Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Bacterial biofilms are surface associated colonies that are of considerable concern and interest to industry, medicine and research. They are resistant to antibiotics, their host’s defences and are able to survive under harsh conditions. Biofilm formation in many bacterial strains are dependent on the production of a polysaccharide intercellular adhesion (PIA), a beta-(1-->6)-N-acetylglucosamine polymer. Vaccines derived from biologically isolated PIA have shown efficacy against clinically isolated strains of E. coli and pathogenic strains of S. aureus in animal models. Accordingly, chemically synthesized neoglycoconjugates based on PIA glycosides will be developed to serve as lead compounds for the development of new antibiotics as well as vaccines against biofilm dependent infections. Described in this thesis is a comprehensive study of the synthesis of PIA oligosaccharides and their deacetylated equivalents, the strategy for installing a stable linker on the free reducing oligosaccharide terminus and finally the conjugation to a model carrier protein for the development of potential neoglycoprotein vaccines.

Bacterial biofilms are surface associated colonies that are of considerable concern and interest to industry, medicine and research. They are resistant to antibiotics, their host’s defences and are able to survive under harsh conditions. Biofilm formation in many bacterial strains are dependent on the production of a polysaccharide intercellular adhesion (PIA), a beta-(1-->6)-N-acetylglucosamine polymer. Vaccines derived from biologically isolated PIA have shown efficacy against clinically isolated strains of E. coli and pathogenic strains of S. aureus in animal models. Accordingly, chemically synthesized neoglycoconjugates based on PIA glycosides will be developed to serve as lead compounds for the development of new antibiotics as well as vaccines against biofilm dependent infections. Described in this thesis is a comprehensive study of the synthesis of PIA oligosaccharides and their deacetylated equivalents, the strategy for installing a stable linker on the free reducing oligosaccharide terminus and finally the conjugation to a model carrier protein for the development of potential neoglycoprotein vaccines.

No description available.

<p> 0-GlcNAc modification is a single N-acetylglucosamine (GlcNAc) modification on Ser or Thr residue on protein. The addition and removal of the 0GlcNAc molecule are controlled by two enzymes (OGT and NCOAT). In this study, I expressed and purified the two enzymes involved in the 0-GlcNAc modification. A method was developed for the synthesis and purification of the peptide substrate YSDSPSTST for in vitro glycosylation and characterized the OGT enzyme activity by the in vitro glycosylation and H3 labeling. A method was developed based on detection of glycosylation peptide by mass spectrometry after separation by capillary liquid chromatography (CapLC). The optimization of mass spectrometry parameters was done using synthesized standard glycopeptide YSDSPSgTST ("Sg" represents 0-GlcNAc modified Serine). The in vitro modification site was determined by CID after alkaline J3-elimination. Furthers experiment could include detection of 0-GlcNAc modification of protein substrate both in vitro and in vivo. This will give a better understanding of the dynamics of 0-GlcNAc modification. </p> / Thesis / Master of Science (MSc)

