As teneurinas (TENs) são de proteínas de transmembrana com significante expressão no sistema nervoso central (SNC). Foi verificado que o último éxon dos genes das TENs codifica um peptídeo com elevada identidade ao fator liberador de corticotropina (CRF) sendo este denominado de peptídeo associado ao terminal carboxila da teneurina (TCAP-1 a 4). Estudos mostram que TCAP-1 controla o comportamento emocional possivelmente por modular as ações mediadas pelo (CRF). A distribuição do TCAP-1 no SNC de primatas poderia subsidiar os seus efeitos funcionais e as possíveis aplicações terapêuticas. O presente trabalho analisou a imunorreatividade e a expressão do RNA mensageiro do TCAP-1 no encéfalo de primatas não-humanos. Cortes frontais do SNC de macacos da espécie Cebus apella (n=3) foram selecionados e utilizados nas técnicas de hibridização in situ e de imuno-histoquímica. Neurônios imunorreativos e que expressam o RNAm para TCAP-1 foram encontrados principalmente em áreas que estão direta ou indiretamente envolvidas na modulação da resposta ao estresse e ansiedade. / Teneurins are transmembrane proteins expressed mainly in the central nervous system (CNS). The last exon of the teneurins exhibits a peptide sequence with high homology to corticotrophin-releasing factor (CRF), named teneurin c-terminal associated peptide (TCAP-1 to 4). TCAP-1 effectively modulates stress induced by CRF. Studies have pointed tha TCAP-1 could have important therapeutic applications in stress disorders. The analysis of TCAP-1 distribution in the primate brain could also give anatomical support to the understanding of its functional effects and possible therapeutic use. Thus, the present study focused on the distribution of neurons exhibiting immunoreactivity and mRNA expression to TCAP-1 in the monkey brain (Cebus apella). Frontal brain sections of three young male Cebus apella monkeys were submitted to immunohistochemistry and in situ hybridization. Immunohistochemistry and in situ hybridization results showed that TCAP-1 is preserved in primate brain, mainly in areas directly or indirectly involved in the modulation of stress and anxiety.

Introdução: A obesidade pode se constituir como fator de risco para o uso de contraceptivos hormonais, especialmente aqueles contendo estrogênios, devido a possíveis efeitos metabólicos indesejados. As alterações metabólicas decorrentes do uso do sistema intrauterino liberador de levonorgestrel (SIU-LNG) foram bem descritas em mulheres com peso normal, mas não em mulheres obesas. Objetivo: Avaliar o efeito do sistema intrauterino liberador de levonorgestrel (SIU-LNG) sobre marcadores ultrassonográficos e laboratoriais de risco metabólico e doença cardiovascular em mulheres obesas. Casuística e Métodos: Estudo clínico randomizado aberto avaliando 106 mulheres obesas (30,0 Kg/m2 e <40 Kg/m2), com idade entre 18 e 40 anos, sem outras comorbidades. As pacientes foram avaliadas antes do início da contracepção e após um ano, sendo realizada randomização simples, para uso de SIU-LNG ou método não hormonal (a paciente poderia escolher entre dispositivo intrauterino de cobre ou preservativo). Foram avaliados: evolução ponderal; circunferência da cintura; volume uterino e ovariano; insulinemia e glicemia de jejum; perfil lipídico; testosterona; sex hormone binding globulin (SHBG); hemograma; aspartato aminotransferase (AST), a alanina aminotransferase (ALT), a fosfatase alcalina e gama glutamil transferase; índice de rigidez da carótida e espessura íntima-média (EIM); dilatação mediada por fluxo da artéria braquial (DMF) e doença hepática gordurosa não alcoólica (DHGNA). Resultados: Nenhuma diferença estatística foi observada na dilatação mediada por fluxo da artéria braquial entre as mulheres em uso de SIU-LNG e métodos não hormonais em um ano de observação. Observou-se uma redução do volume ovariano (-0,5&177;2,6 vs. 0,7&177;2,6 cm3, p=0,03). A hemoglobina aumentou nas usuárias de SIU-LNG (0,4&177;0,8 vs. -0,1&177;0,8 g/dL, p=0,00) juntamente com o hematocrito (1,8&177;2,2 vs. -0,0&177;2,3 %, p=0,00). Não houve alteração do perfil lipídico, glicêmico, da função hepática e doença hepática gordurosa não alcoolica no grupo das usuárias de SIU-LNG e nem das usuárias de métodos não hormonal. Conclusão: Concluiu-se que o SIU-LNG não apresentou efeito metabólico desfavorável comparado a métodos não hormonais após um ano, em mulheres obesas