The anterior cingulate cortex (ACC) expresses high density of Gastrin-releasing peptide (GRP) and GRP receptor mRNA. To address possible function, this investigation used patch clamp recordings in mouse brain slice preparations to evaluate intrinsic properties of ACC neurons and neuronal responses to bath-applied GRP peptide. The ACC neurons were divided according to their morphology, the properties of action potentials and their firing pattern in response to depolarizing current pulses. Two physiological groups of interneurons and three groups of pyramidal neurons were defined. Application of the GRP induced depolarization and increased firing of the interneurons while hyperpolarization and reduced firing in pyramidal neurons. Moreover, activation of GRP receptor facilitated GABAergic neurotransmission via a postsynaptic mechanism. The results suggest that GRP receptor is an important regulator of neuronal circuits in the ACC and may consequently play an important role for ACC neurons in the central processing of high brain function.

No description available.

Mammalian and avian species differ in some appetite-related aspects including how and which neurotransmitters and hormones regulate appetite. The objective of this research was to determine how two satiety-inducing neuropeptides regulate feeding behavior in avian models. Neuropeptide FF (NPFF) was intracerebroventricularly (ICV) injected into Japanese quail and decreased food intake at a dose of 32 nmol. NPFF-injected quail had increased expression levels of hypothalamic melanocortin subtype 3 receptor and decreased expression levels of neuropeptide Y receptor subtype 1 mRNAs compared to vehicle-injected controls. In a second study, gastrin was ICV injected into broiler chicks and decreased food intake at a dose of 500 ng (0.12 nmol). There was increased c-Fos immunoreactivity in the lateral hypothalamus (LH), paraventricular nucleus (PVN), arcuate nucleus, nucleus of the solitary tract, and area postrema at 1 h post-injection. Although a variety of genes were measured in those activated nuclei, there were only differences in melanin-concentrating hormone mRNA in the LH and corticotropin-releasing factor (CRF) mRNA in the PVN, suggesting that CRF signaling was involved in the hypothalamic response to gastrin. However, co-injection of gastrin and astressin, a CRF receptor antagonist did not affect gastrin-induced suppression of food intake, implying that the CRF receptors may not be directly associated with gastrin-induced satiety. Identifying the molecular pathways that mediate the effects of anorexigenic neuropeptides in birds will lead to the development of novel treatment options for appetite-related diseases and increased understanding of factors that affect production efficiency in commercial poultry and survival/resource allocation in wild birds. / Master of Science / Compulsive eating behavior associated with obesity, and anorexia nervosa, are appetite-related disorders for which no effective pharmacological treatment options exist. From an agricultural perspective, understanding what drives eating behavior is important for informing management decisions that affect animal health and welfare. From an evolutionary point of view, understanding the neural mechanisms of appetite in different species is important because of the necessity of appetite for survival. This thesis research focused on elucidating the central mechanisms associated with the actions of two neuropeptides that inhibit food intake. In the first experiment, we explored the mechanisms underlying the appetite suppressive effects of neuropeptide FF (NPFF), a neuropeptide known for its morphine-modulating properties, and found that it decreased food intake in Japanese quail, a bird that is more representative of a bird in the wild than the chicken. NPFF affected the gene expression of several appetite-related factors in the hypothalamus, a region of the brain that regulates appetite, providing insights on the associated molecular pathways. In the next study, we examined the effect of gastrin, a digestive hormone known for its role in regulating gastric acid secretion, in broiler chicks. Broiler chickens are meat-type chickens genetically selected for growth and meat production. Gastrin significantly decreased food intake in broiler chicks, and this was associated with changes in the gene expression of melanin-concentrating hormone and corticotropin-releasing factor in the lateral hypothalamus and paraventricular nucleus of the hypothalamus, respectively. Overall, we were able to provide novel insights regarding the hypothalamic mechanisms that regulate the inhibition of food consumption. These findings may lead to the development of a novel appetite suppressant or stimulant to adjust food consumption in individuals with eating disorders and chickens during specific stages of growth.

