Introdução: Os neuropeptídeos regulam uma variedade de aspectos da função nervosa e neuroendócrina, atuando através da ativação de receptores específicos da membrana celular. No sistemana nervoso central (SNC) os receptores do pepetídeo liberador de gastrina (GRPR) são amplamente expressos, e numerosos efeitos centrais têm sido descritos com a sua ativação, incluindo efeitos sobre a saciedade, regulação do ritmo circadiano, termorregulação, modulação do stress, resposta ao medo, ansiedade e memória. Pesquisas mostram que o bloqueio farmacológico do GRPR em modelos animais leva ao aparecimento de deficits na interação social, padrões restritivos de comportamento e estereotipias motoras, sintomas semelhantes ao comportamento autista em humanos, sugerindo a possibilidade de que o complexo GRP/GRPR possa ter um papel na patogênese do transtorno do espectro autista (TEA). Recentemente, dois estudos não controlados com administração do peptídeo liberador de gastrina (GRP) a 13 crianças com autismo sugeriram que ele é seguro e que possa melhorar alguns sintomas do transtorno, principalmente interação social e sintomas associados à irritabilidade. Objetivos: Comparar a eficácia, segurança, tolerabilidade do GRP em relação ao placebo em sintomas do TEA. Metodologia: Ensaio clínico crossover, randomizado, duplo-cego, controlado por placebo, com uso de GRP 160 picomol/kg por 4 dias consecutivos, em 10 crianças com autismo. Os desfechos foram medidos através da escala Aberrant Behavior Checklist (ABC). Resultados: Todos os participantes eram do sexo masculino, com idade entre 4 e 9 anos. Houve uma redução nos escores da escala ABC e suas subescalas após o uso de GRP e de placebo. Apesar dessa redução ser mais proeminente com o GRP, principalmente nas subescalas Irritabilidade, Comportamento estereotipado e Hiperatividade, não houve diferença estatística entre os resultados (p 0,334). Após uma semana da infusão, 5 crianças apresentavam melhora maior que 25% no escore total da escala ABC com uso de GRP e 2 com uso de placebo, não apresentando diferença estatística (p 0,375). Não houve efeitos adversos, alterações dos sinais vitais ou variações laboratoriais associados ao uso de GRP em nenhum paciente. Conclusões: Os resultados deste estudo, apesar do tamanho reduzido da amostra, reforçam os dados anteriores sobre a segurança do GRP no uso a curto prazo. Apesar de ter ocorrido redução dos escores da escala ABC após uso de GRP, não houve diferença estatística em relação ao placebo. Devido ao desenho crossover e tamanho pequeno da amostra do estudo atual, não foi possível esclarecer a real eficácia do GRP na redução dos sintomas do TEA na infância. Existe a necessidade de novas pesquisas com outros delineamentos e tamanho amostral maior para confirmar a eficácia e segurança do GRP em crianças com autismo. / Introduction: The neuropeptides regulate a variety of aspects of the nervous and neuroendocrine function, acting through activation of specific receptors of the cellular membrane. In system central nervous (CNS) the gastrin-releasing peptide recptors (GRPR) are widely expressed, and numerous central effects have been reported with their activation, including effects on satiety, regulating the circadian rhythm, thermoregulation, stress modulation, response to fear, anxiety and memory. Research has shown that pharmacological blockade of GRPR in animal models leads to the deficits in social interaction, restrictive patterns of behavior and motor stereotypies, autistic symptoms similar to human behavior, suggesting the possibility that the complex GRP/GRPR may have a role in the pathogenesis of autism spectrum disorder (ASD). Recently, two studies are not controlled with the administration of gastrin releasing peptide (GRP) to 13 children with autism suggest that it is safe and can improve some symptoms of the disorder, especially social interaction and symptoms associated with irritability. Objectives: To compare the efficacy, safety, tolerability GRP compared to placebo in ASD symptoms. Methodology: crossover clinical trial, randomized, double-blind, placebo-controlled, using GRP 160 picomol/kg for 4 consecutive days in 10 children with autism. Outcomes were measured by the Aberrant Behavior Checklist scale (ABC). Results: All participants were male, aged between 4 and 9 years. There was a reduction in the scores of the ABC range and its subscales after use GRP and placebo. Despite this reduction be more prominent with the GRP, particularly in subscales Irritability, Stereotypic behavior and Hyperactivity and noncompliance, there was no statistical difference between the results (p 0.334). After a week of infusion, 5 children showed improvement greater than 25% in the total score of the ABC scale in GRP use and 2 with placebo use, however there was no statistical difference (p 0.375). No adverse effects, changes in vital signs or laboratory abnormalities associated with use of GRP in any patient. Conclusions: The results of this study, despite the small sample size, reinforce previous data on the safety of the GRP in the short-term use. Although there was a reduction in ABC scale scores after use of GRP, there was no statistical difference from placebo. Due to the small sample size and design of the current study, it was not possible to clarify the real effectiveness of GRP in reducing the symptoms of ASD in childhood. There is a need for further research with other designs and larger sample size to confirm the efficacy and safety of GRP in children with autism.

