Thesis (Ph. D.)--Ohio State University, 2005. / Title from second page of PDF file. Document formatted into pages; contains xix, 347 p.; also includes graphics. Includes bibliographical references (p. 183-192). Available online via OhioLINK's ETD Center

Thesis (Ph.D.)--Duquesne University, 2006. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p.157-190) and index.

Thesis (M.S.)--Duquesne University, 2009. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 113-138) and index.

B-catenin is a transcriptional co-activator in the Wnt signaling pathway. Upon Wnt stimulation, cytosolic B-catenin translocates into the nucleus where it forms complexes with members of the TCF /LEF family of transcription factors to activate gene transcription. Translocation into the nucleus is followed by transcriptional activation of B-catenin's target genes, which are involved in proliferation, angiogenesis and oncogenesis, is a crucial step in the progression of a subset of cancers. The cellular expression of B-catenin is known to be regulated by phosphorylation. However, the mechanisms(s) responsible for B-catenin nuclear entry is not well understood. Recently, B-catenin was reported to be post-translationally modified by O-glycosylation in breast cancer cells. We investigated whether O-glycosylation regulates the signal transduction properties of the protein. Our results indicated that while there are higher levels of total B-catenin in the nucleus of two prostate cancer cell lines (DU-145 and LNCaP) compared to that in a normal prostate epithelial cell line (PNT1A), most of it was in the unglycosylated form. Also, the normal prostate cell line exhibited higher levels of O-glycosylated B-catenin in both the nucleus and cytosol than what was seen in the two prostate cancer cell lines. We carried out further experiments using PUGNAc, a non-cytotoxic reversible inhibitor of O-GlcNAcase, which causes a time dependent increase in cellular levels of O-glycosylated B-catenin. Treatment of prostate cancer cells with PUGNAc caused a decrease in the expression of B-catenin in the nucleus with increasing cellular O-glycosylation of the protein suggesting that O-glycosylation was hindering B-catenin nuclear translocation. Additional studies showed that O-glycosylation of B-catenin decreased that transcriptional activity of a TopFlash reporter plasmid and the protein expression of two B-catenin target genes. Our results suggest that O-glycosylation of B-catenin may represent a novel mechanism important in the regulation of the nuclear localization and transcriptional activity of B-catenin. / Thesis / Master of Science (MS)

The glycosylation patterns of monoclonal antibodies (Mabs) have become very important in determining therapeutic abilities of many drugs. The thesis studied 3 cell lines producing humanized Mabs in the presence of variable concentrations of the reducing agent dithiothreitol (DTT) to artificially lower the CRP and affect glycan patterns. A new high-throughput hydrophilic interaction chromatography (HILIC) method was developed and used to show a decrease in the Galactosylation Index (GI) of NS0 IgG1 by as much as 50% in cultures with CRP values lower than -100 mV. The shift in GI was unique to NS0 cultures; CHO DP-12 indicated no significant change in GI but did have a 7% increase in fucosylated species in cultures with higher [DTT]. Furthermore no DTT related shifts were observed in any of the CHO EG2-hFc glycans. EG2-hFc did however have an exceptionally high GI of 0.625 compared to GIs of 0.245 in DP-12 and 0.314 in NSO. Another component of the trials determined, using S35 radiolabeling, that the assembly pathway of IgG1 progressed via HC→HC2→HC2LC→HC2LC2 and that the ratio of heavy chain dimer to heavy chain monomer increased greatly over time for cultures with higher DTT concentrations. The increase in heavy chain dimers and lower GI appear to be correlated, possibly due to disruption of the disulfide bonds between LC and HC within the Golgi. This disruption in disulfide bonds affecting galactosyltransferase (GalT) activity is supported by the findings that the partially reduced fragments of IgG1; HC and HC2, are less galactosylated than the HC2LC and whole IgG1 when treated with GalT. When native and agalactosylated EG2-hFc and IgG1 were treated with GalT in vitro, EG2-hFc exhibited an almost 10 fold higher activity level. The cause for the higher activity may be due to overall size difference or point mutations in the Fc region of EG2-hFc. Through the manipulation of CRP, glycan patterns can be influenced however the effect is not universal and must be determined on a per cell line basis. Furthermore, EG2-hFc’s higher GI value may translate into better in vivo activity as a therapeutic and determination of reasons for the high GI may lead to better means for future glycoengineering. / February 2015

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

No description available.

