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.

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.

L-arginine is one of the most versatile amino acids due to the fact that it serves as a precursor for many molecules which have important roles in bodily functions including mammalian reproduction. The current studies sought to further examine the role that L-arginine has in mammalian reproduction utilizing both in vivo and in vitro approaches. In the first study, a novel bioluminescent murine pregnancy model was developed to monitor VEGFR2 transcription activity non-invasively in the fetoplacental unit. Secondly, the effect that dietary L-arginine supplementation has during mouse gestation was examined. L-arginine supplementation increased weight gain during the latter third of gestation, total litter size, number of implantation sites, and litter birth weight. Additionally, L-arginine supplementation increased VEGFR2 transcription activity in the fetoplacental unit which may create a more favorable environment for fetal survival. Moreover, the increased number of implantation sites observed suggests an effect of L-arginine at the level of the endometrium. To this end, the effect that L-arginine has on apoptosis and cell proliferation in an established endometrial cell line was examined. The addition of L-arginine at physiological (200 micromolar) and supra-physiological (800 micromolar) concentrations increased cell proliferation , and this effect was achieved through biosynthesis of polyamines and nitric oxide. L-arginine also decreased the proportion of cells that were experiencing mitochondrial mediated apoptosis, and it was observed that this decrease in mitochondrial mediated apoptosis was concurrent with increased phosphorylation of BAD protein, which induces apoptosis when not phosphorylated. The final study examined the ability of porcine uterine epithelial (PUE) cells to synthesize L-arginine from L-citrulline. L-citrulline was able to support PUE cell proliferation in the absence of L-arginine. Additionally, ASS-1 and ASL, L-arginine synthesizing enzymes, were expressed in PUE cells and were regulated by the presence of L-arginine and L-citrulline, respectively. This data would support the hypothesis that PUE cells may be able to convert L-citrulline to L-arginine. Together, the current findings along with the plethora of relevant literature provide further evidence for the role of L-arginine in mammalian reproduction and allow for new questions to be investigated regarding this particular amino acid’s role in mammalian reproduction.

No description available.

Le travail de la thèse présente une série de (bio)capteurs conductimétriques, à base de la clinoptilolite, pour la détermination de l’ammonium, de l’urée et de la L-arginine. La clinoptilolite, le matériau nanométrique, possédant des propriétés de la sorption intrinsèque et une capacité d’échange cationique vis-à-vis des espèces ammonium, a été d’abord utilisée pour la réalisation d’un microcapteur conductimétrique sélectif à NH4+. Ci-après, une application de ce nanomatériau dans les biocapteurs est favorable pour le fonctionnement dans les solutions tampons multicomposants. Parmi plusieurs variantes de biocapteurs à l’urée à base de la zéolite, la plus intéressante est le biocapteur, dans lequel la couche de la clinoptilolite, déposée sur le transducteur, a été recouverte par le dépôt de la couche de l’uréase et de la zéolite. Pour l’élaboration d’un biocapteur conductimétrique hautement sensible pour la détermination de la L-arginine, l’arginase et l’uréase ont été co-réticulées sur le transducteur. Une détermination quantitative de la L-arginine dans une solution buvable « Arginine Veyron » a montré un fort accord avec les données fournies par le producteur. Une procédure détaillée de l’optimisation du biocapteur conductimétrique pour la détection de la L-arginine dans le sérum bovin a été proposée. La clinoptilolite a été également appliquée comme un modificateur dans la co-immobilisation de l’arginase et l’uréase pour améliorer les caractéristiques analytiques de biocapteur conductimétriques pour la détermination de la L-arginine / Currentwork presents a serie of conductometric (bio)sensors based on clinoptilolite, for ammonium, urea and L-arginine determination. Clinoptilolite, a nanoscale material possessing exceptional sorption and cation-exchange properties toward ammonium species, was initially used for the development of NH4+-selective conductometric microsensor. The clinoptilolite-based microsensor was selective toward ammonium in the presence of interferences that are commonly found along with ammonium in natural waters. Hereafter, an application of this nanomaterial in biosensors is favorable for operation in multicomponent buffer solutions. Among the several variants of the urea biosensors based on zeolite, considerably better characteristics were obtained for the biosensor comprising a clinoptilolite adlayer and an upper layer of immobilized urease and zeolite. In the work, for first time was developed a highly sensitive conductometric biosensor for L-arginine determination based on arginase and urease co-immobilized in a single membrane. The results of a quantitative determination of L-arginine in a drinkable solution “Arginine Veyron”, obtained by the biosensor, were in high correlation with the data provided by the producer. The L-arginine conductometric biosensor was optimized for the serum analysis. Clinoptilolite was also applied as a modifier in co-immobilization of arginase and urease for the improvement of analytical characteristics of the conductometric biosensor for L-arginine determination

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.

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.