Muitas evidências demonstram que o peptídeo liberador de gastrina (GRP) é um fator de crescimento que afeta funções neuroendócrinas, incluindo proliferação e diferenciação celular, comportamento alimentar, formação de memória, respostas a estresses, desenvolvimento de neoplasias, desordens neurológicas e psiquiátricas. Porém, os eventos moleculares pelos quais isso ocorre ainda não são totalmente compreendidos. No presente estudo, nós avaliamos as interações entre o receptor do peptídeo liberador de gastrina (GRPR) e a via de sinalização celular da PKA, tanto na proliferação celular de glioblastoma humano (in vitro) quanto na consolidação da memória no hipocampo de ratos Wistar (in vivo). Mostramos que o GRP age em sinergismo com agentes que estimulam a via do cAMP/PKA, promovendo a proliferação de células de glioblastoma humano, pois o tratamento com GRP combinado com um ativador de adenilil ciclase (AC), forskolin, ou um análogo de cAMP, 8-Br-cAMP, ou um inibidor do tipo IV de fosfodiesterase, rolipram, aumentaram a proliferação das células de U- 138MG, quando avaliadas pelo método de MTT. Nenhum destes compostos teve efeito sozinho. O mRNA de GRPR e a expressão protéica em U-138MG foram detectados pelas técnicas de RT-PCR e imuno-histoquímica. No estudo in vivo a bombesina em baixas doses induziu um aumento na consolidação da memória. O resultado foi potencializado na combinação com um ativador do receptor de dopamina D1/D5 (D1R), além de ser prevenido quando combinado com um inibidor da via da PKA. Os resultados sugerem que GRP e GRPR interagem com a via de sinalização cAMP/PKA tanto na estimulação da proliferação celular em linhagem de câncer humano quanto na modulação da memória no hipocampo de ratos. / Increasing evidence indicates that gastrin-releasing peptide (GRP) acts as an autocrine growth factor for brain tumors as well as been implicated in memory formation, however, underlying molecular events are poorly understood. In the present study, we examined interactions between the GRPR and cellular signaling pathways in influencing memory consolidation in the hippocampus and on proliferation of glioblastoma cell in vitro. We show here that GRP acts synergistically with agents that stimulate the cAMP/PKA pathway to promote proliferation of human gliobastoma cells. Treatment with GRP combined with the adenylyl cyclase (AC) activator forskolin, the cAMP analog 8-Br-cAMP, or the phosphodiesterase type IV (PDE4) inhibitor rolipram increased proliferation of U138-MG cells in vitro measured by MTT assay. None of the compounds had an effect when given alone. GRP receptor (GRPR) mRNA and protein expression in U138-MG cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. We investigated the interactions between the GRPR and the PKA pathway in male Wistar rats. BB-induced enhancement of consolidation was potentiated by co infusion of activators of the dopamine D1/D5 receptor (D1R) pathway and prevented by a PKA inhibitor. The results suggest that GRP and the GRPR interact with the cAMP/PKA signaling pathway in stimulating a cancer cell line proliferation and in memory modulation by hippocampal.