allowed us determine the deleted region from 9.6 kb upstream to 11 kb downstream of PROP with a maximum deletion size of 18.4 kb. Both methods yielded good quality DNA, allowing the amplification of 3 exons of PROP1 gene. The NaCl method showed to be faster and less expensive, resulting in a larger amount of DNA when compared with the commercial kit. In conclusion, we describe a complete deletion of PROP1 gene in two siblings with classical hypopituitarism phenotype associated with hypoplastic or enlarged pituitary gland and standardized the DNA extraction of oral cells with NaCl, which presented lower costs and faster results, when compared with the extraction by a commercial kit indicating that oral swabs are a reliable DNA source for genetic studies.

Orientadores: Denise Engelbrecht Zantut Wittmann, Heraldo Mendes Garmes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T18:57:15Z (GMT). No. of bitstreams: 1 RosellCastillo_Alejandro_M.pdf: 1638622 bytes, checksum: 1b3c715bebcd9913f573b5ef8758046e (MD5) Previous issue date: 2015 / Resumo: Introdução O Hipopituitarismo (HP) é uma doença que tem como condição básica a deficiência na produção ou na ação de qualquer um dos hormônios da adeno-hipófise. Quando isto ocorre em dois ou mais hormônios, é denominado Pan-hipopituitarismo (PH). O HP é conhecidamente associado ao aumento da prevalência de Síndrome metabólica (SM), principalmente em pacientes com deficiência grave de hormônio de crescimento (GH). Apesar da suposta relação entre HP e síndrome metabólica, poucos estudos avaliaram a prevalência da SM e suas características clínico-laboratoriais nos pacientes com PH, geralmente ressaltando apenas a relação com a falência de GH. Objetivos Neste sentido, devido à escassez de informações na literatura, realizamos um estudo em pacientes com Pan-hipopituitarismo investigando as características e as frequências da SM e esteatose hepática (EH), assim como os perfis glicêmico, lipídico e de marcadores inflamatórios e de RI comparados a um grupo controle pareados pela idade, sexo e IMC. Metodologia Estudo transversal em que foram avaliados 41 pacientes com diagnóstico de PH e 37 indivíduos com função hipofisária normal pareados pela idade, sexo e índice de massa corporal. Avaliaram-se dados clínicos, antropométricos, ultrassonográficos (para EH), concentrações séricas de proteína C reativa (PCR) bem como exames laboratoriais que refletem os perfis lipídico, glicêmico e de RI. Resultados As frequências de SM e EH foram 65,9% e 78% nos pacientes com PH, e de 59,5% e 64,9% nos indivíduos do grupo de controle respetivamente. No entanto a frequência do diagnostico de dislipidemia (DLP) foi maior nos pacientes com PH (75,6% vs 51,4%; p=0,026). Os valores de Índice cintura quadril (ICQ) (p<0,001), glicemia (Gli) (p=0,010), insulina (p<0,001), e do índice indicativo de resistência à insulina (Homeostatic Model Assessment, HOMA-IR) (p<0,001) foram maiores nos indivíduos do grupo de controle. Por outro lado as concentrações séricas de PCR (p=0,011) foram maiores nos pacientes com PH. Na comparação entre os pacientes e indivíduos controle com SM, apresentaram-se valores significativamente superiores no grupo de indivíduos com SM, em relação à Gli (p=0,043), hemoglobina glicada (HBGli) (p=0,030), insulina (p<0,001), HOMA-IR (p<0,001), ICQ ( p=0,001). No entanto, os pacientes com PH e SM apresentaram maior frequência do diagnóstico de DLP (P=0,012) e concentrações significativamente superiores de PCR (P=0,028). O índice de massa corporal (IMC), idade e sexo feminino foram fatores de risco independentes para o desenvolvimento de SM nos pacientes com pan-hipopituitarismo, e a hemoglobina glicada nos indivíduos do grupo controle. Conclusões As frequências de síndrome metabólica e esteatose hepática foram semelhantes em pacientes com PH e nos indivíduos controle, por outro lado a frequência do diagnóstico de DLP foi maior nos pacientes com PH. Os indivíduos controle com SM apresentaram maior RI quando comparados aos pacientes com PH e SM. No entanto, os pacientes com PH e SM apresentaram maiores concentrações de PCR, e maior frequência do diagnóstico de DLP. O IMC, idade e sexo feminino foram os fatores de risco independentes para o desenvolvimento da SM nos pacientes com PH e a hemoglobina glicada nos indivíduos do grupo de controle / Abstract: Introduction Hypopituitarism (HP) is a disease that is characterized by the deficiency in