grau I e II, sem outras comorbidades. / Background: Obesity may be a risk factor for the use of hormonal contraceptives, especially those containing estrogens, due to possible unwanted metabolic effects. The metabolic effects of levonorgestrel-releasing intrauterine system (LNG-IUS) have been studied in normal-weight women but not in obese women. Objective: To evaluate the effect of levonorgestrel-releasing intrauterine system on multiple ultrasonographic and laboratorial markers of metabolic and cardiovascular disease risk in obese women. Methods: A randomized clinical trial study evaluating 106 obese women ( 30.0 kg/m2 and <40 kg/m2), aged between 18 and 40 years with desire contraception, without other co-morbidities. Patients were evaluated before the start of contraception and after a year, made simple randomization to use of LNG-IUS or nonhormonal method (the patient could choose between copper intrauterine device or condom). Were evaluated: weight gain; waist circumference; uterine and ovarian volumes; insulinemia and fasting glucose; lipid profile; testosterone; sex hormone binding globulin (SHBG); hemogram; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase and gamma glutamyl transferase; stiffness index and carotid intima-media thickness (IMT); flow-mediated brachial artery dilatation (FMD) and nonalcoholic fatty liver disease (NAFLD). Results: No statistical difference was observed in flow-mediated brachial artery among women using the LNG-IUS and non-hormonal methods in one year of observation dilation. There was a volume reduction (-0.5 &177; 2.6 vs. 0.7 &177; 2.6 cm3, p=0.03). Hemoglobin increased in users of LNG-IUS (0.4 &177; 0.8 vs. -0.1 &177; 0.8 g/dL, p=0.00) together with the hematocrit (1.8 &177; 2.2 vs. -0.0 &177; 2.3%, p=0.00). There was no change in lipid profile, glucose, liver function and nonalcoholic fatty liver disease in the group of users of the LNG-IUS or the users of non-hormonal methods. Conclusion: We conclude that the LNG-IUS showed no adverse metabolic effect compared to non-hormonal methods after one year in grade I and II obese women without other comorbidities.

Made available in DSpace on 2016-03-22T17:27:35Z (GMT). No. of bitstreams: 1 xenia1.pdf: 708937 bytes, checksum: cc885d49bb2a8c9ada32a3218f9e37f3 (MD5) Previous issue date: 2014-01-24 / Objective: To investigate the relationship between peripheral levels of corticotropin releasing hormone (CRH) and interleukin-1&#61538; (IL-1&#61538;) in BD individuals with and without suicide risk (SR) and controls. Methods: 120 young adults (40 controls, 40 BD subjects without SR and 40 BD subjects with SR) were enrolled from a population-based study carried out in the city of Pelotas, (Brazil). BD and SR were assessed with MINI 5.0 and peripheral markers were evaluated by ELISA. Results: Levels of CRH were significantly lower both in BD subjects without SR (p = 0.04) or with SR (p = 0.02) when compared to control. However, levels of IL-1&#61538; were increased in BD subjects with SR (p = 0.05) when compared to control. Socio-demographic and clinical variables, current mood episode and use of psychiatry medication were not associated with changes in these markers. No correlation was found between peripheral levels of CRH and IL-1&#61538; (p = 0.60) in the population or in BD with SR group (p = 0.88) Conclusions: These results suggest that peripheral mechanisms linking stress hormones and the immune system might be critical patterns involved in suicidal behavior associated with BD / Avaliar se existem alterações nos níveis periféricos do hormônio liberador de corticotropina (CRH) e na interleucina-1&#61538; (IL-1&#61538;) em pacientes com transtorno bipolar, com ou sem risco de suicídio comparando com indivíduos sem transtornos de humor. 2.3.2. Objetivos específicos 1. Avaliar os níveis séricos de CRH e IL-1&#61538; em pacientes com diagnóstico de transtorno bipolar comparando com controles sem transtornos de humor; 2. Comparar os níveis séricos de CRH e IL-1&#61538; em pacientes com diagnóstico de transtorno bipolar com e sem risco de suicídio, e controles sem transtornos de humor; 3. Investigar se existe algum efeito da diferença de gênero, idade, atividade física, índice de massa corporal e fatores sócio- demográficos com os níveis de CRH e IL-1&#61538; em pacientes bipolares com e sem risco de suicídio e controles. 4. Verificar se existem alterações nos níveis periféricos de CRH e IL-1&#61538; durante os episódios atuais de humor dentro do transtorno bipolar. 5. Verificar se existem uma correlação entre os níveis periféricos de CRH e IL-1&#61538;

