The master´s thesis deals with the study of the interaction between the polysaccharide hyaluronan of diffrerent molecular weights with the amino acids arginine, lysine, arginine hydrochloride and 6-aminocaproic acid. They are expected interaction between carboxyl groups of hyaluronan and amino groups of amino acids. These interactions were investigated by using ultrasonic spectroscopy, DLS, measuring pH and conductivity. Obtained results were compared with sodium polystyrene sulfonate. With ultrasonic spectroscopy was observed a change of concentration inkrement for titration of amino acid to water or polymers solutions especially for high molecular weight hyaluronan and for NaPSS in combination with 6AKK in concentration range of added amino acid 0–30 mM. The size of this change could mean a degree of interaction between polymers and amino acids. This theory has not been confirmed by other methods. By pH and conductivity measurements interations between arginine and low molecular weight hyaluronan and NaPSS were only confirmed. There was no possibility to make unequivocal conclusions from determination of particle size and zeta potential by DLS. Overall, the issue of the interaction of amino acids with polyanions was proved above expectations complex and will be appropriate to further expand the observations made in this thesis.

De nos jours, la moitié des infections nosocomiales sont liées à la pose de dispositifs médicaux. Dans ce contexte, nous avons développé deux types de revêtements antimicrobiens adaptés au domaine biomédical. Le premier s'appuie sur le greffage d’un peptide comprenant une séquence d’accroche, une séquence antimicrobienne et un site de clivage spécifique d'un pathogène. La séquence antimicrobienne sera alors libérée uniquement en présence du pathogène. Malgré la réussite de l’accroche du peptide, certains paramètres restent encore à optimiser afin d’obtenir un effet antimicrobien. Le deuxième revêtement s’appuie sur la conception d’un film antimicrobien « couche-par-couche » avec l’utilisation de poly(L-arginine) (PAR) et d’acide hyaluronique (HA). L’influence de la taille des chaînes de PAR a été étudiée et seul le film construit à partir de PAR de 30 résidus possède un effet antibactérien. Avec cette PAR, nous avons démontré que HA est le seul polyanion conduisant à des propriétés antibactériennes. Ces propriétés antimicrobiennes sont maintenues lorsque d’autres homopolypeptides cationiques sont associés à HA. / Nowadays, about half of hospital-acquired infections are due to medical devices implantation. In this context, we have developed two types of antimicrobial coatings adapted to the biomedical field. The first one is based on peptide composed of an anchoring sequence, an antimicrobial sequence and a pathogen-specific cleavage site and grafted on the substrate. The antimicrobial site will be released only in the presence of the pathogen through the use of the cleavage site. Despite of the success of peptide grafting, some parameters must be optimized in order to obtain an antimicrobial effect. The second antimicrobial coating concept is based on the layer-by-layer technique by using poly(L-arginine) (PAR) and hyaluronic acid (HA). The effect of the size of PAR chains on the antimicrobial character of the coating was investigated and it is proven that only films composed with PAR of 30 residues present an antibacterial effect. Moreover HA is the only polyanion leading to such antimicrobial multilayer. It is also demonstrated that this antimicrobial properties is maintained when other cationic homopolypeptides are used in association with HA in layer-by-layer films.

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.

