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Circadian rhythms in physiology and behavior are synchronized by a central pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus. Shift work, jet lag and sleep disorders can disrupt circadian rhythms, negatively impacting health and well-being. The SCN pacemaker resets rapidly in response to changes in the daily light cycle, however, adjustment of peripheral oscillators to changing time zones or work shifts is more gradual, leading to internal desynchrony. In addition, many diseases can impair the SCN’s ability to adjust to changes in the light cycle. My research investigated whether combined pharmacological inhibition of neuropeptide Y and serotonin could enhance resetting and attenuate transient cycles in locomotor activity following a sudden change in light exposure. I found that simultaneously blocking neuropeptide Y and serotonin receptors potentiated phase shifts during the late subjective night and significantly reduced transient cycles of locomotor activity in hamsters. Development of treatments that enhance the circadian system’s response to light may alleviate some of the negative health consequences experienced by travelers, shift workers and individuals with disease-related circadian desynchrony.

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / L'accouchement, l'aboutissement ultime de la grossesse, est un processus bien orchestré mettant en œuvre une pléiade de peptides produits par le placenta. Récemment, la mise en évidence d'une relation entre la durée de la grossesse et la concentration plasmatique du facteur de relâche de la corticostimuline (CRF) a permis de proposer un lien entre ce peptide et l'horloge placentaire déterminant la durée de la grossesse. Or, le neuropeptide Y (NPY), un peptide produit abondamment par le placenta tout au long de la grossesse, stimule in vitro la relâche du CRF par le syncytiotrophoblaste. Puisque ce syncytiotrophoblaste est connu pour être, du moins durant le troisième trimestre de la grossesse, la principale origine du CRF circulant, le but de cette thèse est d'étudier les modalités de cet effet du NPY. Les hypothèses principales ayant balisé cette étude ont été: 1) le syncytiotrophoblaste arbore des récepteurs pour le NPY; 2) un mode de relâche du CRF dépendant du calcium est présent dans le syncytiotrophoblaste et 3) la stimulation de la relâche du CRF par le NPY dans les cellules trophoblastiques implique des voies de signalisation en amont et en aval d'une hausse de la concentration du calcium intracellulaire. En premier lieu, étant donné la nature polaire du syncytiotrophoblaste, les études de liaison ont été effectuées en parallèle sur des membranes d'origine apicale (BBM) et basale (BPM) afin de déterminer s'il y a une ségrégation des sites de liaisons pour le NPY. Les résultats obtenus suggèrent l'existence d'une population mixte de sites de liaison du NPY (Y1 et Y3) se retrouvant exclusivement sur les BBM. Par la suite, les voies de signalisation associées aux récepteurs du NPY ont été explorées sur des préparations de BBM ou sur des cellules trophoblastiques en culture primaire. Les résultats obtenus montrent que dans les BBM, l'interaction du NPY avec le récepteur de sous-type Y1 est couplée à l'activation des phospholipases C-P (PLC-P), ces dernières étant responsables, en partie, d'une activation des protéines kinases C (PKCs). L'autre portion de l'activation de ces PKCs étant attribuable à l'activation des kinases de la position D3 des phosphoinositides (PI3-K). Les résultats obtenus à l'aide des cellules trophoblastiques montrent que le NPY entraîne l'activation de la kinase dépendante du calcium et de la calmoduline de type II (CaMK.11) et des MAP kinases de type ERKv2 (ERKv2). En troisième lieu, afin de vérifier lesquelles de ces voies de signalisation sont impliquées dans le contrôle de la relâche du CRF par le NPY, nous avons, dans un premier temps, caractérisé l'habileté des cellules trophoblastiques à sécréter le CRF. Cette étape importante montre que les cytotrophoblastes issus de placentas à terme, acquièrent, en parallèle avec leur différenciation vers le phénotype apparenté au syncytiotrophoblaste, l'habileté de sécréter le CRF. En dernier lieu, nous avons démontré que le NPY, en interagissant avec des récepteurs de type Y1, induit la synthèse et la relâche du CRF par les cellules trophoblastiques. L'augmentation de la synthèse est, en grande partie, attribuable à l'activation des PLC-P, la relâche subséquente du calcium des réserves intracellulaires et à l'activation de la CaMKII. L'augmentation de la relâche est subséquente à l'activation de PKCs indépendantes du calcium, à l'activation de l'axe initié par les PLC-P et à un influx calcique ne passant pas par les L-VOCC. Intéressement, l'activation directe des L­VOCC avec le Bay K8644, bien qu'entraînant l'activation des CaMKII, favorise la relâche du CRF sans influencer la synthèse du peptide. Pris dans leur globalité, ces résultats illustrent bien la pluralité de la signalisation des récepteurs de sous-type Y 1 et la complexité du contrôle de la relâche du CRF par le NPY. De plus, ces résultats sont une étape importante dans la compréhension des mécanismes qui permettent aux peptides interagissant avec un récepteur couplé aux protéines liant les nucléotides guanyliques (protéines G), sensibles à la toxine pertussique (PTX), de réguler la relâche du CRF.

