Les lésions surrénaliennes surviennent dans la population générale à une fréquence d’environ 2-3%. Parmi les anomalies génétiques identifiées jusqu’à présent dans les tumeurs surrénaliennes, les mutations somatiques de β-caténine sont les plus prévalentes. Elles sont présentes dans environ 20% des adénomes et carcinomes cortico-surrénaliens. β-caténine est l’élément central de la voie canonique de WNT qui joue un rôle crucial dans le développement embryonnaire, l’homéostase et la tumourigenèse. Les mutations activatrices de β-caténine conduisent à l’accumulation nucléaire de β- caténine qui interagit avec les TCF/LEF-1 qui active la transcription des gènes cibles. Les gènes cibles de β-caténine, varient et dépendent du contexte cellulaire. Dans la glande surrénale, les gènes cibles de β-caténine sont inconnus. Nous avons effectué des études de microarray qui nous ont permis d’identifier 490 transcrits dérégulés dans les adénomes corticosurrénaliens porteurs de mutations ponctuelles de β-caténine. L’expression aberrante d’ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP et CITED2 dans les adénomes porteurs de mutations de β-caténine a été confirmée par PCR en temps réel. Le traitement des cellules humaines de carcinome cortico-surrénalien H295R (mutation de CTNNB1, Ser45Prol) avec les inhibiteurs de β-caténine/TCF (PKF115-584 et PNU74654) ont confirmé l'implication de β-caténine dans la régulation transcriptionelle d’ISM1, RALBP1, PDE2A, ENC1 et CITED2. En conclusion, nos travaux ont conduit à l’identification de nouveaux gènes cibles de β-catenin impliqués dans la tumourigenèse cortico-surrénalienne. / Adrenal lesions occur in the general population at a prevalence of about 2-3%. Several mutations have been identified in adrenocortical tumours. β-catenin mutations were recently found to be the most frequent genetic alteration in both sporadic adrenocortical adenomas and carcinomas (20-30%). β-catenin is the central player in canonical Wnt signaling which plays a key role in organ/ gland development, maintenance of homeostasis and tumourigenesis. Activation of Wnt signaling by altered regulation of β-catenin levels evokes -catenin accumulation in the nucleus, and interaction with the TCF/LEF-1 proteins that activates the transcription of target genes. These target genes are believed to be highly cell and context specific and are linked to developmental and cell cycling functions. β-catenin target genes in adrenocortical tumours are unknown. Using microarray technology, we found 490 transcripts that are deregulated in adrenocortical adenomas harbouring β-catenin activating mutations in comparison to non mutated adenomas and normal adrenal glands. These genes differ highly in function and many are poorly characterized genes. Differential expression of ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP and CITED2 in adenomas with activating β-catenin mutations was confirmed by real-time PCR. Treatment of human adrenocortical carcinoma cells, H295R (CTNNB1 Ser45Prol), with β-catenin/TCF inhibitors (PKF115-584 and PNU74654) further confirmed the implication of β-catenin on the transcriptional regulation of ISM1, RALBP1, PDE2A, ENC1 and CITED2. In conclusion, we have found new potential β-catenin target genes that may be involved in adrenocortical tumourigenesis.

