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 January 6, 2010). Thesis advisor: M. Cathleen Kuehl-Kovarik. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains viii, 138 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 105-134).

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains xiv, 223 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 187-220).

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.

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.

One of the leading experts in the field of Neuroendocrinology and Neuroimmunmodulation, Samuel Mac Donald McCann, known by all his friends as ‘Don’, passed away in 2007. This article pays tribute to his outstanding scientific contribution and a glimpse on his fascinating personality. A member of the National Academy of Sciences of the United States and pioneer in the field of neuroendocrine regulation, he identified numerous hormones and peptides and set the stage for basic concepts in physiology and clinical medicine. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Indiana University-Purdue University Indianapolis (IUPUI) / Anxiety disorders are estimated to impact 1 in 4 individuals within their lifetime. For some individuals, repeated episodes of the stress response leads to pathological anxiety and depression. The stress response is linked to increased levels of corticotropin-releasing factor (CRF) in the basolateral nucleus of the amygdala (BLA), a putative site for regulating anxiety and associative processes related to aversive emotional memories, and activation of CRF receptors in the BLA of rats produces anxiety-like behavior. Mimicking repeated episodes of the stress response, sub-anxiogenic doses of urocortin 1 (Ucn1), a CRF receptor agonist, are microinjected into the BLA of rats for five consecutive days, a procedure called priming. This results in 1) behavioral sensitization, such that a previously non-efficacious dose of Ucn1 will elicit anxiety-like response after the 3rd injection and 2) the development of a persistent anxiety-like phenotype that lasts at least five weeks after the last injection without any further treatment. Therefore, the purpose of this thesis was to identify mechanisms involved in the Ucn1-priming-induced anxiogenesis. The first a set of experiments revealed that the anxiety-like behavior was not due to aversive conditioning to the context or partner cues of the testing environment. Next, Ucn1-priming-induced gene expression changes in the BLA were identified: mRNA expression for Sst2, Sst4, Chrna4, Chrma4, and Gabrr1 was significantly reduced in Ucn1-primed compared to Vehicle-primed rats. Of these, Sst2 emerged as the primary receptor of interest. Subsequent studies found that antagonizing the Sstr2 resulted in anxiety-like behavior and activation of Sstr2 blocked acute Ucn1-induced anxiety-like responses. Furthermore, pretreatment with a Sstr2 agonist delayed the behavioral sensitization observed in Ucn1-induced priming but did not stop the development of persistent anxiety-like behavior or the Ucn1-priming-induced decrease in the Sstr2 mRNA. These results suggest that the decrease in Sstr2 mRNA is associated with the expression of persistent anxiety-like behavior but dissociated from the mechanisms causing the behavioral sensitization. Pharmacological studies confirmed that a reduced Sstr2 mediated effect in the BLA is likely to play a role in persistent anxiety and should be investigated further.

Lors d’un stress, l’activation de l’axe hypothalamo-hypophyso-surrénalien (HHS) conduit à une augmentation de la production de glucocorticoïdes (tel que la corticostérone) par les glandes surrénales. Le rôle de la corticotropin-releasing hormone (CRH), à l’origine de l’activation de l’axe HHS, est encore méconnu. En effet, les récepteurs à la CRH sont présents aussi bien au niveau du système nerveux central (SNC), notamment au niveau du cervelet, qu’au niveau du système immunitaire (SI). Cela suggère donc une action directe possible de cette hormone sur ces deux systèmes. Au cours de ce projet, nous avons étudié les régulations des SNC et SI lors d’un stress, et plus particulièrement le rôle de la CRH et de ses récepteurs dans ces régulations. Suite à des injections chroniques de corticostérone, mimant un stress, nous avons observé une altération des fonctions locomotrices qui semble être reversée lorsque le CRH-R1 est inhibé avec un antagoniste. Ces premiers résultats permettent de mettre en avant un éventuel rôle de la CRH dans la régulation des fonctions motrices au niveau du cervelet en conditions de stress. En parallèle, d’autres études in vitro réalisées sur des splénocytes murins stimulés avec de la CRH ont montré une diminution de la viabilité des lymphocytes B (LB). Suite à ces résultats, nous avons caractérisé pour la première fois la présence de récepteurs à la CRH sur cette population de LB murins. Ces résultats montrent l’importance de la CRH dans les régulations des SNC et SI en condition de stress et le rôle de cette hormone dans les interactions entre les deux systèmes / In stress conditions, the Hypothalamo-Pituitary-Adrenal (HPA) axis activation leads to an overproduction of glucocorticoïds (such as corticosterone in rodent) by adrenal glands and this activation is well characterized. However, various questions remain about the precise role of corticotropin-releasing hormone (CRH), which is at the beginning of the HPA activation. Indeed, CRH receptors are presents both in central nervous system (CNS), especially in cerebellum, and in immune system (IS). This suggest a possible direct action of this hormone on both system. In this project, we studied the regulations on CNS and IS in stress conditions and more particularly the CRH role and these receptors in these regulations. After chronic corticsterone injections, to mimic a stress, we observed a locomotor alteration which seems to be inverted when CRH-R1 were inhibited with an antagonist. These first results show an possible CRH role in locomotor regulation in cerebellum under stress condition. In parallel, others in vitro studies performed on murine splenocytes stimulated with CRH showed a B lymphocyte (LB) viability decrease. Furthermore, we are the first to characterise the CRH receptors on murine LB. This work show the CRH importance in CNS and IS regulations under stress conditions and its role in interactions between the two systems

[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.

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.