Thèse (Ph. D.)--Université Laval, 2008. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). "June 2006." Includes bibliographical references (leaves 67-75). Also available via the World Wide Web.

nÃo hà / As taxas de morbimortalidade se elevam quando a diarreia està associada à desnutriÃÃo. Entretanto, os mecanismos pelos quais as deficiÃncias nutricionais afetam o intestino sÃo em grande parte desconhecidos. O objetivo desse trabalho foi avaliar alteraÃÃes morfomÃtricas, nas proteÃnas transportadoras de substratos e no transporte intestinal de eletrÃlitos e Ãgua em modelo de desnutriÃÃo em camundongos. Objetivamos ainda, analisar o efeito da toxina do cÃlera (TC) associada ou nÃo à desnutriÃÃo, sobre as proteÃnas transportadoras de substratos, sobre o transporte hidroeletrolÃtico em camundongos e por fim, avaliamos os efeitos de soluÃÃes de reidrataÃÃo oral (SRO) da OMS (SGli) e modificadas com glutamina (SGln), alanil-glutamina(SAla-Gln) e arginina (SArg) nesse transporte. Camundongos (n=20) receberam por 7 dias uma raÃÃo deficiente em proteÃnas, gorduras e minerais (DBR). Segmentos de Ãleo foram obtidos antes e no 7 dia da dieta, para estudos de morfometria, imunohistoquÃmica para as proteÃnas: SGTL-1, PepT-1, CAT-1 e SN-2 e avaliaÃÃo por RT-qPCR da expressÃo RNAm dessas proteÃnas transportadoras. O modelo de perfusÃo intestinal por 75 min em camundongos (n=6) foi utilizado para avaliar o transporte intestinal de Ãgua e eletrÃlitos e para avaliar o papel de soluÃÃes de reidrataÃÃo oral em camundongos nutridos e desnutridos expostos ou nÃo à toxina do cÃlera (1Âg/ml). Animais desnutridos apresentaram perda ponderal, atrofia dos vilos e reduÃÃo na expressÃo por imunofluorescÃncia da SGTL-1. A desnutriÃÃo causou ainda reduÃÃo na expressÃo do RNAm da SGTL-1 e PEPT-1 e aumento na expressÃo do RNAm para o SN-2 no ileo de camundongos. No modelo de perfusÃo intestinal, a desnutriÃÃo aguda aumentou a secreÃÃo intestinal de eletrÃlitos e Ãgua. A TC aumentou a secreÃÃo de eletrÃlitos e Ãgua em modelo de perfusÃo intestinal de camundongos. A TC aumentou a transcriÃÃo para o RNAm dos transportadores intestinais SGTL-1, PEPT-1 e CAT-1, mas nÃo aumentou a transcriÃÃo para o SN-2. As soluÃÃes de reidrataÃÃo com glicose (SGli), glutamina (SGln), alanil-glutamina (SAla-Gln) e arginina (SArg) diminuÃram a secreÃÃo de eletrÃlitos induzida pela TC. Apenas a SGln nÃo conseguiu diminuir significativamente a secreÃÃo de Ãgua induzida pela TC. Apenas a SGli reduziu a secreÃÃo de Ãgua induzida pela TC. SGli, SAla-Gln e SArg, mas nÃo SGln, diminuÃram a secreÃÃo de sÃdio e cloreto induzida pela TC. A desnutriÃÃo associada à diarreia pela TC causou reduÃÃo na transcriÃÃo para o RNAm dos transportadores intestinais SGTL-1, PEPT-1, CAT-1 e SN-2. A desnutriÃÃo associada à TC aumentou a secreÃÃo de Ãgua quando comparado ao grupo nutrido exposto à TC. SGli, SAla-Gln e SArg, mas nÃo SGln, diminuÃram a secreÃÃo de Ãgua induzida pela desnutriÃÃo associada à TC. Todas as soluÃÃes diminuÃram a secreÃÃo de sÃdio e cloreto induzida pela desnutriÃÃo associada à TC. / The morbidity and mortality rates rise when diarrhea is associated with malnutrition. However, the mechanisms by which nutritional deficiencies affect the gut are largely unknown. The aim of this study was to evaluate morphological changes in transport proteins and substrates in intestinal transport of electrolytes and water in malnutrition model in mice. We aim to further analyze the effect of cholera toxin (CT) with or without malnutrition on the carrier proteins substrates on electrolyte transport in mice and finally, we evaluate the effects of oral rehydration solutions (ORS) of OMS (SGli) e modified with glutamine (SGln), alanyl-glutamine (SAla-Gln) and arginine (SArg) in this transport. Mice (n=20) received for 7 days a diet deficient in protein, fat and minerals (DBR). Segments of ileum were obtained before and on day 7 of the diet, for studies of morphology, immunohistochemistry for proteins: SGTL-1, PEPT-1, CAT-1 and SN-2 and evaluated by RT-qPCR of mRNA expression of these transport proteins. The model of intestinal perfusion for 75 min in mice (n=6) was used to evaluate the intestinal transport of water and electrolytes and to evaluate the role of oral rehydration solutions in mice exposed nourished and malnourished or without cholera toxin (1Âg/ml). Malnourished animals showed weight loss, atrophy of the villi and reduced expression by immunofluorescence of SGTL-1. Malnutrition also caused a reduction in mRNA expression SGTL-1 and PEPT-1 and increased mRNA expression for SN-2 in mice ileum. In the model of intestinal perfusion, acute malnutrition increased intestinal secretion of electrolytes and water. CT increased the secretion of electrolytes and water into the intestinal perfusion model mice. SGli, SGln, SAla-Gln and SArg decreased secretion of electrolytes induced by CT. Just SGln failed to significantly decrease water secretion induced by CT. CT increased mRNA transcription for intestinal transporters SGTL-1, PEPT-1 and CAT-1 but not to increased transcription SN -2. Only SGli reduced water secretion induced by CT. SGli, SAla-Gln and SArg, but not SGln , decreased secretion of sodium and chloride induced by CT. The malnutrition associated with diarrhea caused by TC reduction in the transcription of mRNA for intestinal transporters SGT -1, PEPT-1, CAT-1 and SN-2. Malnutrition associated with CT increased the secretion of water when compared to the group fed exposed to TC. SGli , SAla-Gln and SArg, but not SGln, decreased water secretion induced by malnutrition associated with TC. All solutions decreased secretion of sodium and chlorid induced malnutrition associated with TC.

