Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Hydrogen sulfide mediates various physiological functions and along with carbon monoxide and nitric oxide it is an important gaseous signaling molecule. Cellular targets for H2S are proteins, enzymes, transcriptional factors or ion channels. In many cases, the effect of H2S on the regulatory protein is mediated by modifications of its cystein residues. In hemeproteins, the regulation of catalytic activity is induced by formation of the Fe(III)-SH complex or by reduction of the heme iron with subsequent formation of Fe(II)-O2 complex. The effect of Na2S on model sensor heme-containing proteins is presented in this thesis. Protein, isolated from bacterium Anaeromyxobacter sp. strain FW109-5, containing a globine coupled sensor domain and a histidine kinase domain is one of the studied proteins, the second one is protein isolated from bacterium Escherichie coli, containing a globine coupled sensor domain and a diguanylate cyclase domain. The effect of Na2S on both model proteins and their mutants was studied by UV-Vis spectral analysis. Spectra of YddV-HD Y43A were very unique, because thery confirmed formation of a homogenous complex Fe(III)-SH in this protein, whereas only mixtures of varous heme complexes were detected in other tested proteins. Additionally the effect of Na2S on functional domain...

Bordetella pertussis is an important human pathogen that causes an infection disease called whooping cough. This gram-negative bacterium produces an adenylate cyclase toxin (CyaA) that recognizes an integrin receptor CD11b/CD18 present on the surface of myeloid phagocytes and delivers an adenylate cyclase (AC) domain into the cell cytosol. This thesis deals with the endocytic machinery of CyaA and its potential use as a specific marker for endocytosis of the CD11b/CD18 receptor molecule. Detoxified mutant of CyaA, CyaA-AC- , that has the capacity to promote calcium influx as well the potassium efflux, was shown to trigger activation of the integrin receptor CD11b/CD18 followed with endocytic uptake by clathrin-dependent pathway. On the other side, the inactive mutant CyaA-KP-AC- that is unable to provoke integrin activation was endocytosed by clathrin-independent pathway. These results suggest that the various endocytic pathways of the CD11b/CD18 are determined by different conformational states of the receptor molecule.

Morphine is an opioid agonist, which can exert cardioprotective effects under certain conditions. Lipid rafts are considered important platforms for membrane organization of signaling proteins and, therefore, these structures could play a role in the effects of morphine, which acts through the opioid receptors. The aim of this thesis was to investigate the distribution of the main components of the opioid receptor and Gi/o-mediated signaling pathway in lipid rafts isolated from rat myocardium, which was affected by various doses of morphine. Because we used different isolation techniques with different solubilization agents (Triton X-100, CHAPS, cholate and sodium carbonate) for preparation of lipid rafts, it was of interest to characterize more closely these preparations. Another aim of this study was to investigate how different methods of isolating these structures affect activity of the key target enzyme of the opioid signaling pathway, i.e. adenylyl cyclase. The presence of signaling molecules of the Gi/o/AC pathway of the opioid system in membrane rafts was confirmed and the distribution of selected proteins was dependent on the type of extractant. We also observed the effect of morphine on the localization of proteins in lipid rafts. Different extractants provided different degree of...

Thesis advisor: Charles S. Hoffman / Glucose detection in yeast occurs via a cAMP signaling pathway that is similar to that of other signaling pathways in humans. The presence of glucose in the environment ultimately represses, as a result of cAMP signaling, the transcription of the gene fbp1. Adenylate cyclase is known to convert ATP to cAMP, and is thus a central protein in the propagation of the signal. Mutant forms of the adenylate cyclase gene (git2) have been found by the inability for the organism to repress fbp1 transcription in the presence of glucose. In this study, two questions were under investigation. The first was focused on the ability of the mutations to affect the dimerization of the catalytic domain. The second investigated multiple protein-protein interactions in the leucine rich-repeat (LRR) domain of adenylate cyclase. Both domains contain mutations that confer an activation defect, and they are thus are thought to have a relationship. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

