Durante a infecção por Trypanosoma cruzi, o sistema endócrino exerce importante influência no direcionamento da resposta imune do hospedeiro. A prolactina é um dos diversos hormônios envolvidos em uma série de complexas interações com o sistema imunológico, participando diretamente do processo de imunorregulação. As pesquisas relacionadas com a administração de hormônios em modelos experimentais infectados por T. cruzi visam comprovar a modulação dos mecanismos de defesa por estas substâncias e contribuem para a busca de novas terapias auxiliares e melhorias no tratamento convencional da Doença de Chagas. O objetivo deste trabalho foi elucidar o papel da prolactina na modulação do sistema imune durante a fase aguda da doença de Chagas experimental, através da administração desta substância em ratos Wistar jovens infectados com a cepa Y de T.cruzi. O envolvimento e o perfil de ativação das células de defesa durante a infecção aguda foram analisados, utilizando-se ensaios de linfoproliferação, apoptose de linfócitos e análise de marcadores celulares. Além disso, outros parâmetros imunológicos também foram avaliados, entre eles, citocinas intracelulares IFN-?, TNF-?, IL-4 e IL-10, quimiocina MCP-1 e óxido nítrico. Os animais submetidos ao tratamento com prolactina apresentaram redução significativa na parasitemia sanguínea, bem como diminuição na apoptose celular e elevados percentuais de células essenciais na resposta imune do hospedeiro frente ao parasita. Desta forma, os dados obtidos neste trabalho demonstram os efeitos estimulatórios da prolactina e corroboram a ideia da utilização de substâncias imunomoduladoras no desenvolvimento de novas estratégias terapêuticas, que possam promover o equilíbrio da resposta imune do hospedeiro frente à infecção, prevenindo os possíveis danos teciduais observados tanto na fase aguda como crônica da doença de Chagas. / During the course of infection by Trypanosoma cruzi, the host immune system is involved in distinct, complex interactions with the endocrine system, and prolactin (PRL) is one of several hormones involved in immunoregulation. Numerous studies have demonstrated that the modulation of the immune system with the administration of regulatory hormones in different experimental models infected with T. cruzi, are very important inciting the investigation of new therapies for Chagas\' disease. The role of PRL as a possible factor in the modulation of the immune response was the target of this study using as animal model young rats infected with the Y strain of T. cruzi, in the attempt to demonstrate the relationship between the neuroendocrine system and the immune response involved in the acute phase of the experimental Chagas\' disease. For this purpose, some immune parameters were analyzed: dosages of cytokines IFN-?, TNF-?, IL-4, IL-10 and chemokine MCP-1, phenotypic analysis of cell subsets by flow cytometry, production of nitric oxide, lymphoproliferation and apoptosis of splenocytes. Therapy with PRL caused a significant decrease in parasitemia during the acute phase. PRL also triggered an increase in the percentage of cells of the innate and adaptive immune response and decreased apoptosis. Thus, the data obtained in this study corroborate the idea of using substances such as PRL in the development of new therapeutic strategies that can modulate the inflammatory response, contributing to the balance of the host body against infection, preventing the possible tissue damage observed in both acute and chronic Chagas\' disease.

