Effects of {$\alpha$}1, {$\alpha$}2, {$\beta$}3 adrenergic agonists on resting oxygen consumption and effects of {$\alpha$} and {$\beta$}2 adrenergic antagonists on noradrenaline stimulated oxygen consumption of the human peripheral blood mononuclear cells (PBMC) were studied using the Clark oxygen electrode. It was found that, the {$\alpha$}2 agonist increases slightly the resting oxygen consumption in the PBMC while, the {$\beta$}2 and the {$\alpha$} antagonists inhibit the noradrenaline stimulated oxygen consumption considerably. The {$\alpha$}1 and {$\beta$}3 agonists have no effect on the resting oxygen consumption. The NA termogenesis in the PBMC is mediated by {$\beta$}1 (25%), {$\beta$}2 (8%) and {$\alpha$}2 (12%) adrenergic receptors and not by {$\beta$}3 adrenoreceptors. The NA thermogenesis in the PBMC is mediated by different adrenoreceptors than the NA thermogenesis in the brown adipose tissue. The NA thermogenesis of PBMC is independent on the presence of organic substrates in the cultivation medium. Administration of external substrates (glucose, glutamine, methionine) to the medium has no effect on resting and NA stimulated oxygen consumption of PBMC, while administration of cysteine increases NA stimulated oxygen consumption considerably, indicating that acetyl-CoA could be an important substrate for NA thermogenesis in PBMC.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-13T17:36:44Z No. of bitstreams: 1 andersondutrademelo.pdf: 664118 bytes, checksum: 3695f89c95d5ed609cc3f5bd69a7f406 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T12:58:01Z (GMT) No. of bitstreams: 1 andersondutrademelo.pdf: 664118 bytes, checksum: 3695f89c95d5ed609cc3f5bd69a7f406 (MD5) / Made available in DSpace on 2016-10-22T12:58:01Z (GMT). No. of bitstreams: 1 andersondutrademelo.pdf: 664118 bytes, checksum: 3695f89c95d5ed609cc3f5bd69a7f406 (MD5) Previous issue date: 2008-02-22 / O diabetes reduz a secreção de catecolaminas em resposta a variações glicêmicas, acentuando o quadro de descontrole metabólico dos indivíduos doentes. Diversos estudos têm demonstrado que a hiperglicemia é a principal causa dos problemas decorrentes da instalação do diabetes. Desta forma, o objetivo deste trabalho foi analisar o efeito do diabetes sobre o processo de síntese e secreção de catecolaminas e se o tratamento com insulina reverte as modificações causadas pela doença. Métodos e Resultados: Foram usados ratos Wistar, machos, com 60 dias. O diabetes foi induzido pela injeção intravenosa de estreptozotocina na proporção de 50 mg/Kg de animal. O grupo controle recebeu injeção de solução tampão. Para determinar o protocolo de tratamento com insulina, estudamos o padrão de ingestão alimentar e de variações glicêmicas durante um dia, medindo estes dois parâmetros de hora em hora, durante 24h consecutivas. Foi estabelecido o tratamento com insulina NPH humana na dose de 5U diárias, 1 U às 13 h e 4 U às 19h. Após 15 dias da indução, os animais foram sacrificados e as glândulas adrenais foram retiradas. Com a intenção de caracterizar o efeito do diabetes sobre alguns parâmetros bioquímicos correntemente utilizados como marcadores da doença, foram medidos os níveis de frutosamina, triglicerídeos e colesterol e suas frações. Foi quantificado o conteúdo total de catecolaminas e a secreção basal e a estimulada por altas concentrações de potássio, carbamilcolina e cafeína. As catecolaminas foram dosadas por método fluorimétrico. A expressão de tirosina hidroxilase (TH), enzima reguladora da via de síntese de catecolaminas, foi avaliada por Western Blot. A glicemia foi de 82,82 ± 1,24 mg/dl, 405,74 ± 23,35 mg/dl e 103,72 ± 6,79 mg/dl nos animais controle, diabéticos e diabéticos tratados com insulina (DTI). A variação na massa corporal durante o período experimental foi negativa nos ratos diabéticos, ou seja, eles emagreceram 6,1 ± 3,84 g, enquanto que os animais controles e os diabéticos tratados, aumentaram seus pesos, em média, 36,34 ± 1,8 g e 43,32 ± 3,79 g, respectivamente. O diabetes modificou os níveis de colesterol total, LDL e VLDL, modificações que foram corrigidas pelo tratamento com insulina. Não houve diferença, entre controles e diabéticos, nos níveis de triglicérides, frutosamina e LDH. O tratamento com insulina reduziu significativamente os níveis de frutosamina. O conteúdo total de catecolaminas foi 21,14% menor nos diabéticos sem tratamento, quando comparado aos controles (p<0,05). O tratamento com insulina recuperou os estoques de catecolaminas dos ratos diabéticos. A expressão de TH foi similar em todos os grupos experimentais. A secreção basal e a estimulada por altas concentrações de K+ e por carbamilcolina foi reduzida pelo diabetes em 24,3%, 42,28% e 28,9%, respectivamente. Este efeito não foi corrigido pelo tratamento com insulina. A secreção estimulada pela mobilização de Ca2+ de pools intracelulares sensíveis à cafeína não é afetada pelo diabetes. Os nossos resultados nos permitem concluir que o diabetes afeta a secreção basal de catecolaminas e a estimulada via membrana plasmática e que isto não é determinado pela redução dos estoques de catecolaminas, nem é revertido pelo tratamento com insulina exógena. / The diabetes reduces the catecholamine secretion with hypoglycemic episodes, to turning worse the metabolic disorder of diabetic people. Several studies have shown that hyperglycemia has pivotal role in diabetic complication development. This work studied the effect of diabetes on catecholamine synthesis and secretion and the effects of insulin treatment. Methods and results: 60 days old, male Wistar rats were used. The diabetes was induced by a single intravenous injection of streptozotocin (50mg/Kg body weight). The control group received buffer injection. To establish the protocol of insulin treatment the food consumption and the blood glucose levels were measured during 24h from hour to hour. The insulintreated diabetic rats received human NPH insulin at 1pm (1U) and 7pm (4U). After 15 days of the streptozotocin injection, the rats were sacrificed and the adrenal glands withdrew. To evaluate the effect of diabetes and the insulin treatment, fructosamine, triglycerides, total cholesterol, HDL, LDL and VLDL were measured. The total catecholamine content of adrenal gland and the basal and stimulated catecholamine secretion was quantified. The experiments of stimulated catecholamine secretion were performed with high potassium, carbachol and caffeine. The catecholamines measurement was done by fluorimetric method. The expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, was analyzed by western blotting. The glicemia was 82.82 ± 1.24 mg/dl, 405.74 ± 23.35 mg/dl and 103.72 ± 6.79 mg/dl in control, diabetic and insulin-treated diabetic groups, respectively. The body mass of diabetic rats was reduced in 6.1 ± 3.84g and increased on control and insulin-treated diabetic rats, in 36.34 ± 1.8g and 43.32 ± 3.79g, respectively. The diabetes changed total cholesterol, LDL and VLDL plasma levels, alteration reversed by insulin treatment. The triglycerides, fructosamine and LDH levels were not affected by diabetes. The insulin-treated rats showed significant reduction of fructosamine levels. The diabetic rats presented a significant reduction, 21.14% on the catecholamine content when compared to the control group, p<0.05. The insulin treatment recovered the catecholamine stores. The TH expression was similar in all three experimental groups. The diabetes reduced the basal and stimulated catecholamine secretion by 24.3%, 42.28% (high K+) e 28.9% (carbachol).The catecholamine secretion stimulated by mobilization of intracellular Ca2+ pools was not affected by diabetes or insulin treatment. Our results show that diabetes reduces the catecholamine secretion, and this is not consequence of reduction on cell catecholamine stores and it is not reversed by insulin therapy.