secretion or action of any of the anterior pituitary hormones. When it occurs in two or more hormonal axis it is denominated pan-hypopituitarism (PH). PH is associated to and increased prevalence of metabolic syndrome (MS), especially in patients with severe growth hormone (GH) deficiency. Despite the supposed relation of MS and PH, few studies have assessed the prevalence of MS and its clinical and laboratorial characteristics in patients with PH, often only highlighting the association with GH deficiency. Objectives Thus, due to the paucity of information in the literature, we conducted a study in patients with PH investigating the characteristics and the frequencies of MS and hepatic steatosis (HS), as well as fasting glycaemia, lipid profile and inflammatory and insulin resistance markers compared to a control group paired by age, sex and body mass index (BMI). Methodology This was a cross study evaluating 41 patients with PH and 37 individuals with normal pituitary function paired by age, sex and BMI. We evaluated clinical, anthropometric and ultrassonographic (for HS) data as well as serum levels of C-reactive protein (CRP), fasting glycaemia, lipid profile and insulin resistance index. Results The frequencies of MS and HS were 65.9% and 78% in patients with PH and 59.5% and 64.9% in control group individuals, respectively. However, the frequency of dyslipidemia diagnosis was higher in patients with PH (75.6% vs 51.4%; p=0.026). Waist to hip ratio (WHR) (p<0.001), fasting glycemia (p=0.01), fasting insulin (p<0.001) and the HOMA (Homeostatic Model Assessment ¿ HOMA IR) (p<0.001) were higher in control group individuals. On the other hand, CRP (p=0.011) levels were higher in patients with PH. Comparing control group individuals with MS and patients with PH and MS, fasting glycaemia (p=0.043), HbA1c (p=0.03), fasting insulin (p<0.001), HOMA IR (p<0.001) and WHR (p=0.001) were higher in the control group. Patients with PH and MS presented higher frequency of dyslipidemia diagnosis (p=0.012) and CRP levels (p=0.028). Body mass index, age and female sex were independent risk factors for MS in patients with PH, whereas the only significant factor in the control group was HbA1c. Conclusions The frequencies of MS and HS were similar in patients with PH and control group individuals. However, the frequency of dyslipidemia was higher in patients with PH. Control group individuals with MS showed increased insulin resistance when compared to patients with PH and MS. Patients with PH and MS had higher serum concentrations of CRP, and higher frequency of dyslipidemia diagnosis. Body mass index, age and female sex were independent risk factors for MS development in patients with PH whereas only HbA1c was a significant risk factor for MS in control group individuals / Mestrado / Clinica Medica / Mestre em Clinica Medica

Traumatic brain injury (TBI) effects a large number of individuals, both civilians and military personnel, every year. The neuroinflammatory response mounted in the brain following a head injury continues long after the effects of initial subside. While it was initially thought to only occur in moderate or severe TBI, the deleterious effects of this cascade have recently been identified in patients with mild TBI (mTBI). Hypopituitarism is an often underreported condition and can result from TBI of all severity. The long-term sequelae of TBI can manifest in or exacerbate many other comorbidities of brain injury, such as neuroendocrine dysfunction or mental health conditions. Both TBI and hypopituitarism can present with symptoms similar to some psychiatric disorders, or exacerbation comorbid conditions. Veteran patients presenting to their primary care providers with symptoms of irritability, depression, anxiety, or cognitive and behavioral changes may meet criteria to receive diagnoses of psychiatric illnesses prevalent in the military population, while not being evaluated for pituitary dysfunction, and thus receive inadequate treatment. The proposed study aims to identify the prevalence of patients that are receiving psychiatric treatment that have both a history of mTBI and reduced levels of pituitary hormones on serum assays. By identifying a significant portion of this population, future studies can assess the impact that hormonal replacement has on success of psychotherapy, resolution of symptoms, and impact on functional status, among other factors.