This thesis is focused on the development, preclinical evaluation, and optimization of radiotracers for the detection of gastrin-releasing peptide receptor (GRPR)-expressing tumors. The work is divided into three distinct parts: (1) the development of bombesin (BN) antagonist (RM26)-based imaging radiotracers for the detection of GRPR-expressing tumors using different positron emission tomography (PET) and single photon emission computed tomography (SPECT) radionuclides (68Ga, 18F and 111In), (2) the establishment of a method to monitor the ligand-G protein-coupled receptor (GPCR) interaction in real time without requiring purification and stabilization of the receptors, and (3) the evaluation of radiopeptide structure-related factors (length of mini-PEG linker and composition of chelator for metal labeling) affecting the in vitro and in vivo characteristics of RM26-based tracers. We demonstrated the possibility of high-contrast in vivo imaging of GRPR-expressing xenografts despite the physiological expression of GRPR in abdominal organs. Fast radioactivity clearance from the blood and healthy organs, including receptor-positive organs, and long retention in the tumors resulted in high tumor-to-background ratios. A novel real-time assay for measuring the kinetics of the radiotracers targeting GPCR was evaluated. Living cells were used instead of purified receptors in this technology, bringing the developmental work one step closer to the true target environment (imaging in living systems). The comparative study of 68Ga-labeled NOTA-PEGn-RM26 with di-, tri-, tetra- and hexaethylene glycol chains demonstrated that the addition of only a few units of ethylene glycol to the spacer is insufficient to appreciably affect the biodistribution of the radiopeptide. Finally, a comparative study of 68Ga-labeled PEG2-RM26 analogs N-terminally conjugated to NOTA, NODAGA, DOTA or DOTAGA highlighted the influence of the chelator on the targeting properties of the radiopeptide. The main conclusion that can be drawn from this thesis is that 68Ga-NOTA-PEG2-RM26 has favorable biodistribution properties, such as rapid clearance from blood and tissues with physiological GRPR expression levels and long retention in GRPR-expressing tumors, and that this radiopeptide is potentially suitable for initial clinical investigation.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Introdução: A incidência de melanoma maligno (MM) está aumentando mundialmente e o manejo de pacientes com doença avançada representa um difícil problema. Durante décadas, a quimioterapia foi o tratamento padrão no tratamento de pacientes com MM metastático. Entretanto, essa modalidade tem produzido resultados desapontadores. Recentemente, com os avanços no conhecimento sobre os eventos moleculares relacionados ao desenvolvimento do MM, novas drogas dirigidas a alvos moleculares de relevância na doença têm sido identificadas. O peptídeo liberador de gastrina (GRP) é um peptídeo neuroendócrino, o qual possui efeito estimulador no crescimento em vários tipos de neoplasias murinas e humanas. Poucos dados são conhecidos em relação à expressão de receptores de GRP (GRPR) em materiais de biópsia de pacientes portadores de MM. A identificação de uma expressão elevada destes receptores no MM poderá permitir uma maior compreensão sobre a biologia desta neoplasia, bem como embasar estudos com o uso de moduladores desta via de sinalização com potencial ação terapêutica. Objetivos: O objetivo desse estudo foi determinar a expressão de receptores de GRP em biópsias de pacientes com MM, bem como buscar eventuais correlações entre os níveis de expressão de GRPR e fatores prognósticos reconhecidos nesta doença. Métodos: Foi realizado um estudo imuno-histoquímico (IHQ) em blocos fixados em formalina e embebidos em parafina, contendo material de biópsia de 51 pacientes portadores de MM cutâneo. Utilizou-se um anticorpo policlonal de coelho anti-GRPR (OPA1-15619, Affinity Bioreagents, USA). Após a quantificação da expressão de GRPR nas amostras, foram analisadas as diferenças de expressão entre subgrupos prognósticos, com a aplicação do teste exato de Fisher. Resultados: A expressão de GRPR foi demonstrada no citoplasma de 42 das 51 (82,4%) amostras de MM cutâneo. A expressão foi considerada forte em 30 amostras (58,9%). Não foi observada diferença significativa na expressão de GRPR quando foram analisadas amostras de MM em sítio primário versus metastático. Foram correlacionados os escores da expressão de GRPR com os achados patológicos associados ao prognóstico do MM cutâneo primário, não tendo sido detectadas diferenças significativas com relação aos níveis de Clark (p=0,35) e Índice espessura de Breslow (p= 0,175). Conclusão: Nosso trabalho mostrou uma expressão de