by Lau Hoi Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 83-96). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Endocrine control of normal and abnormal growth of prostate --- p.1 / Chapter 1.1.1 --- Androgen regulation of prostate gland --- p.1 / Chapter 1.1.2 --- Estrogen regulation of prostate gland --- p.4 / Chapter 1.2 --- Gonadotropin-releasing hormone plays a central role in reproduction --- p.6 / Chapter 1.2.1 --- GnRH gene --- p.7 / Chapter 1.2.2 --- GnRH receptor --- p.9 / Chapter 1.3 --- Therapeutic strategies using GnRH analogs to treat prostate cancer --- p.12 / Chapter 1.4 --- Expression of GnRH or its receptor in reproductive tissues --- p.12 / Chapter 1.4.1 --- Expression of GnRH in reproductive --- p.13 / Chapter 1.4.2 --- Expression of GnRH and its receptor in pituitary and reproductive tissues --- p.13 / Chapter 1.5 --- Animal models for the study of prostate cancer --- p.15 / Chapter 1.5.1 --- Nobel rat inducible model --- p.15 / Chapter 1.5.2 --- Androgen dependent rat Dunning prostatic tumor --- p.16 / Chapter 1.5.3 --- Androgen-independent prostatic carcinoma line of Noble rat --- p.18 / Chapter 1.6 --- Aim of study --- p.18 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and supply of Nobel rat --- p.20 / Chapter 2.2 --- Induction of dysplasia in Nobel rat prostate gland by long-term treatment with steroids --- p.20 / Chapter 2.2.1 --- Chemicals --- p.20 / Chapter 2.2.2 --- Preparation of steroid hormone-filled Silastic tubings --- p.20 / Chapter 2.2.3 --- Surgical implantation of Silastic® tubings --- p.21 / Chapter 2.2.4 --- Protocols of hormonal treatments --- p.21 / Chapter 2.3 --- Androgen- dependent Dunning rat prostatic adenocarcinoma --- p.22 / Chapter 2.4 --- Androgen- independent prostatic carcinoma line (ALT) of Noble rat --- p.22 / Chapter 2.5 --- Detection of mRNA expression of gonadotropin- releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.23 / Chapter 2.5.1 --- Preparation of tissue for total RNA extraction --- p.23 / Chapter 2.5.2 --- Total RNA extraction --- p.24 / Chapter 2.5.3 --- Reverse-transcription Polymerase Chain Reaction (RT-PCR) --- p.25 / Chapter 2.5.4 --- Purification of DNA fragments from agarose gels --- p.27 / Chapter 2.5.5 --- Subcloning of DNA into vector --- p.27 / Chapter 2.5.6 --- Nucleotide sequencing --- p.30 / Chapter 2.5.7 --- Southern blot analysis --- p.32 / Chapter 2.5.7.1 --- Southern blotting --- p.32 / Chapter 2.5.7.2 --- Preparation of α-32P-dCTP labelled GnRH probe --- p.32 / Chapter 2.5.7.3 --- Hybridization --- p.33 / Chapter 2.6 --- Detection of mRNA expression of gonadotropin-releasing hormone receptor (GnRH-R) in normal and neoplastic rat prostates --- p.34 / Chapter 2.6.1 --- Cloning of GnRH-R cDNA and synthesis of its probe --- p.34 / Chapter 2.6.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates by Southern blot --- p.36 / Chapter 2.6.3 --- Detection of GnRH receptor mRNA expression in Dunning tumor --- p.37 / Chapter 2.6.4 --- Detection of the GnRH receptor mRNA expression in AIT tumor by RT-PCR --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Detection of mRNA expression of gonadotropin-releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.38 / Chapter 3.1.1 --- Reverse -transcription Polymerase Chain Reaction (RT-PCR) --- p.38 / Chapter 3.1.2 --- Purification of DNA fragments amplified by PCR from the agarose gel --- p.38 / Chapter 3.1.3 --- Subcloning of DNA into vector --- p.39 / Chapter 3.1.4 --- Nucleotide sequencing --- p.39 / Chapter 3.1.5 --- Southern-blot analysis --- p.39 / Chapter 3.2 --- Detection of gonadotropin-releasing hormone receptor mRNA expression in normal and neoplastic rat prostates --- p.40 / Chapter 3.2.1 --- Cloning of gonadotropin-releasing hormone receptor (GnRH) cDNA and synthesis of probe from the normal Noble rat pituitary gland --- p.40 / Chapter 3.2.