Endocardial endothelial cells (EECs) can exert a significant influence on cardiac function by releasing various factors such as nitric oxide (NO), prostanoids, endothelin-1 (ET-1) and angiotensin II (Ang II). Recently, results obtained in our laboratory demonstrated the presence of NPY and its receptors, Y[subscript 1] and Y[subscript 2], as well as ET-1 and its receptors, ET[subscript A] and ET[subscript B], at the level of endocardial endothelial cells (EECs). We have also shown that NPY induces a sustained rise in the intracellular calcium level of these cells, and that only right ventricular EECs have the capacity of secreting NPY. Moreover, the evidence in the literature has become plentiful about complex interactions existing between ET-1 and other cardioactive mediators, such as NO and Ang II. Based on the above-mentioned data, the objective of this study was to investigate if a dialogue equally exists between the systems of NPY and ET-1 at the level of human right (hREECs) and left (hLEECs) ventricular EECs. Using the technique of indirect immunofluorescence coupled to 3-D confocal microscopy, as well as ELISA, our results show that increasing concentrations of NPY (10[superscript -15], 10[superscript -10] and 10[superscript -5]M) induce the release of ET-1 from REECs and LEECs in a time- and dose-dependent fashion. However, right ventricular EECs seem to have a higher ET-1 secretory capacity as compared to their left counterparts. Upon the use of selective antagonists for the NPY receptors, Y[subscript 1], Y[subscript 2] and Y[subscript 5], and the ET-1 receptors, ET[subscript A] and ET[subscript B], our results demonstrated that in REECs the NPY-induced release of ET-1 seems to be primarily due to Y[subscript 2] receptor activation, with the subsequent activation of the ET[subscript A] and ET[subscript B] receptors by the released ET-1. On the other hand, in LEECs, the NPY-evoked secretion of ET-1 seems to be mainly the result of Y[subscript 5] receptor activation by NPY. Unlike REECs, the ET-1 released by NPY in this type of cells does not seem to be contributing further to its own release by activation of its ET[subscript A] and ET[subscript B] receptors. Therefore, our results suggest that NPY is a regulator of ET-I secretion at the level of human EECs, and that this secretory process of ET-1 is different between the right and left ventricular cells. Moreover, these results serve to highlight and endorse the important sensory and tuning roles that right and left ventricular EECs possess, respectively. The ability of EECs to contribute to the local as well as systemic release of factors, such as NPY and ET-1, can affect not only the excitation-secretion coupling of EECs and the excitation-contraction coupling of cardiomyocytes, but also the physiological and pathophysiological state of the underlying, heart muscle.

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

In order to elucidate the effects of NPY directly on bone tissue, two different doses of NPY (NPY dose 1= 1X10-5 M and NPY dose 2 = 1X 10&amp / #65533 / 6 M) and NPY dose 2 plus its inhibitor were applied together with hyaluronic acid (HA) into the intramedullary area of right tibia of Wistar rats. HA alone was administered as the control group. On three time points, day one, week one and week two after administration, the tibiae were collected and stored at &amp / #65533 / 20oC for analysis. Evaluation was performed via conventional radiography, dual energy X-ray absorbtiometry (DEXA), quantitative computerized tomography (QCT), three point bending test (TPB) and histology techniques. QCT was used to assess both atomic content and density of both medulla and cortex of tibiae. From DEXA results, it was observed that inhibition of NPY causes an increase in the bone mass from first day to second week. This phenomena was also observed in histology results so that new bone formation in the inhibitor administered bone was encountered at week two. In both medulla and cortex areas&amp / #65533 / atomic content, an increase in average effective atomic number was displayed after administration of NPY plus NPY inhibitor throughout two weeks. In addition, density of medulla of tibiae measured by QCT also revealed an increase in bone mass when inhibitor is applied throughout two weeks. As a result, overall evaluation of data obtained from DEXA, QCT and histological analysis revealed that NPY inhibits bone formation or have a pro-osteoclastic effect / inversely HA displayed osteogenic effect.