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This thesis describes the application of the radioisotope 11C to the synthesis of two analogues of the neurohormone melatonin. The labelled compounds were intended to be used as tracers for the medical imaging technology Positron Emission Tomography (PET). [ 11C]Carbon dioxide, produced in a small on-site cyclotron by the nuclear reaction 14N(p,a)11C, was converted into [11C]CH3COC1 by reaction first with CH3MgBr, followed by reaction with phthaloyl dichloride. The labelled acid chloride was distilled into a solution of an amine, yielding the corresponding 11C-labelled amide, which was purified by a simple solid-phase extraction method. An apparatus was designed and built that allowed the remote synthesis with several hundred millicuries of [11C]C02• The apparatus was mounted in a hot cell and operated remotely with a Macintosh Powerbook programmed in Hypercard. The apparatus and software are generic for these acylation reactions. The individual reaction steps were optimized in terms of reaction time, solvents and equipment; radiosyntheses of a number of purified labelled acetamides were completed in 35 minutes. The radiochemical yields ranged from 15 to 20% with specific activities in the 500 mCi/J..tmol range at the end of the synthesis. 2-Iodo-[11C-acetyl]melatonin (11) and 7-methoxynaphthylenyl-1-ethyl-N-[11Cacetyl] acetamide (15) were synthesized for the first time for PET studies. It was shown that both compounds readily cross the blood-brain-barrier and penetrate into all brain tissues. Specific binding to the melatonin receptors in the suprachiasmatic nuclei of the hypothalamus could not be visualized with either one of the 11C-labelled ligands because of low specific activity and high nonspecific binding. / Thesis / Master of Science (MSc)

G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution. (c) 2006 Elsevier Ltd. All rights reserved.

No hipotálamo, centro regulador de uma variedade de comportamentos inclusive o reprodutivo, são produzidos diversos peptídeos moduladores, dentre eles o hormônio concentrador de melanina [MCH]. Já foi observada presença do MCH também em tecidos periféricos, incluindo os testículos. Por isso buscamos sua presença no ovário de ratas nas fases do ciclo estral, gestantes e lactantes. Para a determinação da presença do peptídeo foram utilizados os métodos de imuno-histoquímica e western blotting, para a detecção do RNAm, a hibridização in situ e RT-PCR, e ensaio imunoenzimático para determinação de níveis séricos hormonais de progesterona, prolactina e MCH. Nossos experimentos indicam a presença do RNAm do ppMCH no ovário, bem como do peptídeo em todas as fases do ciclo reprodutivo, no corpo lúteo e na medula ovariana, resultado quantificado pela técnica de western blotting. Além do mais, observamos que os níveis séricos de MCH são constantes nas fases analisadas e que a contribuição do ovário nesses níveis é significativa. / In the hypothalamus, the regulatory center of a variety of behaviors, including reproductive behavior, several peptides modulating these functions are produced, among them the melanin-concentrating hormone [MCH]. In addition to the brain tissue, its presence in several peripheral tissues, including the testis, has been observed. Therefore, we used rats to investigate the presence of MCH in the estrous cycle phase, during gestation and lactation periods. In order to determine the presence of the peptide, immunohistochemistry and western blotting methods were used. To detect the preproMCH RNAm, we used in situ hybridization and RT-PCR, and immunoenzymatic assay to determine serum levels of progesterone, prolactin and MCH. Our findings demonstrated the presence of ppMCH mRNA in ovary, as well as the peptide, and it was localized in the corpus luteum and ovarian marrow. Furthermore, we observed that serum MCH levels are constant in the analyzed phases and that the contribution of the ovary at these levels is significant.

CAPA-related peptides belong to a family of neuropeptides localized to the central nervous system that can function in diverse roles in the regulation of water and salt homeostasis in insects. These peptides are known to stimulate fluid secretion by Malpighian tubules (MTs) in Dipteran species, thus serving a diuretic function. In contrast, this thesis demonstrates that members of this family of peptides in Rhodnius prolixus serve an anti-diuretic role and have multiple tissue targets, whereby they oppose the activity of diuretic hormones such as serotonin (5-Hydroxytryptamine hydrochloride; 5-HT). I have identified two genes each encoding three peptides in R. prolixus, suggesting this insect is capable of producing a greater number of CAPA-peptides compared to other insects that contain only a single CAPA gene. Interestingly, while the second peptide encoded in each R. prolixus gene (RhoprCAPA-α2/-β2) inhibits the stimulatory effects of serotonin on tissues such as the anterior midgut and Malpighian tubules, it appears the other CAPA-related and pyrokinin-related peptides do not play a major role in inhibiting the effects of serotonin on these tissues. More specifically, serotonin-stimulated fluid secretion by MTs and fluid absorption by the anterior midgut are reduced by the anti-diuretic peptide, RhoprCAPA-α2. In addition, I have also identified a G protein-coupled receptor which likely mediates the anti-diuretic effect associated with RhoprCAPA-α2 and have functionally characterized this receptor in Chinese hamster ovary cells. Spatial transcript expression analysis in fifth-instars reveals a wide distribution of the receptor in tissues associated with the rapid post-gorging diuresis. Thus, my findings suggest that numerous tissues are regulated by the CAPA peptides in R. prolixus. Gene structure and phylogenetic analyses demonstrate that this receptor is the orthologue of the D. melanogaster capa receptor (CG14575) with homologs in other insects. Taken together, my thesis demonstrates that the RhoprCAPA peptides play an integral role in the coordination and maintenance of anti-diuresis in R. prolixus. This mechanism is necessary following the rapid diuresis associated with blood-feeding by this medically-important insect.