Les lésions surrénaliennes surviennent dans la population générale à une fréquence d’environ 2-3%. Parmi les anomalies génétiques identifiées jusqu’à présent dans les tumeurs surrénaliennes, les mutations somatiques de β-caténine sont les plus prévalentes. Elles sont présentes dans environ 20% des adénomes et carcinomes cortico-surrénaliens. β-caténine est l’élément central de la voie canonique de WNT qui joue un rôle crucial dans le développement embryonnaire, l’homéostase et la tumourigenèse. Les mutations activatrices de β-caténine conduisent à l’accumulation nucléaire de β- caténine qui interagit avec les TCF/LEF-1 qui active la transcription des gènes cibles. Les gènes cibles de β-caténine, varient et dépendent du contexte cellulaire. Dans la glande surrénale, les gènes cibles de β-caténine sont inconnus. Nous avons effectué des études de microarray qui nous ont permis d’identifier 490 transcrits dérégulés dans les adénomes corticosurrénaliens porteurs de mutations ponctuelles de β-caténine. L’expression aberrante d’ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP et CITED2 dans les adénomes porteurs de mutations de β-caténine a été confirmée par PCR en temps réel. Le traitement des cellules humaines de carcinome cortico-surrénalien H295R (mutation de CTNNB1, Ser45Prol) avec les inhibiteurs de β-caténine/TCF (PKF115-584 et PNU74654) ont confirmé l'implication de β-caténine dans la régulation transcriptionelle d’ISM1, RALBP1, PDE2A, ENC1 et CITED2. En conclusion, nos travaux ont conduit à l’identification de nouveaux gènes cibles de β-catenin impliqués dans la tumourigenèse cortico-surrénalienne. / Adrenal lesions occur in the general population at a prevalence of about 2-3%. Several mutations have been identified in adrenocortical tumours. β-catenin mutations were recently found to be the most frequent genetic alteration in both sporadic adrenocortical adenomas and carcinomas (20-30%). β-catenin is the central player in canonical Wnt signaling which plays a key role in organ/ gland development, maintenance of homeostasis and tumourigenesis. Activation of Wnt signaling by altered regulation of β-catenin levels evokes -catenin accumulation in the nucleus, and interaction with the TCF/LEF-1 proteins that activates the transcription of target genes. These target genes are believed to be highly cell and context specific and are linked to developmental and cell cycling functions. β-catenin target genes in adrenocortical tumours are unknown. Using microarray technology, we found 490 transcripts that are deregulated in adrenocortical adenomas harbouring β-catenin activating mutations in comparison to non mutated adenomas and normal adrenal glands. These genes differ highly in function and many are poorly characterized genes. Differential expression of ISM1, RALBP1, PDE2A, CDH12, ENC1, PHYHIP and CITED2 in adenomas with activating β-catenin mutations was confirmed by real-time PCR. Treatment of human adrenocortical carcinoma cells, H295R (CTNNB1 Ser45Prol), with β-catenin/TCF inhibitors (PKF115-584 and PNU74654) further confirmed the implication of β-catenin on the transcriptional regulation of ISM1, RALBP1, PDE2A, ENC1 and CITED2. In conclusion, we have found new potential β-catenin target genes that may be involved in adrenocortical tumourigenesis.

Made available in DSpace on 2014-07-29T15:13:56Z (GMT). No. of bitstreams: 1 TESE SAULO FERNANDES MANO DE CARVALHO.pdf: 5897981 bytes, checksum: 9ded1ddc23ddf1eb697350aee8c49480 (MD5) Previous issue date: 2009-12-22 / Adrenalectomy is a highly complex surgical procedure. In order to perform it, surgeons must have a profound knowledge of the anatomophysiologic aspects of the adrenal glands, especially their vascular diversity, as well as full understanding of diagnostic means so as to properly identify their diseases. Open adrenalectomy is the most widely used method in veterinary medicine, while laparoscopic adrenalectomy usually is the method of choice in human medicine. Hyperadrenocorticism, whether hypophysis-dependent or not, is the most the most common adrenal disease. Treatment involves stopping the growth of tumors either surgically or chemically. Treatment of choice will depend on tumor functionality, type and size, as well as on its image. The swine model has proven adequate for surgical procedures, since it possesses standardized experimentation protocols and physiological parameters. Among the main benefits of laparoscopic adrenalectomy versus open adrenalectomy is rapid recovery and diminished hospitalization time. Laparoscopic adrenalectomy is the procedure of choice in human medicine, and it is nowadays considered to be the gold standard. In veterinary medicine more research is needed so as to better establish its advantages over open adrenalectomy. In this study open adrenalectomy was compared to laparoscopic adrenalectomy. 