The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

Deficiência na enzima lisossomal glucocerebrosidase (GCR) resulta na doença de Gaucher. O tratamento atual consiste na administração da enzima exógena, produzida em células CHO. Porém, o medicamento disponível no mercado é extremamente custoso. Neste trabalho, propusemos a clonagem e a expressão da GCR humana em células CHO, visando a obtenção de um clone celular produtor para viabilizar a produção futura da enzima, a um custo menor, no Instituto Butantan. A expressão estável da GCR recombinante foi obtida a partir da transfecção de células CHO-dhfr- com o plasmídeo pED de expressão em células de mamíferos contendo o cDNA da GCR, seguido de amplificação gênica por MTX. A GCR foi detectada no extrato celular (~ 64 kDa) e secretada para o sobrenadante (63-69 kDa) em ensaios de western blotting, usando o anticorpo policlonal anti-GCR gerado neste trabalho. A enzima secretada hidrolisou o substrato 4-MUG e a sua produtividade foi estimada em 5,14 pg/célula/dia para o melhor subclone produtor, selecionado para a produção futura da GCR em larga escala. / Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher\'s disease. Current treatment consists on enzyme replacement therapy by the administration of recombinant GCR produced in CHO cells. However, the medicine available in the market is extremely expensive. In this work, we proposed the cloning and expression of human GCR in CHO cells, in order to obtain a productive cellular clone for future production of GCR enzyme at a lower cost at the Butantan Institute. The stable expression of recombinant GCR was obtained after transfection of CHO-dhfr- cells with pED mammalian expression vector containing the GCR cDNA, followed by gene amplification with MTX. The GCR was detected by western blotting analysis, either as cell-associated (~ 64 kDa) or as secreted forms (63-69 kDa), using the anti-GCR polyclonal antibody produced in this work. The secreted enzyme was active on 4-MUG and was produced at a level of about 5,14 pg/cell/day for the best producer subclone, selected for subsequent steps of GCR production on large scale in next future.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Lafora disease is a neurodegenerative disorder caused by mutations in either the EPM2A or the EPM2B gene that encode a glycogen phosphatase, laforin and an E3 ubiquitin ligase, malin, respectively. A hallmark of the disease is accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle and heart. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein degradation/quality control. We evaluated three arms of protein quality control (the autophagolysosomal pathway, the ubiquitin-proteasomal pathway, and ER stress response) in embryonic fibroblasts from Epm2a-/-, Epm2b-/- and Epm2a-/- Epm2b-/- mice. There was an mTOR-dependent impairment in autophagy, decreased proteasomal activity but an uncompromised ER stress response in the knockout cells. These defects may be secondary to the glycogen overaccumulation. The absence of malin, but not laforin, decreased the level of LAMP1, a marker of lysosomes, suggesting a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. To understand the physiological role of malin, an unbiased diGly proteomics approach was developed to search for malin substrates. Ubiquitin forms an isopeptide bond with lysine of the protein upon ubiquitination. Proteolysis by trypsin cleaves the C-terminal Arg-Gly-Gly residues in ubiquitin and yields a diGly remnant on the peptides. These diGly peptides were immunoaffinity purified using anti-diGly antibody and then analyzed by mass spectrometry. The mouse skeletal muscle ubiquitylome was studied using diGly proteomics and we identified 244 nonredundant ubiquitination sites in 142 proteins. An approach for differential dimethyl labeling of proteins with diGly immunoaffinity purification was also developed. diGly peptides from skeletal muscle of wild type and Epm2b-/- mice were immunoaffinity purified followed by differential dimethyl labeling and analyzed by mass spectrometry. About 70 proteins were identified that were present in the wild type and absent in the Epm2b-/- muscle tissue. The initial results identified 14 proteins as potential malin substrates, which would need validation in future studies.