Phäochromozytome und Paragangliome (PPGL) sind seltene neuroendokrine Tumore, die von Chromaffinzellen im Nebennierenmark (Phäochromozytome, PCC) oder vom extra-adrenalen Paraganglion (Paragangliome, PGL) ausgehen. Patienten mit PPGL zeigen in der Regel Anzeichen und Symptome, die mit der Biosynthese, Speicherung und Sekretion von Katecholaminen (Dopamin, Noradrenalin, Adrenalin) zusammenhängen. Der Katecholamin-Stoffwechsel bei PPGLs wird durch den genetischen Hintergrund bestimmt. Tumoren, die auf pathogene Varianten (PVs) in Genen zurückzuführen sind, die zur Aktivierung von Hypoxie-Signalwegen führen, sind nicht in der Lage, Epinephrin zu synthetisieren, während solche, die auf PVs in Genen zurückzuführen sind, die Kinase-Signalwege aktivieren, zur Epinephrin-Synthese befähigt sind. Dieser genetisch-biochemisch-klinische Phänotyp wurde in der klinischen Forschung nachgewiesen. Der Mechanismus, der der Regulierung der Katecholamin-Biosynthese durch Tumoren aufgrund von PVs in Kinase-Signalisierungsgenen zugrunde liegt, ist jedoch nicht klar. Ziel dieser Arbeit war es, den Mechanismus der Katecholamin-Biosynthese in PPGLs aufgrund von PVs in HRAS, einem mit Kinase-Signalwegen assoziierten Gen, zu untersuchen. Darüber hinaus wurden klinische Merkmale wie Katecholamin-assoziierte Anzeichen und Symptome, die prächirurgische Diagnose und die Entwicklung einer wiederkehrenden Erkrankung bei Patienten mit PPGL untersucht. Um den Katecholamin-Stoffwechsel bei PPGL zu untersuchen, wurden Katecholamine und PNMT-Enzymaktivitäten in 251 PPGL-Geweben gemessen. Anschließend wurden zwei Hotspot-Varianten von Hras, G13R und Q61R, durch CRISPR/Cas9-basiertes Prime Editing in eine Phäochromozytom-Zelllinie (PC12) der Ratte eingeführt. Katecholamine und nachgeschaltete HRAS-Faktoren, die die Epinephrin-Biosynthese regulieren, wurden in den Zellen mit/ohne Hras-PVs gemessen. Wir fanden heraus, dass PPGLs, die auf HRAS-PVs zurückzuführen sind, einen signifikant höheren Epinephrin-Gehalt und PNMT-Enzymaktivitäten aufweisen als solche, die auf PVs in Genen zurückzuführen sind, die mit Hypoxie-Signalwegen in Verbindung stehen. In Zelllinienversuchen steigerten PVs in Hras die Pnmt-Expression zusammen mit einer erhöhten Epinephrin-Synthese. Weitere Experimente zeigten, dass Hras-PVs die Pnmt-Expression durch Phosphorylierung von SP1 über den MAPK-Signalweg hochregulieren. Darüber hinaus verringerten Hras-PVs die Glukokortikoidrezeptorspiegel, wodurch die Empfindlichkeit gegenüber der gGukokortikoid-induzierten Expression von Pnmt reduziert wurde. Drei separate klinische Projekte wurden durchgeführt, um die mit Katecholaminen verbundenen klinischen Merkmale zu verstehen. Das erste Projekt untersuchte die Unterschiede in der klinischen Behandlung und die perioperativen Komplikationen bei Patienten mit Harnblasen-Paragangliom (UBPGL), die vor der Operation diagnostiziert oder fehldiagnostiziert wurden. In diesem Projekt wurde mehr als die Hälfte (53,6 %) der Patienten mit UBPGL vor der Operation nicht diagnostiziert. Wie erwartet wurden Patienten, die vor der Operation fehldiagnostiziert wurden, kaum mit einer alpha-adrenergen Blockade behandelt, und mehr dieser Patienten erlitten während der Operation eine Bluthochdruckkrise und perioperative Komplikationen als Patienten, die vor der Operation richtig diagnostiziert wurden. Eine weitere Analyse ergab, dass bei 34,5 % der Patienten mit Katecholamin-assoziierten Symptomen und/oder Bluthochdruck vor der Operation keine UBPGL diagnostiziert wurde. Darüber hinaus wurde Bluthochdruck als unabhängiger Faktor identifiziert, der mit der präoperativen Diagnose von UBPGL assoziiert ist. Das zweite Projekt untersuchte die Unterschiede im Auftreten von Katecholamin-assoziierten Zeichen und Symptomen bei Patienten mit und ohne metastasiertem PPGL (mPPGL). Wir konnten zeigen, dass das Auftreten von Katecholamin-assoziierten Anzeichen und Symptomen bei Patienten mit mPPGL mit der Produktion von Noradrenalin verbunden war, während es bei Patienten ohne mPPGL mit Epinephrin zusammenhing. Allerdings unterschieden sich die Anzeichen und Symptome bei Patienten mit metastasiertem PPGL nicht signifikant von denen mit nicht-metastasiertem PPGL. Das dritte Projekt untersuchte das Wiederauftreten der Krankheit bei Patienten mit sporadischem PPGL. Wir konnten zeigen, dass ein noradrenerger/dopaminerger Phänotyp des Primärtumors bei Patienten mit sporadischem PPGL ein unabhängiger Prädiktor für ein Wiederauftreten der Erkrankung ist. Darüber hinaus zeigten wir, dass bei 14,7 % dieser Patienten ein Rezidiv auftrat, bei einigen sogar noch 10 oder 15 Jahre nach der Resektion des Primärtumors. Diese Arbeit zeigte anhand von klinischen Tumorgewebedaten und in vitro gentechnisch hergestellten Zellmodellen, dass die Epinephrinbiosynthese bei PPGL vorwiegend durch den genetischen Hintergrund aufgrund von PVs in HRAS reguliert wird. Klinische Studien haben gezeigt, dass das Auftreten von Anzeichen und Symptomen, die prächirurgische Diagnose und das Wiederauftreten der Erkrankung mit dem Katecholaminstoffwechsel bei Patienten mit PPGL in Zusammenhang stehen. Daher muss der Katecholaminstoffwechsel bei der klinischen Behandlung von Patienten mit PPGL unbedingt berücksichtigt werden.:Content III Abbreviations: V List of figures and tables VII Zusammenfassung 1 Summary 3 1 Introduction and outline of the thesis 5 1.1 Pheochromocytomas and paragangliomas 5 1.2 Catecholamine metabolism in PPGLs 5 1.3 Pathogenic variants in the susceptibility genes of PPGLs 7 1.4 The association between genetic pathogenic variants and catecholamine metabolism in PPGLs 8 1.5 Catecholamine-associated clinical manifestations in patients with PPGLs 12 1.6 