Orientador: Maria Emilia Franco Oliveira / Coorientador: Pedro Paulo Maia Teixeira / Coorientador: Fábio Morato Monteiro / Banca: Pietro Sampaio Baruselli / Banca: Lindsay Unno Gimenes / Resumo: Devido a comum ocorrência de regressão prematura (RP) do corpo lúteo (CL) em novilhas após primeira ovulação (OV), os objetivos do estudo foram: 1) acompanhar a dinâmica lútea após indução da OV em novilhas Nelore pré-púberes e 2) determinar diferenças relacionáveis a funcionalidade dessa estrutura. Cinquenta e sete fêmeas (289,61±32,28 kg, ECC de 5,66±0,65 e 17,47±0,81 meses de idade) foram divididas em dois grupos de tratamento para indução da OV. No grupo GP4+GnRH foi utilizado dispositivo intravaginal de progesterona (P4) de 3º uso por 10 dias e, 48 horas após remoção, aplicado 0,02mg de acetato de buserelina (GnRH), e no grupo GGnRH foi utilizado somente o GnRH. Os CLs formados foram acompanhados pela ultrassonografia a cada dois dias até a sua regressão funcional (diminuição do sinal vascular do Doppler colorido e concentrações de P4 abaixo de 1 ng/mL), sendo determinado para cada dia o diâmetro, área, valores numéricos (VPN) e heterogeneidade dos pixels e percentual (%) de vascularização. A velocidade do pico sistólico, velocidade diastólica final, índice de resistência e o índice de pulsatilidade (IP) da artéria ovariana também foram determinados para cada avaliação, além da concentração sérica de P4. Essas características foram comparadas entre os tratamentos, funções dos CLs (duração normal ou regredido prematuramente), dias das avaliações e suas interações, utilizando o procedimento MIXED do programa SAS (p≤0,05). Três animais de cada tratamento não responderam ao ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the common occurrence of premature regression (PR) of the corpus luteum (CL) in heifers after the first ovulation (OV), the aims of this study were to: 1) monitor the luteal dynamics after OV induction in prepubertal Nellore heifers, and 2) determine differences related to the functionality of this structure. Fifty-seven females (BW 289.61±32.28 kg, BCS 5.66±0.65 and 17.47±0.81 months old) were divided into two treatment groups for OV induction. In the group GP4+GnRH, an intravaginal progesterone (P4) device of 3rd use was used for 10 days and, 48 hours after its removal 0.02 mg of buserelin acetate (GnRH) was applied, and in the GGnRH group only GnRH was used. Formed CLs were monitored via ultrasonography every two days until functional regression (decrease of the vascular signal of color Doppler and serum P4 concentrations below 1 ng/mL), being determined for each day the diameter, area, numerical values (NV) and heterogeneity of the pixels, and vascularization percentage (%). The systolic and diastolic peak velocity, resistance and pulsatility index (PI) of the ovarian artery were also determined for each day in addition to the serum P4 concentration. These characteristics were compared between treatments, CLs functions (normal duration or prematurely regressed), days of evaluations and their interactions, using the MIXED procedure of SAS program (p≤0.05). Three animals from each treatment did not respond to the OV inductor (6/57=11%), which determined an ovulation ... (Complete abstract click electronic access below) / Mestre

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism. / Science, Faculty of / Mathematics, Department of / Graduate