Introduction: Impairment of cytosolic calcium ([Ca2+]i) signaling and in particular calcium overload has emerged as a possible unifying mechanism for precipitating acute pancreatitis (AP.) In the L-arginine (L-arg) experimental model of AP, nitric oxide (NO) has been implicated however the disease progression is largely unaffected by nitric oxide synthase (NOS) inhibitors (8). Additionally, L-ornithine (L-orn), a NOS-independent metabolite of L-arg, has been shown to be potent at inducing AP (28). Both L-arg and L-orn activate calcium-sensing like receptors (CaSR) (31) such as the GPRC6a which may be responsible for initiating the [Ca2+]i overload. The aim of this study is to investigate the effects of L-arg and L-orn on pancreatic acinar cells that maybe linked to the pathophysiology of AP. Furthermore to provide an alternative theory to the NO mediated ones, in particular that L-arg induces toxic changes in [Ca2+]i via a GPRC6a like receptor. Methods: Whole pancreata were harvested from male Sprague Dawley rats. Pancreatic acinar cells were isolated by collagenase digestion. [Ca2+]i was measured using fura-2 imaging, and cell viability assessed using physiological CCK. Oxidative stress was measured using dichlorofluorescein (DCF) and cell death was quantified using trypan blue exclusion. Results: L-arg and L-orn (100mM) induced spike-like, reversible increases in [Ca2+]i in 46% and 74% of cells and Ca2+ overload in 11% and 26% respectively. At 500 mM both induced Ca2+ overload in all cells however this was also seen with the osmotic control, mannitol. Isosmotic L-arg and L-orn (100mM) induced only reversible increases in [Ca2+]i. Neither L-arg nor L-orn had significant effects on CCK-evoked [Ca2+]i oscillations. Both L-arg and L-orn induced significant oxidative stress responses (22% and 37% of a maximum response seen with 3mM H202, respectively). Both L-arg and L-orn caused cell death in 76% +/- 4 and 89% +/- 7 at 3 hours respectively, compared to 35% +/- 4 and 40% +/- 3 with controls (Hepes, Glycine). Conclusion: The data suggests that the L-arg and L-orn causes significant increase in oxidative stress and cell death. The data suggests that although changes in [Ca2+]i were induced by both L-arg and L-orn the large concentrations used experimentally are likely to induce significant osmotic effects.