Acute necrotizing pancreatitis (ANP) is a common gastrointestinal cause of emergency admissions in dogs and humans and can lead to a systemic inflammatory response syndrome resulting in multiple organ dysfunction syndrome. Among the various complications associated with ANP, acute lung injury (ALI) or its more severe form, acute respiratory distress syndrome (ARDS), are major contributors leading to high mortality rates associated with severe acute pancreatitis (AP) in human patients. The incidence of ALI/ARDS in ANP dogs is not well characterized. However, signs of respiratory complications have been reported clinically in dogs suffering from AP. The pathophysiology of ANP and its systemic complications in dogs and humans are not well understood. Most of the data related to AP comes from rodent models of AP, which may not always represent the true mechanisms occurring in the lungs of dogs or humans with ANP. I decided to undertake evaluation of pancreas and lungs from dogs (N=21) that died of ANP. The cases were selected through the search of the medical records of the Veterinary Medical Center of the Western College of Veterinary Medicine (WCVM). Six healthy SPCA dogs were used as controls. The histology of pancreas was first graded to record the range of ANP severities within dog cases included in this study. Then, characterization of lung inflammation was done with histological grading and qualitative analysis of immunohistochemical staining for von Willebrand Factor (vWF), Toll-Like Receptor-4 (TLR4), interleukin-6 (IL6), and inducible nitric oxide synthase (iNOS). Quantification of the recruitment of septal macrophages in the lungs, designated as pulmonary intravascular macrophages (PIMs), in ANP dogs was achieved by counting the number of positive cells in alveolar septa using a macrophage antibody (MAC387). The results revealed that dogs suffering from ANP have variable lung inflammation, which was characterized by a significant infiltration of mononuclear phagocyte cells in the alveolar septa of all ANP dogs (median, 138; range 31-935) compared to control dogs (median: 1.5; range 0-16; p < 0.001), which suggested that PIMs are induced in ANP. In addition, robust staining for vWF in alveolar septal capillaries in lungs of ANP dogs suggested a strong microvascular inflammatory response. Finally, TLR4, IL6, and iNOS expression was increased in lungs of ANP dogs compared to control dogs. The second study was to investigate whether PIMs are induced in a mouse model of L-arginine-induced ANP. Therefore, lungs of L-arginine treated mice (n=7 per time point) were evaluated at various time points (24 hours, 72 hours and 120 hours) using histology and immunohistochemical staining for CD68 cells and vWF. Nine control mice were used. Counting of CD68-positive cells in the lungs of mice treated with L-arginine showed increased numbers of mononuclear phagocytes in alveolar septa at every time point (p<0.001). Also, the lung’s vasculature from L-arginine-treated mice showed increased vWF staining. Taken together, the data showed that ANP in dogs caused significant recruitment of PIMs, increased expression of vWF, TLR4, IL-6, and iNOS suggesting presence of lung inflammation. The mouse model of L-arginine-induced ANP also showed recruitment of PIMs and increased vascular expression of vWF suggesting that this model may be relevant to study the mechanisms of PIMs recruitment and their functions in lung physiology associated with ANP.

D-Arginine dehydrogenase (DADH) catalyzes the oxidation of D-arginine to imino arginine using FAD as the cofactor. The enzyme is part of a recently discovered two-enzyme complex from Pseudomonas aeruginosa involved in arginine utilization. Function of the enzyme within the organism is unknown. Work on this enzyme has been undertaken to understand the structure as well as its reaction mechanism so as to eventually assign a function to the enzyme within the physiological context. In the reductive half-reaction 2 e- and 1 H+ are transferred from the amino acid substrate to FAD cofactor. In the oxidative half-reaction the reducing equivalents from the FAD cofactor are passed to an electron acceptor that is yet to be discovered. The enzyme has been established to have no reactivity with O2. Choline oxidase (CHO) from Arthrobacter globiformis is a well characterized member of Glucose-Methanol-Choline Superfamily that reacts with molecular O2. It catalyzes the oxidation of choline to glycine betaine mediated by betaine aldehyde intermediate using FAD as the cofactor and O2 as the oxidant to regenerate oxidized FAD for further reaction. Glycine betaine, the product of the reaction is an important osmolyte that regulates nutrients for plants under stressful conditions. Therefore it is of commercial interest to genetically engineer crops that do not typically possess competent pathways for glycine betaine synthesis. In this dissertation molecular details concerning the reductive half-eaction of DADH and oxidative half-reaction of CHO have been studied using a combination of steady state kinetics, rapid kinetics, pH, multiple substrates, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects or computational approaches. In DADH, the oxidation of amino acid substrate by FAD has been shown to most likely proceed via hydride transfer mechanism in the reductive half-reaction with Glu87, Tyr53, Tyr249 and His48 emerging as key players in substrate binding, catalysis or for up keeping the integrity of the FAD cofactor. In CHO, the oxidative half-reaction proceeds without stabilization of any reaction intermediates with H atom from reduced FAD and H+ from solvent or solvent exchangeable site occurring in the same kinetic step.

