Le cancer du sein est le cancer le plus répandu chez les Canadiennes. Grâce aux avancées technologiques et scientifiques, l'incidence de ce cancer est stable depuis une quinzaine d'année et le taux de mortalité diminue. Le plus important dans le traitement du cancer est un diagnostic précoce et un suivi de la thérapie bien adapté au type de cancer. La tomographie d'émission par positrons (TEP) permet de visualiser la distribution spatiale et temporelle de molécules radiomarquées. Le principal traceur utilisé en oncologie, le 2-deoxy-2-[18]Fluoro-D-deoxyglucose ( 18 [F]FDG), est un analogue du glucose. Il permet de visualiser les tumeurs et d'évaluer le métabolisme du glucose dans les cellules. Par contre, il a une lacune importante : il n'est pas spécifique aux cellules cancéreuses car il marque les cellules qui ont un métabolisme du glucose élevé. Malgré cela, le FDG reste un traceur très utilisé. Cependant, le développement de nouveaux composés spécifiques à certains récepteurs surexprimés dans différents types de cancer reste une idée à exploiter afin d'améliorer l'imagerie TEP. Le neuropeptide Y (NPY) est un peptide de 36 acides aminés qui est un neurotransmetteur très abondant dans le système nerveux central et périphérique. Il a plusieurs rôles physiologiques importants. Il est un stimulateur puissant de la prise de nourriture, un anxiolytique et un puissant vasoconstricteur. Quatre types de récepteurs humains ont été clonés, soient : Y1R, Y2R, Y4R et Y5R. Le récepteur Y1 est présent dans 85% des carcinomes mammaires primaires chez l'humain. Dans les tissus sains, le récepteur Y2 est exprimé de façon prédominante tandis que sur les cellules cancéreuses, c'est l'expression du récepteur YI qui prédomine. Le ratio de l'expression Y1/Y2 pourrait donc être exploité afin de détecter de façon plus précoce le cancer du sein. La bombésine (BBN), un peptide de 14 acides amines, a été découvert chez la grenouille Bombina Bombina . L'équivalent humain du BBN, le GRP possède 27 acides amines. La bombésine se lie à 3 types de récepteurs humains dont le récepteur à relâche de gastrine (GRPR). Il est surexprimé dans 65% des cas de carcinomes canalaires in situ . Le GRPR peut également être utilisé comme marqueur du caractère malin dans les cas de cancer. Un hétérodimère est l'union de deux molécules différentes pour n'en former qu'une seule. Dû au fait que les récepteurs Y1 et GRP ne sont pas surexprimés dans 100% des cas de cancer du sein l'utilisation d'une seule molécule qui ciblerait à la fois les deux récepteurs serait avantageuse pour l'imagerie du cancer du sein. Les avantages à l'utilisation d'un hétérodimère plutôt qu'un mélange de deux composés radioactifs sont : une dose de radioactivité administrée moins grande pour le même résultat et les interactions possibles entre les composés sont évitées. Puisque deux récepteurs surexprimés dans les cas de cancer du sein sont visés du même coup par le peptide, une plus grande captation tumorale est attendue. Le premier objectif de l'étude est de développer un peptide capable de lier spécifiquement le récepteur Y1 du NPY dans les cas de cancer du sein. Le second objectif est de développer un peptide hétérodimère ciblant préférentiellement les récepteurs Y1 et GRP dans les cas de cancer du sein. Les peptides seront combinés à un radio-isotope, le 64 Cu, afin de permettre l'imagerie TEP. Plusieurs techniques seront utilisées pour caractériser les différents peptides incluant des études de compétition in vitro , de stabilité plasmatique ainsi que de biodistribution. Afin d'évaluer tout le potentiel du traceur hétérodimère, une étude comparative entre ce dernier et les deux traceurs monomères sera présentée."--Résumé abrégé par UMI