Statistically based experimental designs were applied to optimise medium constituent for biogas production utilizing N-­‐acetylglucosamine as a carbon source for Clostridium beijerinckii and Clostridium paraputrificum. The important medium constituents influencing total biogas produced, identified by the Plackett and Burman method, were FeSO4.7H2O and initial pH for C. beijerinckii cultures whilst for C. paraputrificum cultures N-­‐acetylglucosamine, L-­‐ cysteine.HCl.H2O and MgCl2. A one factor L-­‐cysteine.HCl.H2O optimization design was applied to investigate the ideal concentration of L-­‐cysteine.HCl.H2O required to achieve an anaerobic environment for optimum C. beijerinckii total biogas production. The Method of Steepest Ascent was then employed to locate the optimal area of the significant medium variables. Using the Box-­‐behnken method, experimental results showed that there were significant linear effects of independent variables, N-­‐acetylglucosamine for C. beijerinckii cultures and for C. paraputrificum cultures N-­‐acetylglucosamine, L-­‐cysteine.HCl.H2O and MgCl2 on total biogas volume. Significant curvature or quadratic effects of N-­‐ acetylglucosamine and L-­‐cysteine.HCl.H2O were identified for C. paraputrificum cultures. There were no significant interaction effects between medium constituent variables on resulting biogas volume. The optimal conditions for the maximum volume of biogas produced for C. beijerinckii cultures were 21 g/l of N-­‐ acetylglucosamine, 0.1 g/l of FeSO4.7H2O and initial pH of 6.11 and for C. paraputrificum were 29 g/l of N-­‐acetylglucosamine, 0.27 g/l of L-­‐ cysteine.HCl.H2O and 0.4 g/l of MgCl2. Using this statistical optimization strategy, the total biogas volume from N-­‐acetylglucosamine utilization increased from 150 ml/l to 6533 ml /l in the C. beijerinckii cultures and 100 ml/l to 5350 ml/l in the C. paraputificum cultures. The maximum yield of bio-­‐hydrogen by C. paraputrificum from N-­‐acetylglucosamine was 2.55 mol of H2 / mol of N-­‐ acetylglucosamine and by C. beijerinckii was 2.43 mol of H2 / mol of N-­‐ acetylglucosamine.

Jaburetox (Jbtx) é um peptídeo de 10 kDa derivado de uma das isoformas de urease de Canavalia ensiformis. Em um estudo anterior realizado com o triatomíneo vetor da doença de Chagas Triatoma infestans, esse peptídeo foi encontrado interagindo com a proteína UDP-N-acetilglicosamina pirofosforilase (UAP), alterando também sua atividade enzimática no sistema nervoso central, in vivo e in vitro. A UAP já foi encontrada em eucariotos, bactérias e vírus, estando relacionada com as rotas de produção de quitina, N-glicosilação e síntese de glicoinositolfosfolipídeos. Assim, o presente trabalho tem três objetivos: i) investigar o efeito de Jbtx sobre a atividade enzimática e a expressão gênica da UAP do inseto modelo Rhodnius prolixus, ii) clonar e expressar a UAP e iii) estudar a UAP filogeneticamente. Para a primeira parte, foram avaliados, no triatomíneo R. prolixus, a atividade enzimática da UAP e o perfil de expressão dessa enzima e da quitina sintase em insetos controles e alimentados com Jbtx. Para a segunda, o cDNA da enzima de R. prolixus foi clonado em vetor pET-15b e expressado em Escherichia coli Rosetta 2. A purificação da enzima recombinante foi feita por cromatografia de afinidade a níquel. Para a terceira parte, foram buscadas sequências de aminoácidos homólogas às da UAP de R. prolixus no servidor pHmmer e foi construída uma árvore filogenética com o método de Máxima Verossimilhança. Os resultados obtidos indicam que o Jbtx aumenta a atividade enzimática da UAP em glândulas salivares, corpo gorduroso e epiderme, enquanto diminui a expressão da UAP em intestino médio anterior, túbulos de Malpighi, glândulas salivares, corpo gorduroso, epiderme e sistema nervoso central, assim como a expressão da quitina sintase nos mesmos órgãos e no intestino médio posterior. Foi obtida uma UAP recombinante de 56 kDa, compatível com peso molecular previsto in silico. A árvore filogenética construída contém 40 sequências, sendo 38 de insetos e 2 sequências de grupo externo. A árvore segue o padrão de evolução dos insetos e foi identificado um novo organismo com potenciais dois genes codificantes de UAP. Esse trabalho apresenta a primeira evidência de que Jbtx altera a expressão gênica em R. prolixus. O resultado obtido pela análise filogenética indica que a UAP é uma enzima ancestral à diversificação em Insecta. / Jaburetox (Jbtx) is a 10 kDa peptide derived from a urease isoform of Canavalia ensiformis. In a previous work with the triatomine vector of Chagas’ disease Triatoma infestans, this peptide was found interacting with the protein UDP-N-acetylglucosamine pyrophosphorylase (UAP), also increasing the UAP enzymatic activity in the central nervous system in vivo and in vitro. UAP has been described in eukaryotes, bacteria and virus, and is involved in chitin production, N-linked glycosylation and glyco inositol phospholipids synthesis pathway. Thus, the present work has three main aims: i) to understand the effect of Jbtx on this enzyme on the model insect Rhodnius prolixus, ii) to clone and express UAP and iii) to study UAP from a phylogenetic point of view. Firstly, UAP enzymatic activity and its expression profile, as well as the chitin synthase expression, were analysed in the triatomine R. prolixus in saline- or Jbtx-fed insects. Secondly, the cDNA from R. prolixus’ UAP was cloned into the pET-15b vector and expressed in Escherichia coli Rosetta 2. The recombinant enzyme was purified through a nickel affinity chromatography. Thirdly, homolog sequences to R. prolixus’ UAP were searched in pHmmer database and a phylogenetic tree was built using the Maximmum Likelihood method. The results obtained indicate that Jbtx increases UAP enzymatic activity in salivary glands, fat body and epidermis, while decreasing UAP’s expression in the anterior and posterior midgut, Malpighian tubules, salivary glands, fat body, epidermis and central nervous system, as well as the chitin synthase expression in the same organs and the posterior midgut. A 56 kDa recombinant UAP was obtained, in agreement with the in silico estimated size. The phylogenetic tree built has 40 sequences, from which 38 are from insects and 2 are from mammals (external group). The tree follows the insect evolution patterns and a new organism containing two potential UAP coding genes was identified. This work presents the first evidence that Jbtx is able to interfere in the gene expression in R. prolixus. The results obtained through phylogenetic analysis shows that UAP is an enzyme ancestral to the diversification in Insecta.