O diabetes mellitus tipo 1 é uma doença metabólica, caracterizada pela desregulação glicêmica, que ocorre devido a um ataque autoimune. A insulinoterapia é o tratamento clássico para o DM1. Contudo, alguns pacientes que apresentam essa doença não respondem de forma eficiente a este tratamento e apresentam episódios frequentes de hipoglicemia severa e despercebida (pacientes hiperlábeis). Essas complicações comprometem de forma significativa a qualidade de vida dessas pessoas. O transplante de ilhotas é uma importante alternativa para o tratamento de pacientes hiperlábeis com DM1. No entanto, essa terapia apresenta restrições como a necessidade de mais de um doador por transplante e significativa morte das ilhotas devido ao estresse provocado pelo procedimento de isolamento, além da morte promovida pelo sistema imune do paciente nos primeiros momentos pós-transplante. A autofagia é um mecanismo de reciclagem de componentes citoplasmáticos que é fundamental para a homeostase celular. Em condições de estresse, este mecanismo é ativado acima do seu nível basal, promovendo a degradação de agregados proteicos e organelas defeituosas, evitando assim, danos celulares que comprometam a viabilidade da célula. Trabalhos realizados por nosso grupo têm mostrado a citoproteção que PRL promove em células-beta, reduzindo a apoptose induzida por citocinas pró-inflamatórias. Também demonstramos o papel essencial de HSPB1 na inibição de apoptose induzida por PRL após o tratamento com citocinas. Além disso, resultados recentes de nosso laboratório mostraram um aumento nos níveis de autofagia em células-beta após sua exposição a citocinas, bem como uma restauração a níveis normais na presença de PRL. Visando um melhor entendimento do papel da PRL na modulação da autofagia em células-beta, o objetivo desse projeto foi estudar se HSPB1 também é essencial no mecanismo de regulação da autofagia induzido por PRL.Para tal, fizemos experimentos em modelos de células-beta MIN6, MIN6 silenciadas para HSPB1 (MIN6-shHSPB1) e MIN6 com sequencia short hairpin aleatória (MIN6- SsC), medindo a morte celular através de ensaios de viabilidade, e ensaios de western blot para avaliar os níveis de marcadores de autofagia e fluxo autofágico (degradação de autofagossomos), tratando as células com citocinas, prolactina e indutores ou inibidores de autofagia. Os resultados mostraram que a modulação da autofagia ocasionada pela prolactina em células-beta se dá, em parte, através de HSPB1. O tratamento com prolactina foi capaz de inibir a morte celular induzida por citocinas, mesmo na presença de cloroquina, um bloqueador de autofagia, o que nos levou a concluir que a autofagia não é uma via envolvida na citoproteção de células beta induzida por PRL. Os resultados gerados nesse estudo contribuíram para uma melhor compreensão dos eventos moleculares induzidos por PRL em células-beta, e poderão permitir a inferência de novas abordagens que melhorem a citoproteção, cultura e transplante dessas células em pacientes com diabetes tipo 1. / Type 1 diabetes mellitus is a metabolic disease characterized by glycemic dysregulation, which occurs due to an autoimmune destruction of beta-cells. Insulin therapy is the gold standard treatment for DM1. However, some DM1 patients do not respond efficiently to this treatment and suffer frequent episodes of severe hypoglycemia unawareness. Since this complication jeopardizes the quality of life of these people, Islet transplantation is a therapeutic alternative indicated to treat these patients. However, besides the lack of enough organ donors, the loss of beta cells during both the isolation as well as the infusion of islets into the recipient induce a great estresse and thus a significant cell death is one of the drawbacks of this procedure. Autophagy is a mechanism of recycling cytoplasmic components and is essential for cellular homeostasis. Under estresse conditions, this mechanism is activated above basal levels, promoting the degradation of protein aggregates and defective organelles, thus avoiding cell damage that could compromise cell viability. Studies carried out by our group have shown not only that PRL promotes cytoprotection in beta-cells, reducing pro-inflammatory cytokines-induced apoptosis, but also that HSPB1 plays an essential role in this inhibition of apoptosis mediated by PRL after treatment with cytokines. Moreover, recent results from our laboratory showed an increase in autophagy levels in beta-cells after exposure to cytokines, as well as a restauration to normal levels in the presence of PRL. In order to better understand the role of PRL in the modulation of autophagy in these cells, the aim of this project is to study whether HSPB1 is also essential in the mechanism of autophagy regulation induced by PRL. Using MIN6 beta cell models where HSPB1 was silenced (MIN6-shHSPB1) or not (MIN6-SsC), we studied cell death by viability assays. Moreover, western blot assays were performed in order to assess levels of autophagy and autophagic flux markers in the cells.Our results showed that HSPB1 in one of the mediators of PRL-induced modulation of autophagy. Nevertheless, since hormonal treatment was still able to inhibit cytokinesinduced cell death even in the presence of chloroquin, an autophagy blocker, we conclude that autophagy is not a signaling pathway involved in PRl-induced beta-cell cytoprotection. Altogether, the results shown in this study may help to increase the knowledge of the molecular events induced by PRL in beta-cells, and may allow to infer new approaches to improve cytoprotection, culture and transplantation of these cells into type 1 diabetic patients.