Oxytocin has established roles in parturition and lactation, but can also be released in response to non-reproductive stimuli, such as hyperosmolarity and stress. As a majority of appetite regulating peptides activate the hypothalamo-pituitary-adrenal stress axis, and oxytocin is also a stress hormone in the rat, it was hypothesized that the oxytocin system in the neurohypophysial axis could be a target for appetite-regulating peptides of central and peripheral origin. The effects of central administration of neuropeptide Y (NPY; a central orexigenic peptide and a central and peripheral neurotransmitter co-released with noradrenaline; n=5 rats) and systemic administration of secretin (a peripheral gut peptide belonging to the family of brain-gut peptides; n=26) and leptin (a peripheral anorexigenic peptide from adipose tissue; n=23) on the electrical activity of SON oxytocin neurones in vivo were studied in urethane-anaesthetized female rats with extracellular recording. Effects were compared with the excitatory responses to cholecystokinin (CCK; a peripheral anorexigenic gut peptide; n=45). Influences of fasting and pregnancy and effects of these peptides on the activity of SON vasopressin neurones were also studied. Results: (1) All the central and peripheral appetite peptides tested increased the electrical activity of SON oxytocin neurones. (a) NPY: Basal firing rate of 3.5 ± 1.05 (mean ± s.e.m) spikes/s was increased by 1 ± 0.45 spikes/s 1min after NPY (basal vs 0-10min post-NPY: P=0.03, paired t-test; n=5). (b) Secretin: Basal rate of 4.1 ± 0.4 spikes/s was increased by 1.7 ± 0.2 spikes/s 2.5min after secretin (basal vs 0-10min post-secretin: P<0.001, paired t-test; n=26). (c) Leptin: Basal rate of 3.4 ± 0.4 spikes/s was increased by 0.4 ± 0.08 spikes/s 1.5min after leptin (basal vs 0-10min post-leptin: P=0.01, paired t-test; n=23). (d) CCK: Basal rate of 3.6 ± 0.3 spikes/s was increased by 1.1 ± 0.15 spikes/s 1min after CCK (basal vs 0-10min post- CCK: P<0.001, Wilcoxon signed rank test; n=45). (2) Secretin induced excitatory responses were greater than to other peptides (P<0.001, Kruskal-Wallis one-way ANOVA on ranks). (3) Secretin dose-dependently increased SON oxytocin neurone electrical activity and peripheral oxytocin release in anaesthetized rats. (4) Intracerebroventricular infusion and microdialysis studies with benoxathian (α1 adrenergic antagonist) revealed that secretininduced excitation of SON oxytocin and vasopressin neurones involves central excitatory noradrenergic pathways. (5) Fasting for 18h did not alter the excitation of SON oxytocin neurones induced by secretin, CCK and leptin. (6) The pathway leading to excitation of oxytocin neurones by CCK was not influenced by prior leptin administration. (7) SON oxytocin neurones were responsive to leptin during late pregnancy. (8) NPY-induced excitation of oxytocin neurones was intact in anaesthetised late pregnant rats, contrasting with attenuated oxytocin secretory responses observed previously in conscious rats. (9) Systemic NPY excited SON oxytocin neurones. (10) Systemic CCK administration either inhibited (77%) or did not affect (23%) SON vasopressin neurones, while leptin had no significant effect, and responses to secretin were predominantly excitatory (67%). Systemic NPY inhibited vasopressin neurones, but central NPY was ineffective. Conclusion: Appetite peptides target SON oxytocin neurones. Postprandially released secretin and leptin might, like CCK, induce peripheral oxytocin release, so as to regulate water and electrolyte homeostasis, which is inevitably disturbed during feeding. Any central release of oxytocin induced by these peptides, might regulate feeding behaviour and satiety. Oxytocin neurone excitation induced by NPY may be relevant during stress responses.

