Gene families in vertebrates often contain more dulicates (paralogs) than in invertebrates. This has been attributed to genome duplications, i.e., tetraploidizations. Two of the gene families that have expanded in vertebrate evolution are the neuropeptide Y (NPY) family of peptides and the neuropeptide Y receptors (NPYR) that are involved in many brain functions including appetite regulation. Two NPYR genes, Y2 and Y7, were cloned in the rainbow trout. Although they arose from a common ancestral gene in early vertebrate evolution, their ligand-binding properties are very similar. Two NPYR genes were cloned in the coelacanth Latimeria chalumnae and found to be orthologs of Y5 and Y6 discovered in mammals. Analyses of gene families close to the NPYR genes in the pufferfishes T. nigroviridis and T. rubripes showed that at least 25 additional gene families had an evolutionary history similar to the NPYR family, thereby providing evidence for fish specific-duplications of these chromosomes. Cloning and phylogenetic analysis of 22 NPYR gene fragments from several ray-finned fishes showed that basal species seem to have the same repertoire as tetrapods. Despite the tetraploidization in the teleost fish lineage, many teleosts seem to have fever genes than the gnathostome ancestor due to gene loss. Only one duplicate seems to have survived. The NPY peptide family was found to have expanded in the teleost tetraploidization with duplicates of both NPY and PYY (peptide YY) in some teleosts. Fourteen neighboring gene families were found to have evolved in a similar manner as the NPY-family genes. Positional information fascilitated orthology assignment of peptide genes in teleost fishes and allowed correction of previously misidentified genes. In summary, the evolutionary history of the NPY and NPYR gene families involve large-scale duplication events coinciding with the proposed tetraploidizations. The appearance of new genes in early vertebrates and in teleost fishes probably had important implications for the evolution of new functions in this system.

Biological function is mainly carried out by a dynamic population of proteins which may be used as markers for disease diagnosis, prognosis, and as a guide for effective treatment. In analogy to genomics, the study of proteins is called proteomics and it is generally performed by two-dimensional gel electrophoresis and mass spectrometric methods. However, gel based proteomics is methodologically restricted from the low mass region which includes important endogenous peptides. Furthermore, the study of endogenous peptides, peptidomics, is compromised by protein fragments produced post mortem during conventional sample handling. In this thesis nanoflow liquid chromatography and mass spectrometry have been used together with improved methods for sample preparation to semi-quantitatively monitor peptides in brain tissue. The proteolysis of proteins and rise of fragments in the low mass region was studied in a time-course study up to ten minutes, where a potential marker for sample quality was found. When rapidly denatured brain tissue was analyzed, the methods enabled detection of hundreds of peptides and identifications of several endogenous peptides not previously described in the literature. The identification process of endogenous peptides has been improved by creating small targeted sequence collections from existing databases. In applications of the MPTP model for Parkinson’s disease the protein and peptide expressions were compared to controls. Several proteins were significantly changed belonging to groups of mitochondrial, cytoskeletal, and vesicle associated proteins. In the peptidomic study, the levels of the small protein PEP-19 was found to be significantly decreased in the striatum of MPTP administered animals. Using imaging mass spectrometry the spatial distribution of PEP-19 was found to be predominant in the striatum and the levels were concordantly decreased in the parkinsonian tissue as verified by immunoblotting.

Two whole genome duplications (2R) occurred early in vertebrate evolution. By using combined information from phylogenetic analyses and chromosomal location of genes, this thesis delineates the evolutionary history of two receptor-ligand systems that expanded by these large scale events. A third whole genome duplication (3R) took place in the teleost fish lineage and has also contributed to the complexity of the gene families. New members of neuropeptide Y (NPY) peptide and receptor families were generated in 2R and 3R. Evolutionary comparisons show that the ancestral teleost fish had four peptides; subsequently, differential losses of the peptide genes occurred. In zebrafish the peptides and receptors display differences in tissue distribution and have  evolved binding preferences. In the frog Silurana tropicalis three peptides and six receptors werev identified, also displaying some differences in tissue distribution and receptor-ligand preferences. The findings in these experimental animals highlight both evolutionary conservation and lineage-specific features of the NPY system. The opioid system consists of four receptors and several peptides originating from four precursors. These results show that the receptor family was formed in 2R and 3R and that 2R together with one local duplication gave rise to the peptide family. The ancestral receptor and peptide genes were located on the same chromosome, suggesting coevolution. The Hox gene clusters, important in early development, provided the first strong evidence for 2R. Several neighboring gene families were analyzed and found to have expanded in 2R and 3R. In depth analyses of insulin-like growth factor binding protein (IGFBP) and voltage-gated sodium channel (SCN) gene families illustrates the importance of local duplications in combination with whole genome duplications in the formation of gene families. These findings provide additional strong evidence for two genome duplications in early vertebrate evolution and show that these events generated many new genes that could evolve new or more specialized functions.