O diabetes mellitus tipo 1 é uma doença metabólica, caracterizada pela desregulação glicêmica, que ocorre devido a um ataque autoimune. A insulinoterapia é o tratamento clássico para o DM1. Contudo, alguns pacientes que apresentam essa doença não respondem de forma eficiente a este tratamento e apresentam episódios frequentes de hipoglicemia severa e despercebida (pacientes hiperlábeis). Essas complicações comprometem de forma significativa a qualidade de vida dessas pessoas. O transplante de ilhotas é uma importante alternativa para o tratamento de pacientes hiperlábeis com DM1. No entanto, essa terapia apresenta restrições como a necessidade de mais de um doador por transplante e significativa morte das ilhotas devido ao estresse provocado pelo procedimento de isolamento, além da morte promovida pelo sistema imune do paciente nos primeiros momentos pós-transplante. A autofagia é um mecanismo de reciclagem de componentes citoplasmáticos que é fundamental para a homeostase celular. Em condições de estresse, este mecanismo é ativado acima do seu nível basal, promovendo a degradação de agregados proteicos e organelas defeituosas, evitando assim, danos celulares que comprometam a viabilidade da célula. Trabalhos realizados por nosso grupo têm mostrado a citoproteção que PRL promove em células-beta, reduzindo a apoptose induzida por citocinas pró-inflamatórias. Também demonstramos o papel essencial de HSPB1 na inibição de apoptose induzida por PRL após o tratamento com citocinas. Além disso, resultados recentes de nosso laboratório mostraram um aumento nos níveis de autofagia em células-beta após sua exposição a citocinas, bem como uma restauração a níveis normais na presença de PRL. Visando um melhor entendimento do papel da PRL na modulação da autofagia em células-beta, o objetivo desse projeto foi estudar se HSPB1 também é essencial no mecanismo de regulação da autofagia induzido por PRL.Para tal, fizemos experimentos em modelos de células-beta MIN6, MIN6 silenciadas para HSPB1 (MIN6-shHSPB1) e MIN6 com sequencia short hairpin aleatória (MIN6- SsC), medindo a morte celular através de ensaios de viabilidade, e ensaios de western blot para avaliar os níveis de marcadores de autofagia e fluxo autofágico (degradação de autofagossomos), tratando as células com citocinas, prolactina e indutores ou inibidores de autofagia. Os resultados mostraram que a modulação da autofagia ocasionada pela prolactina em células-beta se dá, em parte, através de HSPB1. O tratamento com prolactina foi capaz de inibir a morte celular induzida por citocinas, mesmo na presença de cloroquina, um bloqueador de autofagia, o que nos levou a concluir que a autofagia não é uma via envolvida na citoproteção de células beta induzida por PRL. Os resultados gerados nesse estudo contribuíram para uma melhor compreensão dos eventos moleculares induzidos por PRL em células-beta, e poderão permitir a inferência de novas abordagens que melhorem a citoproteção, cultura e transplante dessas células em pacientes com diabetes tipo 1. / Type 1 diabetes mellitus is a metabolic disease characterized by glycemic dysregulation, which occurs due to an autoimmune destruction of beta-cells. Insulin therapy is the gold standard treatment for DM1. However, some DM1 patients do not respond efficiently to this treatment and suffer frequent episodes of severe hypoglycemia unawareness. Since this complication jeopardizes the quality of life of these people, Islet transplantation is a therapeutic alternative indicated to treat these patients. However, besides the lack of enough organ donors, the loss of beta cells during both the isolation as well as the infusion of islets into the recipient induce a great estresse and thus a significant cell death is one of the drawbacks of this procedure. Autophagy is a mechanism of recycling cytoplasmic components and is essential for cellular homeostasis. Under estresse conditions, this mechanism is activated above basal levels, promoting the degradation of protein aggregates and defective organelles, thus avoiding cell damage that could compromise cell viability. Studies carried out by our group have shown not only that PRL promotes cytoprotection in beta-cells, reducing pro-inflammatory cytokines-induced apoptosis, but also that HSPB1 plays an essential role in this inhibition of apoptosis mediated by PRL after treatment with cytokines. Moreover, recent results from our laboratory showed an increase in autophagy levels in beta-cells after exposure to cytokines, as well as a restauration to normal levels in the presence of PRL. In order to better understand the role of PRL in the modulation of autophagy in these cells, the aim of this project is to study whether HSPB1 is also essential in the mechanism of autophagy regulation induced by PRL. Using MIN6 beta cell models where HSPB1 was silenced (MIN6-shHSPB1) or not (MIN6-SsC), we studied cell death by viability assays. Moreover, western blot assays were performed in order to assess levels of autophagy and autophagic flux markers in the cells.Our results showed that HSPB1 in one of the mediators of PRL-induced modulation of autophagy. Nevertheless, since hormonal treatment was still able to inhibit cytokinesinduced cell death even in the presence of chloroquin, an autophagy blocker, we conclude that autophagy is not a signaling pathway involved in PRl-induced beta-cell cytoprotection. Altogether, the results shown in this study may help to increase the knowledge of the molecular events induced by PRL in beta-cells, and may allow to infer new approaches to improve cytoprotection, culture and transplantation of these cells into type 1 diabetic patients.