The neuropeptide Y (NPY) system consists of 3-4 peptides and 4-7 receptors in vertebrates. It has powerful effects on appetite regulation and is involved in many other biological processes including blood pressure regulation, bone formation and anxiety. This thesis describes studies of the evolution of the NPY system by comparison of several vertebrate species and structural studies of the human Y2 receptor, which reduces appetite, to identify amino acid residues involved in peptide-receptor interactions. The NPY system was studied in zebrafish (Danio rerio), western clawed frog (Xenopus tropicalis), and sea lamprey (Petromyzon marinus). The receptors were cloned and functionally expressed and their pharmacological profiles were determined using the native peptides in either binding studies or a signal transduction assay. Some peptide-receptor preferences were observed, indicating functional specialization. A receptor family closely related to the NPY receptors, called the QRFP receptors, was investigated. A QRFP receptor was cloned from amphioxus, Branchistoma floridae, showing that the receptor arose before the origin of the vertebrates. Evolutionary studies demonstrated that the ancestral vertebrate had as many as four QRFP receptors, only one of which remains in mammals today. This correlates with the NPY receptor family, located in the same chromosomal regions, which had seven members in the ancestral vertebrate but only 4-5 in living mammals. Some vertebrates have considerably more complex NPY and QRFP receptor systems than humans and other mammals. Two studies investigated interactions of NPY-family peptides with the human Y2 receptor. Candidate residues, selected based on structural modeling and docking, were mutated to disrupt possible interactions with peptide ligands. The modified receptors were expressed in cultured cells and investigated by measuring binding and functional responses. Several receptor residues were found to influence peptide-receptor interactions, some of which are involved in maintaining receptor structure. In a pilot study, the kinetics of peptide-receptor interaction were found to be very slow, of the order several hours. In conclusion, this thesis clarifies evolutionary relationships for the complex NPY and QRFP peptide-receptor systems and improves the structural models of the human NPY-family receptors, especially Y2. These results will hopefully facilitate drug design for targeting of NPY-family receptors.

by Hinny Shuk-Yee Lam. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 92-117). / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / Table of Contents --- p.v / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Pancreas and Islets of Langerhans --- p.1 / Chapter 1.1.1 --- Islet Hormones and Glucose Balance --- p.3 / Chapter 1.1.2 --- Glucagon and Its Derived Peptides --- p.4 / Chapter A. --- Tissue-specific Post-translational Processing --- p.4 / Chapter B. --- Features of Proglucagon Gene --- p.6 / Chapter 1.1.3 --- Insulin and Features of Its Gene --- p.9 / Chapter 1.2 --- Regulation of Islet Hormone Secretion --- p.12 / Chapter 1.2.1 --- Endocrine Control --- p.12 / Chapter A --- GIP --- p.13 / Chapter B. --- Truncated GLP-1 --- p.13 / Chapter 1.2.2 --- Paracrine Control --- p.14 / Chapter 1.2.3 --- Neuroendocrine Control --- p.15 / Chapter 1.3 --- Neuropeptide Y --- p.16 / Chapter 1.3.1 --- NPY in Central Nervous System --- p.17 / Chapter 1.3.2 --- NPY in Pancreas --- p.17 / Chapter 1.3.3 --- NPY and Islet Hormones --- p.18 / Chapter 1.4 --- Synthesis and Secretion --- p.19 / Chapter 1.5 --- Objectives of Study --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Effects of NPY on Islet Gene Expression --- p.26 / Chapter 2.1.1 --- Tissue Culture --- p.26 / Chapter A. --- Materials --- p.26 / Chapter B. --- Maintenance and Passage --- p.26 / Chapter C. --- Experimental Protocol --- p.28 / Chapter 2.1.2 --- Total RNA Isolation --- p.28 / Chapter A. --- Materials --- p.28 / Chapter B. --- Extraction Using FastPrep System --- p.29 / Chapter C. --- Quantification of RNA --- p.30 / Chapter D. --- Preparation of Reagents --- p.30 / Chapter 2.1.3 --- Northern Blot Analysis --- p.31 / Chapter A. --- Materials --- p.31 / Chapter B. --- Formaldehyde Gel Electrophoresis --- p.32 / Chapter C. --- Transfer