Catecholamine metabolite testing and diagnosis of PPGLs 12 1.7 Clinical treatment of patients with PPGLs 13 1.8 Follow-up for patients with PPGL 14 1.9 Outline of the thesis 15 2 Methods and results 17 2.1 Part 1: Regulation of epinephrine biosynthesis in HRAS-mutant paragangliomas 18 2.2 Part 2: Association of catecholamine metabolism and clinical management of patients with PPGL 67 2.2.1 Publication 1: 67 Differences in clinical presentation and management between pre- and postsurgical diagnoses of urinary bladder paraganglioma: is there clinical relevance? a systematic review 67 2.2.2 Publication 2: 74 Metastatic Pheochromocytoma and Paraganglioma: Signs and Symptoms Related to Catecholamine Secretion 74 2.2.3 Publication 3: 86 Recurrent disease in patients with sporadic pheochromocytoma and paraganglioma 86 3 General discussion 95 4 Conclusion 99 5 References 100 6 Acknowledgements 108 7 List of journal articles and invited presentations 109 8 Appendix 111 / Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors derived from chromaffin cells within the adrenal medulla (pheochromocytomas, PCCs) or extra-adrenal paraganglia (paragangliomas, PGLs). Patients with PPGL normally present signs and symptoms that are associated with catecholamine (dopamine, norepinephrine, epinephrine) biosynthesis, storage, and secretion. Catecholamine metabolism in PPGLs is influenced by the genetic background. Tumors resulting from pathogenic variants (PVs) in genes that lead to activation of hypoxia signaling pathways are unable to synthesize epinephrine, whereas those resulting from PVs in genes that activate kinase signaling are capable of epinephrine synthesis. This genetic-biochemical-clinical phenotype has been revealed in clinical research. However, the mechanism behind the regulation of catecholamine biosynthesis in tumors due to PVs in kinase signaling genes remains unclear. Thus, this thesis aimed to investigate the mechanism of catecholamine biosynthesis in PPGLs due to PVs in HRAS, a gene associated with kinase signaling pathways. In addition, clinical features such as catecholamine-associated signs and symptoms, a presurgical diagnosis, and the development of recurrent disease in patients with PPGL were studied. To investigate the catecholamine metabolism in PPGLs, catecholamines and PNMT enzyme activities of 251 PPGL tissues were measured. Subsequently, two hotspot variants of Hras, G13R and Q61R, were introduced into a rat pheochromocytoma cell line (PC12) using CRISPR/Cas9-based prime editing. The levels of catecholamines and downstream factors of HRAS that regulate epinephrine biosynthesis were measured in the cells with/without Hras PVs. We found that PPGLs resulting from HRAS PVs had significantly higher epinephrine content and PNMT enzyme activities compared to those resulting from PVs in genes associated with hypoxia signaling pathways. Furthermore, in our cell line experiments, PVs in Hras increased Pnmt expression, along with increased epinephrine synthesis. Moreover, further experiments indicated that Hras PVs upregulated Pnmt expression through phosphorylation of SP1 via the MAPK pathway. In addition, Hras PVs decreased glucocorticoid receptor levels, thereby reducing sensitivity to glucocorticoid-induced expression of Pnmt. Three separate clinical projects were performed to better understand the clinical features associated with catecholamines. The first project investigated the differences in clinical management and per-operative complications between patients with urinary bladder paraganglioma (UBPGLs) who were correctly diagnosed and those who were misdiagnosed before surgery. In this project, more than half (53.6%) of the patients with UBPGL were not diagnosed before surgery. As expected, patients who were misdiagnosed before surgery received limited treatment with alpha-adrenergic blockade, resulting in a higher incidence of hypertension crisis during surgery and perioperative complications compared to patients diagnosed before surgery. Further analysis indicated that 34.5% of these patients presenting with catecholamine-associated symptoms and/or hypertension were not diagnosed with UBPGL before surgery. In addition, we identified hypertension as an independent factor associated with pre-surgical diagnosis of UBPGLs. The second project analyzed differences in the presentation of catecholamine-associated signs and symptoms in patients with and without metastatic PPGL (mPPGL). We showed that the presentation of catecholamine-associated signs and symptoms was associated with the production of norepinephrine in patients with mPPGL, whereas in non-mPPGL patients, it was associated with epinephrine. However, the signs and symptoms in patients with metastatic PPGLs did not significantly differ from those in patients with non-metastatic PPGL. The third project analyzed recurrent disease in patients with sporadic PPGL. We showed that a noradrenergic/dopaminergic phenotype of primary tumors was an independent predictor of recurrent disease among patients with sporadic PPGL. In addition, we showed that 14.7% of these patients experienced recurrent disease, with some cases occurring even 10 or 15 years after the resection of their primary tumors. This thesis, through the use of clinical tumor tissue data and in vitro genetically engineered cell models, indicated that epinephrine biosynthesis was predominantly regulated by the genetic background in PPGLs with PVs in HRAS. Additionally, clinical studies showed that the presentation of signs and symptoms, a pre-surgical diagnosis, and the presence of recurrent disease were associated with catecholamine metabolism in patients with PPGL. It is therefore imperative to