32 swines, divided into four groups of eight animals each, underwent surgery; being it that one group underwent open and the other laparoscopic adrenalectomy, with their respective control groups (sham). Parameters such as surgical time, body temperature, haematocrit, surgical intercurrences and XXXXX, as well as physiological, hormonal and metabolic responses were evaluated in young swines, both in open and laparoscopic adrenalectomy. There were no significant differences in surgical times. Most frequent intercurrences were accidents with intestinal loops, lesions to adrenal vessels and renal haematoma. Blood losses were not significant and, even though there was hypothermia it did not lead to any unfavorable clinical outcome. Deambulation time was longer for the open adrenalectomy group, although the difference was not significant. Significant differences were found in leucometry values of the laparoscopic sham group, which were higher when compared to laparoscopic groups; SpO2 of the G3 group was lower than that of the other groups, and PCR of the laparoscopic groups were lower postoperatively. There were no significant differences in mean blood pressure in all of four groups. Laparoscopy is a reliable technique for experimental adrenalectomy in swines and can be used as a reference for surgical treatment of adrenal diseases in other animal species. Metabolic parameters did not show significant differences. These results indicated that the laparoscopic technique is beneficial over open surgery, and that it can be considered a safe procedure for other species. Adrenalectomy is a highly complex surgical procedure. In order to perform it, surgeons must have a profound knowledge of the anatomophysiologic aspects of the adrenal glands, especially their vascular diversity, as well as full understanding of diagnostic means so as to properly identify their diseases. Open adrenalectomy is the most widely used method in veterinary medicine, while laparoscopic adrenalectomy usually is the method of choice in human medicine. Hyperadrenocorticism, whether hypophysis-dependent or not, is the most the most common adrenal disease. Treatment involves stopping the growth of tumors either surgically or chemically. Treatment of choice will depend on tumor functionality, type and size, as well as on its image. The swine model has proven adequate for surgical procedures, since it possesses standardized experimentation protocols and physiological parameters. Among the main benefits of laparoscopic adrenalectomy versus open adrenalectomy is rapid recovery and diminished hospitalization time. Laparoscopic adrenalectomy is the procedure of choice in human medicine, and it is nowadays considered to be the gold standard. In veterinary medicine more research is needed so as to better establish its advantages over open adrenalectomy. In this study open adrenalectomy was compared to laparoscopic adrenalectomy. 32 swines, divided into four groups of eight animals each, underwent surgery; being it that one group underwent open and the other laparoscopic adrenalectomy, with their respective control groups (sham). Parameters such as surgical time, body temperature, haematocrit, surgical intercurrences and XXXXX, as well as physiological, hormonal and metabolic responses were evaluated in young swines, both in open and laparoscopic adrenalectomy. There were no significant differences in surgical times. Most frequent intercurrences were accidents with intestinal loops, lesions to adrenal vessels and renal haematoma. Blood losses were not significant and, even though there was hypothermia it did not lead to any unfavorable clinical outcome. Deambulation time was longer for the open adrenalectomy group, although the difference was not significant. Significant differences were found in leucometry values of the laparoscopic sham group, which were higher when compared to laparoscopic groups; SpO2 of the G3 group was lower than that of the other groups, and PCR of the laparoscopic groups were lower postoperatively. There were no significant differences in mean blood pressure in all of four groups. Laparoscopy is a reliable technique for experimental adrenalectomy in swines and can be used as a reference for surgical treatment of adrenal diseases in other animal species. Metabolic parameters did not show significant differences. These results indicated that the laparoscopic technique is beneficial over