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation, and they are: (i) discovery of piperidine derivatives as T-type calcium channel inhibitors for the treatment of epilepsy and neuropathic pain and as protein disulfide isomerase inhibitors for the treatment of influenza viral infection; (ii) discovery of peptide-based calcitonin gene-related peptide receptor antagonists for the treatment of inflammatory pain; and (iii) synthesis of substituted 6-(dimethylamino)-2-phenylisoindolin-1-ones for the inhibition of luciferase. T-type calcium channels are important regulators of nervous system, and upregulated T-type calcium channel activities have been found to link to various types of neurological disorders, such as epilepsy and neuropathic pain. To discover novel T-type calcium channel blockers, a series of 1,4-disubstituted piperidine derivatives were designed and synthesized. Among them, compound 1-4 was found to be a good T-type calcium channel inhibitor with an IC₅₀ of 1 nM for Ca[subscript v]3.2 inhibition. It also showed 86% suppression of seizure induced death in mice and good in vivo analgesic effects on both thermal and mechanical pain thresholds in Spared Nerve Injury rat models. Therefore 1-4 can potentially be used as a T-type calcium channel blocker in the treatment of epilepsy and neuropathic pain. Influenza is a respiratory viral infection. Since viruses rely on host cell proteins for their entry, survival and replication, development of drugs targeting host cell proteins has identified as an effective strategy in controlling viral infections. We synthesized a series of 1,4-disubstituted piperidine derivatives for the inhibition of protein disulfide isomerase enzyme and influenza. Among them, 1-29 was found to possess strong anti-influenza activity (EC₅₀ = 2.5 µM). This suggests the potential use of piperidine scaffold in designing anti-influenza drugs in future. Calcitonin gene-related peptide (CGRP) receptor antagonism has been identified as a successful approach for the treatment of inflammatory pain. Therefore, a novel class of peptide antagonists of CGRP receptor was synthesized and screened for their binding affinities to the CGRP receptor and their analgesic effects on inflammatory-induced pain in rats. Among them, peptide 2-3 showed a higher binding affinity towards the CGRP receptor than previously reported peptide antagonists and exhibited analgesic effects up to 2 h in both Aδ and c-fiber pain tests. Therefore 2-3 indicates its potential use as a CGRP receptor antagonist in the treatment of inflammatory pain. Firefly luciferase is commonly used as a reporter in cells expressing a luciferase gene or its enzymatic activity under the control of a promoter of interest to assess its transcriptional activity. It has been found that some molecules such as molecules with carboxylic acid moiety can directly inhibit luciferase activity in cells. However, it is suggested that carboxylic acid moiety of the compounds may also be associated with side reactions in cells. Therefore, to study whether carboxylic acid moiety causes side effects, we designed two probe molecules, 3-1 and 3-2. Synthesis of probe molecule 3-2 is discussed. Synthesis of probe molecule 3-1 and further investigation of its luciferase inhibition will therefore be useful to understand the toxicity of carboxylic acid containing drugs in future.

