Meduloblastoma é o tumor intracranial mais comum em crianças, provavelmente derivado de células precursoras da camada granular externa do cerebelo durante seu desenvolvimento. O tratamento padrão consiste em cirurgia, radioterapia e quimioterapia, que produzem graves sequelas nos pacientes e garantem uma sobrevida baixa, o que demonstra a necessidade de novas alternativas terapêuticas para a doença. Evidências demonstram que o receptor do peptídeo liberador de gastrina (GRPR) está superexpresso em diversos tumores humanos, assim como seu agonista (GRP) pode atuar como um fator de crescimento autócrino em tumores cerebrais. No presente estudo, avaliamos a expressão de GRPR e o efeito de seus agonistas, bombesina (BB) e GRP, além do antagonista RC-3095, sobre a viabilidade celular de linhagens de meduloblastoma humano DAOY, D283 e ONS76. Mostramos que meduloblastomas, apesar de expressarem GRPR, não têm sua viabilidade celular afetada por agonistas e antagonista desse receptor. Uma vez que há evidências de que BDNF (fator neurotrófico derivado de cérebro) esteja relacionado à diferenciação celular em meduloblastomas, também avaliamos o efeito de BDNF sobre a viabilidade celular das linhagens de meduloblastoma humano. As linhagens DAOY e D283 tiveram sua viabilidade celular reduzida pela presença de BDNF. Uma vez que a via da PKA tem sido implicada na iniciação e progressão de vários tumores, também avaliamos o efeito de rolipram, um inibidor de fosfodiesterase tipo IV, sobre a viabilidade celular das linhagens de meduloblastoma humano, sendo que rolipram reduziu a viabilidade celular de todas as linhagens estudadas. Os receptores de BDNF e a via da PKA podem, portanto, ser alvos moleculares promissores para o desenvolvimento de novas terapias para meduloblastomas. / Medulloblastoma is the most common intracranial tumor in children and is believed to arise from the precursor cells of the external granule layer of the developing cerebellum. The standard treatment, consisting of surgery, craniospinal radiotherapy and chemotherapy, produces severe sequelae in patients and provides a poor overall survival, indicating the need for new therapeutic alternatives for treating this disease. Evidences show that the gastrin releasing peptide receptor (GRPR) is overexpressed in various human tumors and its agonist (GRP) can act as an autocrine growth factor in brain tumors. In the present study, we evaluated GRPR expression, as well as the effect of its agonists, bombesin (BB) and GRP, and its antagonist RC-3095, over cell viability of the human medulloblastoma cell lines DAOY, D283 and ONS76. We found that medulloblastomas, in spite of expressing GRPR, do not have its viability affected by the presence of agonists and antagonist of this receptor. Since there are evidences that BDNF (brain-derived neurotrophic factor) is related to cell differentiation in medulloblastomas, we also evaluated the effect of BDNF over the viability of medulloblastoma cell lines. The viability of the cell lines DAOY and D283 was reduced by the presence of BDNF. Since the PKA pathway has been implicated in the initiation and progression of various tumors, we also evaluated the effect of rolipram, a phosphodiesterase IV inhibitor, over the viability of the same medulloblastoma cell lines and we found that rolipram inhibited the viability of all the cell lines studied. BDNF receptors, as well as the PKA pathway, may be therefore promising molecular targets for the development of new therapies for treating medulloblastomas.

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.

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.

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.

Prolactin-releasing peptide (PrRP) is a member of the family of RF-amide peptides. These peptides have typical C-terminal sequence -Arg-Phe-NH2 and similar biological effects. PrRP was discovered as an endogenous ligand of an orphan receptor GPR10 while searching for a factor responsible for a prolactin secretion. This effect was not later confirmed and nowadays, PrRP is mainly considered as an anorexigenic peptide. This is supported by a fact that PrRP and GPR10 deficient mice suffer from hyperphagia and late-onset obesity. Besides GPR10, PrRP is bound to NPFF2 receptor whose endogenous ligand is neuropeptide FF (NPFF). In this study, the PrRP's analogues modified at the N-terminus with fatty acids of different lenghts were tested in vitro on binding and activation MAPK/ERK1/2 signalling pathway. In in vivo experiments on food intake, the central anorexigenic effects of lipidized PrRP-analogues were tested provided their crossing blood brain barrier. Binding studies showed that all analogues bound to rat pituitary RC-4B/C cells with high affinity, analogues containing fatty acid with Ki of one order of magnitude lower than native PrRP. High affinity was also confirmed for binding to cells overexpressing GPR10 receptor and cell membranes with overexpressed NPFF2 receptor. All tested analogues...

