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

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

A fundamental question in the neurobiological study of drug addiction concerns the mechanisms mediating the motivational effects of chronic drug withdrawal. According to one theory, drugs of abuse activate opposing motivational processes after both acute and chronic drug use. The negative experience of withdrawal is the opponent process of chronic drug use that drives relapse to drug-seeking and -taking, making the identification of the neurobiological substrates mediating withdrawal an issue of central importance in addiction research. In this thesis, I identify the involvement of the neurotransmitters dopamine (DA) and corticotropin-releasing factor (CRF) in the opponent motivational a- and b-processes occurring after acute and chronic nicotine administration. I report that acute nicotine stimulates an initial aversive a-process followed by a rewarding opponent b-process, and chronic nicotine stimulates a rewarding a-process followed by an aversive opponent b-process (withdrawal). These responses can be modeled using a place conditioning paradigm. I demonstrate that the acute nicotine a-process is mediated by phasic dopaminergic activity and the DA receptor subtype-1 (D1R) but not by tonic dopaminergic activity and the DA receptor subtype-2 (D2R) or CRF activity, and the opponent b-process is neither DA- nor CRF-mediated. I also demonstrate that the chronic nicotine a-process is DA- but not CRF-mediated, and that withdrawal from chronic nicotine (the b-process) decreases tonic but not phasic DA activity in the ventral tegmental area (VTA), an effect that is D2R- but not D1R-mediated. I show that a specific pattern of signaling at D1Rs and D2Rs mediates the motivational responses to acute nicotine and chronic nicotine withdrawal, respectively, by demonstrating that both increasing or decreasing signaling at these receptors prevents the expression of the conditioned motivational response. Furthermore, I report that the induction of nicotine dependence increases CRF mRNA in VTA DA neurons, and that blocking either the upregulation of CRF mRNA or the activation of VTA CRF receptors prevents the anxiogenic and aversive motivational responses to withdrawal from chronic nicotine. The results described in this thesis provide novel evidence of a VTA DA/CRF system, and demonstrate that both CRF and a specific pattern of tonic DA activity in the VTA are necessary for the aversive motivational experience of nicotine withdrawal.

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

No description available.

BDNF/TrkB são descritos em diversas neoplasias onde iniciam sinais mitogênicos, facilitam o crescimento tumoral, previnem apoptose e regulam angiogênese e metástase. Outros fatores de crescimento também são importantes para tumorigênese, como GRP/GRPR e EGF/EGFR. O objetivo geral deste trabalho foi investigar o papel de BDNF/TrkB em câncer colorretal avaliando possíveis interações com GRPR e EGFR. Verificamos que BDNF e seu receptor, TrkB, estão presentes em amostras de pacientes com câncer colorretal esporádico, e os níveis de BDNF encontram-se mais elevados no tecido neoplásico que no tecido adjacente ao tumor. O tratamento com RC- 3095, um antagonista de GRPR, na linhagem celular de câncer colorretal humana, HT-29, causa diminuição nos níveis de NGF secretados pelas células e aumento de BDNF em relação ao controle não tratado. RC-3095 inibe a proliferação e viabilidade celular das linhagens HT-29 (EGFR positiva) e SW-620 (EGFR negativa), embora apenas em HT-29 ocorra um aumento significativo na expressão de mRNA de BDNF. Por isso, um anticorpo monoclonal anti-EGFR, cetuximabe, foi combinado a RC-3095, nas células HT-29, sendo capaz de prevenir tal aumento, sugerindo que este efeito seja mediado por EGFR. Os tratamentos com um inibidor de Trks, K252a (1000 nM) ou com cetuximabe (10 nM) também inibem a proliferação celular. Entretanto, a combinação de BDNF a cetuximabe previne este efeito, enquanto que a combinação de doses não efetivas de K252a (10 nM) à cetuximabe (1 nM) inibe a proliferação celular de HT- 29. Além disso, cetuximabe também causa aumento na expressão de mRNA de TrkB e BDNF, após 600 minutos de tratamento. Nossos resultados sugerem que a inibição da proliferação celular in vitro ou do crescimento tumoral in vivo devem acontecer através do bloqueio combinado entre GRPR e TrkB em células de câncer colorretal EGFR positivas, e que BDNF também esteja envolvido em mecanismos de resistência a fármacos. Por isso, o bloqueio de BDNF / TrkB pode emergir como potencial alvo antitumoral. / BDNF / TrkB are described in various cancers where they participate in tumor growth, apoptosis, angiogenesis and metastasis. Furthermore, other growth factors are also important to tumorigenesis as GRP/GRPR and EGF/EGFR. Therefore, the aim of this study was to investigate the role of BDNF/TrkB in colorectal cancer evaluating the interactions with GRPR and EGFR. We found that BDNF and its receptor, TrkB, are present in samples from patients diagnosed with sporadic colorectal cancer, and BDNF levels were higher in tumor tissue compared to adjacent tumor tissue. Treatment with RC-3095, GRPR antagonist, in human colorectal cancer cell line, HT-29 caused a decrease in NGF levels secreted by cells, and generated increase of BDNF when compared to untreated control. RC-3095 inhibited the proliferation and cell viability in HT-29 (EGFR positive) and SW-620 (EGFR negative), but only HT-29 cells showed a significant increase in BDNF mRNA expression. Therefore, a monoclonal anti-EGFR antibody, cetuximab was combined with RC-3095 in HT-29 cells, and was able to prevent such an increase, suggesting that this effect is mediated by EGFR. The treatment with a Trk inhibitor, K252a (1000 nM) or cetuximab (10 nM), inhibited cell proliferation. However, the combination of BDNF with cetuximab prevented this effect, whereas the combination of ineffective doses of K252a (10 nM) with cetuximab (1 nM) still inhibited cell proliferation of HT-29. Furthermore, cetuximab also caused an increase in BDNF and TrkB mRNA expression, 600 minutes after treatment. In summary, our results suggest that inhibition of cell proliferation in vitro or tumor growth in vivo must occur between the combination of GRPR and TrkB in EGFR positive colorectal cancer cells, and that BDNF is also involved in drug resistance mechanisms. Therefore, blockage of BDNF / TrkB may emerge as potential antitumor target.