During the past few decades, obesity has risen significantly in the United States with recent estimates showing that 65% of Americans are overweight and 30% are obese. This increase is a major cause for concern because obesity is linked to many secondary health consequences that include type II diabetes, heart disease, and cancer. Current approaches to the obesity problem primarily have focused on controls of food intake and have been largely unsuccessful. Food, however, almost always has to be acquired (foraging) and frequently is stored for later consumption (hoarding). Therefore, a more comprehensive approach that includes studying the underlying mechanisms in human foraging and food hoarding behaviors could provide an additional target for pharmaceutical or behavioral manipulations in the treatment and possibly prevention of obesity. Neuropeptide Y (NPY) is a particular peptide that provides a potent orexigenic drive to alter foraging, food hoarding (appetitive ingestive behaviors) and food intake (consummatory ingestive behaviors) in variety of species. NPY is predominantly produced in the arcuate nucleus of the hypothalamus (ARC) and has extensive efferent projections throughout the brain. Two target nuclei of ARC-NPY, the paraventricular nucleus of the hypothalamus (PVH) and perifornical area (PFA), have been shown to mediate the effect of NPY on food intake in laboratory rats and mice, but nothing is known about the effect of ARC-NPY on foraging and food hoarding. In addition, the action of specific NPY receptor subtypes within these two nuclei for these behaviors is unknown. Even though ARC-NPY is one of the main sources of input into the PVH and PFA, it is not known if this NPY fiber projection mediates alterations in appetitive and consummatory ingestive behaviors. Therefore, the purpose of this dissertation is to test 1) if NPY within the PVH or PFA controls appetitive, as well as, consummatory ingestive behaviors, 2) if NPY Y1 receptors within the PVH or PFA differentially control appetitive or consummatory ingestive behaviors, and 3) if NPY from the ARC is necessary for the control of appetitive and consummatory ingestive behaviors.

The mechanism of weight gain due to treatment with olanzapine, a serotonin receptor antagonist, has not been fully understood. Weight gain and food intake are under the control of neuropeptides/hormones, POMC (proopiomelanocortin), CART (cocaine and amphetamine regulated transcript), AgRP (Agouti-related peptide) and NPY (neuropeptide Y) that are synthesized and secreted from the arcuate nucleus (ARC) of hypothalamus. In this study, the altereration of the ARC neuropeptide/hormone levels both in humans and rats were determined as one of the weight gain mechanism. To examine olanzapine&rsquo / s weight gain effects, male first attack psychotic patients (pre-treatment), were hospitalized and treated for 4 -weeks (post-treatment), (n = 22), and healthy control group (n = 26) were included to the study. Case-control association design was used to analyze the changes in body mass index (BMI), peripheral leptin and the ARC neuropeptides levels. In patients, after 4-weeks of the olanzapine treatment / BMI and the waist circumference were significantly increased with average weight gain of 4.33 kg. In pre-treatment group, NPY levels were significantly lower while &alpha / -MSH, the anorexigenic product of POMC levels were significantly higher vs. control. At post-treatment, both leptin and NPY levels were significantly increased but the CART levels did not change. To further understand the underlying mechanism of olanzapine induced weight gain, the drug was orally administrated to 10 healthy male Wistar rats to analyze both the hypotalamic gene expression and peripheral levels of those candidate neuropeptides. In rats food consumption was increased and hypotalamic mRNA levels of NPY, AgRP and POMC were decreased while CART levels did not show any alteration. Consistent with the expression data, circulating levels of NPY, AgRP and &alpha / -MSH decreased significantly but CART levels were also reduced unexpectedly. In conclusion, it may be presumed that the antagonistic effect of olanzapine on the ARC neurons might be the basis for a disregulation of the neurohormones secretion which may cause weight gain in the treated psychotic patients.

No description available.

In this thesis, we have advanced a set of distinct bioinformatic and computational tools to address the structure and function of proteins. Using data mining of the protein data bank (PDB), we have collected statistics connecting the propensity between the protein sequence and the secondary structure. This new tool has enabled us to evaluate new structures as well as a family of structures. A comparison of the wild type staphylococcal nuclease to various mutants using the proposed tool has indicated long-range conformational deviations spatially distant from the mutation point. The energetics of protein unfolding has been studied in terms of the forces observed in molecular dynamics simulations. An adaptive integration of the steered molecular dynamics is proposed to reduce ground state dominance by the rare low energy trajectories on the estimated free energy profile. The proposed adaptive algorithm is utilized to reproduce the potential of mean force of the stretching of decaalanine in vacuum at lower computational cost. It is then used to construct the potential of mean force of this transition in solvent for the first time as to observe the hydration effect on the helix-coil transformation. Adaptive steered molecular dynamics is also implemented to obtain the free energy change during the unfolding of neuropeptide Y and to confirm that the monomeric form of neuropeptide Y adopts halical-hairpin like pancreatic-polypeptide fold.