consider catecholamine metabolism in the clinical management of patients with PPGL.:Content III Abbreviations: V List of figures and tables VII Zusammenfassung 1 Summary 3 1 Introduction and outline of the thesis 5 1.1 Pheochromocytomas and paragangliomas 5 1.2 Catecholamine metabolism in PPGLs 5 1.3 Pathogenic variants in the susceptibility genes of PPGLs 7 1.4 The association between genetic pathogenic variants and catecholamine metabolism in PPGLs 8 1.5 Catecholamine-associated clinical manifestations in patients with PPGLs 12 1.6 Catecholamine metabolite testing and diagnosis of PPGLs 12 1.7 Clinical treatment of patients with PPGLs 13 1.8 Follow-up for patients with PPGL 14 1.9 Outline of the thesis 15 2 Methods and results 17 2.1 Part 1: Regulation of epinephrine biosynthesis in HRAS-mutant paragangliomas 18 2.2 Part 2: Association of catecholamine metabolism and clinical management of patients with PPGL 67 2.2.1 Publication 1: 67 Differences in clinical presentation and management between pre- and postsurgical diagnoses of urinary bladder paraganglioma: is there clinical relevance? a systematic review 67 2.2.2 Publication 2: 74 Metastatic Pheochromocytoma and Paraganglioma: Signs and Symptoms Related to Catecholamine Secretion 74 2.2.3 Publication 3: 86 Recurrent disease in patients with sporadic pheochromocytoma and paraganglioma 86 3 General discussion 95 4 Conclusion 99 5 References 100 6 Acknowledgements 108 7 List of journal articles and invited presentations 109 8 Appendix 111

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O estado nutricional e hormonal em fases iniciais de desenvolvimento (gestação e lactação) está relacionado a alterações epigenéticas, que podem levar ao desenvolvimento de doenças. A obesidade infantil está relacionada com a ocorrência da obesidade na idade adulta, resistência à insulina e maior risco cardiometabólico. Em estudos experimentais, a superalimentação neonatal causa obesidade e aumenta o risco de doenças cardiovasculares. Estes animais apresentam obesidade visceral, hiperfagia, hiperleptinemia e hipertensão na idade adulta. Previamente, demonstramos que a hiperleptinemia neonatal causa hiperfunção da medula adrenal e microesteatose na idade adulta. No presente estudo avaliamos a função adrenal de ratos adultos obesos no modelo de superalimentação neonatal por redução do tamanho da ninhada e a sensibilidade as catecolaminas no tecido adiposo visceral (TAV) e no fígado. Ao nascimento todas as ninhadas tiveram seu número de filhotes ajustados para 10. Para induzir a superalimentação neonatal, o tamanho da ninhada foi reduzido de dez para três filhotes machos no terceiro dia de lactação até o desmame (SA), enquanto que o grupo controle permaneceu com 10 filhotes durante toda a lactação. Após o desmame, os ratos tiveram livre acesso à dieta padrão e água até 180 dias (1 animal de cada ninhada, n = 7). O TAV e as glândulas adrenais foram pesadas. As contrações hormonais séricas, o conteúdo hepático de glicogênio e triglicerídeos foram avaliados por kits comerciais. O conteúdo e a secreção de catecolaminas adrenais foram avaliados utilizando o método do trihidroxindol. O conteúdo dos hormônios eixo hipotálamo-hipófise-córtex adrenal, das enzimas da via de síntese das catecolaminas na glândula adrenal, ADRB2 no fígado e ADRB3 no TAV foram determinados por Western blotting ou imunohistoquímica. As diferenças foram consideradas significativas quando p <0,05. Aos 180 dias de vida, o grupo SA apresentou maior massa corporal (+15%), maior consumo alimentar (+15%) e maior adiposidade visceral (+79%). Os hormônios do eixo hipotálamo-hipófise-córtex-adrenal não foram alterados. O grupo SA apresentou maior expressão de tirosina hidroxilase e de DOPA descarboxilase (+31% e 90%, respectivamente); conteúdo de catecolaminas adrenais (absoluta: 35% e relativa: 40%), e secreção de catecolaminas, tanto basal quanto estimulada por cafeína (+35% e 43%, respectivamente). O conteúdo ADRB3 no TAV não foi alterado nos grupo SA, entretanto o ADRB2 no fígado apresentou-se menor (-45%). O grupo SA apresentou maior conteúdo de glicogênio e triglicerídeos no fígado (+79% e +49%, respectivamente), além de microesteatose. A superalimentação neonatal resulta em hiperativação adrenomedular e aparentemente está associada a preservação da sensibilidade às catecolaminas no VAT. Adicionalmente sugerimos que o maior conteúdo de glicogênio e triglicerídeos hepático seja devido a menor sensibilidade as catecolaminas. Tal perfil pode contribuir para a disfunção metabólica hepática e hipertensão arterial que são características deste modelo de obesidade programada. / Nutritional and hormonal status at early phases of development are related to epigenetic changes, promoting disease development. Childhood overweight is related with late obesity, insulin resistance and higher cardiometabolic risk. Rats overfed during lactation show higher visceral adiposity, hyperphagia, leptin resistance and hypertension in adulthood. Previously, we demonstrated that neonatal hyperleptinemia is associated with adrenal medullary hyperfunction and liver steatosis at adulthood. Here, we evaluated the adrenal function and liver tissue of adult obese rats that were overfed during lactation. To induce early overfeeding, the litter size was reduced from ten to three male pups at the third day of lactation until weaning (SL). Control group had ten rats per litter (NL). After weaning, rats had free access to standard diet and water until 180 days old (1 animal from each litter, n=7). Significant differences had p<0.05. The SL group presented higher adrenal catecholamine content (absolute: +35% and relative: +40%), tyrosine hydroxylase (+31%), DOPA decarboxylase (+90%) protein contents, basal and caffeine-induced catecholamine in vitro secretion (+35% and +43%, respectively). However, hormones of the hypothalamic-pituitary-adrenal cortex axis were unchanged. The &#946;3-adrenergic receptor content in visceral adipose tissue was unchanged in SL rats, but the &#946;2-adrenergic receptor in the liver was lower (-45%). SL group showed higher glycogen and triglycerides contents in liver (+79% and +49%, respectively), which showed microesteatosis. Although the neonatal overfeeding leads to higher adrenomedullary function, adult obese SL rats have a dysfunction in hepatic &#946;2-adrenergic receptor, which can contribute for the hepatic dysfunction characteristic of liver obesity complications.