GRPR em amostras de MM cutâneo na vasta maioria dos casos (82,4%). Em 30 amostras (58.3%), a expressão de GRPR foi considerada de forte intensidade. Não houve associação entre a intensidade de expressão de GRPR quando comparadas amostras de sítio primário versus metastático, níveis de Clark ou índices de Breslow. Este estudo é um dos primeiros na literatura a descrever uma expressão de GRPR elevada em amostras obtidas de pacientes portadores de MM cutâneo. Estudos subsequentes, preferencialmente com um maior número amostral, são necessários para confirmar estes achados e permitir melhor análise da expressão deste receptor em distintos subgrupos prognósticos. Se confirmados, os nossos dados podem justificar a realização de estudos que explorem novas estratégias terapêuticas utilizando agentes moduladores da expressão de GRPR em pacientes com MM refratário ao tratamento convencional. / Background: The incidence of malignant melanoma (MM) is increasing worldwide and the management of patients with disseminated disease is a difficult problem. Chemotherapy was the treatment of choice in metastatic melanomas for many decades. However, this option produces disappointing results. Recently, the better understanding about molecular events related do the development of MM allowed the development of new drugs directed against specific molecular targets. The gastrin-releasing peptide (GRP) is a neuroendocrine peptide shown to have growth-stimulatory effects on many types of murine and human cancers. Few data are available about GRP receptor (GRPR) expression in MM. The understanding about the molecular biology of MM may allow the identification of novel intracellular pathways of relevance in this disease, and potential GRPR modulators of therapeutic application in patients with refractory MM. Objectives: The aim of this study was to determine the GRPR expression in biopsy samples of patients with cutaneous MM, as well as to correlate its expression with known prognostic factors of relevance in this disease. Methods: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsy samples from 51 patients with cutaneous MM. A rabbit polyclonal anti-GRPR antibody (OPA1-15619, Affinity Bioreagents, USA) was used. Following the quantification of GRPR expression in the samples, the differences in GRPR expression among distinct prognostic MM subgroups were analyzed, using the Fisher´s test. Results: GRPR immunoexpression was demonstrated in cytoplasm of 42/51 (82.4%) cutaneous MM cases. It was strongly expressed in 30 (58.9%) of the samples. No significant differences between GRPR expression neither in relation to the primary or metastatic site, nor among known prognostic subgroups Clark´s level (p=0.35) and Breslow index ( p= 0.175) was observed. Conclusion: Our study has demonstrated the occurrence of a high GRPR expression in tumor specimens obtained from patients with cutaneous MM (82,4%). In 30 samples, a strong intensity of expression was documented (58.3%). No correlation was observed between the level of GRPR expression in primary or metastatic sites, nor for distinct Clark´s levels or Breslow index. This is one of the first studies demonstrating a high GRPR expression in tumor samples from patients with MM. Further studies are warranted, preferably including a larger patient population, to allow a better analysis of the expression of these receptors in different prognostic subgroups. If these observations are confirmed, the therapeutic use of GRPR inhibitors should be considered in patients with advanced MM who failed conventional treatments.

Introdução: A incidência de melanoma maligno (MM) está aumentando mundialmente e o manejo de pacientes com doença avançada representa um difícil problema. Durante décadas, a quimioterapia foi o tratamento padrão no tratamento de pacientes com MM metastático. Entretanto, essa modalidade tem produzido resultados desapontadores. Recentemente, com os avanços no conhecimento sobre os eventos moleculares relacionados ao desenvolvimento do MM, novas drogas dirigidas a alvos moleculares de relevância na doença têm sido identificadas. O peptídeo liberador de gastrina (GRP) é um peptídeo neuroendócrino, o qual possui efeito estimulador no crescimento em vários tipos de neoplasias murinas e humanas. Poucos dados são conhecidos em relação à expressão de receptores de GRP (GRPR) em materiais de biópsia de pacientes portadores de MM. A identificação de uma expressão elevada destes receptores no MM poderá permitir uma maior compreensão sobre a biologia desta neoplasia, bem como embasar estudos com o uso de moduladores desta via de sinalização com potencial ação terapêutica. Objetivos: O objetivo desse estudo foi determinar a expressão de receptores de GRP em biópsias de pacientes com MM, bem