Instituto Dr. Josà Frota / A epitelizaÃÃo, contraÃÃo e deposiÃÃo da matriz de tecido conjuntivo constituem mecanismos pelos quais as feridas cicatrizam. Estudos demonstraram que L-arginina favorece a cicatrizaÃÃo, aumentando a sÃntese de colÃgeno. Objetivou-se, neste trabalho, estudar os efeitos da suplementaÃÃo de L-arginina sobre concentraÃÃes in vivo de glicose, piruvato, lactato e corpos cetÃnicos no sangue e em cicatriz de retalho miocutÃneo, como tambÃm verificaÃÃo da pressÃo de ruptura de cicatriz intestinal. Quarenta e oito ratos brancos, machos, da linhagem Wistar, foram divididos em dois grupos: (G-1/Controle) e (G-2/Experimento). Ambos foram submetidos à confecÃÃo de retalho miocutÃneo e dorsal, suturado com nylon monofilamentado nÂ5.0 e tambÃm, submetidos a laparotomia mediana e, jejunotomia de dois centÃmetros na borda antemesentÃrica seguida de sutura imediata com fio poliglecaprone 25, nÂ5.0. No pÃs-operatÃrio os animais do G1/C receberam suplementaÃÃo alimentar de caseinato de cÃlcio (fonte protÃica intacta de alto valor biolÃgico, 5% do VCT/dia). Os animais do G-2/E receberam suplementaÃÃo alimentar com L-arginina (5% do VCT/dia). Cada grupo foi subdividido em trÃs subgrupos e apÃs cada 7,14 e 30 dias foram coletadas amostras de sangue e de segmento de Ãrea cicatricial do retalho miocutÃneo para estudos enzimÃticos com dosagem de metabÃlitos, como tambÃm realizado teste de pressÃo de rotura no segmento intestinal contendo cicatriz. A queda das concentraÃÃes sanguÃneas de piruvato, lactato e corpos cetÃnicos no 14 pÃs-operatÃrio, no grupo E em relaÃÃo ao grupo C, sugere maior utilizaÃÃo destes metabÃlitos nos tecidos perifÃricos por possÃvel aÃÃo anabÃlica da oferta de L-arginina. O aumento das concentraÃÃes tissulares, no retalho miocutÃneo, de corpos cetÃnicos no 30 dia pÃs-operatÃrio nos animais tratados com suplementaÃÃo alimentar de L-arginina traduziu provÃvel aumento de captaÃÃo desses metabÃlitos pelos tecidos em cicatrizaÃÃo. Observou-se um aumento da pressÃo de rotura no 14 pÃs-operatÃrio do G-2/E em relaÃÃo ao G-1/C. Os resultados sugerem que suplementaÃÃo de L-arginina tem efeito sobre concentraÃÃes sÃricas de substratos e sobre cicatrizaÃÃo cutÃnea, muscular e de intestino delgado. / Cicatrization, contraction and deposition of connective tissue matrix constitute healing mechanisms for the development of the wound scar. Studies have demonstrated that L-arginine favors cicatrization, increasing the synthesis of collagen. The aim of this paper was to study the effects of L-arginine upon the concentrations of glucose, pyruvate, lactate and ketone bodies, in vivo, in blood, miocutaneous skin graft scar and on the pressure of rupture of intestinal surgical scar in rats. Forty-eight white Wistar male rats, were distributed in 2 groups: (G1/control) and G2/experiment). A pedunculated miocutaneous skin graft was raised on the dorsum of all rats followed by immediate suturing with nylon 5-0. Jejunotomies were also performed on the antimesenteric border of the small intestine. Bowel sutures were carried out utilizing synthetic material (poliglecaprone 25). Oral supplementation of L-arginine (5% of TCI/day) was delivered via gavage to G-2/Experiment rats beginning 12 hours following surgical procedures daily. G-1/Control animals were fed sodium caseinate instead. Blood samples were collected 7, 14 and 30 days later. Measurement of intestinal rupture pressure was carried out at the same intervals, following blood samples collection. Decreased concentrations of pyruvate lactate and ketone bodies in the 14th postoperative day suggest greater utilization by peripheral tissues due to a possible anabolic effect of L-arginine. Increased ketone bodies concentrations in the miocutaneous graft in the 30th postoperative day in L-arginine treated rats may be due to increased uptake of these metabolites by healing tissues. Group G-2/E intestinal rupture pressure was greater than G-1/C on the 14th day. These results suggest that oral supplementation of L-arginine has effects upon blood concentrations of metabolites and on the cutaneous, muscular and small intestine cicatrization

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.

<p>Cardiovascular disease (CVD) is the major cause of death in patients with chronic renal failure (CRF). Endothelial function and oxidative stress (OS) have previously been shown to be important in the pathogenesis of CVD. In this thesis, the endothelium-dependent vasodilatation (EDV) and OS were investigated in the patients with CRF. Also the influence of L-arginine, erythropoietin and diclofenac on EDV were evaluated in patients with CRF. </p><p>Patients with CRF were found to be characterized by a defect EDV even after correction for traditional cardiovascular risk factors. This impairment was related to the degree of renal failure. </p><p>Measurement of OS markers in CRF patients demonstrated that these patients were in a state of OS compared to healthy controls. The most informative indices to evaluate the degree of OS in CRF were: oxidized glutathione (GSSG) level, ratio between oxidized and reduced glutathione (GSSG/GSH ratio), lag phase of lipoprotein fraction (LPF) and baseline diene conjugation level of LPF. </p><p>Simultaneously investigated OS markers and EDV demonstrated a relationship between OS and EDV in patients with CRF. EDV was positively correlated with total antioxidative activity, reduced glutathione (GSH) and lag phase of LDL. </p><p>Local infusion of L-arginine as a substrate for nitric oxide synthesis and diclofenac as an inhibitor of cyclooxygenase-derived vasoconstrictive agents augmented EDV in patients CRF. In contrast, the erythopoietin treatment (both acute and long-term) impaired EDV in CRF patients. </p><p>In conclusion, patients with CRF have increased levels of OS markers and impaired endothelial vasodilatory function. These factors may be important with respect to the high morbidity and mortality of CVD found in patients with CRF. One possible mechanism to reduce CVD in patients with CRF is to improve endothelial function and eliminate OS. Locally administrated L-arginine and diclofenae improved EDV but erythropoietin administration impaired EDV in patients with CRF. </p>