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O estado nutricional e hormonal em fases iniciais de desenvolvimento (gestação e lactação) está relacionado a alterações epigenéticas, que podem levar ao desenvolvimento de doenças. A obesidade infantil está relacionada com a ocorrência da obesidade na idade adulta, resistência à insulina e maior risco cardiometabólico. Em estudos experimentais, a superalimentação neonatal causa obesidade e aumenta o risco de doenças cardiovasculares. Estes animais apresentam obesidade visceral, hiperfagia, hiperleptinemia e hipertensão na idade adulta. Previamente, demonstramos que a hiperleptinemia neonatal causa hiperfunção da medula adrenal e microesteatose na idade adulta. No presente estudo avaliamos a função adrenal de ratos adultos obesos no modelo de superalimentação neonatal por redução do tamanho da ninhada e a sensibilidade as catecolaminas no tecido adiposo visceral (TAV) e no fígado. Ao nascimento todas as ninhadas tiveram seu número de filhotes ajustados para 10. Para induzir a superalimentação neonatal, o tamanho da ninhada foi reduzido de dez para três filhotes machos no terceiro dia de lactação até o desmame (SA), enquanto que o grupo controle permaneceu com 10 filhotes durante toda a lactação. Após o desmame, os ratos tiveram livre acesso à dieta padrão e água até 180 dias (1 animal de cada ninhada, n = 7). O TAV e as glândulas adrenais foram pesadas. As contrações hormonais séricas, o conteúdo hepático de glicogênio e triglicerídeos foram avaliados por kits comerciais. O conteúdo e a secreção de catecolaminas adrenais foram avaliados utilizando o método do trihidroxindol. O conteúdo dos hormônios eixo hipotálamo-hipófise-córtex adrenal, das enzimas da via de síntese das catecolaminas na glândula adrenal, ADRB2 no fígado e ADRB3 no TAV foram determinados por Western blotting ou imunohistoquímica. As diferenças foram consideradas significativas quando p <0,05. Aos 180 dias de vida, o grupo SA apresentou maior massa corporal (+15%), maior consumo alimentar (+15%) e maior adiposidade visceral (+79%). Os hormônios do eixo hipotálamo-hipófise-córtex-adrenal não foram alterados. O grupo SA apresentou maior expressão de tirosina hidroxilase e de DOPA descarboxilase (+31% e 90%, respectivamente); conteúdo de catecolaminas adrenais (absoluta: 35% e relativa: 40%), e secreção de catecolaminas, tanto basal quanto estimulada por cafeína (+35% e 43%, respectivamente). O conteúdo ADRB3 no TAV não foi alterado nos grupo SA, entretanto o ADRB2 no fígado apresentou-se menor (-45%). O grupo SA apresentou maior conteúdo de glicogênio e triglicerídeos no fígado (+79% e +49%, respectivamente), além de microesteatose. A superalimentação neonatal resulta em hiperativação adrenomedular e aparentemente está associada a preservação da sensibilidade às catecolaminas no VAT. Adicionalmente sugerimos que o maior conteúdo de glicogênio e triglicerídeos hepático seja devido a menor sensibilidade as catecolaminas. Tal perfil pode contribuir para a disfunção metabólica hepática e hipertensão arterial que são características deste modelo de obesidade programada. / Nutritional and hormonal status at early phases of development are related to epigenetic changes, promoting disease development. Childhood overweight is related with late obesity, insulin resistance and higher cardiometabolic risk. Rats overfed during lactation show higher visceral adiposity, hyperphagia, leptin resistance and hypertension in adulthood. Previously, we demonstrated that neonatal hyperleptinemia is associated with adrenal medullary hyperfunction and liver steatosis at adulthood. Here, we evaluated the adrenal function and liver tissue of adult obese rats that were overfed during lactation. To induce early overfeeding, the litter size was reduced from ten to three male pups at the third day of lactation until weaning (SL). Control group had ten rats per litter (NL). After weaning, rats had free access to standard diet and water until 180 days old (1 animal from each litter, n=7). Significant differences had p<0.05. The SL group presented higher adrenal catecholamine content (absolute: +35% and relative: +40%), tyrosine hydroxylase (+31%), DOPA decarboxylase (+90%) protein contents, basal and caffeine-induced catecholamine in vitro secretion (+35% and +43%, respectively). However, hormones of the hypothalamic-pituitary-adrenal cortex axis were unchanged. The &#946;3-adrenergic receptor content in visceral adipose tissue was unchanged in SL rats, but the &#946;2-adrenergic receptor in the liver was lower (-45%). SL group showed higher glycogen and triglycerides contents in liver (+79% and +49%, respectively), which showed microesteatosis. Although the neonatal overfeeding leads to higher adrenomedullary function, adult obese SL rats have a dysfunction in hepatic &#946;2-adrenergic receptor, which can contribute for the hepatic dysfunction characteristic of liver obesity complications.