Several lactic acid bacteria contain β-galactosidases. Beta galactosidases catalyze lactose hydrolysis and transfer acceptor sugars onto galactose, producing galactooligosaccharides. The aim of this work was to exploit β-galactosidases of lactic acid bacteria as crude cell extracts to produce novel oligosaccharides with mannose, N-acetylglucosamine, and fucose. Of 17 strains of lactic acid bacteria, transferase activity was the strongest in crude cell extracts of Lactobacillus delbrueckii subsp. bulgaricus, followed by Streptococcus thermophilus, Lactobacillus animalis, and Lactobacillus reuteri in a buffered 19% (w/w) lactose solution. Incorporation of 6 % (w/w) glycerol increased transferase activity and enzyme stability at higher incubation temperatures. Incorporation of 10% (w/w) mannose, N-acetylglucosamine and fucose as acceptor sugars yielded three distinct oligosaccharides with mannose and two with N-acetylglucosamine and fucose, with the composition confirmed using gas chromatography-mass spectrometry. This is the first public report indicating production of oligosaccharides containing N-acetylglucosamine and fucose from β-galactosidases of lactic acid bacteria.

Cardiovascular disease is the leading cause of morbidity and mortality in developed nations, and heart disease is predicted to remain the leading killer for the foreseeable future. Acute myocardial infarctions—nearly 1.1 million annually occurring in the U.S. alone—are the major cardiovascular disease subgroup. Current treatments for myocardial infarction are limited to interventions that serve to mitigate the initial insult, but clinical applications to protect or regenerate damaged myocardium are lacking. This dissertation examines three therapeutic systems to treat the infarcted heart. First, the decoration of a polymeric nanoparticle with N-acetylglucosamine for the uptake of anti-­apoptotic therapeutics to ameliorate cardiomyocyte cell death. Second, novel dendrimeric structure architecture to allow for regio­selected decoration of ligands to induce angiogenesis. Third, exosomes secreted from hypoxic cardiac progenitor cells as a naturally derived therapeuticfor angiogenesis and anti-fibrosis, and to provide bio-inspired clues for future therapies.

No description available.