Introdução: Os neuropeptídeos regulam uma variedade de aspectos da função nervosa e neuroendócrina, atuando através da ativação de receptores específicos da membrana celular. No sistemana nervoso central (SNC) os receptores do pepetídeo liberador de gastrina (GRPR) são amplamente expressos, e numerosos efeitos centrais têm sido descritos com a sua ativação, incluindo efeitos sobre a saciedade, regulação do ritmo circadiano, termorregulação, modulação do stress, resposta ao medo, ansiedade e memória. Pesquisas mostram que o bloqueio farmacológico do GRPR em modelos animais leva ao aparecimento de deficits na interação social, padrões restritivos de comportamento e estereotipias motoras, sintomas semelhantes ao comportamento autista em humanos, sugerindo a possibilidade de que o complexo GRP/GRPR possa ter um papel na patogênese do transtorno do espectro autista (TEA). Recentemente, dois estudos não controlados com administração do peptídeo liberador de gastrina (GRP) a 13 crianças com autismo sugeriram que ele é seguro e que possa melhorar alguns sintomas do transtorno, principalmente interação social e sintomas associados à irritabilidade. Objetivos: Comparar a eficácia, segurança, tolerabilidade do GRP em relação ao placebo em sintomas do TEA. Metodologia: Ensaio clínico crossover, randomizado, duplo-cego, controlado por placebo, com uso de GRP 160 picomol/kg por 4 dias consecutivos, em 10 crianças com autismo. Os desfechos foram medidos através da escala Aberrant Behavior Checklist (ABC). Resultados: Todos os participantes eram do sexo masculino, com idade entre 4 e 9 anos. Houve uma redução nos escores da escala ABC e suas subescalas após o uso de GRP e de placebo. Apesar dessa redução ser mais proeminente com o GRP, principalmente nas subescalas Irritabilidade, Comportamento estereotipado e Hiperatividade, não houve diferença estatística entre os resultados (p 0,334). Após uma semana da infusão, 5 crianças apresentavam melhora maior que 25% no escore total da escala ABC com uso de GRP e 2 com uso de placebo, não apresentando diferença estatística (p 0,375). Não houve efeitos adversos, alterações dos sinais vitais ou variações laboratoriais associados ao uso de GRP em nenhum paciente. Conclusões: Os resultados deste estudo, apesar do tamanho reduzido da amostra, reforçam os dados anteriores sobre a segurança do GRP no uso a curto prazo. Apesar de ter ocorrido redução dos escores da escala ABC após uso de GRP, não houve diferença estatística em relação ao placebo. Devido ao desenho crossover e tamanho pequeno da amostra do estudo atual, não foi possível esclarecer a real eficácia do GRP na redução dos sintomas do TEA na infância. Existe a necessidade de novas pesquisas com outros delineamentos e tamanho amostral maior para confirmar a eficácia e segurança do GRP em crianças com autismo. / Introduction: The neuropeptides regulate a variety of aspects of the nervous and neuroendocrine function, acting through activation of specific receptors of the cellular membrane. In system central nervous (CNS) the gastrin-releasing peptide recptors (GRPR) are widely expressed, and numerous central effects have been reported with their activation, including effects on satiety, regulating the circadian rhythm, thermoregulation, stress modulation, response to fear, anxiety and memory. Research has shown that pharmacological blockade of GRPR in animal models leads to the deficits in social interaction, restrictive patterns of behavior and motor stereotypies, autistic symptoms similar to human behavior, suggesting the possibility that the complex GRP/GRPR may have a role in the pathogenesis of autism spectrum disorder (ASD). Recently, two studies are not controlled with the administration of gastrin releasing peptide (GRP) to 13 children with autism suggest that it is safe and can improve some symptoms of the disorder, especially social interaction and symptoms associated with irritability. Objectives: To compare the efficacy, safety, tolerability GRP compared to placebo in ASD symptoms. Methodology: crossover clinical trial, randomized, double-blind, placebo-controlled, using GRP 160 picomol/kg for 4 consecutive days in 10 children with autism. Outcomes were measured by the Aberrant Behavior Checklist scale (ABC). Results: All participants were male, aged between 4 and 9 years. There was a reduction in the scores of the ABC range and its subscales after use GRP and placebo. Despite this reduction be more prominent with the GRP, particularly in subscales Irritability, Stereotypic behavior and Hyperactivity and noncompliance, there was no statistical difference between the results (p 0.334). After a week of infusion, 5 children showed improvement greater than 25% in the total score of the ABC scale in GRP use and 2 with placebo use, however there was no statistical difference (p 0.375). No adverse effects, changes in vital signs or laboratory abnormalities associated with use of GRP in any patient. Conclusions: The results of this study, despite the small sample size, reinforce previous data on the safety of the GRP in the short-term use. Although there was a reduction in ABC scale scores after use of GRP, there was no statistical difference from placebo. Due to the small sample size and design of the current study, it was not possible to clarify the real effectiveness of GRP in reducing the symptoms of ASD in childhood. There is a need for further research with other designs and larger sample size to confirm the efficacy and safety of GRP in children with autism.