Orientadores: José Roberto Lovadino, Debora Alves Nunes Leite Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T00:14:41Z (GMT). No. of bitstreams: 1 VieiraJunior_WaldemirFrancisco_M.pdf: 2625418 bytes, checksum: 9b4424cf9945e3e37e733389e7cacc32 (MD5) Previous issue date: 2015 / Resumo: Objetivo: Avaliar o efeito de dentifrícios com diferentes princípios ativos aplicados previamente ao clareamento dental com peróxido de hidrogênio 35% (PH) nas propriedades físicas e cromáticas do esmalte, conteúdo mineral e na morfologia superficial. Métodos: Blocos de esmalte bovino foram submetidos a protocolos de tratamento na máquina simuladora de escovação (n = 10): 1) escovação com água destilada e exposição ao gel placebo (PLA - controle negativo) ou 2) gel clareador (PH - controle positivo), escovação com diferentes dentifrícios previamente ao clareamento com PH, sendo: 3) dentifrício com nitrato de potássio e fluoreto de sódio (PN), 4) com monoflúorfosfato de sódio (FT), à base de 5) arginina 8%, Pro-Argin¿ (PA), 6) arginina 1,5%, Neutraçúcar¿ (SAN), ou 7) com vidro bioativo NovaMin¿ (NM). No capítulo 1 foi realizada nos tempos inicial e final a análise da rugosidade superficial (Ra) e cor pelo sistema Cie L*a*b* (?E, ?L, ?a e ?b). No tempo final foi avaliada a microdureza superficial (SMH) e a microdureza em profundidade (CSMH - 10, 25, 50, 75 e 100?m). No capítulo 2, a concentração iônica de fósforo no gel [P] foi analisada e determinada o nível elementar (%) de Ca, Na, P e proporção entre Ca / P por espectroscopia de energia dispersiva por raios-x (EDS) e a morfologia de superfície por microscopia eletrônica de varredura (MEV). Os dados foram submetidos a teste Proc-Mixed de medidas repetidas (Ra), ANOVA (SMH, Nível elementar, [P] e Cor), ANOVA de parcelas subdivididas (CSMH) e teste de Tukey (p<0,05). Resultados: O clareamento dental (PH) diminuiu os valores de SMH e CSMH, entretanto os dentifrícios diminuíram o efeito da perda de CSMH e aumentaram a SMH (NM > PA = SAN > outros grupos). O clareamento aumentou a Ra em comparação ao PLA, todos os grupos tiveram aumento da Ra, exceto NM que não diferiu de PLA. Os valores de ?E, ?L e ?b não diferiram entre os grupos experimentais e o controle positivo (PH). O grupo PH aumentou a perda de fósforo [P], entretanto NM não diferiu de PLA. PH diminuiu os valores de % Ca em comparação PLA, no entanto PA não diferiu de PLA. MEV, apenas os grupos PH e FT demonstraram alteração de morfologia. Conclusão: O clareamento dental alterou as propriedades e conteúdo mineral do esmalte, entretanto a utilização prévia de dentifrícios preveniu os efeitos negativos, sem interferir na eficácia do tratamento. O dentifrício com vidro bioativo demonstrou potencial efeito benéfico na terapia clareadora / Abstract: Objective: Evaluate the effect of toothpastes with different active agents applied prior to dental bleaching with 35% hydrogen peroxide on enamel properties, whiteness effectiveness, enamel mineral content and morphology surface. Methods: 70 enamel blocks (4x4x2 mm) were submitted to in vitro treatment protocols in tooth-brushing machine (n=10): 1) with distilled water and exposure to placebo gel (PLA) or 2) bleaching with 35% hydrogen peroxide (HP); brushing with different toothpaste was performed prior the bleaching treatment (HP), being: 3) potassium nitrate toothpaste (PN) containing NaF, 4) conventional MFP fluoridated toothpaste (FT), 5) arginine-carbonate (8% arginine) based toothpaste (PA), 6) arginine-carbonate (1.5% arginine) based toothpaste (SAN) and 7) toothpaste containing bioactive glass (NM). In chapter 1, the color changes were characterized using the Cie L*a*b* system (?E, ?L, ?a and ?b) and roughness (Ra) analysis was performed before and after treatments, surface microhardness (SMH) and cross- sectional microhardness (CSMH) were analyzed after treatments. In chapter 2, the phosphorus concentration in gel ([P]) was performed and the elemental levels (%) of Ca, Na, P and proportion between Ca/P were determined by Energy Dispersive X- ray Spectrometer (EDS) and morphology surface by Scanning Electron Microscopy (SEM). Data were analyzed PROC MIXED (Ra), ANOVA (SMH, %, [P], ?E, ?L, ?a and ?b), ANOVA split plot (CSMH) and Tukey's test post hoc (p<0.05). Results: ?E, ?L and ?b were statically similarity in all bleached groups. After bleaching, SMH and CSMH decreased in HP and increased signi?cantly in the treatment groups (SMH) versus the negative and positive control (NM > PA = SAN > all others groups) or decreased HP effects (CSMH). Ra increased in all bleached groups, with exception in NM that did not differ to PLA. HP increased the [P] loss, however only NM group application prior to HP did not differ of PLA. EDS analysis showed that HP decreased the % Ca values differing to PLA, the % P was increased in bleaching groups, however the PA group did not differ to PLA. SEM analysis presented the decrease of demineralization effect in groups submitted the toothpaste application prior to HP, with exception the HP and MFP group that demonstrated morphology alteration. Conclusion: Dental bleaching affect the enamel properties and mineral content however toothpastes showed a potential effect in reducing the bleaching effects on the enamel properties without influence on the whiteness effectiveness. Furthermore, the toothpaste containing bioactive glass showed potential beneficial effect for bleaching therapy / Mestrado / Dentística / Mestre em Clínica Odontológica

No description available.