BDNF/TrkB são descritos em diversas neoplasias onde iniciam sinais mitogênicos, facilitam o crescimento tumoral, previnem apoptose e regulam angiogênese e metástase. Outros fatores de crescimento também são importantes para tumorigênese, como GRP/GRPR e EGF/EGFR. O objetivo geral deste trabalho foi investigar o papel de BDNF/TrkB em câncer colorretal avaliando possíveis interações com GRPR e EGFR. Verificamos que BDNF e seu receptor, TrkB, estão presentes em amostras de pacientes com câncer colorretal esporádico, e os níveis de BDNF encontram-se mais elevados no tecido neoplásico que no tecido adjacente ao tumor. O tratamento com RC- 3095, um antagonista de GRPR, na linhagem celular de câncer colorretal humana, HT-29, causa diminuição nos níveis de NGF secretados pelas células e aumento de BDNF em relação ao controle não tratado. RC-3095 inibe a proliferação e viabilidade celular das linhagens HT-29 (EGFR positiva) e SW-620 (EGFR negativa), embora apenas em HT-29 ocorra um aumento significativo na expressão de mRNA de BDNF. Por isso, um anticorpo monoclonal anti-EGFR, cetuximabe, foi combinado a RC-3095, nas células HT-29, sendo capaz de prevenir tal aumento, sugerindo que este efeito seja mediado por EGFR. Os tratamentos com um inibidor de Trks, K252a (1000 nM) ou com cetuximabe (10 nM) também inibem a proliferação celular. Entretanto, a combinação de BDNF a cetuximabe previne este efeito, enquanto que a combinação de doses não efetivas de K252a (10 nM) à cetuximabe (1 nM) inibe a proliferação celular de HT- 29. Além disso, cetuximabe também causa aumento na expressão de mRNA de TrkB e BDNF, após 600 minutos de tratamento. Nossos resultados sugerem que a inibição da proliferação celular in vitro ou do crescimento tumoral in vivo devem acontecer através do bloqueio combinado entre GRPR e TrkB em células de câncer colorretal EGFR positivas, e que BDNF também esteja envolvido em mecanismos de resistência a fármacos. Por isso, o bloqueio de BDNF / TrkB pode emergir como potencial alvo antitumoral. / BDNF / TrkB are described in various cancers where they participate in tumor growth, apoptosis, angiogenesis and metastasis. Furthermore, other growth factors are also important to tumorigenesis as GRP/GRPR and EGF/EGFR. Therefore, the aim of this study was to investigate the role of BDNF/TrkB in colorectal cancer evaluating the interactions with GRPR and EGFR. We found that BDNF and its receptor, TrkB, are present in samples from patients diagnosed with sporadic colorectal cancer, and BDNF levels were higher in tumor tissue compared to adjacent tumor tissue. Treatment with RC-3095, GRPR antagonist, in human colorectal cancer cell line, HT-29 caused a decrease in NGF levels secreted by cells, and generated increase of BDNF when compared to untreated control. RC-3095 inhibited the proliferation and cell viability in HT-29 (EGFR positive) and SW-620 (EGFR negative), but only HT-29 cells showed a significant increase in BDNF mRNA expression. Therefore, a monoclonal anti-EGFR antibody, cetuximab was combined with RC-3095 in HT-29 cells, and was able to prevent such an increase, suggesting that this effect is mediated by EGFR. The treatment with a Trk inhibitor, K252a (1000 nM) or cetuximab (10 nM), inhibited cell proliferation. However, the combination of BDNF with cetuximab prevented this effect, whereas the combination of ineffective doses of K252a (10 nM) with cetuximab (1 nM) still inhibited cell proliferation of HT-29. Furthermore, cetuximab also caused an increase in BDNF and TrkB mRNA expression, 600 minutes after treatment. In summary, our results suggest that inhibition of cell proliferation in vitro or tumor growth in vivo must occur between the combination of GRPR and TrkB in EGFR positive colorectal cancer cells, and that BDNF is also involved in drug resistance mechanisms. Therefore, blockage of BDNF / TrkB may emerge as potential antitumor target.

