No description available.

Cystic fibrosis (CF) is one of the relatively common inherited diseases caused by a mutation in the gene encoding for CFTR protein, which forms a chloride channel that significantly affects ion homeostasis and the associated fluid management of the cell. This disease mainly affects the respiratory and digestive systems, being the most life-threatening in the respiratory tract. Patients with CF suffer from frequent and recurrent respiratory infections that lead to the development of chronic inflammation and gradual destruction of lung tissue. These lung infections, which are caused mostly by the opportunistic pathogen Pseudomonas aeruginosa, are the most common cause of morbidity and mortality in these patients. At present, antibiotics are used in the treatment of Pseudomonas aeruginosa infections, but new methods of antibacterial therapy need to be found to overcome the development of resistance. In addition to active immunization of CF patients against Pseudomonas aeruginosa, their passive immunization with specific chicken antibodies directed against this pathogen offers promising possibilities. This dissertation thesis is aimed to verify the prophylactic potential of hen IgY antibodies against the virulence factor Pseudomonas aeruginosa - lectin PAIIL, and to further develop an experimental...

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

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Human C-reactive protein (CRP), injected intravenously into mice or produced inside mice by a human transgene, protects mice from death following administration of lethal numbers of Streptococcus pneumoniae. The protective effect of CRP is due to reduction in the concentration of bacteria in the blood. The exact mechanism of CRP-dependent killing of pneumococci and the partners of CRP in this process are yet to be defined. The current efforts to determine the mechanism of action of CRP in mice are directed by four known in vitro functions of CRP: 1. the ability of pneumococcal C-polysaccharide-complexed CRP to activate complement pathways, 2. the ability of CRP to bind to Fcγ receptors on phagocytic cells, 3. the ability of CRP to bind to immobilized complement regulator protein factor H which can also be present on pneumococci, and, 4. the ability of CRP to interact with dendritic cells. CRP-treated dendritic cells may well be as host-defensive as CRP alone. An interesting condition for the protective function of CRP is that CRP must be given to mice within a few hours of the administration of pneumococci. CRP does not protect mice if given later, suggesting that CRP works prophylactically but not as a treatment for infection. However, full knowledge of CRP may lead to the development of CRP-based treatment strategies to control pneumococcal infection. Also, because CRP deficiency in humans has not yet been reported, it becomes important to investigate the deficiency of the mechanism of action of CRP in CRP-positive individuals.

Les oligosaccharides de chitine (COs) jouent des rôles majeurs chez les plantes. Alors que les COs longs (6-8 unités saccharidiques) sont des éliciteurs c’est-à-dire qu’ils activent leurs mécanismes de défenses vis-à-vis de microorganismes pathogènes, les COs courts (4-5 unités saccharidiques) participeraient à l’établissement de symbioses avec des microorganismes bénéfiques permettant une meilleure assimilation des nutriments du sol. Afin de mieux comprendre comment les plantes discriminent ces signaux moléculaires, il est nécessaire de disposer de molécules pures aux structures chimiques parfaitement contrôlées pour caractériser les récepteurs protéiques mis en jeu. Dans le cadre de cette thèse nous nous sommes intéressés à la synthèse de COs de degré de polymérisation contrôlé et leur modification pour obtenir de nouvelles sondes d’affinité. Pour cela un plan d’expérience a été développé afin d’optimiser la production de COs et plus particulièrement d’oligosaccharides longs par hydrolyse enzymatique de chitine avec une enzyme commerciale, le lysozyme du blanc d’œuf. Par la suite, un nouveau type de sonde d’affinité permettant le marquage spécifique de protéines affines des COs a été mis au point. Nous avons en effet montré pour la première fois que des glycosides de triazinyle peuvent être efficacement utilisés pour introduire un groupe fluorescent sur une protéine interagissant avec l’oligosaccharide sans aucune activation chimique ou physique extérieure. Après avoir démontré la preuve de concept avec des lectines, une sonde d’activité permettant de mesurer l’activité de chitinases par fluorescence et de réaliser leur marquage dans le même temps a été synthétisée. Ces nouveaux outils devraient permettre de progresser dans la caractérisation des récepteurs de COs chez les plantes mais également permettre à terme de découvrir de nouvelles protéines lectines ou enzymes qui interagissent avec les sucres. / Chitinoligosaccharides (COs) play major roles in plants. While long COs (6-8 saccharide units) are elicitors activating plant defense mechanisms against pathogenic microorganisms, short COs (4-5 saccharide units) would participate in the establishment of symbioses with beneficial microorganisms allowing better assimilation of soil nutrients. Identifying the receptors involved in these processes to understand how plants discriminate these signal molecules requires having access to pure molecules with well-defined degrees of polymerization. In this thesis, we focused on the synthesis of well-defined COs and their modification to obtain new affinity probes. For this purpose, a design of experiments was developed in order to optimize the production of COs and more particularly of long ones by enzymatic hydrolysis of chitin with a commercial enzyme, hen egg-white lysozyme. Subsequently, a new type of affinity-based probe allowing the specific labeling of CO-binding proteins has been developed. We have shown for the first time that triazinyl glycosides can be effectively used to introduce a fluorescent group on an oligosaccharide-binding protein without any external chemical or physical activation. After demonstrating the proof of concept with lectins, a fluorescent activity-based probe allowing continuous assay of chitinases and their labeling at the same time was synthesized. These new tools offer exciting perspectives for the characterization of CO receptors in plants as well as for the discovery of new lectins and carbohydrate-active enzymes.