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates --- p.42 / Chapter 3.2.3 --- Detection of GnRH receptor mRNA expression in rat Dunning tumor by PCR --- p.43 / Chapter 3.2.4 --- Detection of GnRH receptor mRNA expression in AIT tumor --- p.43 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Detection of mRNA expression of gonadotropin-releasing releasing hormone(GnRH) in normal and neoplastic rat prostates --- p.69 / Chapter 4.1.1 --- Expression of GnRH mRNA in normal Nobel rat prostate gland --- p.69 / Chapter 4.1.2 --- Expression of GnRH mRNA in dysplastic Nobel rat prostate --- p.71 / Chapter 4.1.3 --- Expression of GnRH mRNA in androgen-dependent rat Dunning prostatic tumor --- p.72 / Chapter 4.1.4 --- Expression of GnRH mRNA in AIT tumor --- p.74 / Chapter 4.2 --- Detection of GnRH receptor in normal and dysplastic rat prostates --- p.75 / Chapter 4.2.1 --- Negative expression of GnRH receptor in normal and dysplastic Nobel in rat prostates --- p.75 / Chapter 4.2.2 --- Positive expression of GnRH receptor mRNA in rat Dunning tumor --- p.77 / Chapter 4.2.3 --- Negative expression of GnRH receptor mRNA in ALT tumor --- p.78 / Chapter Chapter 5 --- Summary and Conclusions --- p.80 / References --- p.83

A homeostase energética é controlada por fatores neurais, endócrinos, adipocitários e intestinais. O sistema nervoso central (SNC) recebe sinalização de fatores periféricos e exerce uma função fundamental no controle da homeostase energética, estando bem estabelecido que existem populações neuronais que expressam neuropeptídeos que medeiam efeitos específicos na ingestão e/ou gasto energético. O fator liberador de corticotrofina (CRF), além de seus efeitos no controle da atividade do eixo hipotálamo-hipófise-adrenal, tem sido descrito como potente neuropeptídeo anorexígeno, modulando a ingestão alimentar e o gasto energético. Foi observado que a síntese de CRF é influenciada pela leptina, que atuaria aumentando a ativação de neurônios produtores de CRF no núcleo paraventricular (PVN). Os hormônios gonadais também participam na regulação da ingestão alimentar, do peso e da composição corporal. O efeito anorexígeno do estradiol é mediado pela ativação de receptores presentes nas áreas envolvidas no controle da homeostase energética. Em trabalho prévio de nosso laboratório foi observado que o menor ganho de peso e ingestão alimentar com o tratamento com estradiol em ratas ovariectomizadas está associado à maior expressão de RNAm de CRF no PVN. Dessa forma, este trabalho visa esclarecer a participação do CRF nos efeitos do estradiol no controle da homeostase energética. Para tanto, foram utilizadas ratas Wistar adultas, pesando entre 200-230g, provenientes do Biotério Central do Campus de Ribeirão Preto USP. Todos os animais foram submetidos à cirurgia de ovariectomia bilateral. Em todos os experimentos, houve três grupos de animais: ratas ovariectomizadas (OVX), ratas ovariectomizadas com reposição de estradiol (OVX+E) e ratas ovariectomizadas com dieta pareada ao grupo OVX+E (OVX+DP). Durante os oitos dias de cada experimento, estes animais receberam injeção subcutânea de cipionato de estradiol (10 g/Kg peso corporal, Grupo OVX+E) ou veículo (óleo de milho: 0,2 mL/rata, Grupos OVX e OVX+DP) entre 8h e 10h. Para avaliarmos a participação do CRF nos efeitos da leptina nos animais castrados com e sem reposição de estradiol, foi realizado o tratamento com injeção central de leptina (10g/5L) com e sem injeção central prévia de antagonista de CRF (antisauvagina-30). Observamos que o tratamento com cipionato de estradiol causa a redução na ingestão alimentar e no ganho de peso corporal. Ainda, quando realizamos a administração central de leptina há anorexia, perda de peso corporal, aumento na expressão de UCP-1 no BAT e na ativação neuronal no ARQ. Esses efeitos são revertidos quando realizamos administração central prévia do antagonista de CRF-R2. Os dados obtidos sugerem que o estradiol aumenta a sensibilidade à leptina, sendo este efeito mediado, pelo menos em parte, pelo receptor tipo 2 do CRF. / Energy homeostasis is controlled by neural, endocrine, adipocyte and gut factors. Central nervous system plays a key role in the control of energy homeostasis; it receives signals from peripheral factors and it is well established that the hypothalamus contains neuronal populations that express important neuropeptides to the control of food intake and energy expenditure. Besides its action in the control of hypothalamus-pituitary-adrenal axis, corticotropin releasing factor (CRF), has been described as an anorexigenic neuropeptide, modulating food intake and energy expenditure. It was shown that CRF synthesis is influenced by leptin, which would act increasing