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

INTRODUÇÃO: O diagnóstico diferencial da síndrome de Cushing (SC) ACTH-dependente é um dos maiores desafios da endocrinologia, devido ao comportamento clínico e laboratorial semelhante de alguns tumores carcinóides com a doença de Cushing (DC). Assim, testes dinâmicos de secreção de ACTH e cortisol têm sido utilizados com o objetivo de identificar respostas que sejam preditivas e específicas no diagnóstico diferencial. O padrão dessas respostas é atribuído à superexpressão de receptores; entretanto, poucos estudos foram realizados para comprovar tal associação. O objetivo deste estudo foi verificar se a secreção de ACTH e cortisol em resposta aos testes do CRH humano (hCRH), da desmopressina, e do peptídeo liberador do GH (GHRP-6) é dependente da magnitude de expressão dos seus respectivos receptores (CRHR1, AVPR1B e GHSR-1a) em amostras de tumores de pacientes portadores da SC ACTH-dependente. CASUÍSTICA E MÉTODOS: Entre 2002 e 2004, foram avaliados 22 pacientes (20 com DC e dois com Secreção Ectópica de ACTH [SEA], carcinóide de pulmão e timo), idade mediana de 32 anos (15-54 anos), sendo 18 do sexo feminino e quatro do sexo masculino, provenientes da Disciplina de Endocrinologia e Metabologia da Faculdade de Medicina da Universidade de São Paulo. Os pacientes foram submetidos aos testes do hCRH (100 µg), desmopressina (10 µg) e GHRP-6 (1 µg/kg) com dosagens de ACTH e cortisol e também de GH no caso do GHRP-6. Vinte e um indivíduos controles, pareados por sexo e idade, foram submetidos ao teste do GHRP-6. Durante o ato operatório, fragmentos de tumor foram coletados para posterior extração do RNA total. O estudo da expressão foi feito por meio de PCR quantitativo em tempo real dos genes CRHR1, AVPR1B e GHSR-1a em relação ao GAPDH. Fragmentos de tecidos normais (hipófise, pulmão e timo) procedentes de necropsias foram utilizados como controles. RESULTADOS: Observamos maior expressão de GHSR-1a nos pacientes responsivos ao GHRP-6, tanto naqueles com DC quanto no paciente com carcinóide pulmonar. Não houve maior expressão dos receptores CRHR1 e AVPR1B nos pacientes com DC responsivos aos respectivos testes, observando-se, no entanto, uma forte associação entre respostas in vivo e a expressão desses receptores nos pacientes com SEA. As concentrações de ACTH e cortisol induzidas pela administração de GHRP-6 foram mais elevadas nos pacientes com DC quando comparados aos controles, havendo, no entanto, superposição entre as respostas. Observamos também elevação dos níveis séricos de GH nos indivíduos controles e, em menor intensidade, nos pacientes com DC. CONCLUSÕES: Houve maior expressão do receptor GHSR-1a em pacientes com SC ACTH-dependente responsivos ao GHRP-6, estabelecendo-se uma relação direta entre a expressão do receptor e a resposta in vivo ao secretagogo, tanto em pacientes com DC quanto nos portadores de SEA. Uma associação entre a expressão dos receptores CRHR1 e AVPR1B com a resposta in vivo aos respectivos secretagogos foi observada nos pacientes com SEA e não nos pacientes com DC. Tendo em vista a resposta ao GHRP-6 em paciente com SEA, limita-se o uso desse peptídeo no diagnóstico diferencial da SC ACTH-dependente. / INTRODUCTION: The differential diagnosis of ACTH-dependent Cushing\'s syndrome (CS) is one of the major challenges in endocrinology, especially in view of the similar clinical and laboratorial behavior between some carcinoid tumors and Cushing\'s disease (CD). Hence, dynamic tests of ACTH and cortisol release have been carried out with the aim to identify predictive and specific responses for this differential diagnosis. The pattern of the responses has been attributed to receptors overexpression, yet few studies have been undertaken to confirm such association. The aim of the present study was to verify whether ACTH and cortisol release in response to human CRH (hCRH), desmopressin, and GH releasing peptide (GHRP-6) depends on the magnitude of expression of their respective receptors (CRHR1, AVPR1B e GHSR-1a) in samples of tumors from patients with ACTH-dependent CS. PATIENTS AND METHODS: Twenty two patients (20 with CD and 2 with Ectopic ACTH Syndrome [EAS], lung and thymus carcinoid tumors) from the Division of Endocrinology and Metabolism of University of Sao Paulo School of Medicine, median age of 32 years (15-54 years), being 18 females and 4 males, were evaluated between 2002 and 2004. The patients were submitted to dynamic tests with hCRH (100 µg), desmopressin (10 µg) and GHRP-6 (1 µg/kg), with measurement of ACTH and cortisol levels, and also of GH in the case of GHRP-6 stimulation. Twenty one age and sex-matched controls were submitted to the GHRP-6 test. During surgery, tumor fragments were collected and subsequently processed for total mRNA extraction. Gene expression of CRHR1, AVPR1B and GHSR-1a relative to GAPDH was quantitated by real-time qPCR. Tissue samples of normal pituitary, lung and thymus from necropsy were used as controls. RESULTS: Greater expression of GHSR-1a was observed in patients responsive to the GHRP-6 test, both in those with CD and in the one with pulmonary carcinoid tumor. No enhanced expression of receptors CRHR1 and AVPR1B was found in CD patients responsive to the respective dynamic tests, yet there was a strong association between the in vivo responses and the expression of those receptors in the two patients with EAS. GHRP-6 -induced ACTH and cortisol release was more marked in patients with CD as compared with control individuals, but there was overlap of the responses. GH stimulation was observed in control individuals and, to a lesser extent, in patients with CD. CONCLUSIONS: There was greater expression of GHSR-1a in patients with ACTH-dependent CS who responded to GHRP-6, establishing a direct association between receptor gene expression and the in vivo response to the secretagogue in both CD patients and those with EAS. An association between expression of CRHR1 and AVPR1B and the in vivo response to the respective secretagogues was found in patients with EAS but not in those with CD. In view of the response to GHRP-6 in a patient with EAS, we considered the use of this peptide in the differential diagnosis of ACTH-dependent CS of limited value.