INTRODUÇÃO. O termo Lesão Pulmonar Aguda (LPA) é usado para descrever a resposta pulmonar à lesão que ocorre diretamente ou indiretamente nos pulmões. A quebra da barreira alvéolo-capilar determina o influxo de líquido rico em proteínas para dentro dos espaços alveolares, sendo necessária a reabsorção desse líquido no processo de resolução da LPA. A infusão intravenosa de Ácido Oleico (AO) em ratos provoca agudamente edema alveolar difuso e focos hemorrágicos intra-alveolares, sendo um bom modelo de indução. Estudos relatam que o Azul de Metileno (AM) atenua tais lesões, com efeito protetor, no tecido pulmonar, e reduz o edema presente na LPA em animais com sepse através da inibição da guanilato ciclase solúvel (GCs), uma enzima ativadora da via NO-GMPc. OBJETIVO. Estudar a repercussão da inibição da GCs pelo AM na permeabilidade capilar pulmonar ministrando-o antes e após a indução da lesão pulmonar por AO. MÉTODO. Ratos Wistar foram divididos em 5 grupos: Sham com infusão de salina em bolus; AM com infusão de AM por 2h; AO com infusão de AO em bolus, AM/AO com infusão de AM por 2h, sendo que, após 5 min do início, recebeu AO simultaneamente em bolus e AO/AM com infusão de AO em bolus e, após 2h, infusão de AM por mais 2h. Após 4h foi realizada a coleta de materiais (sangue, lavado bronco-alveolar e tecido pulmonar) para análise do NO plasmático e tecidual, gasometria arterial, cálculo do peso úmido/peso seco (PU/PS) e histologia do tecido pulmonar de todos os grupos. A estatística utilizada foi a análise de variância (one-way ANOVA) com p<0.05. RESULTADO. Não foi encontrado hipoxemia grave após 4h de lesão. O grupo AO apresenta um aumento no número de proteínas no lavado bronco-alveolar e na relação PU/PS comparado aos grupos controle: Sham e AM, confirmando a presença de lesão e alteração de permeabilidade pulmonar. Os grupos tratados com AM apresentaram melhora na permeabilidade pulmonar, porém, apenas o grupo pré-tratamento (AM/AO) apresentou diferença estatística na redução do extravasamento de proteínas no lavado. Não foram encontradas diferenças significativas no NO plasmático e tecidual. Na microscopia, a congestão capilar foi intensa, acompanhada de múltiplos focos de edema alveolar, exsudato intra-alveolar proteico, áreas de hemorragia e infiltrado inflamatório neutrofílico, tanto no interstício quanto nos septos alveolares.Os grupos tratados com AM apresentaram diminuição das áreas de edema, exsudação e hemorragia, porém, com maior evidência no grupo AM/AO. CONCLUSÃO. O AM diminui a permeabilidade pulmonar quando administrado de maneira precoce amenizando os danos causados pela LPA induzida por AO. / BACKGROUND. The term Acute Lung Injury (ALI) is used to describe the response to lung injury that occurs directly or indirectly in the lungs. The rupture of the alveolar - capillary barrier determines the influx of protein-rich fluid into the alveolar spaces, the reabsorption of this fluid in the process of resolving the ALI is required. Intravenous infusion of oleic acid (OA) in rats acutely causes diffuse alveolar edema and intra-alveolar hemorrhagic foci, being a good role model induction. Studies have reported that Methylene Blue (MB) attenuates such injuries, with a protective effect in lung tissue and reduce edema present in the ALI present in with sepsis by inhibition of soluble guanylate cyclase (sGC), an activator of the enzyme NO- cGMP pathway. OBJECTIVE. To study the effect of inhibition of sGC by MB in pulmonary capillary permeability ministering to the before and after induction of lung injury by OA. METHOD. Wistar rats were divided into 5 groups: Sham infused with saline bolus, MB infused with MB for 2hrs, OA infused with OA bolus, MB/OA infused with MB for 2hrs, and after 5 min from the beginning, simultaneously received OA bolus and OA/MB infused with OA bolus and after 2hrs, MB infusion for 2hrs. After 4hrs the collection of materials was performed (blood, bronchoalveolar lavage and lung tissue) for analysis of plasma and tissue NO, arterial blood gases, calculation of the wet weight/dry weight (WW/DW) and histology of lung tissue from all groups. The statistic used was analysis of variance (one-way ANOVA) with p<0.05. RESULTS. Not found severe hypoxemia after 4hrs of injury. The OA group shows an increase in the number of proteins in bronchoalveolar lavage and in WW/DW ratio compared to the control groups: Sham and MB, confirming the presence of injury and alterations of lung permeability. The groups treated with MB showed improvement in lung permeability, however, only the pretreatment group (MB/OA) showed statistical significance in reducing the leakage of protein in the lavage. No significant differences were found in plasma and tissue NO. In microscopy, capillary congestion was intense, accompanied by multiple foci of alveolar edema, intra-alveolar proteinaceous exudates, areas of hemorrhage and neutrophilic inflammatory infiltrate in both the interstitium and in the alveolar septa. The groups treated with MB showed reduction in areas of edema, exudation and hemorrhage, however, most obviously in MB/OA group. CONCLUSION. The MB decreases lung permeability when administered as early as possible, mitigating the damage caused by OA-induced ALI.