Les travaux de cette thèse ont porté sur le contrôle des neurones à GHRH dans des conditions physiologiques et pathologiques. Le but étant de caractériser les mécanismes cellulaires et moléculaires impliqués dans le fonctionnement ou dérégulations du réseau de neurones à GHRH. Ces neurones sont les principaux stimulateurs de la libération de l’hormone de croissance (GH). Nous avons d’abord montré que l’axe de la croissance et de l’appétit peuvent être régulés indépendamment au niveau de l’hypothamus. En effet, la ghréline, seule hormone produite par le tractus gastro-intestinal et connue pour stimuler la libération de GH en agissant principalement sur les neurones GHRH, stimule ces derniers de manière uniquement directe. Ces effets sont indépendants de ceux qu’elle exerce sur les neurones voisins à NPY, orexigéniques. De plus, la ghréline et les GHS (agonistes sélectifs du récepteur de la ghréline) ne changent pas le mode de décharge électrique des neurones à GHRH ni ne les synchronise. Enfin, ces effets ne présentent pas de dimorphisme sexuel. Dans un second temps, la somatostatine, principal inhibiteur de l’axe GH, induit un rythme d’activité électrique des neurones à GHRH médié par les récepteurs de sous-type SST1 et SST2. Ces effets sont donc temps-dépendants, et aussi sexuellement dimorphiques. Ils sont probablement impliqués dans la modulation de la pulsatilité ultradienne de la libération de GH. Enfin, après un traumatisme crânien, nous observons un déficit de la libération de GH qui apparaît tôt et est soutenu, comme ceux observés chez l’humain. Aucune inflammation ni changement histologique n’a été observe dans l’hypophyse. Cependant, l’inflammation, impliquant une réaction tanycytaire, microgliale, astrocytaire, est présente dans le noyau arqué et l’éminence médiane (EM), ou sont respectivement présents les corps cellulaires et terminaisons des neurones à GHRH. Ceci est lié à des changements morpho-fonctionnels de l’EM (augmentation perméabilité, rupture des barrières tanycytaires). Aucun changement n’a été observé dans le noyau périventriculaire, où sont localisés les neurones à somatostatine. Enfin, les propriétés électriques passives des neurones à GHRH ne sont pas modifiées. En conclusion, une dérégulation de leur activité au niveau des terminaisons nerveuses doit expliquer les défauts posttraumatiques de libération de GH. / The works of this thesis were interested in the control of the hypothalamic GHRH neurons in physiological and pathological conditions. The goal was to clarify the molecular and cellular mechanisms involved in the control or impairments of GHR neuronal network functions. These neurons are the main stimulators of the GH release. We first showed that the hypothalamic growth axis could be regulated independently from the feeding network. Indeed, GHRH neurons are directly stimulated by ghrelin, which is the only hormone produced by the gastrointestinal tract known to stimulate the GH release through acting mainly on GHRH neurons. These effects are independent from its orexigenic effects exerted on the neighbourings NPY neurons. In addition, ghrelin and GHS (synthetic ghrelin receptor agonists) don’t change neither the firing rate of GHRH neurons, nor synchronize them. These effects are not gender-dependant; by contrast, Somatostatin, the major GH axis inhibitor, generates a sexual dimorphic and rhythmic inhibition of the GHRH neurons electrical activity mediated by its SST1 and SST2 receptors subtypes. These effects are so time-dependant direct and indirect effects and can probably be involved in the generation of the ultradian rhythm of the GH release. After a traumatic brain injury, we found an early and sustained deficiency of the GH release, like those observed in human. No pathological changes are visible in the pituitary gland. Inflammation occurs at the arcuate nucleus, and mainly at the median eminence levels; it involves a strong astrocyte reaction, tanycytes, and microglial and (or) infiltrated immune cells activations. These changes elicit morpho-functional impairments of the median eminence, permeability and leakage of the tanycyte barrier between the blood, CSF and Arc; at the opposite, nothing occur at the periventricular level, where are located SST neurons. Neither the number of GHRH neurons, neither their passive electrophysiological properties changed. Impairments of the activities of the GHRH nerve terminals, maybe associated to impairments of their regulated activity, must explain a GH deficiency.