Não é claro como o receptor de glicocorticóide (GR) contrarregula a atividade do STAT5 na transição da gestação para a lactação. Dexras1 é uma pequena proteína G ativada por dexametasona (DEX), que regula morfologia celular, crescimento, etc. Neste estudo detectamos a expressão de Dexras1 em células beta de ilhotas pancreáticas. DEX induz a expressão de Dexras1 em células RINm5F, que está aumentada na gestação e diminuída na lactação. A expressão protéica de 11<font face=\"Symbol\">&#946HSD1, enzima ativadora de glicocorticóides (GCs), segue esse perfil. A ligação tanto do GR como do STAT5, analisada por ChIP assay, ao promotor do gene da Dexras1 aumentada por DEX é revertida por prolactina (PRL), e está diminuída e aumentada na gestação e lactação, respectivamente. DEX induz a associação ao GR, fosforilação e translocação nuclear do STAT5b. O silenciamento gênico de Dexras1 promoveu aumento da secreção de insulina, e aumentou os níveis de pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 e PKA. Sendo assim, a regulação de Dexras1 por PRL e GCs contribui para a secreção de insulina característica do periparto. / It is not clear how glucocorticoid receptor (GR) counteracts STAT5 activity during the transition of pregnancy to lactation. Dexras1 is a small G protein activated by dexamethasone (DEX) that controls cell morphology, growth, etc. In the present study we detected Dexras1 expression in pancreatic beta cell. DEX induces Dexras1 expression in RINm5F cells, which is increased in pregnancy and decreased in lactation. The expression of 11<font face=\"Symbol\">&#946HSD1, the glucocorticoids (GCs) activating enzyme, followed Dexras1 profile in pancreatic islet. Both GR and STAT5b bindings to Dexras1 gene promoter, analyzed by ChIP assay, are increased by DEX and PRL counteracts this effect. Both bindings are decreased in pregnancy and increased in lactation. DEX induces STAT5b association to GR, phosphorylation and nuclear translocation. Dexras1 knockdown using small interference RNA (si-RNA) promoted an increase in insulin secretion, as well as increased levels of pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 and PKA. Thus, Dexras1 regulation by PRL and GCs contributes to insulin secretion during peripartum.