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No. of bitstreams: 1 santiagotavarespaes.pdf: 1214222 bytes, checksum: 624191b9f520fa9270585bbfc50073f4 (MD5) Previous issue date: 2013-12-11 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente estudo objetivou: 1) Verificar se o procedimento da adrenodemedulação contribui para potencializar o efeito da dieta rica em carboidratos simples no desenvolvimento da obesidade; 2) Comparar os efeitos do exercício físico crônico, iniciado aos 35 dias de vida, sobre a glicemia e colesterol total de ratos obesos e controles adrenodemedulados ou falso operados; 3) Avaliar se a diminuição do conteúdo das catecolaminas exerce influência no desempenho físico. Os animais foram distribuídos em 2 grupos controles e obesos, e divididos em 4 subgrupos falso operados e adrenodemedulados, exercitados ou sedentários. A indução da obesidade pelo consumo de ração rica em carboidrato iniciou-se aos 21 dias de vida e a adrenodemedulação ocorreu aos 22 dias de vida. O protocolo de exercício físico realizado foi o de corrida em esteira com intensidade e volume incrementais, durante 8 semanas, 5 vezes por semana. Os animais foram eutanasiados aos 92 dias de vida. Avaliou-se o consumo alimentar, peso corporal, índice de Lee, gorduras perigonadal e retroperitoneal, catecolaminas, teste de esforço, glicemia e colesterol. Na análise inferencial, foi realizada uma análise de variância (ANOVA) de três fatores com o intuito de testar os efeitos da dieta, adrenodemedulação e treinamento físico sobre as variáveis dependentes, seguido da ANOVA de um fator para comparar a diferença entre essas variáveis. Os animais com obesidade induzida por dieta rica em carboidratos apresentaram valores de consumo alimentar mais elevados que os animais controles, o que culminou num aumento 25% maior de peso corporal cujo reflexo foi observado pelo índice de Lee mais elevados que os animais com dieta padrão; essas diferenças foram independentes da adrenodemedulação. Ademais, tanto os valores de gordura perigonadal e retroperitoneal quanto glicemia e colesterol total se mostraram aumentados nos animais obesos, sendo que, para essas variáveis a adrenodemedulação mostrou-se capaz de influenciar significativamente no potencial aumento dos valores apresentados. Por outro lado o treinamento físico mostrou-se capaz de atenuar tais valores. As catecolaminas dos animais adrenodemedulados mostraram-se aproximadamente 100 vezes mais baixas que os grupos falso operados, dado que demonstra a efetividade da cirurgia; entretanto, o desempenho no teste de esforço dos animais adrenodemedulados não se mostrou diferente de seus pares falso operados, exercitados ou não. Portanto, concluímos que a variável desempenho físico não parece ter sido influenciada pela presença das catecolaminas adrenais. Além disso, sugerimos que a dieta rica em carboidratos quando associada à ausência das catecolaminas adrenais promove maior adiposidade em ratos. O treinamento físico atenua a composição corporal, glicemia e colesterol total independentemente da dieta. Contudo, a adrenodemedulação parece impedir uma redução mais pronunciada dos níveis de glicemia e colesterol. / The present study aimed to: 1) Verify if the adrenaldemedullation procedure contributes to potentiate the effect of high-sugar diet during the development of obesity; 2) Compare the effects of chronic exercise, initiated at 35 days of life on blood glucose and total cholesterol of obese and controls sham or adrenaldemedullated rats; 3) Evaluate if the lack of adrenal catecholamine influences physical performance. Animals were distributed into 2 groups of obese and lean rats, and then divided into 4 subgroups sham and adrenaldemedullated, exercised or sedentary. At 21 days old, obesity was induced by high-sugar diet and, adrenaldemedullation was performed at 22 days of life. Exercise protocol performed was treadmill running with incremental intensity and volume, 5 times a week during 8 weeks. Animals were euthanized at 92 days and it were evaluated food intake, body weight, Lee index, retroperitoneal and perigonadal fat pads, total adrenal catecholamine content, performance test, blood glucose and total cholesterol. It was used three-way ANOVA to test the effects of diet, exercise training and adrenaldemedullation on dependent variables followed by one-way ANOVA to compare the difference among variables. High-sugar diet obese groups presented higher values of food intake than control animals, resulting in an increase of 25 % on body weight and higher Lee Index independent of adrenaldemedullation. Moreover, retroperitoneal and perigonadal fat pad, blood glucose and total cholesterol values were enhanced in high-sugar obese animals and potentiated by adrenaldemedullation. On the other hand, physical training was able to attenuate those parameters. Adrenaldemedullated rats, control and obese ones, showed 100 times lower adrenal catecholamine than sham groups, which demonstrates the effectiveness of the surgery. Therefore, the variable physical performance does not appear to be influenced by the presence of adrenal catecholamine. Moreover, we suggest that the high-carbohydrate diet when associated to the absence of adrenal catecholamines promotes greater adiposity in rats. Physical training reduces body composition, blood glucose and total cholesterol levels regardless of diet. However, adrenaldemedullation seems to prevent a more pronounced reduction of blood glucose and cholesterol on trained rats.

The one-humped or the dromedarian camel is a pseudo-ruminant mammal, well adapted to the hot and dry climates of the desert. Its ability to withstand torrid heat and extreme desiccation is of paramount importance to its survival. The studies presented in this thesis were designed to investigate and document the effect of dehydration in the presence or absence of angiotensin II (Ang II) AT1 receptor blocker (losartan) on blood constituents, electrolytes, hormones, neurotransmitters as well as liver and kidney enzymes in a subset of dehydrated camels and to compare them with hydrated camels. Additionally, we studied the response of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) and revealed for the first time the cardiac storage form of BNP in the camel heart. Dehydration induced significant increments in packed cell volume (PCV), white blood cells (WBC), gamma glutamyl-transferase (GGT), serum sodium, creatinine and urea levels, and a doubling in plasma cortisol and arginine vasopressin (AVP) levels. At the same time dehydration caused significant decrease in body weights, plasma insulin like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3), and a 50% decrement in ANP and BNP levels. Moreover, dehydration with and without losartan resulted in significant changes in stress hormones and anti-oxidants in plasma, liver and kidney homogenates. Losartan on one hand enhanced the effect of dehydration resulting in significant increases in sodium, creatinine and urea levels. In addition losartan raised the  binding affinity of Ang II AT2 receptors in the small intestine with 8-fold and with 16-fold for liver AT1 receptors, indicating that Ang II AT1 and AT2 receptor binding sites were present in camel's small intestine while only AT1 receptor binding sites were found in the camel liver. One the other hand losartan resulted in significant decrease in body weights impaired the rise in anti-diuretic hormone and reduced aldosterone level. Finally, we showed that the proBNP is the storage form of BNP in the camel heart.