como buscar eventuais correlações entre os níveis de expressão de GRPR e fatores prognósticos reconhecidos nesta doença. Métodos: Foi realizado um estudo imuno-histoquímico (IHQ) em blocos fixados em formalina e embebidos em parafina, contendo material de biópsia de 51 pacientes portadores de MM cutâneo. Utilizou-se um anticorpo policlonal de coelho anti-GRPR (OPA1-15619, Affinity Bioreagents, USA). Após a quantificação da expressão de GRPR nas amostras, foram analisadas as diferenças de expressão entre subgrupos prognósticos, com a aplicação do teste exato de Fisher. Resultados: A expressão de GRPR foi demonstrada no citoplasma de 42 das 51 (82,4%) amostras de MM cutâneo. A expressão foi considerada forte em 30 amostras (58,9%). Não foi observada diferença significativa na expressão de GRPR quando foram analisadas amostras de MM em sítio primário versus metastático. Foram correlacionados os escores da expressão de GRPR com os achados patológicos associados ao prognóstico do MM cutâneo primário, não tendo sido detectadas diferenças significativas com relação aos níveis de Clark (p=0,35) e Índice espessura de Breslow (p= 0,175). Conclusão: Nosso trabalho mostrou uma expressão de GRPR em amostras de MM cutâneo na vasta maioria dos casos (82,4%). Em 30 amostras (58.3%), a expressão de GRPR foi considerada de forte intensidade. Não houve associação entre a intensidade de expressão de GRPR quando comparadas amostras de sítio primário versus metastático, níveis de Clark ou índices de Breslow. Este estudo é um dos primeiros na literatura a descrever uma expressão de GRPR elevada em amostras obtidas de pacientes portadores de MM cutâneo. Estudos subsequentes, preferencialmente com um maior número amostral, são necessários para confirmar estes achados e permitir melhor análise da expressão deste receptor em distintos subgrupos prognósticos. Se confirmados, os nossos dados podem justificar a realização de estudos que explorem novas estratégias terapêuticas utilizando agentes moduladores da expressão de GRPR em pacientes com MM refratário ao tratamento convencional. / Background: The incidence of malignant melanoma (MM) is increasing worldwide and the management of patients with disseminated disease is a difficult problem. Chemotherapy was the treatment of choice in metastatic melanomas for many decades. However, this option produces disappointing results. Recently, the better understanding about molecular events related do the development of MM allowed the development of new drugs directed against specific molecular targets. The gastrin-releasing peptide (GRP) is a neuroendocrine peptide shown to have growth-stimulatory effects on many types of murine and human cancers. Few data are available about GRP receptor (GRPR) expression in MM. The understanding about the molecular biology of MM may allow the identification of novel intracellular pathways of relevance in this disease, and potential GRPR modulators of therapeutic application in patients with refractory MM. Objectives: The aim of this study was to determine the GRPR expression in biopsy samples of patients with cutaneous MM, as well as to correlate its expression with known prognostic factors of relevance in this disease. Methods: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsy samples from 51 patients with cutaneous MM. A rabbit polyclonal anti-GRPR antibody (OPA1-15619, Affinity Bioreagents, USA) was used. Following the quantification of GRPR expression in the samples, the differences in GRPR expression among distinct prognostic MM subgroups were analyzed, using the Fisher´s test. Results: GRPR immunoexpression was demonstrated in cytoplasm of 42/51 (82.4%) cutaneous MM cases. It was strongly expressed in 30 (58.9%) of the samples. No significant differences between GRPR expression neither in relation to the primary or metastatic site, nor among known prognostic subgroups Clark´s level (p=0.35) and Breslow index ( p= 0.175) was observed. Conclusion: Our study has demonstrated the occurrence of a high GRPR expression in tumor specimens obtained from patients with cutaneous MM (82,4%). In 30 samples, a strong intensity of expression was documented (58.3%). No correlation was observed between the level of GRPR expression in primary or metastatic sites, nor for distinct Clark´s levels or Breslow index. This is one of the first studies demonstrating a high GRPR expression in tumor samples from patients with MM. Further studies are warranted, preferably including a larger patient population, to allow a better analysis of the expression of these receptors in different prognostic subgroups. If these observations are confirmed, the therapeutic use of GRPR inhibitors should be considered in patients with advanced MM who failed conventional treatments.