Introdução: Os neuropeptídeos regulam uma variedade de aspectos da função nervosa e neuroendócrina, atuando através da ativação de receptores específicos da membrana celular. No sistemana nervoso central (SNC) os receptores do pepetídeo liberador de gastrina (GRPR) são amplamente expressos, e numerosos efeitos centrais têm sido descritos com a sua ativação, incluindo efeitos sobre a saciedade, regulação do ritmo circadiano, termorregulação, modulação do stress, resposta ao medo, ansiedade e memória. Pesquisas mostram que o bloqueio farmacológico do GRPR em modelos animais leva ao aparecimento de deficits na interação social, padrões restritivos de comportamento e estereotipias motoras, sintomas semelhantes ao comportamento autista em humanos, sugerindo a possibilidade de que o complexo GRP/GRPR possa ter um papel na patogênese do transtorno do espectro autista (TEA). Recentemente, dois estudos não controlados com administração do peptídeo liberador de gastrina (GRP) a 13 crianças com autismo sugeriram que ele é seguro e que possa melhorar alguns sintomas do transtorno, principalmente interação social e sintomas associados à irritabilidade. Objetivos: Comparar a eficácia, segurança, tolerabilidade do GRP em relação ao placebo em sintomas do TEA. Metodologia: Ensaio clínico crossover, randomizado, duplo-cego, controlado por placebo, com uso de GRP 160 picomol/kg por 4 dias consecutivos, em 10 crianças com autismo. Os desfechos foram medidos através da escala Aberrant Behavior Checklist (ABC). Resultados: Todos os participantes eram do sexo masculino, com idade entre 4 e 9 anos. Houve uma redução nos escores da escala ABC e suas subescalas após o uso de GRP e de placebo. Apesar dessa redução ser mais proeminente com o GRP, principalmente nas subescalas Irritabilidade, Comportamento estereotipado e Hiperatividade, não houve diferença estatística entre os resultados (p 0,334). Após uma semana da infusão, 5 crianças apresentavam melhora maior que 25% no escore total da escala ABC com uso de GRP e 2 com uso de placebo, não apresentando diferença estatística (p 0,375). Não houve efeitos adversos, alterações dos sinais vitais ou variações laboratoriais associados ao uso de GRP em nenhum paciente. Conclusões: Os resultados deste estudo, apesar do tamanho reduzido da amostra, reforçam os dados anteriores sobre a segurança do GRP no uso a curto prazo. Apesar de ter ocorrido redução dos escores da escala ABC após uso de GRP, não houve diferença estatística em relação ao placebo. Devido ao desenho crossover e tamanho pequeno da amostra do estudo atual, não foi possível esclarecer a real eficácia do GRP na redução dos sintomas do TEA na infância. Existe a necessidade de novas pesquisas com outros delineamentos e tamanho amostral maior para confirmar a eficácia e segurança do GRP em crianças com autismo. / Introduction: The neuropeptides regulate a variety of aspects of the nervous and neuroendocrine function, acting through activation of specific receptors of the cellular membrane. In system central nervous (CNS) the gastrin-releasing peptide recptors (GRPR) are widely expressed, and numerous central effects have been reported with their activation, including effects on satiety, regulating the circadian rhythm, thermoregulation, stress modulation, response to fear, anxiety and memory. Research has shown that pharmacological blockade of GRPR in animal models leads to the deficits in social interaction, restrictive patterns of behavior and motor stereotypies, autistic symptoms similar to human behavior, suggesting the possibility that the complex GRP/GRPR may have a role in the pathogenesis of autism spectrum disorder (ASD). Recently, two studies are not controlled with the administration of gastrin releasing peptide (GRP) to 13 children with autism suggest that it is safe and can improve some symptoms of the disorder, especially social interaction and symptoms associated with irritability. Objectives: To compare the efficacy, safety, tolerability GRP compared to placebo in ASD symptoms. Methodology: crossover clinical trial, randomized, double-blind, placebo-controlled, using GRP 160 picomol/kg for 4 consecutive days in 10 children with autism. Outcomes were measured by the Aberrant Behavior Checklist scale (ABC). Results: All participants were male, aged between 4 and 9 years. There was a reduction in the scores of the ABC range and its subscales after use GRP and placebo. Despite this reduction be more prominent with the GRP, particularly in subscales Irritability, Stereotypic behavior and Hyperactivity and noncompliance, there was no statistical difference between the results (p 0.334). After a week of infusion, 5 children showed improvement greater than 25% in the total score of the ABC scale in GRP use and 2 with placebo use, however there was no statistical difference (p 0.375). No adverse effects, changes in vital signs or laboratory abnormalities associated with use of GRP in any patient. Conclusions: The results of this study, despite the small sample size, reinforce previous data on the safety of the GRP in the short-term use. Although there was a reduction in ABC scale scores after use of GRP, there was no statistical difference from placebo. Due to the small sample size and design of the current study, it was not possible to clarify the real effectiveness of GRP in reducing the symptoms of ASD in childhood. There is a need for further research with other designs and larger sample size to confirm the efficacy and safety of GRP in children with autism.