CRF neuron activation in the paraventricular nucleus (PVN). Gonadal hormones also participate in the regulation of food intake, body weight and body composition. Estradiol anorexigenic effect is mediated by specific receptors located in areas involved in the control of energy homeostasis. It was previously demonstrated that the reduction of food intake and body weight gain in ovariectomized treated rats is associated with an increase in CRF mRNA expression in the PVN. The present study aimed to investigate the role of CRF on estradiol regulation of energy homeostasis. Wistar female rats, weighing 200 230g, were bilaterally ovariectomized and divided into three groups: ovariectomized rats (OVX), ovariectomized rats treated with estradiol (OVX+E) and ovariectomized rats pair-fed with OVX+E rats (OVX+PF). The animals received daily subcutaneous injections of either estradiol cypionate (10 g/Kg bw, OVX+E) or vehicle (corn oil, OVX, OVX+PF) between 8 10 am, during 8 days. To evaluate the role of CRF on leptins effects we performed intracerebroventricular (icv) injection of recombinant leptin (10g/5L) with or without previous icv treatment with CRF-R2 antagonist (ansauvagin-30). We observed that estradiol replacement in OVX rats induced lower food intake and body weight gain. Leptin icv treatment reduced food intake, body weight gain and increased UCP-1 expression in brown adipose tissue and neuronal activation in the arcuate nucleus. These effects were abolished with previous icv administration of CRF-R2 antagonist. In conclusion, our data suggest that estradiol increases central sensitivity to leptin and this effect is mediated, at least in part, by CRF type 2 receptor.

La vasopressine (AVP) et la corticolibérine (CRF) agissent de manière synergique lors de la réponse aux stimuli stressants. Elles régulent de manière concertée la sécrétion d'adrénocorticotrophine hypophysaire et la libération de catécholamines surrénalienne. Dans ces deux structures, les isoformes de récepteurs présents sont les récepteurs V1B et CRF1. Nous avons démontré que deux mécanismes moléculaires sous-tendent la synergie fonctionnelle de l'AVP et du CRF : un croisement des voies de seconds messagers propres à chacun des récepteurs d'une part, et une modification de leurs propriétés pharmacologiques résultant de leur interaction (hétérodimérisation) d'autre part. Pour valider ce dernier mécanisme, nous avons recherché des formes naturelles ou mutées de récepteurs à l'AVP et au CRF conservant leurs propriétés de couplage aux protéines G, mais incapables d'hétérodimériser, et avons analysé les conséquences de cette rupture d'hétérodimérisation sur leur aptitude à agir en synergie. Grâce à une approche de mutagénèse dirigée, nous avons commencé à résoudre la question des portions de récepteurs engagées dans l'hétérodimérisation. Les résultats obtenus apportent les premières évidences permettant de comprendre la synergie AVP/CRF au niveau moléculaire, et particulièrement le rôle de l'hétérodimérisation. L'hétérodimère V1B/CRF1 pourrait être impliqué dans le stress et ses états pathologiques que sont l'anxiété et la dépression. Nous montrons que les récepteurs V1B et CRF1 sont co-exprimés dans les neurones de certaines structures cérébrales régulant ces phénomènes comportementaux. Démontrer l'existence de l'hétérodimère V1B/CRF1 dans des tissus natifs sera la prochaine étape de ce travail. Si elle est validée, le complexe V1B/CRF1 pourra être considéré comme une cible pharmacologique de première importance dans le traitement de l'anxiété et de la dépression. Travail soutenu par l'Institut de Recherches SERVIER et la Fondation pour la Recherche Médicale. / Vasopressin (AVP) and Corticotropin-Releasing Factor (CRF) are involved in the stress response, mainly by regulating ACTH secretion from the pituitary and by increasing catecholamine and corticosteroids secretion from the adrenal medulla. In these two structures, AVP and CRF have been shown to act in synergism via V1B and CRF1 receptors. Recently, our group demonstrated that such synergism operates via both second messenger crosstalk and putative mechanism involving receptors heterodimerization. To further validate this last original mechanism, we monitored the influence of receptor heterodimerization selectivity and of receptor heterodimerization disruption on functional synergism. We also deciphered receptor dimers interface by synthesizing receptor mutants that do not heterodimerize anymore.These results give clues to the comprehension of AVP/CRF synergism at the molecular level and trigger the potential role of receptors heterodimerization in stress-related behaviours. Indeed both V1B and CRF1 are also co-expressed in neurons of relevant brain area. Establishing the physical association of V1B/CRF1 as heterodimers in native tissue, the next step of our project, would be of considerable importance.Work supported by SERVIER (France) an d the FRM.