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

O esbranquiçamento é o principal problema pós-colheita em beterraba minimamente processada. Esse problema consiste no ressecamento das células superficiais do produto, resultando no aspecto esbranquiçado decorrente do bloqueio das células túrgidas e íntegras das camadas inferiores. As etapas de corte e a manipulação durante o preparo dos produtos minimamente processados (PMPs) podem ser consideradas fonte de estresse abiótico, que estimula a formação de espécies reativas de oxigênio (EROs). O acúmulo de EROs provoca alterações bioquímicas e estruturais nas células, ocasionando alterações visuais e nutricionais que afetam diretamente a qualidade comercial. Aditivos alimentares são utilizados para retardar a degradação dos PMPs, mantendo-os frescos e adequados para o consumo por mais tempo. Ácidos orgânicos e aminoácidos são aditivos do tipo GRAS (Generally recognized as safe), ou seja, seguros para alimentos e são amplamente utilizados em produtos hortícolas. O objetivo deste trabalho foi avaliar a eficácia da arginina no controle do esbranquiçamento em beterraba minimamente processada e comparar com os demais aditivos utilizados comercialmente. Desse modo inicialmente foi determinada a combinação mais eficaz de dose e pH de aplicação da arginina, com avaliação de quatro doses (0, 10, 25 e 50 mM) combinadas com três faixas de pH (inalterado, pH 6,0 e pH 7,0). A aplicação de arginina apresenta resultados promissores em relação ao retardo do esbranquiçamento em beterraba minimamente processada, principalmente para o tratamento com 25 mM de arginina em pH 7,0. Além disso, esse tratamento também reduz a atividade respiratória, atividade de enzimas oxidativas (POD e PPO) e promove a manutenção de compostos bioativos importantes para a hortaliça (Betalaínas, compostos fenólicos e flavonois) por 12 dias após o processamento. Na segunda etapa, quatro aditivos (arginina, cisteína, ácido cítrico e ácido ascórbico) e água destilada, como tratamento controle, foram comparados quanto à eficácia na conservação de beterrabas minimamente processadas. O tratamento com arginina promove os melhores resultados quanto a manutenção do aspecto visual e de compostos bioativos em relação aos demais aditivos. O modo de ação diferenciado da arginina favorece a redução do estresse oxidativo, estendendo a vida útil do produto. / White blush is the main postharvest issue in minimally processed red beet. This issue consists in dehydration of superficial cell layers of the product, resulting in a whitening aspect caused by the blockage of turgid and intact cells on the layers below. During the minimal processing preparation, cutting and manipulation of minimally processed products (MPPs) are considered source of abiotic stress, which induce the formation of reactive oxygen species (ROS). The accumulation of ROS causes biochemical and structural changes that directly affects the commercial quality. Food additives are used to slow the degradation of MPPs by keeping them fresh and suitable for consumption. Organic acids and amino acids are GRAS (Generally reconized as safe) additives widely used in vegetables. This study aimed to evaluate the efficacy of arginine in controlling white blush in minimally processed redbeet and compare with additives used commercially. Thus, initially it was determined the best combination of four doses of arginine (0, 10, 25 and 50 mM) with three pH ranges (unchanged, pH 6,0 and pH 7,0). Arginine treatment presents promising results regarding the white blush delay in minimally processed redbeet. In addition, this additive also reduces the respiratory activity, activity of oxidative enzymes (POD and PPO) and maintains the important bioactive compounds (betalains, phenolic compounds and flavonols) for 12 days after processing. In the second step, four additives (arginine, cysteine, citric acid and ascorbic acid) and distilled water, as control treatment, were compared for efficacy in conservation of minimally processed beets. Arginine treatment promotes the best results regarding the maintenance of the visual aspect and bioactive compounds content in relation to the other additives. The different mode of action of arginine favors the reduction of oxidative stress, extending the shelf life of the product.

[pt] Este trabalho teve como objetivo o estudo das interações entre a cisplatina, que é usada como medicamento quimioterápico e dois aminoácidos de importância biológica: ácido guanidoacético (Gaa) e arginina (Arg). Por esta razão, procurou-se trabalhar in vitro com condições próximas ao meio biológico utilizando, em função disto, apenas água deionizada como solvente. Para isto, foram testados vários procedimentos de síntese que resultaram em quatro compostos diferentes: dois com o Gaa e dois com a arginina. Estes compostos foram caracterizados pelas seguintes técnicas: análise elementar (espectrometria de absorção atômica e CHN), condutimetria, análise termogravimétrica, ressonância magnética nuclear, difração de pó e espectroscopia de infravermelho. Foi possível verificar que tanto o Gaa como a arginina comportam-se como monodentados e complexam-se com a cisplatina através do átomo de nitrogênio da amina. / [en] The aim of this work is the study of the interactions between cis-platinum, which is employed as a chemotherapic drug, and two amino acids of biological importance: guanidinoacetic acid (Gaa) and arginine (Arg). In order to work in conditions as similar as possible of the biological environment, this work was done in vitro, using only deionized water as a solvent. With this purpose, several synthesis procedures were tested which resulted in four different compounds: two with Gaa and two with arginine. These compounds were characterized through the following techniques: elementary analysis (atomic absorption spectrometry and CHN), conductimetry, thermogravimetric analysis, nuclear magnetic resonance, powder diffraction and infrared spectroscopy. It was possible to verify that both Gaa and arginine behaved as monodentates ligands and complexed with the cis-platinum through the amine´s nitrogen atom.