Muitas evidências demonstram que o peptídeo liberador de gastrina (GRP) é um fator de crescimento que afeta funções neuroendócrinas, incluindo proliferação e diferenciação celular, comportamento alimentar, formação de memória, respostas a estresses, desenvolvimento de neoplasias, desordens neurológicas e psiquiátricas. Porém, os eventos moleculares pelos quais isso ocorre ainda não são totalmente compreendidos. No presente estudo, nós avaliamos as interações entre o receptor do peptídeo liberador de gastrina (GRPR) e a via de sinalização celular da PKA, tanto na proliferação celular de glioblastoma humano (in vitro) quanto na consolidação da memória no hipocampo de ratos Wistar (in vivo). Mostramos que o GRP age em sinergismo com agentes que estimulam a via do cAMP/PKA, promovendo a proliferação de células de glioblastoma humano, pois o tratamento com GRP combinado com um ativador de adenilil ciclase (AC), forskolin, ou um análogo de cAMP, 8-Br-cAMP, ou um inibidor do tipo IV de fosfodiesterase, rolipram, aumentaram a proliferação das células de U- 138MG, quando avaliadas pelo método de MTT. Nenhum destes compostos teve efeito sozinho. O mRNA de GRPR e a expressão protéica em U-138MG foram detectados pelas técnicas de RT-PCR e imuno-histoquímica. No estudo in vivo a bombesina em baixas doses induziu um aumento na consolidação da memória. O resultado foi potencializado na combinação com um ativador do receptor de dopamina D1/D5 (D1R), além de ser prevenido quando combinado com um inibidor da via da PKA. Os resultados sugerem que GRP e GRPR interagem com a via de sinalização cAMP/PKA tanto na estimulação da proliferação celular em linhagem de câncer humano quanto na modulação da memória no hipocampo de ratos. / Increasing evidence indicates that gastrin-releasing peptide (GRP) acts as an autocrine growth factor for brain tumors as well as been implicated in memory formation, however, underlying molecular events are poorly understood. In the present study, we examined interactions between the GRPR and cellular signaling pathways in influencing memory consolidation in the hippocampus and on proliferation of glioblastoma cell in vitro. We show here that GRP acts synergistically with agents that stimulate the cAMP/PKA pathway to promote proliferation of human gliobastoma cells. Treatment with GRP combined with the adenylyl cyclase (AC) activator forskolin, the cAMP analog 8-Br-cAMP, or the phosphodiesterase type IV (PDE4) inhibitor rolipram increased proliferation of U138-MG cells in vitro measured by MTT assay. None of the compounds had an effect when given alone. GRP receptor (GRPR) mRNA and protein expression in U138-MG cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. We investigated the interactions between the GRPR and the PKA pathway in male Wistar rats. BB-induced enhancement of consolidation was potentiated by co infusion of activators of the dopamine D1/D5 receptor (D1R) pathway and prevented by a PKA inhibitor. The results suggest that GRP and the GRPR interact with the cAMP/PKA signaling pathway in stimulating a cancer cell line proliferation and in memory modulation by hippocampal.