Les récepteurs Erythropoietin-producing hepatocyte (EPH) constituent la plus grande famille de récepteurs à activité tyrosine kinase transmembranaires. Leur activité kinase peut être induite par leurs ligands, les éphrines (EFN). Une fois activés, ces récepteurs sont impliqués dans la régulation de la fonction cellulaire par transduction antérograde ou rétrograde du signal EPH-EFN. Au cours de la dernière décennie, nos études ont démontré que les EPH / EFN jouent un rôle important dans la régulation de la pression artérielle par la modulation de la contractilité des cellules musculaires lisses vasculaires (VSMC). EPHB6, EFNB1 et EFNB3 ont un effet négatif sur la contractilité des VSMC et la pression artérielle, tandis que EPHB4 et EFNB2 montrent un effet positif. La famille EPH / EFN est donc un nouveau système yin et yang qui ajuste finement l'homéostasie de la pression artérielle. Nous avons également constaté que les catécholamines urinaires de 24 h sont réduites chez les souris mâles EPHB6 knockout (KO), suggérant que l’EPHB6 régule la pression artérielle non seulement via les VSMC mais aussi par la sécrétion de catécholamine (CAT). La régulation de CAT par l’EPHB6 dépend de la testostérone car (1) les niveaux réduits de CAT ne sont pas observés chez les souris femelles EPHB6 KO ; et (2) la castration chez les souris mâles EPHB6 KO ramène la CAT à des niveaux normaux. Durant ma thèse, nous avons étudié le mécanisme impliqué dans la régulation de la sécrétion et de la synthèse des catécholamines chez les cellules chromaffines des glandes surrénales (AGCC) par la voie de signalisation de l’EPHB6. En ex vivo, la teneur totale en épinéphrine et la sécrétion d'épinéphrine déclenchée par l'acétylcholine (ACh) sont toutes deux réduites dans les glandes surrénales venant des souris KO mâles mais pas dans celles venant des femelles ou de mâles castrés. Ensuite, nous avons observé une diminution de l’afflux de Ca2+ dépendant de l'ACh dans les AGCC venant des souris mâles EPHB6 KO, ce qui découle de l'effet non-génomique de la testostérone. En appliquant le patch clamping de cellules entières sur les AGCC, nous avons démontré que la diminution d’afflux de Ca2+ dans ces cellules est causée par l’augmentation des courants de potassium à grande conductance activé par le calcium (BK). En utilisant l'enregistrement ampérométrique, nous avons constaté que la sécrétion de CAT par les AGCC est compromise en l'absence d'EPHB6. Nous avons également observé une diminution du désassemblage de la F-actine corticale dans les AGCC venant de souris mâles KO associée à une diminution de l'exocytose des vésicules contenant es catécholamines. Ces deux phénomènes n’ont pas été observés chez les femelles KO ni chez les mâles castrés. Des études complémentaires ont montré que le désassemblage défectueux de la F-actine dans les AGCC est régulé par la signalisation inverse de l'EPHB6 à l'EFNB1 via deux voies de signalisations différentes : la voie du membre A de la famille des homologues Ras (RHOA) et la voie de la tyrosine kinase proto-oncogène de la famille Src (FYN) / proto-oncogène c-ABL / la calponine monooxygénase associée aux microtubules et le domaine LIM contenant 1 (MICAL-1). En outre, nous avons observé que la diminution de la teneur totale en épinéphrine dans la glande surrénale venant des souris mâles KO est causée par une expression altérée de la tyrosine hydroxylase (TH), qui est l’enzyme limitant la vitesse dans la biosynthèse des CAT. L'effet non génomique de la testostérone a également participé dans ce processus. Nous avons révélé que la signalisation inverse d'EPHB6 à EFNB1 contribue à la surexpression de TH dans les AGCC par l’augmentation de son niveau de transcription. La voie en aval de cette signalisation inverse implique la petite famille Rac GTPase 1 (RAC1) / MAP kinase kinase 7 (MKK7) / c-Jun N-terminal kinase (JNK) / proto-oncogène c-Jun / activator protein 1 (AP1) / réponse de croissance précoce 1 (EGR1). Ces travaux démontrent pour la première fois un rôle spécifique de la famille EPH / EFN dans la régulation de la biologie médullaire de la glande surrénale. La signalisation rétrograde d’EPHB6 via EFNB1 régule la synthèse et la sécrétion des catécholamines de concert avec la testostérone dans les AGCC. / Erythropoietin-producing hepatocyte (Eph) receptors are the largest family of cell surface transmembrane receptor tyrosine kinases. Their kinase activity can be activated by their ligands, ephrins (EFNs), and involved in cell function regulation through either EPH-EFN forward or reverse signaling transduction. In the last decade, we have revealed the previously unknown function of EPHs/EFNs in the regulation of blood pressure by modulating the contractility of vascular smooth muscle cells (VSMCs). EPHB6, EFNB1, and EFNB3 have a negative effect on the VSMCs contractility and blood pressure, while EPHB4 and EFNB2 show a positive effect instead. Thus, EPH/EFN family is a novel yin and yang system that finely tunes blood pressure homeostasis. EPHB6 also targets cells responsible for catecholamine (CAT) secretion in addition to the VSMCs, since we found that the 24-h urine catecholamines are reduced in male EPHB6 knockout (KO) mice. This phenotype in EPHB6 KO mice is testosterone-dependent because the reduced CAT levels are not observed in female KO mice; castration in KO male mice reverts the CAT levels to a normal range. In this research, we investigated the mechanism for the regulation of catecholamine secretion and synthesis in adrenal gland chromaffin cells (AGCCs) by EPHB6 signaling. In ex vivo, the total content of epinephrine and the acetylcholine (ACh)-triggered epinephrine secretion were both reduced in the adrenal gland from KO male but not female or castrated mice. Then, we found a reduced ACh-dependent Ca2+ influx in AGCCs from male EPHB6 KO mice, and this effect depended on the non-genomic effect of testosterone. The results of whole-cell patch clamping on AGCCs indicated that the enhanced large-conductance calcium-activated potassium (BK) currents were responsible for the reduced Ca2+ influx in these cells. Using amperometry recording, we found that CAT secretion by AGCCs was compromised in the absence of EPHB6. The cortical F-actin disassembly in AGCCs from KO male but not female or castrated mice was reduced, accompanied by decreased catecholamine vesicle exocytosis. Further study showed such defective F-actin disassembly in AGCCs was regulated by the reverse signaling from EPHB6 to EFNB1 via the Ras homolog family member A (RHOA) and proto-oncogene Src family tyrosine kinase (FYN)/proto-oncogene c-ABL/microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways. Further, we observed that the reduced total content of epinephrine in the adrenal gland from male KO mice was caused by impaired expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in CAT biosynthesis. The non-genomic effect of testosterone was also involved in this process. We revealed that the reverse signaling from EPHB6 to EFNB1 contributed to the up-regulation of TH expression in AGCCs by enhancing its transcription. The downstream pathway of this reverse signaling involved Rac family small GTPase 1 (RAC1)/MAP kinase kinase 7 (MKK7)/c-Jun N-terminal kinase (JNK)/ proto-oncogene c-Jun/activator protein 1 (AP1)/early growth response 1 (EGR1). The present research, for the first time, revealed the specific role of the EPH/EFN family on the regulation of the adrenal gland medullary biology. The EPHB6 reverse signaling through EFNB1 in concert with testosterone regulates the catecholamine synthesis and secretion in AGCCs.