O zinco é um elemento de grande importância para o desenvolvimento intra-uterino e é usado durante a gravidez para auxiliar o crescimento fetal. A arginina, um aminoácido dibásico, além de ser necessário para o crescimento, apresenta importantes funções biológicas e fisiológicas. Alguns autores mostraram que a ingestão de arginina melhora a resposta imune. Assim, o objetivo desse trabalho foi investigar o papel da suplementação alimentar de L-arginina e zinco na infecção chagásica experimental de ratas prenhes. Para tal, foram utilizadas ratas Wistar infectadas pela cepa Y de Trypanosoma cruzi, no 3º dia da prenhez e tratadas com L-arginina ou sulfato de zinco. Alguns parâmetros foram avaliados como a dosagem de óxido nítrico, citocinas (IFN-?, TNF-?, IL-2, TGF-?, IL-4, IL-10), corticosterona, número de macrófagos, linfoproliferação de esplenócitos, parasitemia e análise histopatológica do coração, placentas e fetos. A administração de zinco e arginina causou redução do parasitismo sanguíneo e cardíaco, ocasionou um aumento significativo nas concentrações de NO e na proliferação de esplenócitos. Além disso, contribuiu para o melhor desenvolvimento fetal. Dessa forma, podemos sugerir que a suplementação com estes elementos pode ser utilizada como uma substância imunomoduladora alternativa e, juntamente com a terapia anti-parasitária específica, minimizar as agressões ao hospedeiro, evitando a administração por longos períodos da droga tripanocida que sabidamente produz sérios efeitos colaterais e teratogênicos. / Zinc is an important element for the intrauterine development and during pregnancy is used to assist fetal growth. Arginine, a dibasic amino acid, besides its importance in the growth process, has important biological and physiological functions. Some authors have reported that daily intake of arginine improves the immune response. The objective of this study was to investigate the role of dietary supplementation of L-arginine and zinc in experimental Chagas disease in pregnant rats. To this end, we used Wistar rats infected with the Y strain of Trypanosoma cruzi, on the 3rd day of pregnancy and treated with L-arginine or zinc sulfate. Some parameters were evaluated such as nitric oxide, cytokines (IFN-?, TNF-?, IL-2, TGF-?, IL-4, IL-10), corticosterone, number of macrophages, lymphocyte proliferation of splenocytes, parasitemia and histopathology of the heart, placenta and fetus. The administration of arginine and zinc caused a reduction in blood and heart parasitism, with a significant increase in the concentrations of NO and proliferation of splenocytes. Moreover, it contributed to a better fetal development. Therefore, we suggest that supplementation with these elements may be used as an alternative immunomodulating substance and together with the specific anti-parasitic therapy minimize the aggressions against the host, avoiding the administration of trypanocidal drugs over long periods that is known to produce serious side effects and teratogenic effects.