L’hypertension artérielle et l’obésité sont deux composantes conjointement reliées du syndrome métabolique. Les récepteurs de l’ANP (GCA) et de l’oxyde nitrique (GCs) ont des propriétés diurétiques, natriurétiques, vasodilatatrices et sont liés au contrôle de la pression. Des études récentes ont démontré leur implication dans l’obésité. Hypothèse : Une différence génétique au niveau du gène GCA pourrait contribuer à des différences physiologiques. La composante lipidique et/ou sodique de la diète pourrait influencer la fonction rénale, cardiaque et les valeurs anthropométriques différemment chez les souches congéniques. Objectifs : (1) Déterminer l’effet de la composante lipidique et sodique des diètes; (2) Évaluer l’influence de GCA sur la réponse physiologique des souches congéniques; (3) Expliquer les mécanismes physiologiques procurant une réduction de la pression artérielle chez la souche SM9. Méthodologie : Des modèles congéniques du rat Dahl (DSS) hypertendu, nourri avec une diète riche en gras (HF) ou normale (NF), ont été utilisés pour démontrer l’impact d’un segment chromosomique d’origine normotendue. Résultats : La souche SM9 a une prise de poids plus importante que SM12 et DSS sur diète HF malgré un apport alimentaire équivalent. La souche SM9 présente également un ratio masse adipeuse/masse maigre plus élevé que SM12 et DSS. Nous n’avons observé aucune augmentation de la pression artérielle en réponse à la diète HF pour les 3 souches malgré une augmentation du dommage rénal pour les 3 souches. Le dommage rénal est plus important chez DSS que pour les 2 congéniques. La réponse diurétique à l’ANP est plus élevée chez SM9 et est influencée par le contenu en sel dand la diète. La perte glomérulaire plus importante chez le rat DSS semble compensée par une augmentation de la réponse à l’ANP par les glomérules résiduels. Il y a une corrélation entre l’activité de GCA en réponse à l’ANP, les niveaux d’ARNm et le nombre de répétition du dinucléotide TA dans son promoteur. Le rat DSS présente une hypertrophie cardiaque plus importante que les deux souches congénique et ceci n’est pas modifié par la diète HF. Conclusion : Nos études ont permis de mettre en évidence un effet génétique impliquant le segment chromosomique normotendu contenant GCA dans la réponse à une diète HF chez le rat DSS. / Hypertension and obesity are two related components of the metabolic syndrome. The ANP receptor (GCA) and nitric oxide receptor (sGC) have diuretic, natriuretic, vasodilatory properties, and are linked to blood pressure control. Furthermore a recent study has demonstrated the implication of GCA and sGC in the development of obesity. Hypothesis: A genetic difference in GCA gene could contribute to physiological differences. The differencial lipid and/or sodium composition of the diet could influence the renal, cardiac and anthropometric values. Objectives: (1) To determine the effect of fat and sodium on the physiological parameters; (2) To evaluate the influence of GCA on the physiological response of the congenic rat; (3) To explain the mechanisms of the blood pressure reduction in SM9 rats. Methodology: Congenic model of DSS rat, fed with either high fat (HF) or normal (NF) diet, were used to demonstrate the impact of a chromosome segment from normotensive origin on physiological functions. C2SM9 contains GCA and sGC from normotensive origin while C2SM12 harbours only sGC from normotensive origin. Results: HF diet had negative feature on body composition, renal damage, creatinine clearance and inhibited the diuretic/natriuretic effect of ANP. The normotensive segment including GCA and sGC has reduced the blood pressure, improve the renal damage and increased the diuretic/natriuretic capacity of SM9 in response to ANP injection when compared to SM12 and DSS. GCA mRNA and the clearance receptor ratio were reduced in SM9 in the renal cortex and retroperitoneal fat. SM12 and SM9, containing the chromosomal segment that includes sGC, improve their lipid profile compared with DSS. Conclusion: Our results suggested a compensatory increase in the GCA levels for SM12 and DSS that is insufficient to improve their pathophysiologic status as observed in SM9. HF diet increases the metabolic syndrome in those rats.

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.