The involvement of corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY) in the pathophysiology of anxiety and anxiety-related disorders is well established. The objective of this study is to explore the genetic variations in the CRH and NPY genes in a well-documented behavioral animal model, the vervet monkey (Chlorocebus aethiops sabaeus), in order to uncover a possible association between these polymorphisms and behavioral traits quantitatively extracted following analysis of social behavior and responses to novelty challenges. / The vervet CRH and NPY genes were amplified and sequenced; the priority was given to the regions expanding from -1kb upstream of the transcription initiation site (where most of the regulatory elements are found in both genes) through the second exon. / Polymorphism discovery analysis revealed the presence of 9 vervet CRH SNPs and 9 vervet NPY SNPs; the SNPs are relatively evenly distributed across the regions covered. An association between one intronic NPY SNP and "defensive aggression" was detected. / These results are coherent with other reports implicating NPY in defensive aggressive behavior, and support the notion that fear responses are fundamental behavioral traits for the dissection of anxiety.

Little is known regarding the neurophysiological mechanisms by which the neuropeptide prokineticin 2 (PK2) regulates circadian rhythms. Using whole-cell electrophysiology, we have investigated a potential role for regulation of neuronal excitability by PK2 on neurons of the area postrema (AP), a medullary structure known to influence autonomic processes in the central nervous system. In current-clamp recordings, focal application of 1µM PK2 reversibly influenced the excitability of the majority of dissociated AP cells tested, producing both depolarizations (38%) and hyperpolarizations (28%) in a concentration-dependent manner. Slow voltage ramps and ion substitution experiments revealed a PK2-induced Cl- current was responsible for membrane depolarization, while hyperpolarizations were the result of inhibition of an inwardly rectifying non-selective cation current. In contrast to these differential effects on membrane potential, nearly all neurons that displayed spontaneous activity responded to PK2 with a decrease in spike frequency. These observations are in accordance with voltage-clamp experiments showing that PK2 caused a leftward shift in Na+ channel activation and inactivation gating. Lastly, using post hoc single cell RT-PCR technology, we have shown that 7 out of 10 AP neurons depolarized by PK2 were enkephalin-expressing cells. The observed actions on enkephalin neurons indicate PK2 may have specific inhibitory actions on this population of neurons in the AP acting to reduce their sensitivity to incoming signals. These data suggest that PK2 regulates the level of AP neuronal excitability and may impart a circadian influence on AP autonomic control. / Thesis (Master, Physiology) -- Queen's University, 2009-08-18 11:18:05.977

Social insects have complex forms of social organization. Molecular mechanisms involved in the regulation of their reproduction are not fully understood. This dissertation investigated the vitellogenin receptor (VgR), short neuropeptide F (sNPF) receptor, and two insulin receptors (InRs) in the red imported fire ant Solenopsis invicta, focusing on their possible roles in the regulation of queen reproduction. Knowledge of these receptors may provide novel ways to manipulate either reproductive castes or overall reproductive outcome, diminishing the fire ant impact as invasive pest. Fire ant virgin queens have more abundant VgR (SiVgR) transcripts than newly-mated queens, but limited egg formation. To elucidate whether queen maturation involved changes in SiVgR expression, we investigated both virgin and mated queens. In both queens, immunofluorescence analysis of ovaries revealed differential SiVgR localization in early and late stage oocytes; however, mated queens showed higher SiVgR expression than virgin queens. In virgin queens, the SiVgR signal was first observed at the oocyte membrane beginning at day 12 post-emergence, coinciding with the maturation period required before a mating flight. SiVgR silencing in virgins through RNA interference abolished egg formation, demonstrating that SiVgR is involved in queen ovarian development pre-mating. The sNPF and insulin signaling pathways have been implicated in the regulation of food intake and body size, and these peptides also play a gonadotropic role in the ovaries of some insect species. To elucidate the sites of action of the sNPF peptide(s), the sNPF receptor tissue expression and cellular localization were analyzed in the queen brain, subesophageal ganglion (SEG), and ovaries by immunofluorescence. Results suggest that the sNPF signaling cascade may be involved in diverse functions, and the sNPF peptide(s) may act in the brain and SEG as neurotransmitter(s) or neuromodulator(s), and in the ovaries as neurohormone(s). In addition, to elucidate the role of insulin signaling pathway in the fire ant, two putative InRs were cloned. Transcriptional expression analyses show that the receptor abundance was negatively correlated with body size and nutrition status in fire ant immatures. In queens, the expression of InRs in different queen tissues correlates with tissue requirements for queen reproductive physiology and behaviors.