Introdução: A incidência de melanoma maligno (MM) está aumentando mundialmente e o manejo de pacientes com doença avançada representa um difícil problema. Durante décadas, a quimioterapia foi o tratamento padrão no tratamento de pacientes com MM metastático. Entretanto, essa modalidade tem produzido resultados desapontadores. Recentemente, com os avanços no conhecimento sobre os eventos moleculares relacionados ao desenvolvimento do MM, novas drogas dirigidas a alvos moleculares de relevância na doença têm sido identificadas. O peptídeo liberador de gastrina (GRP) é um peptídeo neuroendócrino, o qual possui efeito estimulador no crescimento em vários tipos de neoplasias murinas e humanas. Poucos dados são conhecidos em relação à expressão de receptores de GRP (GRPR) em materiais de biópsia de pacientes portadores de MM. A identificação de uma expressão elevada destes receptores no MM poderá permitir uma maior compreensão sobre a biologia desta neoplasia, bem como embasar estudos com o uso de moduladores desta via de sinalização com potencial ação terapêutica. Objetivos: O objetivo desse estudo foi determinar a expressão de receptores de GRP em biópsias de pacientes com MM, bem como buscar eventuais correlações entre os níveis de expressão de GRPR e fatores prognósticos reconhecidos nesta doença. Métodos: Foi realizado um estudo imuno-histoquímico (IHQ) em blocos fixados em formalina e embebidos em parafina, contendo material de biópsia de 51 pacientes portadores de MM cutâneo. Utilizou-se um anticorpo policlonal de coelho anti-GRPR (OPA1-15619, Affinity Bioreagents, USA). Após a quantificação da expressão de GRPR nas amostras, foram analisadas as diferenças de expressão entre subgrupos prognósticos, com a aplicação do teste exato de Fisher. Resultados: A expressão de GRPR foi demonstrada no citoplasma de 42 das 51 (82,4%) amostras de MM cutâneo. A expressão foi considerada forte em 30 amostras (58,9%). Não foi observada diferença significativa na expressão de GRPR quando foram analisadas amostras de MM em sítio primário versus metastático. Foram correlacionados os escores da expressão de GRPR com os achados patológicos associados ao prognóstico do MM cutâneo primário, não tendo sido detectadas diferenças significativas com relação aos níveis de Clark (p=0,35) e Índice espessura de Breslow (p= 0,175). Conclusão: Nosso trabalho mostrou uma expressão de GRPR em amostras de MM cutâneo na vasta maioria dos casos (82,4%). Em 30 amostras (58.3%), a expressão de GRPR foi considerada de forte intensidade. Não houve associação entre a intensidade de expressão de GRPR quando comparadas amostras de sítio primário versus metastático, níveis de Clark ou índices de Breslow. Este estudo é um dos primeiros na literatura a descrever uma expressão de GRPR elevada em amostras obtidas de pacientes portadores de MM cutâneo. Estudos subsequentes, preferencialmente com um maior número amostral, são necessários para confirmar estes achados e permitir melhor análise da expressão deste receptor em distintos subgrupos prognósticos. Se confirmados, os nossos dados podem justificar a realização de estudos que explorem novas estratégias terapêuticas utilizando agentes moduladores da expressão de GRPR em pacientes com MM refratário ao tratamento convencional. / Background: The incidence of malignant melanoma (MM) is increasing worldwide and the management of patients with disseminated disease is a difficult problem. Chemotherapy was the treatment of choice in metastatic melanomas for many decades. However, this option produces disappointing results. Recently, the better understanding about molecular events related do the development of MM allowed the development of new drugs directed against specific molecular targets. The gastrin-releasing peptide (GRP) is a neuroendocrine peptide shown to have growth-stimulatory effects on many types of murine and human cancers. Few data are available about GRP receptor (GRPR) expression in MM. The understanding about the molecular biology of MM may allow the identification of novel intracellular pathways of relevance in this disease, and potential GRPR modulators of therapeutic application in patients with refractory MM. Objectives: The aim of this study was to determine the GRPR expression in biopsy samples of patients with cutaneous MM, as well as to correlate its expression with known prognostic factors of relevance in this disease. Methods: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsy samples from 51 patients with cutaneous MM. A rabbit polyclonal anti-GRPR antibody (OPA1-15619, Affinity Bioreagents, USA) was used. Following the quantification of GRPR expression in the samples, the differences in GRPR expression among distinct prognostic MM subgroups were analyzed, using the Fisher´s test. Results: GRPR immunoexpression was demonstrated in cytoplasm of 42/51 (82.4%) cutaneous MM cases. It was strongly expressed in 30 (58.9%) of the samples. No significant differences between GRPR expression neither in relation to the primary or metastatic site, nor among known prognostic subgroups Clark´s level (p=0.35) and Breslow index ( p= 0.175) was observed. Conclusion: Our study has demonstrated the occurrence of a high GRPR expression in tumor specimens obtained from patients with cutaneous MM (82,4%). In 30 samples, a strong intensity of expression was documented (58.3%). No correlation was observed between the level of GRPR expression in primary or metastatic sites, nor for distinct Clark´s levels or Breslow index. This is one of the first studies demonstrating a high GRPR expression in tumor samples from patients with MM. Further studies are warranted, preferably including a larger patient population, to allow a better analysis of the expression of these receptors in different prognostic subgroups. If these observations are confirmed, the therapeutic use of GRPR inhibitors should be considered in patients with advanced MM who failed conventional treatments.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G1-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho β-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of β-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of β-catenin and CREB pathways.