<p>Parkinson’s disease (PD) is a complex neurodegenerative movement disorder involving protein misfolding, mitochondrial dysfunction, and oxidative stress. The current dissertation, motivated by a lack of valid biomarkers and sustainable therapies, examined the potential application of a novel target for therapeutics and diagnostics of PD — the multifunctional, heat-shock like protein Catecholamine-Regulated Protein 40 (CRP40). The goal of this program of research was to elucidate further the implications of CRP40 in PD using a variety of molecular biology, bioinformatics, and clinical approaches through integrative collaborations with academia, government, and industry partners to translate scientific findings into real world solutions. Chapters 2 and 3 explored the potential therapeutic use and structure-function relationships of CRP40 through elucidating the smallest functional piece of this protein that was six times smaller, and validating a negative control for these experiments (Heat-Shock Protein 47). These initiatives could eventually lead to a small drug that could cross the blood-brain barrier and be targeted to the specific brain regions affected in PD. Chapter 4 examined the potential mechanisms of CRP40, and suggested that this protein may protect neurons from oxidative stress, maintain energy levels, and mitochondrial homeostasis, with important future implications for a variety of disorders. Finally, Chapter 5 presented compelling evidence for the potential use of CRP40 as a valid biomarker for early detection of PD and monitoring of disease progression. Overall, findings suggest that CRP40 may be a critical target for future breakthroughs in the diagnosis and treatment of PD.</p> / Doctor of Science (PhD)

L’Astyanax mexicanus est un poisson téléostéen utilisé dans les études de microévolution. Au sein de la même espèce, il existe plusieurs populations. Des poissons de surface (SF), vivant en banc dans les rivières d’Amérique Centrale et plusieurs populations cavernicoles (CF), vivant dans l’obscurité des grottes mexicaines. La majorité des populations cavernicoles sont indépendantes et ont dérivé des populations de surface depuis environ un million d’années. Les CF sont aveugles et dépigmentés. D’un point de vue comportemental, les CF ont, entre autres, perdu l’agressivité qui est un caractère important des SF. Ce travail caractérise finement les différences comportementales entre poissons de surface et cavernicoles, et analyse les modifications des circuits neuronaux qui sont à l’origine de la perte du comportement agressif chez les poissons cavernicoles. Les tests d’agressivité montrent que deux SF s’attaquent dix fois plus que deux CF. La distribution temporelle des attaques diffère également entre les populations. En effet, la majorité des attaques entre CF ont lieu lors des premières minutes du test, alors que chez les SF la fréquence des attaques augmente au cours du temps. L’étude de l’agressivité de SF rendu aveugle ou dans le noir montre que la perte d’agressivité chez les CF n’est pas due au phénotype aveugle. De plus, l’agressivité des poissons hybrides suggère que le comportement agressif des SF est génétiquement codé. Après des expériences pharmacologiques utilisant des composés interférant avec le système sérotoninergique ou soumettant les SF et les CF à différents régimes alimentaires, nous faisons l’hypothèse selon laquelle la perte d’agressivité des CF est une adaptation à la vie cavernicole, ceux-ci recherchant en permanence de la nourriture. En revanche, l’agressivité des SF est étroitement reliée à la hiérarchie existant au sein du banc, la dominance étant elle-même liée en partie à la concentration de la sérotonine présente au niveau du raphé. La concentration de sérotonine est inversement corrélée à l’agressivité. D’un point de vue neuroanatomique, l’organisation du système sérotoninergique est similaire entre les populations. Cependant, l’un des noyaux hypothalamiques est plus large chez les CF et contient plus de neurones. Par ailleurs, l’agressivité et le taux de sérotonine sont connus pour être inversement corrélés chez les vertébrés. De fait, des traitements pharmacologiques augmentant le taux de sérotonine diminuent l’agressivité des SF et modifient leur profil d’agressivité, mimant celui des CF. Cependant, le système sérotoninergique n’est pas le seul à être modifié dans le système nerveux des CF. En réalité, les dosages HPLC montrent que l’ensemble des systèmes aminergiques est amplifié chez les CF, conduisant à l’apparition d’un phénotype « hyper-aminergique ». Les études neuro-anatomiques et enzymologiques des systèmes sérotoninergiques et catécholaminergiques montrent que des variations du nombre de neurones et de l’activité des enzymes de dégradation des neurotransmetteurs aminergiques convergent vers cette amplification et sont à l’origine de ce phénotype. Par ailleurs, et en parallèle, les méthodes de transgénèses stables ont été testées chez l’Astyanax. Cette étude montre que l’utilisation des méganucléases et des transposons sont utilisables chez notre poisson modèle et permettent l’établissement de lignées transgéniques. Cet outil permettra de tester à l’avenir l’importance et la fonction des gènes d’intérêt lors du développement du système nerveux, ainsi que lors de la mise en place des comportements. / Astyanax mexicanus is a teleost fish model used for evolution studies. Among the same species, there are several populations of sighted surface fish (SF) that live in Mexican rivers and at least twenty nine populations of blind cavefish (CF) that live in perpetual darkness. Several of these cave populations are independently-evolved, and they derived from surface fish-like ancestors. CF have lost their eyes and their pigmentation, and they have also evolved a number of behavioral traits. Most CF populations have lost the aggressive behavior that is a trademark of their SF counterparts. Here we characterized behavioural differences between SF and CF and we investigated the modifications of neural networks responsible for the loss of aggressiveness in cavefish. We first characterized aggressive behavior in Astyanax. Using an “intruder assay”, we found that SF not only attack ten times more during a one hour period, but also show a significantly different pattern in the temporal distribution of their attacks: while two CF attack mostly during the first minutes, SF attack more and more frequently as time goes by during the test. Then we demonstrated that the loss of aggressiveness in CF is not due to their blind phenotype, and using hybrids and independently-evolved populations of CF we could suggest that aggressive behavior in SF is genetically-encoded. After pharmacological experiments using compounds modulating serotonin levels or using SF and CF receiving different food regimes, we hypothesized that the loss of aggressiveness of CF may correspond to an adaptation to cave life, as they spend most of their time looking for food. On the other hand, the aggressiveness of SF is closely connected to the hierarchy within the school, dominance itself being mainly due to the levels of serotonin in the raphe. Thus, serotonin levels are inversely correlated with agonistic behavior. Neuroanatomical analyses on SF and CF brains showed an identical organization of their serotonin neuronal networks, but one of the serotonergic hypothalamic nucleus was significantly larger in CF and contained more neurons. Treatments of embryos with cyclopamine, an inhibitor of Sonic hedgehog (Shh) signaling, showed that this enlargement is induced by the Shh signaling pathway, itself known to be amplified during the development of the CF. In fact, the serotonergic system is not only one that is changed in the nervous system of CF. HPLC measurements showed that all aminergic systems are amplified in CF, which show a sort of “hyper-aminergic” phenotype. Studies comparing the neuroanatomy of aminergic systems and the activity of amine-degrading enzymes between SF and CF showed that variations in both the number of neurons and the activity of degrading enzymes converge towards an amplification of aminergic neurotransmission and are responsible for the phenotype. In parallel, we established transgenesis methods in Astyanax. We showed that techniques using meganuclease or transposons are valuable with our fish species to generate transgenic lines. This tool will be used to test the importance and function of genes of interest in the development of the nervous system and associated behaviors.