The dramatic feeding-related activities of the Chagas' disease vector, Rhodnius prolixus are under neurohormonal regulation of serotonin and various neuropeptides. One such family of neuropeptides, the insect kinins, possesses diuretic, digestive and myotropic activities in many insects. In R. prolixus, they co-localize with the corticotropin-releasing factor (CRF)-like diuretic hormone (DH) in neurosecretory cell bodies and their abdominal neurohaemal sites. Additionally, kinins are present in endocrine cells of the midgut and are known to stimulate hindgut and midgut contractions. Through the experimentation presented in this dissertation, the cloning and spatial expression of the R. prolixus kinin (Rhopr-kinin) transcript is described. Physiological bioassays demonstrate the myostimulatory effects of selected Rhopr-kinin peptides and also illustrate the augmented responses of hindgut contractions to co-application of Rhopr-kinin and Rhopr-CRF/DH. The irreversible effects of two synthetic kinin analogs on the hindgut relative to the native kinins also exhibit the prospective biotechnological significance of this study.

The dramatic feeding-related activities of the Chagas' disease vector, Rhodnius prolixus are under neurohormonal regulation of serotonin and various neuropeptides. One such family of neuropeptides, the insect kinins, possesses diuretic, digestive and myotropic activities in many insects. In R. prolixus, they co-localize with the corticotropin-releasing factor (CRF)-like diuretic hormone (DH) in neurosecretory cell bodies and their abdominal neurohaemal sites. Additionally, kinins are present in endocrine cells of the midgut and are known to stimulate hindgut and midgut contractions. Through the experimentation presented in this dissertation, the cloning and spatial expression of the R. prolixus kinin (Rhopr-kinin) transcript is described. Physiological bioassays demonstrate the myostimulatory effects of selected Rhopr-kinin peptides and also illustrate the augmented responses of hindgut contractions to co-application of Rhopr-kinin and Rhopr-CRF/DH. The irreversible effects of two synthetic kinin analogs on the hindgut relative to the native kinins also exhibit the prospective biotechnological significance of this study.

CAPA-related peptides belong to a family of neuropeptides localized to the central nervous system that can function in diverse roles in the regulation of water and salt homeostasis in insects. These peptides are known to stimulate fluid secretion by Malpighian tubules (MTs) in Dipteran species, thus serving a diuretic function. In contrast, this thesis demonstrates that members of this family of peptides in Rhodnius prolixus serve an anti-diuretic role and have multiple tissue targets, whereby they oppose the activity of diuretic hormones such as serotonin (5-Hydroxytryptamine hydrochloride; 5-HT). I have identified two genes each encoding three peptides in R. prolixus, suggesting this insect is capable of producing a greater number of CAPA-peptides compared to other insects that contain only a single CAPA gene. Interestingly, while the second peptide encoded in each R. prolixus gene (RhoprCAPA-α2/-β2) inhibits the stimulatory effects of serotonin on tissues such as the anterior midgut and Malpighian tubules, it appears the other CAPA-related and pyrokinin-related peptides do not play a major role in inhibiting the effects of serotonin on these tissues. More specifically, serotonin-stimulated fluid secretion by MTs and fluid absorption by the anterior midgut are reduced by the anti-diuretic peptide, RhoprCAPA-α2. In addition, I have also identified a G protein-coupled receptor which likely mediates the anti-diuretic effect associated with RhoprCAPA-α2 and have functionally characterized this receptor in Chinese hamster ovary cells. Spatial transcript expression analysis in fifth-instars reveals a wide distribution of the receptor in tissues associated with the rapid post-gorging diuresis. Thus, my findings suggest that numerous tissues are regulated by the CAPA peptides in R. prolixus. Gene structure and phylogenetic analyses demonstrate that this receptor is the orthologue of the D. melanogaster capa receptor (CG14575) with homologs in other insects. Taken together, my thesis demonstrates that the RhoprCAPA peptides play an integral role in the coordination and maintenance of anti-diuresis in R. prolixus. This mechanism is necessary following the rapid diuresis associated with blood-feeding by this medically-important insect.