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD / GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

Neuropeptides, such as substance P (SP) and bombesin/gastrin-releasing peptide (BN/GRP), and neurotrophins are involved in neuro-immunomodulatory processes and have marked trophic, growth-promoting and inflammation-modulating properties. The impact of these modulators in rheumatoid arthritis (RA) is, however, unclear. An involvement of the innervation, including the peptidergic innervation, is frequently proposed as an important factor for arthritic disease. Many patients with RA, but not all, benefit from treatment with anti-TNF medications. The studies presented here aimed to investigate the roles of neuropeptides, with an emphasis on BN/GRP and SP, and neurotrophins, especially with attention to brain-derived neurotrophic factor (BDNF), in human and murine knee joint tissue. The expression patterns of these substances and their receptors in synovial tissue from patients with either RA or osteoarthritis (OA) were studied in parallel with the levels of these factors in blood and synovial fluid from patients with RA and from healthy controls. Correlation studies were also performed comparing the levels of neuropeptides with those of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)]. Furthermore, the impact of anti-TNF treatment on the levels of BDNF in blood was investigated. In a murine model of RA, the expression of these substances on articular chondrocytes along with their expression in synovial tissue was investigated. The expression of BN/GRP in human synovial tissue was confined to fibroblast-like and mononuclear-like cells whereas SP was detected in nerve-related structures. Receptors for these neuropeptides (GRP-R and NK-1R) were frequently present in blood vessel walls, and on fibroblast-like and mononuclear-like cells. The expression of BDNF and its receptors, p75 neurotrophin receptor and TrkB, was mainly confined to nerve structures. The levels of SP, and particularly those of BN/GRP, in synovial fluid and peripheral blood correlated with the levels of pro-inflammatory cytokines. There were clearly more correlations between SP-BN/GRP and inflammatory parameters than between BDNF and these factors. Plasma levels of BDNF were decreased following anti-TNF-treatment. In the joints of the murine model, there was a marked expression of neurotrophins, neurotrophin receptors and NK-1R/GRP-R in the articular chondrocytes. The expression was down-regulated in the arthritic animals. A neurotrophin system was found to develop in the inflammatory infiltrates of the synovium in the arthritic mice. The results presented suggest that there is a local, and not nerve-related, supply of BN/GRP in the human synovial tissue. Furthermore, BN/GRP and SP have marked effects in the synovial tissue of patients with RA, i.e., there were abundant receptor expressions, and these neuropeptides are, together with cytokines, likely to be involved in the neuro-immunomodulation that occurs in arthritis. The observations do on the whole suggest that the neuropeptides, rather than BDNF, are related to inflammatory processes in the human knee joint. A new effect of anti-TNF treatment; i.e., lowering plasma levels of BDNF, was observed. Severe arthritis, as in the murine model, lead to a decrease in the levels of neurotrophin, and neurotrophin and neuropeptide receptor expressions in the articular cartilage. This fact might be a drawback for the function of the chondrocytes. Certain differences between the expression patterns in the synovial tissue of the murine model and those of human arthritic synovial tissue were noted. It is obvious that local productions in the synovial tissue, nerve-related supply in this tissue and productions in chondrocytes to different extents occur for the investigated substances.