L’hypertension artérielle et l’obésité sont deux composantes conjointement reliées du syndrome métabolique. Les récepteurs de l’ANP (GCA) et de l’oxyde nitrique (GCs) ont des propriétés diurétiques, natriurétiques, vasodilatatrices et sont liés au contrôle de la pression. Des études récentes ont démontré leur implication dans l’obésité. Hypothèse : Une différence génétique au niveau du gène GCA pourrait contribuer à des différences physiologiques. La composante lipidique et/ou sodique de la diète pourrait influencer la fonction rénale, cardiaque et les valeurs anthropométriques différemment chez les souches congéniques. Objectifs : (1) Déterminer l’effet de la composante lipidique et sodique des diètes; (2) Évaluer l’influence de GCA sur la réponse physiologique des souches congéniques; (3) Expliquer les mécanismes physiologiques procurant une réduction de la pression artérielle chez la souche SM9. Méthodologie : Des modèles congéniques du rat Dahl (DSS) hypertendu, nourri avec une diète riche en gras (HF) ou normale (NF), ont été utilisés pour démontrer l’impact d’un segment chromosomique d’origine normotendue. Résultats : La souche SM9 a une prise de poids plus importante que SM12 et DSS sur diète HF malgré un apport alimentaire équivalent. La souche SM9 présente également un ratio masse adipeuse/masse maigre plus élevé que SM12 et DSS. Nous n’avons observé aucune augmentation de la pression artérielle en réponse à la diète HF pour les 3 souches malgré une augmentation du dommage rénal pour les 3 souches. Le dommage rénal est plus important chez DSS que pour les 2 congéniques. La réponse diurétique à l’ANP est plus élevée chez SM9 et est influencée par le contenu en sel dand la diète. La perte glomérulaire plus importante chez le rat DSS semble compensée par une augmentation de la réponse à l’ANP par les glomérules résiduels. Il y a une corrélation entre l’activité de GCA en réponse à l’ANP, les niveaux d’ARNm et le nombre de répétition du dinucléotide TA dans son promoteur. Le rat DSS présente une hypertrophie cardiaque plus importante que les deux souches congénique et ceci n’est pas modifié par la diète HF. Conclusion : Nos études ont permis de mettre en évidence un effet génétique impliquant le segment chromosomique normotendu contenant GCA dans la réponse à une diète HF chez le rat DSS. / Hypertension and obesity are two related components of the metabolic syndrome. The ANP receptor (GCA) and nitric oxide receptor (sGC) have diuretic, natriuretic, vasodilatory properties, and are linked to blood pressure control. Furthermore a recent study has demonstrated the implication of GCA and sGC in the development of obesity. Hypothesis: A genetic difference in GCA gene could contribute to physiological differences. The differencial lipid and/or sodium composition of the diet could influence the renal, cardiac and anthropometric values. Objectives: (1) To determine the effect of fat and sodium on the physiological parameters; (2) To evaluate the influence of GCA on the physiological response of the congenic rat; (3) To explain the mechanisms of the blood pressure reduction in SM9 rats. Methodology: Congenic model of DSS rat, fed with either high fat (HF) or normal (NF) diet, were used to demonstrate the impact of a chromosome segment from normotensive origin on physiological functions. C2SM9 contains GCA and sGC from normotensive origin while C2SM12 harbours only sGC from normotensive origin. Results: HF diet had negative feature on body composition, renal damage, creatinine clearance and inhibited the diuretic/natriuretic effect of ANP. The normotensive segment including GCA and sGC has reduced the blood pressure, improve the renal damage and increased the diuretic/natriuretic capacity of SM9 in response to ANP injection when compared to SM12 and DSS. GCA mRNA and the clearance receptor ratio were reduced in SM9 in the renal cortex and retroperitoneal fat. SM12 and SM9, containing the chromosomal segment that includes sGC, improve their lipid profile compared with DSS. Conclusion: Our results suggested a compensatory increase in the GCA levels for SM12 and DSS that is insufficient to improve their pathophysiologic status as observed in SM9. HF diet increases the metabolic syndrome in those rats.

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.