onto Nylon Membrane --- p.33 / Chapter D. --- Labeling of cDNA Probes --- p.34 / Chapter E. --- Hybridization and Autoradiography --- p.35 / Chapter F. --- Preparation of Reagents --- p.36 / Chapter 2.1.4 --- Preparation of cDNA Probe --- p.37 / Chapter A. --- Materials --- p.37 / Chapter B. --- Preparation of Competent Cells --- p.37 / Chapter C. --- Transformation --- p.38 / Chapter D. --- Plasmid DNA Isolation --- p.39 / Chapter E. --- Restriction Enzyme Digestion --- p.41 / Chapter F. --- Agarose Gel Electrophoresis --- p.42 / Chapter G. --- Isolation of DNA Fragments --- p.42 / Chapter H. --- Preparation of Reagents --- p.43 / Chapter 2.1.5 --- Data Analysis --- p.46 / Chapter 2.2 --- Effects of NPY on Cytosolic Calcium --- p.46 / Chapter 2.2.1 --- Tissue Culture --- p.47 / Chapter 2.2.2 --- Confocal Laser Scanning Microscopy --- p.47 / Chapter A. --- Materials --- p.47 / Chapter B. --- Loading of Dye --- p.48 / Chapter C. --- Cytosolic Calcium Measurement --- p.49 / Chapter D. --- Preparation of Reagents --- p.49 / Chapter Chapter 3 --- Results --- p.51 / Chapter 3.1 --- Studies on Islet Gene Expression --- p.51 / Chapter 3.1.1 --- Effect of NPY on Proglucagon Expression --- p.51 / Chapter A. --- Effect at 11 mM Glucose --- p.51 / Chapter B. --- Effect at 5 mM Glucose --- p.52 / Chapter 3.1.2 --- Effect of NPY on Proinsulin Expression --- p.52 / Chapter 3.1.3 --- "Effect of PYY, PP and FSK on Proglucagon Expression" --- p.53 / Chapter 3.2 --- Studies on Cytosolic Calcium --- p.65 / Chapter 3.2.1 --- Features of InRlG9 Cells --- p.65 / Chapter 3.2.2 --- Effect of NPY on Cellular Calcium Level --- p.66 / Chapter Chapter 4 --- Discussion --- p.77 / Chapter Chapter 5 --- References --- p.92

<p>The three evolutionarily related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are ligands to at least five G-protein coupled receptors in mammals, which are denoted by numbers. NPY has many physiological effects including stimulation of appetite and regulation of circadian rhythm and blood pressure. This work describes the ancient origin of the NPY receptor genes as deduced from molecular cloning of six receptors in four distantly related vertebrate species. Three of the receptors have been functionally expressed <i>in vitro</i> to determine ligand binding properties. </p><p>The first Y2 receptor from any non-mammalian species was cloned from the chicken. The receptor was found to exhibit substantial structural and pharmacological differences to mammalian Y2, but showed similar anatomical distribution. </p><p>A receptor was cloned in a primitive vertebrate, an agnathan fish, the river lamprey <i>Lampetra fluviatilis</i>. Phylogenetic analyses indicated that it represents an orthologue to the ancestor of Y4 and the teleost subtypes Yb and Yc. </p><p>Three NPY receptors were cloned from a shark, the spiny dogfish <i>Squalus acanthias</i>. These were found to correspond to the three mammalian subtypes Y1, Y4 and y6, and was thereby the first complete Y1 subfamily in any species outside the mammalian lineage. This suggests that all three receptor subtypes arose in the common ancestor of sharks and mammals 420-450 million years ago. </p><p>The sixth described receptor was cloned from the zebrafish, <i>Danio rerio</i>, and was shown to have equal identity to all three mammalian Y1 subfamily receptors. Phylogenetic analyses including the shark and lamprey sequences suggested that Yb may represent a fourth Y1 subfamily gene.</p><p>It has previously been found that the genes for Y1, Y4 and y6 are located on separate chromosomes. Taken together, these results show that the NPY receptor family expanded by chromosomal duplications early in vertebrate evolution, prior to the origin of gnathostomes. This work will be important for the determination of the time points for the origin of the many functions of NPY as well as for the understanding of the processes that shaped the vertebrate genome.</p>

