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1	Reverse genetic analysis of SPARC function in vertebrate embryogenesis Gilmour, Darren T. January 1995 (has links) No description available. 572.8 Secreted protein acidic rich in cysteine
2	Clonagem e expressão heteróloga do antígeno SsaA de Staphylococcus saprophyticus e avaliação da secreção durante interação com macrófagos / Cloning and heterologous expression of the staphylococcus saprophyticus antigen SsaA and evaluation of secretion during interaction with macrophages Rosa, Isabella I. R. 28 June 2016 (has links) Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-09-14T17:40:15Z No. of bitstreams: 2 Dissertação - Isabella Inês Rodrigues Rosa - 2016.pdf: 1910068 bytes, checksum: 8f551746fe8c21347507aecf98f02609 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-09-14T17:41:15Z (GMT) No. of bitstreams: 2 Dissertação - Isabella Inês Rodrigues Rosa - 2016.pdf: 1910068 bytes, checksum: 8f551746fe8c21347507aecf98f02609 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-14T17:41:15Z (GMT). No. of bitstreams: 2 Dissertação - Isabella Inês Rodrigues Rosa - 2016.pdf: 1910068 bytes, checksum: 8f551746fe8c21347507aecf98f02609 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-06-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Staphylococcus saprophyticus is a pathogenic bacterium of the urinary tract and the main etiological agent of urinary tract infections by Gram-positive bacteria. Although S. saprophyticus potentially can cause serious infections such as pyelonephritis, septicemia, endocarditis and nephrolithiasis, and also multidrug resistance has been reported, not much is known about the mechanisms used by this bacterium during infection. Secreted proteins might be essential on those mechanisms if their role is accomplished during phagocytosis by their assistance of an active infection in phagocytic cells, protecting against oxidative stress and increasing the persistence of bacterial cells within phagocytes, and / or causing lysis of the host cell. Recently our group identified the immunogenic protein SsaA in the secretome of S. saprophyticus. This protein had been previously identified in S. aureus proteome, and it appears to be controlled by regulatory systems for known virulence factors. It also presents similarities with lytic proteins and proteins that assist the persistence within phagocytic cells. However, no approach had analyzed the contribution of SsaA during infection, therefore, through the construction a cloning vector containing the S. saprophyticus gene ssaA, heterologous expression of the recombinant protein and the production of specific polyclonal antibodies, it was able to verify the interaction of SsaA and proteins from macrophages infected by bacterial cells. Through immunofluorescence microscopy, it was verified that the dispersion of SsaA is not limited to phagocytic cells but it was throughout their cytoplasm after internalization of the bacterium. These findings together with other evidence in the literature suggest that SsaA is used during infection by S. saprophyticus, more specifically during phagocytosis. Further approaches are required to confirm if SsaA has a lytic activity and also characterize this protein as a virulence factor, contributing to elucidate strategies used by S. saprophyticus during infection in the human host. / Staphylococcus saprophyticus é uma bactéria patogênica do trato urinário e principal agente etiológico de infecções urinárias por bactérias Gram-positivas. Apesar de potencialmente S. saprophyticus ocasionar infecções graves como pielonefrite, septicemia, nefrolitíase e endocardite, e relatos de multirresistência a antibióticos, relativamente pouco é conhecido sobre os mecanismos utilizados por esta bactéria durante infecção. Proteínas secretadas podem ser essenciais nesses mecanismos se seus papéis forem durante fagocitose auxiliando uma internalização ativa na célula fagocítica, protegendo contra o estresse oxidativo e aumentando a persistência de células bacterianas no interior de fagócitos, e/ou causando lise da célula hospedeira. Recentemente nosso grupo identificou a proteína imunogênica SsaA no secretoma de S. saprophyticus. Essa proteína já havia sido identificada em S. aureus como altamente imunogênica, e parece estar relacionada a fatores de virulência como o Antígeno Imunodominante A (IsaA), a proteína Urease, a hidrolase de peptideoglicano LytM, a Proteína Repressora de Toxinas (Rot) e a proteína de choque térmico HslU. Contudo, poucos estudos conseguem identificar a proteína SsaA secretada e nenhum analisa sua contribuição durante infecção por bactérias do gênero Staphylococcus. Através da construção de um vetor de clonagem contendo o gene ssaA de S. saprophyticus, expressão heteróloga da proteína recombinante e produção de anticorpos policlonais específicos, foi possível verificar a interação entre SsaA e proteínas de macrófagos infectados por células de S. saprophyticus. Através de microscopia de imunofluorescência, foi verificado que a secreção de SsaA não é limitada à fagossomos, mas esta proteína é dispersa em todo o citoplasma da célula fagocítica após internalização de células bacterianas. Os resultados encontrados sugerem que SsaA é utilizada por S. saprophyticus durante infecção, especificamente durante fagocitose. Estudos posteriores serão necessários para confirmar se SsaA possui atividade lítica e caracteriza-la como fator de virulência, contribuindo para elucidar estratégias utilizadas por S. saprophyticus durante infecção no hospedeiro humano. Staphylococcus saprophyticus Proteína secretada Infecção Fagocitose Clonagem molecular Staphylococcus saprophyticus Secreted protein Infection Phagocytosis Molecular cloning BIOQUIMICA::BIOLOGIA MOLECULAR
3	Olfactory ensheathing cell mediated mechanisms of neurite outgrowth and axon regeneration Witheford Richter, Miranda 11 1900 (has links) The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration. Spinal cord injury Transplantation Corticospinal Axon guidance Nervous system Proteomics Cell adhesion molecules Plasma membrane Astrocytes Glia Rubrospinal Lesion Angiogenesis Tyrosine hydroxylase Regeneration Olfactory system Osteonectin In vitro
4	Olfactory ensheathing cell mediated mechanisms of neurite outgrowth and axon regeneration Witheford Richter, Miranda 11 1900 (has links) The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration. Spinal cord injury Transplantation Corticospinal Axon guidance Nervous system Proteomics Cell adhesion molecules Plasma membrane Astrocytes Glia Rubrospinal Lesion Angiogenesis Tyrosine hydroxylase Regeneration Olfactory system Osteonectin In vitro
5	Olfactory ensheathing cell mediated mechanisms of neurite outgrowth and axon regeneration Witheford Richter, Miranda 11 1900 (has links) The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration. / Medicine, Faculty of / Graduate Spinal cord injury Transplantation Corticospinal Axon guidance Nervous system Proteomics Cell adhesion molecules Plasma membrane Astrocytes Glia Rubrospinal Lesion Angiogenesis Tyrosine hydroxylase Regeneration Olfactory system Osteonectin In vitro
6	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760
7	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760

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