Les lingots de Si-PV élaborés par solidification dirigée en creuset présentent des propriétés électriques dégradées dans les zones en contact avec le creuset (red zones). Dans ce contexte, le travail répond à une double problématique. Tout d’abord nous étudions l’influence de la pureté du creuset sur la qualité du silicium et l’étendue de la red zone, et apportons des éléments de compréhension sur les phénomènes physiques à l’origine de cette dernière. Pour cela, des lingots de taille laboratoire cristallisés dans des creusets de différentes puretés sont analysés électriquement et chimiquement. A partir de la compréhension des mécanismes mis en jeu, la deuxième problématique est de développer un creuset innovant permettant de réduire la pollution du lingot de silicium par le creuset et le revêtement, tout d’abord à l’échelle du laboratoire puis sur des lingots de taille semi-industrielle de 60 kg en vue d’un transfert de technologie à l’échelle industrielle. Des cellules photovoltaïques sont fabriquées à partir de lingots cristallisés dans des creusets en silice frittée utilisés dans l’industrie et des creusets innovants, afin de comparer leurs rendements de conversion et valider les effets bénéfiques du creuset innovant. / The majority of silicon used for PV applications is crystallized via directional solidification in silica crucibles with suitable coatings. The obtained ingots exhibit, however, poor electrical properties near the crucible walls (red zones). Until present, the physical mechanisms leading to this degradation are both unclear and unresolved. This thesis addresses exactly these two points. It analyses the root causes leading to the electrical degradation and it proposes an innovative crucible to limit it. An electrical and chemical quantitative study is performed to determine the influence of the purity of the crucible on the quality of the obtained silicon. Specifically, the extent of the red zone is analyzed in great detail in laboratory-scale ingots crystallized in crucibles of different purity. Once the role of impurities present in the crucible is determined, an innovative crucible is proposed and tested. Its scope is to minimize impurity diffusion from the crucible and its coating to the silicon. As proof of concept, laboratory scale (3 kg) and semi-industrial scale (60 kg) ingots are crystallized in this novel crucible and in a standard, reference crucible. The semi-industrial ingots are further used to fabricate solar cells. Characterization of the solar cells validates the beneficial effects of the innovative crucible with respect to the standard one.