Electroconvulsive therapy (ECT) is one of the oldest and most effective treatments for depression; however, its biological underpinnings are poorly understood. Brain-derived neurotrophic factor (BDNF) and the hypothalamic-pituitary-adrenal (HPA) axis are two chemical messenger systems implicated in the antidepressant action and cognitive side effects of ECT. The Wistar-Kyoto (WKY) strain is a genetic model of depression that shows biological, cognitive, behavioural, and treatment-response abnormalities, making it potentially a useful model in which to investigate the underpinnings of the action of electroconvulsive stimulus (ECS: the amimal model of ECT). In addition, the WKY presents a potentially useful model for translational research on depression. The WKY strain is particularly valuable for the measurement of serum BDNF protein, for which the association with antidepressant treatments is much less clear (mostly stemming from investigations in humans) than that between brain BDNF and antidepressant treatments in rodent studies. The three studies presented add insight into the biological and behavioural effects of ECS. The first study (chapter 2) found no evidence of increased (R)-[11C]rolipram binding (an indirect marker of cyclic-adenosine monophosphate, cAMP) in the brain, despite significant increases of brain BDNF protein expression after repeated ECS. The second study (chapter 3) demonstrated the validity of the WKY strain in the investigation of ECS. Relative to Wistar controls, WKY showed similar antidepressant and cognitive effects (despite some abnormal behavioural responses), immediate but not sustained increases in brain BDNF protein, and a novel finding of increased extra-hypothalamic CRF after 5 daily ECS. The final study (chapter 4) demonstrated baseline strain differences in serum (WKY < Wistar) but not brain BDNF and, in both strains, no change in serum BDNF despite significant changes in brain BDNF after repeated ECS treatment. Preliminary results from a human pilot study investigating similar measures in a small group of people receiving ECT for depression are also presented. The results of this body of work advance our understanding of the activation and role of brain and serum measures of BDNF and the HPA axis in ECS/ECT, and raise important issues in the translation of research from basic science to the human condition of depression.

Electroconvulsive therapy (ECT) is one of the oldest and most effective treatments for depression; however, its biological underpinnings are poorly understood. Brain-derived neurotrophic factor (BDNF) and the hypothalamic-pituitary-adrenal (HPA) axis are two chemical messenger systems implicated in the antidepressant action and cognitive side effects of ECT. The Wistar-Kyoto (WKY) strain is a genetic model of depression that shows biological, cognitive, behavioural, and treatment-response abnormalities, making it potentially a useful model in which to investigate the underpinnings of the action of electroconvulsive stimulus (ECS: the amimal model of ECT). In addition, the WKY presents a potentially useful model for translational research on depression. The WKY strain is particularly valuable for the measurement of serum BDNF protein, for which the association with antidepressant treatments is much less clear (mostly stemming from investigations in humans) than that between brain BDNF and antidepressant treatments in rodent studies. The three studies presented add insight into the biological and behavioural effects of ECS. The first study (chapter 2) found no evidence of increased (R)-[11C]rolipram binding (an indirect marker of cyclic-adenosine monophosphate, cAMP) in the brain, despite significant increases of brain BDNF protein expression after repeated ECS. The second study (chapter 3) demonstrated the validity of the WKY strain in the investigation of ECS. Relative to Wistar controls, WKY showed similar antidepressant and cognitive effects (despite some abnormal behavioural responses), immediate but not sustained increases in brain BDNF protein, and a novel finding of increased extra-hypothalamic CRF after 5 daily ECS. The final study (chapter 4) demonstrated baseline strain differences in serum (WKY < Wistar) but not brain BDNF and, in both strains, no change in serum BDNF despite significant changes in brain BDNF after repeated ECS treatment. Preliminary results from a human pilot study investigating similar measures in a small group of people receiving ECT for depression are also presented. The results of this body of work advance our understanding of the activation and role of brain and serum measures of BDNF and the HPA axis in ECS/ECT, and raise important issues in the translation of research from basic science to the human condition of depression.