Haloperidol é um antagonista de receptor D2 de dopamina freqüentemente utilizado na clínica para tratamento de pacientes esquizofrênicos. Haloperidol aumenta a liberação de prolactina pela glândula pituitária anterior, e a prolactina é um fator capaz de modular a atividade do sistema imune. Grupos de seis ratos machos e fêmeas receberam tratamento agudo de 2mg/kg de haloperidol (E1), tratamento prolongado com haloperidol (E2) (2mg/kg/dia por 21 dias); retirada abrupta do tratamento prolongado com haloperidol (E3), ou ainda tratamento agudo com 6mg/kg de haloperidol (E4). Os animais controle receberam tratamento similar com veículo de diluição (grupos C1, C2 e C3 respectivamente). Neste trabalho, o tratamento prolongado com haloperidol (E2) aumentou a atividade de macrófagos, aumentando o espraiamento, a fagocitose e a liberação de NO em ratos machos e fêmeas. O tratamento agudo com 2mg/kg de haloperidol (E1) não foi capaz de alterar a atividade de macrófagos, porém a dose de 6mg/kg (E4) aumentou o espraiamento e a fagocitose. Os tratamentos agudo (E1) e prolongado (E2) com haloperidol aumentaram os níveis plasmáticos de corticosterona e de prolactina tanto em machos quanto em fêmeas. Os macrófagos de ratos machos e fêmeas apresentam os mesmos padrões de alterações após os tratamentos com haloperidol, exceto para produção de H2O2 que foi maior apenas para as fêmeas dos grupos C3 e E3. Discutiu-se de que maneira o haloperidol induz ativação de macrófagos, se por uma forma indireta através do aumento nos níveis de prolactina, ou alternativamente, sendo esta ativação uma conseqüência da ação direta do haloperidol sobre os receptores de dopamina dos macrófagos. / Haloperidol is a receptor D2 antagonist frequently used in the treatment of schizophrenic patients. Haloperidol increased prolactin release from anterior pituitary gland, and prolactin modulates immune system activity. Groups of six male and female rats received acute 2mg/kg haloperidol treatment (E1), long-term (E2) haloperidol treatment (2mg/kg/day for 21 days); abrupt withdrawal of long-term (E3) haloperidol treatment, or acute 6mg/kg haloperidol treatment (E4). Control rats were treated similarly, but with vehicle (groups C1, C2 and C3 respectively). In this work long-term haloperidol treatment (E2) increased macrophage spreading, phagocytosis and NO release in male and female rats. Acute 2mg/kg haloperidol treatment (E1) didn?t change macrophage activity, however, the 6mg/kg dose (E4) increased macrophage spreading and phagocytosis. Corticosterone and prolactin serum levels were increased after acute (E1) and long-term (E2) haloperidol treatments in male and female rats. Macrophage of male and female rats presented the same pattern of alterations after acute and long-term haloperidol treatments, except for production of H2O2 that was larger just for the females of the groups C3 and E3. Haloperidol-induced macrophage activation was discussed in the light of a possible indirect effect through prolactin increments in rats, or, alternatively, as a consequence of a direct action of macrophage dopamine receptor.

Fung Chun Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 117-139). / Abstracts in English and Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of figures --- p.vi / List of tables --- p.viii / List of abbreviations --- p.ix / Chapter Chapter I --- General introduction --- p.1 / Chapter 1.1 --- Different fish habitats with various salinities --- p.1 / Chapter 1.2 --- Osmotic challenges faced by teleosts --- p.2 / Chapter 1.3 --- High ionic strength results in DNA damage and excess water gain --- p.3 / Chapter 1.4 --- Osmoregulatory organs and mechanisms for osmotic balance --- p.4 / Chapter 1.5 --- Different tolerance to salinity changes --- p.8 / Chapter 1.6 --- Effective communication among osmoregulatory organs --- p.9 / Chapter 1.7 --- Introduction to GH and PRL --- p.9 / Chapter 1.8 --- Structure of the GHR and PRLR --- p.10 / Chapter 1.9 --- Hypoosmoregulatory action of GH/IGF-I axis in teleosts --- p.11 / Chapter 1.10 --- Hyperosmoregulatory action of PRL in teleosts --- p.11 / Chapter Chapter II --- Research rationale --- p.13 / Chapter 2.1 --- Physiological importance of osmoregulation in fish --- p.13 / Chapter 2.1.1 --- Energy metabolism --- p.13 / Chapter 2.1.2 --- Growth --- p.14 / Chapter 2.1.3 --- Immunity --- p.14 / Chapter 2.1.4 --- Reproduction --- p.15 / Chapter 2.2 --- Aquaculture importance --- p.15 / Chapter 2.3 --- Unknown molecular regulatory mechanism of hormones during salinity changes in fish --- p.16 / Chapter 2.4 --- Animal model --- p.17 / Chapter Chapter III --- In vivo studies of sbGHR and sbPRLR expression in osmoregulatory organs in response to salinity changes --- p.18 / Chapter 3.1 --- Introduction --- p.18 / Chapter 3.1.1 --- Dynamic change of GH level during salinity changes --- p.18 / Chapter 3.1.2 --- Dynamic change of PRL level during salinity changes --- p.19 / Chapter 3.1.3 --- In vitro studies of GH and PRL release from teleost pituitary in response to extracellular osmolality changes --- p.20 / Chapter 3.1.4 --- Biological actions of GH and PRL through the GHR and PRLR --- p.21 / Chapter 3.2 --- Materials and methods --- p.23 / Chapter 3.3 --- Results --- p.28 / Chapter 3.4 --- Discussion --- p.36 / Chapter 3.4.1 --- Plasma osmolality change during salinity changes --- p.36 / Chapter 3.4.2 --- Gene expression after HSW exposure --- p.38 / Chapter 3.4.3 --- Ionic mediators of the gene expression --- p.43 / Chapter 3.4.4 --- Gene expression after BW exposure --- p.44 / Chapter 3.4.5 --- Dynamic changes of the GHR and PRLR in response to salinity changes --- p.45 / Chapter 3.4.6 --- Regulation of the gene expression in response to salinity changes --- p.46 / Chapter Chapter IV --- Gene expression of sbGHR in liver during salinity changes --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.1.1 --- Responses of the somatotropic axis to salinity changes in fish --- p.49 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.3 --- Results --- p.56 / Chapter 4.4 --- Discussion --- p.60 / Chapter 4.4.1 --- Inhibition of GHR and IGF-I gene expression in liver during HSW exposure --- p.60 / Chapter 4.4.2 --- Downregulation of GHR gene expression by hyperosmotic stress --- p.62 / Chapter 4.4.3 --- Growth retardation of fish during hyperosmotic environment --- p.64 / Chapter Chapter V --- Gene expression studies of sbPRLR in gill organ culture --- p.68 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.1.1 --- Functions of gill in fish osmoregulation --- p.68 / Chapter 5.1.2 --- Gill culture as a model for osmoregulation studies --- p.69 / Chapter 5.2 --- Materials and methods --- p.70 / Chapter 5.3 --- Results --- p.71 / Chapter 5.4 --- Discussion --- p.73 / Chapter Chapter VI --- Regulation of gene expression of sbGHR in liver during hyperosmotic stress: promoter studies --- p.75 / Chapter 6.1 --- Introduction --- p.75 / Chapter 6.1.1 --- What is a promoter? --- p.75 / Chapter 6.1.2 --- Promoter studies of GHR gene --- p.76 / Chapter 6.2 --- Materials and methods --- p.78 / Chapter 6.3 --- Results --- p.85 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter VII --- General discussion and future perspectives --- p.111 / References --- p.117

Also, the branchial hsp70 levels in fish following chronic salinity acclimation and abrupt hypo-osmotic exposure to 6 ppt were assessed by Western blotting. Upon chronic salinity acclimation, the lowest branchial hsp70 level was found in fish cultured in an iso-osmotic salinity of 12 ppt and the highest was in 50 ppt and 6 ppt environments. Freshwater acclimation resulted in return to lower hsp70 level. The results indicated that iso-osmotic salinity would bring about the least stress level while 50 ppt and 6 ppt were the most stressful salinities to Sparus sarba as indicated by using hsp70 expression as a biomarker of stress. Compared to 50 ppt and 6 ppt, the stress level of fish in fresh water was lower. On the other hand, Sparus sarba exhibited a significant increase in branchial hsp70 level immediately after abrupt hypo-osmotic exposure to 6 ppt when compared with seawater fish sampled at the same time point and increased hsp70 level was sustained throughout the sampling period, indicating the exposure was stressful to the fish. / In the present study, pituitary and serum levels of prolactin in a marine teleost, Sparus sarba, chronically acclimated to various salinities: fresh water (0 ppt), hypo-osmotic (6 ppt), iso-osmotic (12 ppt), normal seawater (33 ppt) and hypersaline (50 ppt) or abruptly exposed to a hypo-osmotic environment of 6 ppt were quantified by the developed peptide-based indirect ELISAs. Progressive increases in pituitary and serum prolactin were found as chronic salinity acclimation progressed from seawater to fresh water. Also, prolactin secretion was immediately induced by abrupt hypo-osmotic exposure to 6 ppt and remained significantly elevated up to 5 days post-exposure to 6 ppt. The results underline the importance of prolactin in marine teleosts kept in fresh water or waters of low salinity. However, there was no significant difference in pituitary prolactin during the course of the abrupt hypo-osmotic