Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Also available online.

Obesity is a growing public health issue with few effective treatments. Many of the human genes involved in weight regulation have homologues in other species. In Drosophila melanogaster, 11-cis-vaccenyl acetate (cVA) is a pheromone shown to affect both aggression and feeding behaviour. The aim of this study was to investigate the effect of cVA exposure on gene expression in wild type flies and to confirm crosses with genetic knockouts in the cVA pathway. The genes studied in the wild type flies were Akh (homologue of glucagon), Dilp2,-3 and -5 (homologues of insulin), Dsk (homologue of cholecystokinin), sNPF (homologue of neuropeptide Y) and TβH. The knockout genes studied were Or65a, Or67d and TβH. RNA was extracted from whole heads, cDNA synthesis was performed and the cDNA was then used in SYBR Green RT-qPCR. The knockout genes were not confirmed, due to methodological problems. The expression of Dilp3, Dilp5 and Dsk were significantly lower in the experimental flies, the expression of the other genes where not affected. The results indicate that exposure to cVA affects the expression of some of the neuropeptides involved in weight regulation. Due to the methodological problems experienced, a recommendation is to confirm the data using different reagents.

Although the use of atypical antipsychotic drugs is successful in the treatment of schizophrenia, they cause complications in the long term use that is mainly weight gain. In this study, circulating levels of hypothalamic neuropeptides/hormones, which are related to appetite regulation / neuropeptide Y (NPY), alpha melanocyte stimulating hormone (&alpha / -MSH), cocaine and amphethamine regulated transcript (CART) and plus leptin in male schizophrenic patients who were treated with an atypical antipsychotic drug, risperidone, which is a serotonin antagonist, for 4 weeks was investigated. Based on the hypothesis that the risperidone treatment might alter the circulating levels of those neuropeptides through the serotonergic antagonism, it results in the weight gain. Leptin plasma levels were increased in the risperidone treated patients accompanying by weight gain vs controls and NPY, &alpha / -MSH, CART levels were decreased in the patients before the treatment but they were not changed after treatment. To determine alterations of those candidate genes mRNA expression levels, male Wistar rats were orally administered with risperidone for 4-weeks. Rat studies show that the mRNA expression and plasma levels of POMC, AgRP, and NPY were decreased but CART mRNA levels were increased while their plasma levels were decreased unexpectedly. In conclusion, the serotonergic antagonism of risperidone on POMC neurons may cause increase in appetite / and hence, increased weight gain and leptin levels, even in a short term trial.

The thesis is a study of the effects of radiation on the salivary glands in an experimental and a clinical study. Irradiation is a cornerstone in the management of head and neck cancer and is as other modalities of cancer treatment, afflicted with adverse reactions. An optimal radiotherapy regime is limited by the sensitivity of the normal tissues with regard to early and late effects. In certain cases the early effects can be so troublesome that it will cause interruption in the irradiation and questioning of the curative intention. Although DNA is the lethal target, other parts of the cell have been proposed as sensitive targets to irradiation. Different in vitro secretory models and quantitative morphological characterization and immunohistochemical evaluation of neuropeptides were performed in rat salivary glands after irradiation. The irradiation was given unilaterally or bilaterally once a day for a five-day schedule with 6 MV photons (total dose 20, 30, 35, 40, 45 Gy) or a two fractions regime in five days with a total dose of 24 or 32 Gy. The contralateral gland served as a control for unilaterally treated animals and parallel analyses were done 10 days or 180 days following the last irradiation dose. An early, dose-dependent effect of fractionated irradiation on noradrenaline-stimulated potassium fluxes (86Rb+ fluxes) was demonstrated. In contrast, the exocytotic amylase release displayed no obvious alterations, and morphologically no changes were seen. Regarding late effects (180 days) the noradrenaline-stimulated electrolyte secretion was decreased at least for the higher doses of irradiation. Amylase content and loss of acini was also dose-dependently decreased. At 10 days after bilateral irradiation there was a marked increase in the expression of the neuropeptides substance P, leu-enkephalin and bombesin in the ganglionic cells associated with the submandibular glands and in nerve fibers of the glandular parenchyme. In addition, a clinical prospective evaluation of 25 patients was performed before, during radiotherapy and 6, 12 and 18 months after the end of treatment. A great interindividual variation in the recovery was demonstrated with regard to salivary flow rate. Irradiation doses about 40-50 Gy caused generally reversible changes; sometimes salivary secretion was almost completely restored 6-18 months after the end of radiotherapy. Doses exceeding 65 Gy induced almost irreversible alterations. Even if DNA is the target for the lethal effect of irradiation, other constituents, such as the cell membrane or neuropeptide expression can be significantly affected by irradiation and cause important physiological changes. / <p>S. 1-43: sammanfattning, s. 47-164: 6 uppsatser</p> / digitalisering@umu