No description available.

Isovaleric Acid (IVA) is a 5-carbon branched chain fatty acid present in fermented foods and produced by the fermentation of leucine by colonic bacteria. IVA activates G-protein coupled receptors such as FFAR2, FFAR3, and OR51E1 known to be expressed on enteric neurons and enteroendocrine cells. We previously reported that the shorter, straight chain fatty acids acetate, propionate and butyrate, differentially affect colonic propulsion; however, the effect of branched chain fatty acids on gastrointestinal motility is unknown. We hypothesize that IVA relaxes smooth muscle in a cAMP/PKA dependent manner by direct action on smooth muscle cells. IVA will also decrease peristalsis and encourage retention of luminal contents. This thesis investigates the effect of IVA on smooth muscle tension and peristaltic activity in isolated colon and individual smooth muscle cells. Colon segments from C57BL/6J mice were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with 10 μM acetylcholine (ACh) and the effects of IVA at several concentrations were measured in the absence and presence of Nitric Oxide Synthase inhibitor L-N-nitroarginine (L-NNA), neuronal action potential inhibitor tetrodotoxin (TTX), and adenylate cyclase inhibitor SQ22536. To study individual live cells, mouse smooth muscle was isolated from colon, suspended in smooth muscle buffer, and after contraction with ACh were relaxed with micromolar concentrations of IVA. For peristalsis studies, whole colonic segments isolated from C57BL/6J were catheterized and placed horizontally in organ baths with circulating Krebs buffer. The colon was clamped on the anal end, and a solution (5 μL per mm of colon length) of either Krebs buffer or 50 mM IVA was delivered from the oral end to the lumen. Video of the peristalsis was then analyzed for diameter, changes in diameter, velocity of diameter changes along the length of the colon, normalized to the anatomical changes in the proximal region. IVA in concentrations of 10 mM to 50 mM relaxed the ACh-induced contraction in a sigmoidal fashion. In separate studies, L-NNA nor TTX affected the ability of IVA to inhibit relaxation. SQ22536 inhibited IVA induced relaxation in longitudinal colon compared to vehicle control. In isolated cells, SQ22536 and PKA inhibitor H-89 inhibited IVA-induced relaxation. In peristalsis studies, 50 mM IVA in Krebs buffer changed the character of the peristaltic action by increasing proximal diameter, inhibiting contractions in the proximal end of the colon, and decreasing overall velocity of peristaltic contractions in the proximal region. The data indicate that the branched chain fatty acid IVA causes a concentration-dependent relaxation of colonic smooth muscle that is direct to the smooth muscle and independent of neuronal activity. This relaxation is cAMP/PKA dependent. In addition to the direct relaxation of smooth muscle, intraluminal IVA decreased overall colonic propulsive activity and encouraged retention of the luminal contents. We conclude that the ingestion and production of branched chain fatty acids could affect overall GI motility and is an area for study in dietary and therapeutic control of bowel activity.

Abstract not available