Hipopituitarismo congênito é caracterizado por deficiência hormonal múltipla devido a mutações de fatores de transcrição envolvidos na embriogênese hipofisária. As células-tronco estão presentes na hipófise e são caracterizadas por dar origem a uma célula progenitora e uma célula indiferenciada por divisão assimétrica. Estão envolvidas na hipófise em processos de alta demanda metabólica em diferentes fases da vida. Em estudos prévios, observou-se o acúmulo dos marcadores de células-tronco Sox2 e Nr2e1 no camundongo Ames, que apresenta mutação no gene Prop1. O Sox2 é o marcador consenso de células-tronco na hipófise enquanto que o Nr2e1, nunca antes caracterizado na hipófise, é essencial para a manutenção de células-tronco e neogenese no cérebro. A perda de função deste gene pode causar agressão e falta de instinto materno em camundongos. Com isso, o objetivo desse projeto foi utilizar o animal modelo zebrafish para avaliar o papel repressor do gene prop1 e caracterizar o gene nr2e1 bem como, confirmar se o mesmo está envolvido com a diferenciação terminal na hipófise, e sua interferência no comportamento do animal mutado. O zebrafish se encaixa adequadamente nesse projeto pois é de fácil manutenção, econômico e com rápido desenvolvimento. No presente estudo criou-se 2 modelos de zebrafish utilizando-se a técnica de edição genômica conhecida como CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) para nocautear os genes prop1 e nr2e1. Esta técnica permite uma interrupção específica e substituição de bases no genoma, resultando em uma alta especificidade, baixa toxicidade celular e é herdável. O zebrafish homozigoto com mutação no gene nr2e1 se desenvolve e reproduz como o animal controle, porém apresenta um comportamento mais exploratório quando comparado com o animal selvagem e o heterozigoto. A imunofluorescência para o anticorpo Sox2 no animal mutado mostrou se diferente do selvagem, pois apresenta um aumento da expressão temporal e o mesmo não se colocaliza com o Nr2e1. A imunofluorescência feita com os hormônios não se mostrou diferente entre o mutado e o selvagem. Conclui-se diante dos achados de normalidade do desenvolvimento, fertilidade, ausência de co-localização com o gene Sox2 e presença de hormônios como Tsh, Fsh e Gh, que o gene nr2e1 não é crucial na diferenciação terminal na hipófise porém o animal mutado apresenta um comportamento diferente do animal selvagem. Os resultados da caracterização do zebrafish com mutação no gene prop1 ainda estão em andamento devido a dificuldade de se estabelecer essa linhagem / Congenital hypopituitarism is characterized by multiple hormone deficiencies due to mutations in transcription factors involved in pituitary embryogenesis. Stem cells, which by definition can each give rise to a progenitor and an undifferentiated cell by asymmetric division, are present in the pituitary gland and are important during periods of high metabolic demand in different phases of life. In previous studies, the accumulation of the stem cell markers Sox2 and Nr2e1 was observed in the Ames mouse, which harbors a mutation in Prop1. Sox2 is the consensus stem cell marker in the pituitary gland, while the role of Nr2e1 in the pituitary development has not been characterized although it is essential for neural stem cell maintenance and neogenesis in the brain and its loss of function causes pathological aggression and lack of maternal instinct in mice. In this project, the zebrafish animal model was used to characterize the role of nr2e1, to confirm whether this gene can be involved in the pituitary terminal differentiation, and to determine the effects of this gene on animal behavior. The zebrafish is a particularly appropriate model for use in this project because it is easy to maintain, is economical, and has a rapid metabolism and growth rate. In the present study, we created 2 zebrafish models by knocking out prop1 and nr2e1 using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome-editing technique. This technique enables highly specific gene/reading frame interruption and/or base substitution in the genome, with low cellular toxicity and high heritability. Zebrafish with homozygous nr2e1 mutations develop and reproduce similarly to wild-type zebrafish, but present a more exploratory behavioral pattern compared to wild-type and heterozygous zebrafish. Based on immunofluorescence, Sox2 expression was higher in the mutant zebrafish than in the wild type and was not co-localized with Nr2e1 expression. Hormone expression did not differ between wild-type and mutant zebrafish. We conclude that nr2e1 is not crucial in the terminal differentiation of the hormone-forming pituitary gland; however, it induces a distinct behavioral phenotype at the larval stage. Analyses of zebrafish harboring a prop1 mutation are ongoing owing to issues with the establishment of the lineage