Interdigestive myoelectric activity in rat intestine has been recorded and characterized. The interdigestive pattern of activity can be disrupted by oral glucose and high doses of the duodenal ulcerogen cysteamine. Intravenous glucose had no effect on the interdigestive myoelectric pattern, nor did high doses of porcine motilin. Attempts were made to develop a hybridoma cell line secreting antibodies that would recognize rat Intestinal immunoreactive motilin (IR-M). The murine myeloma cell line NS1 was fused with murine B-cells primed against porcine motilin. One hundred of the monoclonal cell lines produced secreted monoclonal antibodies that recognized porcine motilin. Attempts to identify a cell line secreting antibodies with the ability to stain rat intestinal tissue, however, produced only negative results. Rat intestinal IR-M has been characterized with respect to immunocytochemistry (ICC), radioimmunoassay (RIA), and chromatographic properties. The biological activity of partially purified rat intestinal IR-M has also been evaluated utilizing a rabbit isolated duodenal muscle strip preparation. Five different antisera and one monoclonal antibody directed against natural porcine motilin were utilized in an effort to detect IR-M containing cells in rat intestinal tissues. A variety of techniques were employed including tissue fixation with either Bouins, paraformaldehyde, or benzoquinone. In addition a variety of staining methods including, fluorescein conjugated second antibody, peroxidase-antiperoxidase or peroxidase conjugated second antibody techniques were used. All methods using these antibodies failed to detect IR-H in the rat small intestine. Porcine motilin was able to displace ¹²⁵I-motilin from antisera 13-3, 72X and M03. These antisera were utilized in a motilin RIA to evaluate acid extracts of rat intestinal tissue for IR-M. Only antisera 13-3 and 72X were capable of detecting IR-M in gut extracts, and these antisera gave different distributions of IR-M In the proximal small bowel. Rat intestinal tissue was extracted into 2% trifluoroacetic acid and the soluble fraction clarified by centrifugation. This acid extracted material was precipitated with sodium chloride then dissolved in methanol at pH 6.0. Methanol soluble material was precipitated with ether and the ether precipitate then dissolved in water and chromatographed on Sep-Pak C₁₈ cartridges (Waters). Sep-Pak cartridges were eluted with 50% acetonitrile: 0.1% TFA. The 50% eluate was then fractionated further using cation exchange, gel filtration and reverse phase high pressure liquid chromatography (HPLC). Rat intestinal IR-M peaks from cation exchange chromatography on SP-Sephadex-C25 (Pharmacia) were concentrated and examined for contractility in a rabbit duodenal muscle strip preparation. Purification after SP-Sephadex-C25 was approximately 20 fold. Desensitization of rabbit duodenum to porcine motilin could be demonstrated by pre-treatment with motilin. Contractile activity of partially purified rat intestinal IR-M was not inhibited by pretreatment with motilin. Chromatography on Bio-Gel P-10 (Biorad) eluted with 0.2M acetic HPLC, using a linear gradient of water/acetonitrile (10-45% acetonitrile in 30 min), rat intestinal IR-M did not co-elute with natural porcine motilin. In conclusion, the molecular weight of rat intestinal IR-M appeared to be similar to porci ne motilin as these two substances demonstrated co-elution on gel permeation chromatography. The lack of co-elution with porcine motilin on HPLC indicates that other molecular characteristics of rat intestinal IR-M, such as hydrophobicity, are not similar to porcine motilin. Furthermore, partially purified rat intestinal IR-M did induce a contractile response in rabbit duodenal muscle strips but porcine motilin did not desensitize this preparation to the contractile activity of rat intestinal extracts. This suggests that the contractile activity of these two compounds is induced via different receptor mechanisms. It is concluded that the immunoreactive motilin found in rat intestinal extracts does not resemble natural porcine motilin in structure or biological activity. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In press. Paper 5: Manuscript.