No description available.

Avaliou-se a expressão do gene mbl2 em 79 crianças e suas mães HIV positivas com o objetivo de avaliar a sua influência na transmissão vertical. Os pacientes divididos em dois grupos: crianças HIV positivas e suas mães (n=18) e crianças HIV negativas e suas mães (n=61) foram avaliados pelo CH50 e AP50 (ensaios hemolíticos), dosagem e avaliação funcional da MBL, ativação da cascata terminal do complemento (ELISA) e o gene mbl2 (PCR). Os resultados não mostraram diferença significante entre os níveis séricos, atividade funcional e o gene da MBL entre os grupos, excluindo a sua influência sobre a transmissão materno-fetal do HIV / It was evaluated the mbl2 gene expression in 79 children and their HIV positive mothers with the aim to evaluate its influence on mother-to-child HIV. The patients were divided in two groups: HIV positive children and their mothers (n=18) and HIV negative children and their mothers (n=61) were evaluated by CH50 and AP50 (hemolytic assays); levels and functional MBL and terminal complement cascade (ELISA) and mbl2 gene (PCR). The results didn't show significant difference amons serum levels, functional activities and MBL gene between the groups, excluding the influence in the mother-to child HIV transmission.

Avaliou-se a expressão do gene mbl2 em 79 crianças e suas mães HIV positivas com o objetivo de avaliar a sua influência na transmissão vertical. Os pacientes divididos em dois grupos: crianças HIV positivas e suas mães (n=18) e crianças HIV negativas e suas mães (n=61) foram avaliados pelo CH50 e AP50 (ensaios hemolíticos), dosagem e avaliação funcional da MBL, ativação da cascata terminal do complemento (ELISA) e o gene mbl2 (PCR). Os resultados não mostraram diferença significante entre os níveis séricos, atividade funcional e o gene da MBL entre os grupos, excluindo a sua influência sobre a transmissão materno-fetal do HIV / It was evaluated the mbl2 gene expression in 79 children and their HIV positive mothers with the aim to evaluate its influence on mother-to-child HIV. The patients were divided in two groups: HIV positive children and their mothers (n=18) and HIV negative children and their mothers (n=61) were evaluated by CH50 and AP50 (hemolytic assays); levels and functional MBL and terminal complement cascade (ELISA) and mbl2 gene (PCR). The results didn't show significant difference amons serum levels, functional activities and MBL gene between the groups, excluding the influence in the mother-to child HIV transmission.

DCL-1 (CD302) is a type I transmembrane C-type lectin receptor, which is expressed on monocytes, macrophages, granulocytes and dendritic cells. However, its extracellular domain lacks the amino acids motives essential for carbohydrate binding in the presence of calcium ions, suggesting that it does not have the classic binding capacity found in other C-type lectin receptors such as the mannose receptor. No exogenous or endogenous ligands have been identified yet, though. Due to internal colocalization with F-actin we can assume, that this unconventional lectin receptor plays a role not only in endocytosis and phagocytosis but also in the cell adhesion and migration. The receptor DCL-1 was first identified as a genetic fusion partner of human DEC-205 multilectin receptor in Hodgkin's lymphoma cell lines. The experimental part of this thesis deals with the characterization of disulfide bonds and data acquisition for validation of DCL-1 crystal structure. First the production and refolding conditions were optimized to obtain the highest amount of DCL-1 protein, precisely its extracellular domain. These optimal conditions were used to prepare the protein for in-gel digestion using specific endopeptidases in the presence of cystamine followed by LC-MS analysis. DCL-1 disulfide bonds were determined by comparing...