Cyclic-di-GMP, a common bacterial second messenger, has been thought to help develop virulence and biofilms in bacteria, most specifically in Salmonella Typhimurium. By being able to dysregulate cyclic-di-GMP production, virulence may be better combatted. STM1987, an L-arginine-responsive diguanylate cyclase with a periplasmic sensory domain, dimerizes and generates the bacterial second messenger cyclic-di-GMP in response to the amino acid L-arginine in a pathway that also requires the periplasmic L-arginine-binding protein ArtI. Their biochemical responses to L-arginine and when they dimerize could help clarify this pathway, so I sought to develop a periplasmic dimerization sensor to better monitor these biochemical interactions. Similar to STM1987, the ToxR transcriptional regulator from Vibrio cholera is also activated by dimerization. By switching out the periplasmic domain of ToxR for the periplasmic regions of interest, I can better evaluate the cyclic-di-GMP response to L-arginine. This research aims to find the specific responses in this pathway to be able to use this in combatting bacterial virulence. I was able to successfully show that the STM1987 periplasmic domain dimerizes in response to L-arginine, providing an important insight into this signaling pathway.

Agmatine, an endogenous amine derived from decarboxylation of l-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study, we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to 2 h immobilization stress daily for 7 days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with β-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50 mg/kg/day), i.p.). Likewise, endogenous agmatine levels measured by high-performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92 to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that the administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism.

OBJECTIVE - Exercise training has been shown to restore vasodilation to nitric oxide synthase (NOS) activation in arterioles distal to coronary artery occlusion. Because reactive oxygen species are generated during NOS uncoupling and the production of vasodilator H2O2 is increased during exercise in patients with coronary disease, we proposed that H2O2 may contribute to the restoration of vasodilation in porcine coronary occlusion model. METHODS AND RESULTS - Left circumflex (LCX) coronary artery of miniature swine was progressively occluded for 8 weeks followed by exercise training (EX; 5 days/wk treadmill) or sedentary (SED) protocols for 12 weeks. Arterioles were isolated from distal LCX and nonoccluded left anterior descending (LAD) artery for in vitro study. Vasodilation to NOS activators adenosine and ionomycin was impaired in SED LCX, but not LAD, arterioles. This impairment was restored by L-arginine. NO production induced by adenosine was also reduced in SED LCX arterioles. EX had no effect on LAD arterioles but improved NO production and restored dilation of LCX arterioles. NOS blockade (L-NAME) inhibited vasodilation to NOS activators in LAD (SED & EX) arterioles but was ineffective in SED LCX arterioles. In EX LCX arterioles, vasodilation to NOS activators was slightly inhibited by L-NAME but abolished by catalase. H2O2 production was markedly increased by adenosine in EX LCX arterioles. CONCLUSIONS - This study demonstrates that endothelium-dependent NO-mediated dilation is impaired in SED LCX arterioles and that EX training restores the impaired function. It appears that H2O2, in addition to NO, contributes significantly to EX-induced restoration of endothelium-dependent dilation of coronary arterioles distal to occlusion.

Protein arginine methylation is an essential post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Type I PRMTs transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to the arginine residues and catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Type II PRMTs generate MMA and symmetric dimethylarginine (SDMA). PRMT-catalyzed methylation is involved in many biological processes and human diseases when dysregulated. As the predominant PRMT, PRMT1 catalyzes an estimated 85% of all protein arginine methylation in vivo. Nevertheless, the product specificity of PRMT1 remains poorly understood. A few articles have been published regarding the kinetic mechanism of PRMT1, yet with controversial conclusions. To gain more insights into the product specificity of PRMT1, we dissected the active site of PRMT1 and identified two conserved methionines (Met-48 and Met-155) significant for the enzymatic activity and the product specificity. These two methionines regulate the final product distribution between MMA and ADMA by differentially affecting the first and second methyl transfer step. Current data show that Met-48 also specifies ADMA formation from SDMA. To further understand the kinetic mechanism of PRMT1, we developed a double turnover experiments to conveniently assay the processivity of the two-step methyl transfer. Using the double turnover experiments, we observed that PRMT1-catalyzed dimethylation is semi-processive. The degree of processivity depends on the substrate sequences, which satisfies the controversy between the distributive or partially processive mechanisms previously reported. We are using transient kinetics and single turnover experiments to further investigate the mechanism of PRMT1. Interestingly, during these studies, we found that PRMT1 may incur oxidative damage and the histidine affinity tag influences the protein characteristics of PRMT1. These studies have given important insights into the product specificity and kinetic mechanism of PRMT1, and provided a strong foundation for future studies on PRMT1.