Made available in DSpace on 2014-06-11T19:33:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-14Bitstream added on 2014-06-13T19:44:13Z : No. of bitstreams: 1 pereira_jg_dr_jabo.pdf: 2012412 bytes, checksum: a771199b8c53c2ad3c164715147a19cf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O Kinosternon scorpioides é uma tartaruga de água doce conhecida como jurará. O objetivo do presente trabalho foi identificar serotonina, gastrina, enteroglucagon e motilina em esôfago, estômago e duodeno de K.scorpioides por meio da técnica de imunohistoquímica. Os resultados mostraram que as células imunorreativas à serotonina estavam presentes no epitélio na região caudal do esôfago, nas glândulas gástricas das regiões cárdica, fúndica e pilórica. As células produtoras de gastrina foram identificadas em grande quantidade entre as células da região pilórica e no epitélio de revestimento do duodeno, e em número reduzido na região cárdica. O enteroglucagon foi identificado na superfície dos cílios, no epitélio de revestimento da mucosa esofágica, nas fibras nervosas da lâmina própria e submucosa; no estômago estava localizado entre as células das glândulas cárdicas e fúndicas em número moderado, e aparentemente em maior quantidade nas pilóricas. No duodeno, o enteroglucagon foi identificado na superfície dos microvilos, no epitélio de revestimento, nas fibras nervosas da lâmina própria e submucosa. A motilina foi identificada apenas entre as fibras nervosas dos órgãos estudados. Em conclusão, as células imunorreativas à serotonina, gastrina, enteroglucagon e motilina foram identificadas em todos os órgãos estudados. Entretanto, nos diferentes segmentos houve diferença quanto à distribuição e frequência dessas células / Kinosternon scorpioides is a freshwater turtle known as “jurará”. The aim of this work was to identify peptides serotonin, enteroglucagon, gastrin and motilin in esophagus, stomach and duodenum by immunohistochemical techniques. The results showed that immunoreactive cells for serotonin were presented in the epthitelium of distal region of the esophagus, in glands of cardiac, fundic and pyloric region of the stomach and in the surface of duodenum epithelium. These cells were classified as open and closed type. The cells immunoreactive for gastrin were abundant in pyloric region and duodenum epitheliun, few numerous in cardiac region and absent in the esophagus. Cells rich in enteroglugagon were identified in the cilia surface, in the epithelium of esophagus, among nervous fibers of lamina propria and submucosa; in the stomach, these cells were observed in small number cardiac and fundic glands, however in pyloric region they were abundant. In duodenum, they were observed in microvillus surface, epithelium and nervous fibers of both lamina propria and submucosa. On the other hand, cells that produce motilin were observed just among lamina propia and submucosa. In conclusion, cells rich in serotonin, gastrin, enteroglugagon and motilin were found in all studied organs. However it was observed differences in the segments regarding distribution and frequency of these cells

Orientador: Célio Raimundo Machado / Banca: Ana Lucia Abreu-Silva / Banca: Cláudio César Fonseca / Banca: Márcia Rita Fernandes Machado / Banca: Nadia Delistoianov / Resumo: O Kinosternon scorpioides é uma tartaruga de água doce conhecida como jurará. O objetivo do presente trabalho foi identificar serotonina, gastrina, enteroglucagon e motilina em esôfago, estômago e duodeno de K.scorpioides por meio da técnica de imunohistoquímica. Os resultados mostraram que as células imunorreativas à serotonina estavam presentes no epitélio na região caudal do esôfago, nas glândulas gástricas das regiões cárdica, fúndica e pilórica. As células produtoras de gastrina foram identificadas em grande quantidade entre as células da região pilórica e no epitélio de revestimento do duodeno, e em número reduzido na região cárdica. O enteroglucagon foi identificado na superfície dos cílios, no epitélio de revestimento da mucosa esofágica, nas fibras nervosas da lâmina própria e submucosa; no estômago estava localizado entre as células das glândulas cárdicas e fúndicas em número moderado, e aparentemente em maior quantidade nas pilóricas. No duodeno, o enteroglucagon foi identificado na superfície dos microvilos, no epitélio de revestimento, nas fibras nervosas da lâmina própria e submucosa. A motilina foi identificada apenas entre as fibras nervosas dos órgãos estudados. Em conclusão, as células imunorreativas à serotonina, gastrina, enteroglucagon e motilina foram identificadas em todos os órgãos estudados. Entretanto, nos diferentes segmentos houve diferença quanto à distribuição e frequência dessas células / Abstract: Kinosternon scorpioides is a freshwater turtle known as "jurará". The aim of this work was to identify peptides serotonin, enteroglucagon, gastrin and motilin in esophagus, stomach and duodenum by immunohistochemical techniques. The results showed that immunoreactive cells for serotonin were presented in the epthitelium of distal region of the esophagus, in glands of cardiac, fundic and pyloric region of the stomach and in the surface of duodenum epithelium. These cells were classified as open and closed type. The cells immunoreactive for gastrin were abundant in pyloric region and duodenum epitheliun, few numerous in cardiac region and absent in the esophagus. Cells rich in enteroglugagon were identified in the cilia surface, in the epithelium of esophagus, among nervous fibers of lamina propria and submucosa; in the stomach, these cells were observed in small number cardiac and fundic glands, however in pyloric region they were abundant. In duodenum, they were observed in microvillus surface, epithelium and nervous fibers of both lamina propria and submucosa. On the other hand, cells that produce motilin were observed just among lamina propia and submucosa. In conclusion, cells rich in serotonin, gastrin, enteroglugagon and motilin were found in all studied organs. However it was observed differences in the segments regarding distribution and frequency of these cells / Doutor