exposure experiment. The results may indicate that prolactin might be secreted rapidly from pituitary in large quantities to cope with abrupt exposure to a low-salinity environment. / In the present study, the effects of pharmacological drugs on prolactin levels in pituitary and serum of Sparus sarba were investigated. An increase in prolactin synthesis and release but a decrease in branchial hsp70 expression were found after treatment with sulpiride, a DA-D2 receptor antagonist. In contrast, a reduction in prolactin levels in pituitary and serum but an elevation in hsp70 level in gill were observed following administration of bromocriptine, a DA-D2 receptor agonist. Since hsp70 expression indicates the stress levels, the results of these studies supported the notion that increased prolactin synthesis and release might be related to a reduced stress state and prolactin might have a protective effect on stress tolerance in fish. / Lastly, the role of prolactin in regulating apoptosis in Sparus sarba branchial cells was examined. Successful induction of apoptosis was indicated by an increase in the apoptotic parameter caspase-3 activity in primary cultures of Sparus sarba branchial cells treated with camptothecin, a specific inducer of apoptosis. In this study, prolactin was shown to be anti-apoptotic in Sparus sarba branchial cells as co-treatment with ovine prolactin (oPRL) and camptothecin has been observed to attenuate the elevated caspase-3 activity in gill cell primary cultures. Also, prolactin was found to protect the branchial cells from apoptosis by maintaining the hsp70 level in the cells treated with camptothecin. / The objectives of the present study were to investigate the roles of prolactin in salinity adaptation, hsp70 expression and apoptosis in silver sea bream (Spaurs sarba). Firstly, specific peptide-based indirect ELISAs were developed for pituitary and serum prolactin of Sparus sarba. These assays had been validated by parallelism between the dilution response curves using serially diluted pituitary homogenate and serum sample with the standard curves of the synthetic peptide derived from the amino acid sequence of black sea bream (Acanthopagrus schlegelii ) prolactin. / Ng, Ho Yuen Andus. / "September 2007." / Adviser: N. Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4567. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 143-189). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

All these findings can help us to elucidate the mechanisms for the fish to detect changing osmotic conditions and transform signals to osmoregulatory responses. / In the first part of the study, PRL and PRL-releasing peptide (PrRP) cDNAs have been isolated from euryhaline silver sea bream. The PRL cDNA consists of 1360 bp encoding 212 amino acids whereas the PrRP cDNA contains 631 bp encoding prepro-PrRP with 122 amino acids. PRL mRNA was uniquely expressed in sea bream pituitary but PrRP mRNA was expressed in a variety of tissues. Expression levels of both PRL and PrRP mRNA have been examined in sea bream adapted to different salinities (0, 6, 12, 33 and 50 ppt). In pituitary, both PRL and PrRP mRNA were synchronized in their expression, being significantly higher in fish adapted to low salinities (0 and 6 ppt), but the expression profile of hypothalamic PrRP in different salinities was different. These data suggested that PrRP may possibly act as a local modulator in pituitary rather than a hypothalamic factor for regulating pituitary PRL expression in silver sea bream. / In the second part of the study, silver sea bream abruptly transferred from 33 to 6 ppt exhibited a remarkable pituitary PRL secretion following closely with the temporal changes in serum osmolality and ion levels. In order to investigate the direct effect of extracellular osmolality to pituitary PRL secretion, sea bream pituitary cells were dispersed and exposed to a medium with reduced ion levels and osmolality. PRL released from pituitary cells was found to be significantly elevated. In hyposmotic exposed anterior pituitary cells, cell volume exhibited a 20% increase when exposed to a medium with a 20% decrease in osmolality. These enlarged pituitary cells did not shrink until the surrounding hyposmotic medium was replaced, a phenomenon suggesting an osmosensing ability of silver sea bream PRL cells for PRL secretion in response to a change in extracellular osmotic pressure. / In the third part, olfactory rosette in the nasal cavity was surgically removed from silver sea bream adapted to 6 ppt and 33 ppt and mRNA expression of PRL and PrRP in silver sea bream were measured. The elevated pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish were abolished by this olfactory lamellectomy. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change but those in 33 ppt-adapted fish increase significantly after olfactory lamellectomy. These data suggest a possible osmosensing role of the olfactory system for regulation of PRL expression during hypo-osmotic acclimation of the fish. Besides, calcium-sensing receptor (CaSR) was cloned and its mRNA expression in olfactory system, as shown in other fish species previously, was investigated. However, no CaSR expression could be detected in olfactory rosette and nerve but its expression was demonstrated in osmoregulatory tissues and brain. There was no significant difference in CaSR mRNA expression in pituitary, kidney and anterior intestine of fish adapted to different salinities. These studies could not provide conclusive evidence to correlate CaSR with osmosensing in silver sea bream. / The present study used silver sea bream (Sparus sarba ) as a euryhaline fish model to investigate the regulation of prolactin (PRL) expression and secretion in fish adapted to different salinities. / Kwong, Ka Yee. / Adviser: Norman Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3248. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 154-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

"June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 162-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Haloperidol é um antagonista de receptor D2 de dopamina freqüentemente utilizado na clínica para tratamento de pacientes esquizofrênicos. Haloperidol aumenta a liberação de prolactina pela glândula pituitária anterior, e a prolactina é um fator capaz de modular a atividade do sistema imune. Grupos de seis ratos machos e fêmeas receberam tratamento agudo de 2mg/kg de haloperidol (E1), tratamento prolongado com haloperidol (E2) (2mg/kg/dia por 21 dias); retirada abrupta do tratamento prolongado com haloperidol (E3), ou ainda tratamento agudo com 6mg/kg de haloperidol (E4). Os animais controle receberam tratamento similar com veículo de diluição (grupos C1, C2 e C3 respectivamente). Neste trabalho, o tratamento prolongado com haloperidol (E2) aumentou a atividade de macrófagos, aumentando o espraiamento, a fagocitose e a liberação de NO em ratos machos e fêmeas. O tratamento agudo com 2mg/kg de haloperidol (E1) não foi capaz de alterar a atividade de macrófagos, porém a dose de 6mg/kg (E4) aumentou o espraiamento e a fagocitose. Os tratamentos agudo (E1) e prolongado (E2) com haloperidol aumentaram os níveis plasmáticos de corticosterona e de prolactina tanto em machos quanto em fêmeas. Os macrófagos de ratos machos e fêmeas apresentam os mesmos padrões de alterações após os tratamentos com haloperidol, exceto para produção de H2O2 que foi maior apenas para as fêmeas dos grupos C3 e E3. Discutiu-se de que maneira o haloperidol induz ativação de macrófagos, se por uma forma indireta através do aumento nos níveis de prolactina, ou alternativamente, sendo esta ativação uma conseqüência da ação direta do haloperidol sobre os receptores de dopamina dos macrófagos. / Haloperidol is a receptor D2 antagonist frequently used in the treatment of schizophrenic patients. Haloperidol increased prolactin release from anterior pituitary gland, and prolactin modulates immune system activity. Groups of six male and female rats received acute 2mg/kg haloperidol treatment (E1), long-term (E2) haloperidol treatment (2mg/kg/day for 21 days); abrupt withdrawal of long-term (E3) haloperidol treatment, or acute 6mg/kg haloperidol treatment (E4). Control rats were treated similarly, but with vehicle (groups C1, C2 and C3 respectively). In this work long-term haloperidol treatment (E2) increased macrophage spreading, phagocytosis and NO release in male and female rats. Acute 2mg/kg haloperidol treatment (E1) didn?t change macrophage activity, however, the 6mg/kg dose (E4) increased macrophage spreading and phagocytosis. Corticosterone and prolactin serum levels were increased after acute (E1) and long-term (E2) haloperidol treatments in male and female rats. Macrophage of male and female rats presented the same pattern of alterations after acute and long-term haloperidol treatments, except for production of H2O2 that was larger just for the females of the groups C3 and E3. Haloperidol-induced macrophage activation was discussed in the light of a possible indirect effect through prolactin increments in rats, or, alternatively, as a consequence of a direct action of macrophage dopamine receptor.