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.

Distribution of prolactin-releasing peptide-like immunoreactivity (PrRP- LI) was investigated in the rat medulla with the use of a rabbit polyclonal antiserum against the human PrRP-31 peptide. PrRP-positive neurons were noted mainly in two areas of the caudal medulla: ventrolateral reticular formation and commissural nucleus of the nucleus of the solitary tract (NTS), corresponding to the A1 and A2 areas. PrRP-LI neurons were absent in the medulla rostral to the area postrema. Double-labeling the sections with PrRP antisera and tyrosine hydroxylase (TH) monoclonal antibodies revealed extensive colocalization of PrRP- and TH-like immunoreactivity (TH-LI) in neurons of the A1 and A2 areas. Our results show that PrRP-LI is expressed in a population of A1 and A2 noradrenergic neurons of the rat caudal medulla.

No description available.

Neuropeptides, such as substance P (SP) and bombesin/gastrin-releasing peptide (BN/GRP), and neurotrophins are involved in neuro-immunomodulatory processes and have marked trophic, growth-promoting and inflammation-modulating properties. The impact of these modulators in rheumatoid arthritis (RA) is, however, unclear. An involvement of the innervation, including the peptidergic innervation, is frequently proposed as an important factor for arthritic disease. Many patients with RA, but not all, benefit from treatment with anti-TNF medications. The studies presented here aimed to investigate the roles of neuropeptides, with an emphasis on BN/GRP and SP, and neurotrophins, especially with attention to brain-derived neurotrophic factor (BDNF), in human and murine knee joint tissue. The expression patterns of these substances and their receptors in synovial tissue from patients with either RA or osteoarthritis (OA) were studied in parallel with the levels of these factors in blood and synovial fluid from patients with RA and from healthy controls. Correlation studies were also performed comparing the levels of neuropeptides with those of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)]. Furthermore, the impact of anti-TNF treatment on the levels of BDNF in blood was investigated. In a murine model of RA, the expression of these substances on articular chondrocytes along with their expression in synovial tissue was investigated. The expression of BN/GRP in human synovial tissue was confined to fibroblast-like and mononuclear-like cells whereas SP was detected in nerve-related structures. Receptors for these neuropeptides (GRP-R and NK-1R) were frequently present in blood vessel walls, and on fibroblast-like and mononuclear-like cells. The expression of BDNF and its receptors, p75 neurotrophin receptor and TrkB, was mainly confined to nerve structures. The levels of SP, and particularly those of BN/GRP, in synovial fluid and peripheral blood correlated with the levels of pro-inflammatory cytokines. There were clearly more correlations between SP-BN/GRP and inflammatory parameters than between BDNF and these factors. Plasma levels of BDNF were decreased following anti-TNF-treatment. In the joints of the murine model, there was a marked expression of neurotrophins, neurotrophin receptors and NK-1R/GRP-R in the articular chondrocytes. The expression was down-regulated in the arthritic animals. A neurotrophin system was found to develop in the inflammatory infiltrates of the synovium in the arthritic mice. The results presented suggest that there is a local, and not nerve-related, supply of BN/GRP in the human synovial tissue. Furthermore, BN/GRP and SP have marked effects in the synovial tissue of patients with RA, i.e., there were abundant receptor expressions, and these neuropeptides are, together with cytokines, likely to be involved in the neuro-immunomodulation that occurs in arthritis. The observations do on the whole suggest that the neuropeptides, rather than BDNF, are related to inflammatory processes in the human knee joint. A new effect of anti-TNF treatment; i.e., lowering plasma levels of BDNF, was observed. Severe arthritis, as in the murine model, lead to a decrease in the levels of neurotrophin, and neurotrophin and neuropeptide receptor expressions in the articular cartilage. This fact might be a drawback for the function of the chondrocytes. Certain differences between the expression patterns in the synovial tissue of the murine model and those of human arthritic synovial tissue were noted. It is obvious that local productions in the synovial tissue, nerve-related supply in this tissue and productions in chondrocytes to different extents occur for the investigated substances.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 25, 2010). Vita. Thesis advisor: Charles J. Smith. "December 2009" Includes bibliographical references.