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) and the active ingredient in Prozac®, is found in the environment and disrupts feeding and metabolism in exposed fish. The objective of this research was to investigate the mechanisms involved in the feeding and metabolism disruption in the model goldfish (Carassius auratus). Two short-term waterborne fluoxetine exposures (7- and 14-days) were performed using two environmentally relevant doses of fluoxetine (0.5 and 1 μg/L) and metabolic effects at the level of the brain, liver, serum and bile in goldfish were investigated. Abundances of mRNA transcripts coding for six feeding neuropeptides were examined to determine which may be involved in the initial neural changes associated with decreased appetite in goldfish. The 7-day fluoxetine exposure at 1 μg/L caused corticotropin-releasing factor (CRF) mRNA levels to increase by 2-fold in female hypothalamus and telencephalon, indicating that CRF may be one of the first of the feeding neuropeptides to be altered. Six hepatic miRNAs were also evaluated in the goldfish liver that were previously associated with fluoxetine exposure in zebrafish (Danio rerio). Following the 7-day exposure at 1 μg/L, miR-22b, miR-140, miR-210, miR-301a and miR-457b levels increased in the female goldfish liver by 4-6 fold. The 14-day fluoxetine exposure at 1 μg/L caused 2-fold increases in miR-210, miR-301a, miR-457b and let-7d in male goldfish liver. These miRNAs were associated with the down-regulation of anabolic metabolic pathways in zebrafish, indicating a conservation of miRNA and fluoxetine effect between fish species. Serum and bile metabolite profiles of fluoxetine exposed goldfish were evaluated using ultra performance liquid chromatography coupled to quadrupole time of flight mass spectrometry. Following the 14-day exposure at 1 μg/L, the bile metabolite profiles of male goldfish were significantly different from controls as detected by cluster analysis and fluoxetine was tentatively identified in the serum. No other discriminant metabolites were identified as of yet. The data presented suggest that fluoxetine causes metabolic disruption in goldfish at multiple organ levels. Because of the widespread detection of fluoxetine and other emerging SSRIs in the aquatic environment, future research is required to firmly establish this pharmaceutical class as a metabolic and endocrine disrupting chemical.

Prolactin-releasing peptide (PrRP) is a member of the family of RF-amide peptides. These peptides have typical C-terminal sequence -Arg-Phe-NH2 and similar biological effects. PrRP was discovered as an endogenous ligand of an orphan receptor GPR10 while searching for a factor responsible for a prolactin secretion. This effect was not later confirmed and nowadays, PrRP is mainly considered as an anorexigenic peptide. This is supported by a fact that PrRP and GPR10 deficient mice suffer from hyperphagia and late-onset obesity. Besides GPR10, PrRP is bound to NPFF2 receptor whose endogenous ligand is neuropeptide FF (NPFF). In this study, the PrRP's analogues modified at the N-terminus with fatty acids of different lenghts were tested in vitro on binding and activation MAPK/ERK1/2 signalling pathway. In in vivo experiments on food intake, the central anorexigenic effects of lipidized PrRP-analogues were tested provided their crossing blood brain barrier. Binding studies showed that all analogues bound to rat pituitary RC-4B/C cells with high affinity, analogues containing fatty acid with Ki of one order of magnitude lower than native PrRP. High affinity was also confirmed for binding to cells overexpressing GPR10 receptor and cell membranes with overexpressed NPFF2 receptor. All tested analogues...

The hypothalamic hormones, thyrotrophin-releasing hormone (TRH) and gonadotrophin-releasing hormone (GnRH), play pivotal roles in the growth and sexual maturation of chickens. In chickens, TRH regulates the release and synthesis of thyrotrophin (TSH) and also acts as a growth hormone-releasing factor. GnRH stimulates the release and synthesis of gonadotrophins (LH and FSH). TRH and GnRH are released and stored in the median eminence, and both hormones are transported into the pituitary gland via the hypophysial portal circulation. TRH and GnRH exert their physiological functions by binding to their specific receptors (TRH receptor and GnRH receptor, respectively) on the surface of cells in the pituitary gland. The activated receptors couple to guanine nucleotide-binding regulatory proteins (G proteins), Gq and/or G11, which in turn triggers the secondary messenger [1,2- diacylglycerol (DAG) and inositoltrisphosphate (IP3)] signalling cascade. The signalling generates the physiological effects of the hormones. The TRH-R and GnRH-R are members of G-protein coupled receptor (GPCR) family. The objective of this thesis was to clone and characterise the chicken TRH and GnRH receptors as useful tools for investigating the regulatory roles of TRH and GnRH receptors in the growth and sexual maturation of chickens. In addition, sequence information of the receptors would potentially assist in elucidating the binding sites and the molecular nature of the processes involved in receptor activation.

Cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species and a decreased expression of enzymatic antioxidants. Prodrugs inducing additional oxidative stress can selectively induce apoptosis in cancer cells already having a high level of intrinsic oxidative stress. This study focused on the rational design and in vitro evaluation of a novel prodrug ester, (4- [(nitrooxy)methyl]phenyl-N-acetyl-L-alaninate or NPAA) activated by acylamino-acidreleasing enzyme (AARE, EC 3.4.19.1) to yield a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. NPAA shares structural features with both nitric oxide donating aspirin (NO-ASA), a wellcharacterized QM releasing anticancer prodrug, and N-acetyl-L-alanine-4-nitroanailide (AANA), a known specific substrate for AARE. AARE is a serine peptidase that is overexpressed in some tumors and cancer cell lines. The overall approach was to first predict the 3-dimensional structure of both rat (rAARE) and human AARE (hAARE) and then use the resulting low-resolution models to determine if NPAA was a plausible prodrug by estimating its affinity to hAARE and rAARE in comparison to AANA. The AARE models were constructed using a bioinformatic-based protein structure prediction webserver (I-TASSER) followed by energy minimization and refinement. The resulting models were subjected to a variety of structural quality assessments. The optimal models of hAARE and rAARE were found to have similar three-dimensional structures with a ß- propeller domain and an a/ß-hydrolase domain containing an exopeptidase catalytic site with active site residue distances typically found in serine peptidases. Protein-ligand docking studies showed that both AANA and NPAA could bind to the exopeptidase catalytic site of the hAARE and rAARE models with reasonable affinities and in a region with a highly druggable pocket. In order to validate the in silico results, NPAA was synthesized, purified, physically characterized and evaluated for its in vitro ability to deplete GSH in the presence of rAARE. As anticipated, NPAA was found to deplete GSH and this effect was completely blocked by diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, including rAARE. These studies support further efforts to optimize the design of QM releasing anticancer prodrugs targeted to AARE. Moreover, the molecular models presented here could be useful for the rational design of AARE inhibitors, which could also be exploited as potential anticancer agents.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.

No description available.

Please read the abstract in the section 00front of this document / Thesis (DPhil (Psychiatry))--University of Pretoria, 2003. / Psychiatry / unrestricted

Despite the reputation of carbon monoxide (CO) as a silent killer, new evidence suggests that this gaseous molecule has anti-inflammatory, anti-cancer and vasoprotective properties. Unfortunately, little is known about the role of CO in the body. However, proteins present in mitochondria are believed to be important targets. We previously synthesized a class of novel and structurally modifiable flavonol-based CO-releasing molecules (CORMs). Flavonols are commonly found in fruits and vegetables. The base structure, Flav-1, is fluorescent, exhibits low toxicity, and releases CO after exposure to visible light. Previous reports indicate that addition of a triphenylphosphonium (TPP) tail allows chemical structures to enter mitochondria. We hypothesized that addition of a TPP tail of two or eight carbons in length to Flav-1 (Mito-Flav-C2 or -C8) would facilitate targeting of mitochondria, and thus, localized light-induced CO release. Toxicity of these TPP-tailed molecules was determined in human umbilical vein endothelial cells (HUVECs) and lung epithelial carcinoma cells (A549), using standard cell viability assays. Evaluation of toxicity using the MTT assay revealed lower toxicity of Flav-1 in HUVECs compared to A549 cells, but addition of the TPP tails increased toxicity in both cell lines. However, unlike the MTT assay, Flav-1 with and without tails had similar toxicity when measured in HUVECs by the lactate dehydrogenase assay. Photo-degradation experiments were performed by exposing cells until their light emission was undetectable by using lasers in the near-UV and within the visible light spectrum. Localization of the compounds was observed using a confocal microscope by co-staining with MitoTracker Red (MTR) and Hoechst to visualize the mitochondria and nucleus, respectively. Fluorescence microscopy images of cells treated with Mito-Flav-C2 or -C8 revealed an increase in uptake, compared to Flav-1, plus co-localization with MTR, which suggests mitochondrial localization. A549 cells exposed to laser light or a full spectrum of light lost all fluorescence of Mito-Flav-C2, which indicates CO release. This work highlights the successful synthesis of the first mitochondria targeting CORMs, and that CO release is achievable using different light sources. Moreover, these TPP-tailed CORMs will allow for controlled and localized release of CO to further study its physiological targets.