The computational ability of principal neurons and interneurons in the brain and their ability to work together in concert are thought to underlie higher order cognitive processes such as learning, memory, and attention. Dendrites play a very important role in neuronal information processing because they receive and integrate incoming input and can undergo experience-dependent changes that will alter the future output of the neuron. Here, I have used whole-cell patch clamp recordings and fluorescent Ca2+-imaging to examine the modulation of dendritic excitability in principal neurons of the rat and human hippocampus and neocortex. First, I determined that dendrites of dentate granule cells of the hippocampus are tuned to high frequencies of both afferent input and backpropagating action potentials. Under these conditions they are also capable of generating regenerative dendritic activity that can propagate to the soma, which is prone to modulation. In particular, Neuropeptide Y (NPY) Y1 receptors can decrease frequency-dependent dendritic Ca2+ influx. Dopamine D1 receptors (D1Rs) have an opposite effect; they potentiate frequency-dependent dendritic excitability. These two neuromodulators also have an opposing effect on plasticity, with dopamine acting to induce, and NPY acting to inhibit long-term potentiation (LTP). Parallel observations of D1-induced LTP and an NPY-mediated decrease in dendritic excitability in rodents were complemented by findings in human dentate granule cells. Second, I examined the role of NPY receptors on dendrites of layer 5 pyramidal neurons. In these neurons I found that NPY acts post-synaptically on distal dendrites via the Y1 receptor to inhibit frequency-dependent Ca2+-currents, similar to the findings in dentate granule cells. NPY also decreased regenerative Ca2+ currents caused by the appropriate pairing of pre- and post-synaptic input. Together, these observations demonstrate that the role of NPY in the hippocampus and neocortex is not solely as an anti-epileptic agent. NPY release, likely to occur during high frequency oscillatory activity, can act locally to limit dendritic excitability, which can have a profound effect on plasticity. In the dentate gyrus, NPY can inhibit a D1R induced increased dendritic excitability and resultant changes in synaptic strength. These findings will further the understanding of dendritic information processing in the hippocampus and neocortex.

The three evolutionarily related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are ligands to at least five G-protein coupled receptors in mammals, which are denoted by numbers. NPY has many physiological effects including stimulation of appetite and regulation of circadian rhythm and blood pressure. This work describes the ancient origin of the NPY receptor genes as deduced from molecular cloning of six receptors in four distantly related vertebrate species. Three of the receptors have been functionally expressed in vitro to determine ligand binding properties. The first Y2 receptor from any non-mammalian species was cloned from the chicken. The receptor was found to exhibit substantial structural and pharmacological differences to mammalian Y2, but showed similar anatomical distribution. A receptor was cloned in a primitive vertebrate, an agnathan fish, the river lamprey Lampetra fluviatilis. Phylogenetic analyses indicated that it represents an orthologue to the ancestor of Y4 and the teleost subtypes Yb and Yc. Three NPY receptors were cloned from a shark, the spiny dogfish Squalus acanthias. These were found to correspond to the three mammalian subtypes Y1, Y4 and y6, and was thereby the first complete Y1 subfamily in any species outside the mammalian lineage. This suggests that all three receptor subtypes arose in the common ancestor of sharks and mammals 420-450 million years ago. The sixth described receptor was cloned from the zebrafish, Danio rerio, and was shown to have equal identity to all three mammalian Y1 subfamily receptors. Phylogenetic analyses including the shark and lamprey sequences suggested that Yb may represent a fourth Y1 subfamily gene. It has previously been found that the genes for Y1, Y4 and y6 are located on separate chromosomes. Taken together, these results show that the NPY receptor family expanded by chromosomal duplications early in vertebrate evolution, prior to the origin of gnathostomes. This work will be important for the determination of the time points for the origin of the many functions of NPY as well as for the understanding of the processes that shaped the vertebrate genome.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 261-288).