No description available.

Diss. Linköping : Univ., 2003.

Thesis (M.S.)--University of Delaware, 2009. / Principal faculty advisor: Robert W. Mason, Dept. of Biological Sciences. Includes bibliographical references.

Gaucher disease, the most common lysosomal storage disorder, results from a deficiency in the enzyme glucocerebrosidase. Inherited as an autosomal recessive disorder, Gaucher disease is clinically heterogeneous with both non-neuronopathic (Type 1) and neuronopathic (Types 2 and 3) subtypes. Although over 100 mutations in the glucocerebrosidase (GBA) gene have been identified, there still exists a poor correlation between individual genotypes and observed phenotypes, particularly for the neuronopathic subtypes. Using DNA isolated from archival tissue samples and standard molecular biology techniques, two novel and two rare mutations were identified in three individuals with neuronopathic Gaucher disease. One mutation identified only in aboriginals of Cree descent was further characterized by heterologous expression in baculovirus-infected Sf9 cells and displayed moderate levels of residual enzyme activity, despite the corresponding disease severity observed. Heterologous expression studies were extended to examine systems for high-level glucocerebrosidase expression for biochemical analysis and biotherapeutics. While the methylotrophic yeast Pichia pastoris was found to express minimal amounts of human glucocerebrosidase even when selected for high gene copy number, stable transfected Sf9 cells were found to produce functional glucocerebrosidase at a level of 1.0–1.3mg/L of cell culture. A subsequent analysis of synonymous codon usage bias in Pichia pastoris identified a significant difference in codon choice between the expression host and the GBA gene. Codon optimization studies using a 5′ fragment of the GBA gene fused to a luciferase reporter gene found that alterations in both G+C content and codon bias increased expression levels 7.5 to 10 fold. This suggests that codon optimization of the entire GBA gene could significantly improve production levels of this important enzyme in Pichia pastoris and other expression hosts. / Graduate

No description available.

No description available.

We investigated whether human lysosomal hydrolases, in common with secretory and plasma membrane glycoproteins, associate with the ER chaperone calnexin. Neither $ alpha$- or $ beta$-chains of $ beta$-hexosaminidase A, cathepsin D, nor the endogenous proteases cathepsins B or L associated with calnexin in COS-I cells. Hex $ alpha$-chains misfolded due to either the incorporation of azetidine-2-carboxylic acid, treatment with dithiothreitol, or the presence of a Tay-Sachs Disease mutation (leading to retention of Hex A $ alpha$-chains in the ER) also did not associate with calnexin. Chemical-crosslinking reagents or long-term labeling also failed to show a Hex A $ alpha$-chain association with calnexin. Lysosomal hydrolases also did not associate with the ER chaperone calreticulin. Surprisingly, $ alpha$-L-iduronidase and Hex A $ alpha$-chains associated with calnexin when overexpressed using a CMV promoter. The segregation of lysosomal hydrolases from secretory proteins thus occurs at an earlier stage than predicted. Hydrolase folding appears to be controlled by a pathway different from that used by secretory and plasma membrane glycoproteins.

xx, 277 leaves : ill. ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1974) from the Dept. of Plant Physiology, University of Adelaide

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D. 1974) from the Dept. of Plant Physiology, University of Adelaide.