open surgery, and that it can be considered a safe procedure for other species. / Adrenalectomia é um procedimento operatório de alto grau de complexidade. Para sua execução o cirurgião deve ter conhecimento dos aspectos anatomofisiológicos das glândulas adrenais, especialmente sua diversidade vascular, deve ainda ter pleno entendimento sobre os recursos diagnósticos necessários para identificação das suas enfermidades. Adrenalectomia aberta é o método mais utilizado em medicina veterinária, enquanto na medicina humana a abordagem laparoscópica é a de eleição. O hiperadrenocorticismo, que pode ser hipófise dependente ou não, é enfermidade adrenal de maior frequência. Seu tratamento consiste na contenção da evolução dos tumores por meio de fármacos ou cirúrgico. A escolha do tratamento depende da funcionalidade, tipo e tamanho do tumor e da caracterização dos estudos de imagem. O modelo suíno mostrou-se adequado para a execução dos procedimentos operatórios, visto possuir protocolo de experimentação e parâmetros fisiológicos padronizados. Dentre os principais benefícios da adrenalectomia laparoscópica (AL) frente à adrenalectomia aberta (AA) destaca-se a rápida recuperação dos pacientes com diminuição do tempo de internação. A adrenalectomia laparoscópica é o procedimento de escolha em medicina humana, sendo caracterizada atualmente como padrão ouro. Em medicina veterinária, ainda há necessidade de melhor caracterização dos seus resultados afim de que seus benefícios em relação à adrenalectomia aberta possam ser conhecidos. Neste estudo foi comparada a AA com a AL. Foram operados 32 suínos, divididos em quatro grupos de oito animais, sendo um grupo submetido à AA e outro grupo à AL, com seus respectivos grupos controle (sham). Foram avaliados parâmetros referentes a tempo operatório, temperatura corporal, hematócrito, intercorrências operatórias e tempo de deambulação. Bem com as respostas fisiológicas, hormonais e metabólicas oriundas da adrenalectomia laparoscópica e aberta em suínos jovens. Não houve diferença significativa entre os tempos operatórios. As intercorrências mais frequentes foram acidentes em alças intestinais, lesões em vasos adrenais e hematoma renal. As perdas sanguíneas não foram significativas e apesar de ter ocorrido hipotermia esta não teve repercussão clínica. O tempo de deambulação foi maior para o grupo AA, mas sem diferença significativa em relação ao AL. As diferenças significativas encontradas se relacionaram aos valores de leucometria, os quais apresentaram elevação no grupo sham laparotômico em relação aos grupos laparoscópicos; a SpO2 do grupo G3 foi inferior aos demais grupos; os valores de PCR dos grupos laparoscópicos foram menores no pós operatório. Não houve diferença significativa na pressão arterial média dos quatro grupos. A laparoscopia para adrenalectomia experimental em suínos é uma técnica confiável podendo servir como referência para o tratamento cirúrgico nas outras espécies animais. Os parâmetros metabólicos analisados não apresentaram diferenças significativas. Estes resultados indicaram benefícios da abordagem laparoscópica em relação à aberta, podendo ser considerado um procedimento seguro para utilização em outras espécies.

ARMC5 est une protéine qui contient sept motifs Armadillo répétitifs organisés en tandem et un domaine BTB. Nous avons observé que cette protéine était fortement exprimée dans les organes lymphoïdes, les glandes surrénales et le cerveau. Les souris avec une délétion d’Armc5 (souris KO) étaient de petite taille, et présentaient une diminution de la prolifération et la différenciation des lymphocytes T. L’absence d’ARMC5 entraînait une déficience de la réponse immunitaire médiée par les lymphocytes CD4+ et CD8+ dans les modèles expérimentaux d’encéphalomyélite auto-immune et d’infection au virus de la chorioméningite lymphocytaire, respectivement. Par la suite, plusieurs études ont révélé que la mutation ARMC5 était associée à l’hyperplasie macronodulaire bilatérale primitive des surrénales (HMBPS), qui représente une cause rare du syndrome de Cushing. Nous avons ensuite confirmé que l’hyperplasie des glandes surrénales s’était développée chez les souris KO âgées, et qu’elle s’accompagnait d’une légère augmentation des taux sériques de glucocorticoïdes. Comme ARMC5 ne présentait pas d’activité enzymatique, il était probable qu’elle faisait appel à d’autres protéines pour exercer sa fonction. Nous avons identifié plusieurs protéines qui se liaient à ARMC5, et plus particulièrement le complexe ARMC5/Cullin3 qui formait une ubiquitine ligase (E3) spécifique de la sous-unité RPB1 de l’ARN polymérase II. ARMC5 contrôlait