A L-arginina é reconhecida como um nutriente de fundamental importância na resposta imune, apesar de seus efeitos serem, por vezes, considerados inconstantes. O autoimplante esplênico tem sido proposto como alternativa à esplenectomia total isolada, mas existem preocupações quanto à eficácia do restabelecimento da resposta imune, haja vista que o paciente pode permanecer com risco aumentado de desenvolvimento de infecção fulminante pós esplenectomia, mesmo após a regeneração morfológica do órgão. O objetivo deste estudo foi avaliar a participação da suplementação dietética com L-arginina em subpopulações linfocitárias no sangue, no baço e nos autoimplantes esplênicos de ratos submetidos a esplenectomia isolada ou combinada com autoimplante esplênico. Foram utilizados 42 ratos Sprague-Dawley machos, randomicamente distribuídos em seis grupos: 1 Controle operação simulada; 2 esplenectomia total; 3 esplenectomia total combinada com autoimplante esplênico; 4 Controle operação simulada, com suplementação de L-arginina; 5 esplenectomia total, com suplementação de L-arginina; e 6 esplenectomia total combinada com autoimplante esplênico, com suplementação de L-arginina. Os animais dos grupos 4, 5 e 6 receberam suplementação de L-arginina, uma vez ao dia, durante 15 dias anteriores a coleta sangüínea realizada imediatamente antes dos procedimentos operatórios (semanas 0 e 12). A dose utilizada foi de 1,0 g/kg/dia, administrada por via intragástrica em bolus. As avaliações foram realizadas por meio de hemograma e citometria de fluxo. A análise estatística utilizou testes paramétricos e nãoparamétricos, sendo p<0,05 considerado para a rejeição da hipótese nula. A suplementação com L-arginina acarretou elevação da contagem relativa e absoluta de neutrófilos periféricos, 12 semanas após a realização de esplenectomia total combinada com autoimplante esplênico. A esplenectomia total ocasionou diminuição da contagem relativa de linfócitos T totais, T CD4+ e T CD8&#946; no sangue, mas a suplementação dietética com L-arginina evitou a diminuição do percentual de células T totais e T CD8&#946; no sangue dos animais submetidos a autoimplante esplênico. Tanto a realização de autoimplante esplênico como a suplementação de L-arginina previnem a diminuição da subpopulação de linfócitos T CD4+ no sangue periférico, fato que usualmente ocorre após realização de esplenectomia total. Houve maior proliferação de células brancas / g de tecido nos autoimplante esplênico dos animais suplementados, porém a suplementação não influenciou a contagem de linfócitos T, T CD4+ e B de zona marginal de baço. A suplementação do aminoácido L-arginina após a realização de esplenectomia total combinada com autoimplante esplênico em ratos foi capaz de reverter alterações observadas em algumas das subpopulações linfocitárias, ocasionadas pela esplenectomia. / L-arginine is recognized as a nutrient of fundamental importance in immune response, although its effects are sometimes considered unstable. The splenic autoimplants has been proposed as an alternative to total splenectomy isolated, but there are concerns about the effectiveness of the restoration of the immune response, considering that the patient can remain at increased risk of developing overwhelming postsplenectomy infection, even after morphological regeneration of the organ. The aim of this study was to determine the role of dietary supplementation with L-arginine in lymphocyte subsets in blood, spleen, and splenic auto-transplantation in rats subjected to total splenectomy alone or in combination with splenic autotransplantation. Forty two male Sprague-Dawley rats, were randomly divided into six groups: 1 - Control sham operation, 2 total splenectomy 3 total splenectomy combined with splenic auto-implants, 4 - Control - sham operation, with L-arginine supplementation, 5 total splenectomy, supplemented with L-arginine, and 6 total splenectomy combined with splenic auto-implants, supplemented with L-arginine. Animals in groups 4, 5 and 6 were supplemented with L-arginine, once daily for 15 days before blood sample was collected immediately before the operative procedures (weeks 0 and 12). The dose was 1.0 g / kg / day administered by intragastric bolus. The laboratory evaluations were made by blood count and flow cytometry. Statistical analysis used parametric tests and nonparametric, p<0.05 was considered to reject the null hypothesis. Supplementation with L-arginine led to increase in relative and absolute count of peripheral neutrophils, 12 weeks after completion of total splenectomy combined with splenic auto-implants. Total splenectomy caused a decrease in relative count of T lymphocytes, CD4+ and CD8&#946; in blood, but dietary supplementation with L-arginine prevented the decrease in the percentage of total T cells and CD8&#946; in the blood of animals subjected to splenic auto-transplantation. Both the completion of splenic auto-implants and the L-arginine supplementation may prevent the decrease of the subpopulation of CD4+ T cells in peripheral blood, a fact that usually occurs after completion of total splenectomy. There was a greater proliferation of white blood cells / g of tissue in the splenic autoimplants supplemented animals, but supplementation did not influence the T lymphocyte counts, CD4+ T and B splenic marginal zone. Supplementation of Larginine after performing total splenectomy combined with splenic autotransplantation in rats was able to reverse some of the changes observed in lymphocyte subsets, caused by splenectomy.