In Drosophila, neuropeptides have regulatory roles in development, growth, metabolism and reproduction. This study focused on GABA and the neuropeptides Drosophila tachykinin (DTK), short neuropeptide F (sNPF), adipokinetic hormone (AKH), corazonin (CRZ) and Drosophila insulin-like peptides (DILPs) as possible regulators of metabolic stress responses and homeostasis. We showed that metabotropic GABAB receptors (GBRs) are expressed on brain insulin producing cells (IPCs), suggesting an inhibitory regulation of these cells by GABA. Knockdown of GBR on IPCs shortened lifespan and stress resistance, altered carbohydrate and lipid metabolism at stress (paper I). We showed that three different neuropeptides; DTK, sNPF and ITP, are co-expressed in five pairs of adult neurosecretory cells (paper II). ITP-knock down was not studied yet, but sNPF- and DTK-knock down flies showed decreased stress resistance at desiccation and starvation and decreased water levels at desiccation, suggesting that these peptides are involved in water homeostasis during stress conditions. sNPF was previously shown to affect feeding, growth and DILP expression via the IPCs, but it was not known which sNPF-expressing neurons are responsible for these actions. We could identify a specific set of bilateral neurons (DLPs) that co-express sNPF and corazonin that target the IPCs. We showed that these peptides co-released from DLPs regulate DILP transcription and probably release in the adult Drosophila brain and thus have roles in regulation of stress resistance and metabolism (paper III). AKH signaling was previously shown to affect hemolymph carbohydrate levels and lipid stores in Drosophila. Insulin (DILP) signaling and AKH signaling are suggested to have opposing effects on lipid and sugar metabolism in Drosophila. We studied the possible functional relationship between these two systems; do they mutually regulate each other?  Our results suggest action of DILPs via the Insulin Receptor on the IPCs and the AKH producing cells, but we could not provide evidence for AKH action on IPCs or AKH cells (paper IV). / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Epub ahead of print. Paper 4: Manuscript.</p>