le processus d’ubiquitination de RPB1 qui, par conséquent, s’accumulait dans plusieurs organes majeurs : les glandes surrénales, les ganglions lymphatiques, le cerveau, les poumons, le foie, etc. chez la souris KO. Ces résultats démontrent un rôle clé de l’ubiquitine ligase dans la dégradation de la protéine RPB1. Une accumulation similaire a également été observée dans les tissus hyperplasiques des surrénales provenant de patients atteints d’HMBPS et porteurs de la mutation ARMC5, ce qui souligne la pertinence clinique de nos résultats de recherche fondamentale dans les maladies humaines. Un défaut de dégradation de RPB1 augmentait le pool d’ARN polymérase II. Par ailleurs, nous avons identifié un groupe de gènes fortement surexprimés dans les glandes surrénales déficientes en ARMC5, parmi lesquels figurent les gènes effecteurs qui seraient impliqués dans l’hyperplasie des surrénales chez les souris KO et l’HMBPS chez les patients porteurs de la mutation ARMC5. Finalement, nous avons montré que la délétion ou la mutation d’Armc5 augmentait considérablement le risque des anomalies du tube neural chez les souris et les humains. Chez les patients souffrant de myéloméningocèle, nous avons constaté neuf différentes mutations faux-sens délétères, dont une diminuait l’interaction entre ARMC5 et RPB1. L’augmentation du pool d’ARN polymérase II dans les cellules précurseurs neurales (CPN), causée par la délétion ARMC5, influençait un groupe particulier de gènes, dont certains (p. ex. Folh1) seraient susceptibles de participer au développement du tube neural. En résumé, l’association ARMC5 et Cullin3 forme un complexe E3 qui cible RPB1 provoquant son ubiquitination et sa dégradation. En absence d’un tel mécanisme, on observe une perturbation de l’homéostasie de l’ARN polymérase II, qui mène à une diminution de la réponse immunitaire médiée par lymphocytes T, le développement d’HMBPS et un risque accru d’anomalies du tube neural. / ARMC5 protein contains seven tandem Armadillo repeats and one BTB domain. We observed that Armc5 was highly expressed in the lymphatic organs, adrenal glands, and brain. Armc5 knockout (KO) mice were small in size and exhibited compromised T cell proliferation and differentiation. The absence of ARMC5 resulted in an impairment of the CD4 + cell- and CD8 + cell-mediated immune response in the experimental autoimmune encephalomyelitis model and lymphocytic choriomeningitis virus infection model, respectively. Subsequently, several studies revealed that ARMC5 mutations were related to primary bilateral macronodular adrenal hyperplasia (PBMAH), which is a rare cause of Cushing’s syndrome. We then confirmed that adrenal gland hyperplasia was indeed developed in aged Armc5 KO mice with mildly increased serum glucocorticoid levels. Since ARMC5 did not exhibit enzymatic activity, its function likely depends on the interaction with other proteins. We identified several proteins that binds to ARMC5, most notably ARMC5 binding to Cullin3, forming a ubiquitin ligase (E3) specific for RNA polymerase II subunit I (RPB1). ARMC5 regulated the ubiquitination of RPB1, and its deletion resulted in RPB1 accumulation in major organs (e.g., adrenal glands, lymph nodes, brain, lung, and liver), indicating the critical role of this E3 in RPB1 degradation. A similar accumulation was also found in hyperplasia tissues from adrenal glands of PBMAH patients carrying ARMC5 mutations, underscoring the clinical relevance of our basic research findings in human disease. Defective degradation of RPB1 led to an enlarged RNA polymerase II (Pol II) pool. In addition, we have identified a group of genes strongly upregulated in KO adrenal glands, including the effector genes which would be involved in adrenal gland hyperplasia in Armc5 KO mice and PBMAH patients carrying ARMC5 mutation. Finally, we have shown that deleting or mutating Armc5 significantly augments the risk of neural tube defects in mice and humans. In patients with myelomeningocele, we found nine deleterious missense mutations in ARMC5, one of which weakened the interaction between ARMC5 and RPB1. The enlarged Pol II pool in Armc5 KO neural precursor cells (NPCs) influenced a particular group of genes, some of which (e.g., Folh1) are thought to be involved in the development of the neural tube. In summary, ARMC5 and CUL3 form an E3 complex, which targets RPB1 causing its ubiquitination and degradation. In the absence of such a mechanism, there is a disturbance of RNA polymerase II homeostasis, which leads to a decrease in the T cell-mediated immune response, the development of PBMAH and an increased risk of neural tube defects.