Two groups of neuropeptides (secretin and gonadotropin-releasing hormone groups, GnRH) and their receptors are of considerable interest because they are highly conserved in structure during evolution and have important physiological effects. To study the role of these neuropeptides during development, the zebrafish was selected initially as a model because the embryo is transparent and accessible for genetic manipulation. Later, a mouse model was added to evaluate the effects of permanent gene loss. The purpose of this thesis was to 1) characterize the structure and function of several peptides and receptors within the secretin group that had not been previously identified for zebrafish, 2) examine brain development in zebrafish after gene knock down of an important peptide in each of the secretin and GnRH groups, 3) identify the pattern and location of expression of the GnRH receptors in zebrafish embryos, and 4) study the functional importance of the GnRH receptor in a mouse line deficient in the receptor. The secretin superfamily of hormones includes 10 structure-related polypeptides in mammals, but fewer in zebrafish. In this thesis it was discovered that within the secretin group, zebrafish have two peptides, peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), in addition to two receptors, PHI receptor and growth hormone-releasing hormone-like peptide (GHRH-LP) receptor that were not previously identified. After isolation of the cDNA for each, the signaling pathways were characterized in vitro by transfection of the receptors into COS cells. The PHI-R was activated by PHI but not by VIP or pituitary adenylate cyclase-activating polypeptide (PACAP). The GHRH-LP receptor was activated by GHRH-LP 1 and GHRH but not by GHRH-LP2. A novel observation was that the PAC, receptor is activated by both PACAP and VIP, whereas the PAC, receptor is usually specific to PACAP. Also novel was evidence that a VPAC2 receptor in zebrafish was structurally and functionally a PHI receptor. In examining the role of PACAP in early brain development in zebrafish, morpholinos were used to knockdown PACAP1 and PACAP2 in the zygote. The study revealed that loss of PACAP leads to change in the expression of brain markers. GnRH and its receptors (GnRH-R) are key regulators of reproduction and sexual behaviour. In zebrafish, morpholino-induced knock down of GnRH revealed that GnRH is critical in eye and brain development by affecting transcription factors and/or secreted factors: pax2.1, fgf8, pax6.1 and mab. Further, the GnRH receptor was expressed at 24h post fertilization showing that GnRH and its receptor could influence early development. To determine whether the GnRH receptor is critical at all stages of reproduction, a mouse line was created in which the GnRH receptor was disrupted using the gene trap method. This model has a similar phenotype to the clinical syndrome of hypogonadotropic hypogonadism. In mice, GnRH receptor disruption results in small sexual organs, low gonadotropin and steroid hormone levels, failure of sexual maturation, and lack of reproduction. Also, the GnRH receptor may be important in the fetal testis. This thesis provides evidence that 1) in evolution the PAC, receptor may have responded to both PACAP and VIP in fish, 2) zebrafish have a PHI receptor rather than a VPAC2 receptor, 3) PACAP and GnRH peptides have a fundamental role in early brain development, 4) the GnRH receptor is not only important in reproduction but unexpectedly, in zebrafish embryos is localized in hindbrain motor neurons and fibers, which are involved normally in movement and escape, and 5) GnRH receptor-disrupted mice should be a valuable model for understanding reproduction and clinical approaches to hypogonadotropic hypogonadism.

Signal processing in the visual system is mediated by classic neurotransmission and neuropeptidergic modulatory pathways. In Dipteran insects, especially in the fruitfly Drosophila melanogaster, the morphology of the visual system is very well described. However neurotransmitter and neuropeptidergic circuits within the optic lobe neuropil are only partially known. Using several transgenic fly lines and antibodies we determined the localization of the classical neurotransmitters GABA, acetylcholine and glutamate in the visual system, and their putative targets via detecting several neurotransmitter receptors. We paid particular attention to the peripheral neuropil layer called the lamina, where the light signals are filtered, channeled and amplified (Paper I). We discovered four new types of efferent tangential neurons branching distally to the lamina. Among them was the first neuropeptidergic neuron (LMIo) in this region of Drosophila. The LMIo expresses myoinhibitory peptide (MIP) and has its cell body located close to the main lateral clock neurons that express the neuropeptide pigment-dispersing factor (PDF)(Paper II). Since in other Dipteran species PDF is expressed in processes distally to the lamina, we performed comparative anatomical studies of the MIP, PDF, Ion Transport Peptide (ITP) and serotonin (5-HT) distribution in the visual system of the flies Drosophila and Calliphora. Our data suggest that PDF signaling distal to the lamina of the blowfly might be replaced by MIP signaling in the fruitfly, while ITP and 5-HT expression is conserved in the two species (Paper III). Serotonin is crucial in light adaptation during the daily light-dark cycles. We analyzed putative serotonergic circuits in the lamina. We found that LMIo neurons express the inhibitory receptor 5-HT1A, while 5-HT1B and 5-HT2 are both expressed in the epithelial glia of the lamina. Another novel wide-field neuron with lamina branches expresses the excitatory serotonin receptor 5-HT7. Our studies have identified a fairly complex neuronal circuitry in the tangential plexus above the lamina. (Paper IV). Finally we tested circadian locomotor activity rhythms in flies with the GABAB receptor knocked down on the lateral PDF-expressing clock neurons. We observed significant changes in the activity periods and diminished strength of rhythmicity during DD suggesting a modulatory role of GABA in clock function (Paper V). / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- T cells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell???mediated inflammatory responses and showed reduced levels of the Th1 cell???promoting cytokine interleukin 12 and reduced interferon ?? production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicated that this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- T cells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell???mediated inflammatory responses and showed reduced levels of the Th1 cell???promoting cytokine interleukin 12 and reduced interferon ?? production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicated that this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system.