The main goal in biopharmaceutical production is achieving high volumetric productivity while maintaining product quality (i.e. glycosylation). The objectives of this project were to explore the use of dielectric analysis in the early detection of cell demise and to analyze the impact of nucleotide / nucleotide sugar precursor feedings in biopharmaceutical production and glycosylation. Measurements of changes in the polarizability of individual cells can be performed in a dielectrophoretic (DEP) cytometer designed at the University of Manitoba. In this instrument the trajectory of individual cells was tracked according to their polarizability and recorded as a force index (FI). The identified sub-populations from a batch bioreactor and apoptosis-induced cultures were correlated with the fluorescent markers of apoptosis analyzed in a flow cytometer. Discrete cell sub-populations were identified as cells passed through the various stages of apoptosis. In the batch and the starvation culture the early changes in the measured FI of cells correlated with the Annexin V fluorescent assay associated with early phase apoptosis. For the oligomycin and staurosporine cultures changes in the FI could be correlated to modifications in the mitochondrial metabolism linked with early apoptosis for both inducers. In fed-batch experiments 10 mM galactose alone or 20 mM galactose in combination with 1 mM uridine or 1 mM uridine + 8 μM MnCl2 was added to the basal and feed medium for two CHO cell lines to determine their impact on the biopharmaceutical production and the glycosylation process. The results showed that the addition of all three precursors combined increased UDP-Gal, which increased and maintained the galactosylation index during the bioprocess for CHO-EG2 and CHO-DP12 cultures by 25.4% and 37.9%, respectively, compared to the non-supplemented fed-batch culture. In both cell lines saturation was reached when a further increase in the UDP-Gal concentration did not increase the galactosylation. A negative impact on cell growth was observed with the uridine addition in the CHO-EG2 culture, which was linked to the CHO-EG2 cell line being DHFR-/-. This work presents a dielectric detection method to monitor early changes in the cell metabolism and information for shifting and maintaining galactosylation during biopharmaceutical production. / February 2016

It was of interest to clone key genes involved in O-glycosylation with a view to using reverse genetics to establish their function. The Candida homolog of the S. cerevisiae MNT1 gene (Hausler and Robbins, 1992) was cloned by heterologous probing of a genomic DNA library. The CaMNT1 gene was found to be regulated differentially in response to the environment and exhibited a transitory increase in the level of transcription during early germ tube formation. Low stringency Southern analysis of C. albicans genomic DNA identified several CaMNT1 homologs suggesting CaMNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases. In order to demonstrate that specific glycosyl residues were actively involved in the host-fungus interaction, the CaMNT1 gene was disrupted in two strains using the ura-blaster technique. Disruption at the CaMNT1 locus led to a 90% reduction in -1,2-mannosyltransferase activity when -methyl mannoside was used as an acceptor, but had no obvious influence on viability, growth rate, germ tube formation or proteinase production. CaMnt1 appears to be involved in O-glycosylation since the Camnt1 null mutant strain accumulated intracellularly the O-glycosylated enzyme chitinase. Mannosyltransferase-deficient Camnt1 mutants were significantly reduced in their ability to adhere to human buccal epithelial cells in vitro and were attenuated in virulence in systemic models of candidosis. O-linked mannan may therefore be important for direct interactions with epithelial surfaces or for the stabilization and function of cell surface adhesins. The low virulence potential displayed by Camnt1 mutants clearly demonstrates the important role glycosylation plays in the virulence of C. albicans. Given that O-glycosylation differs significantly between yeast and man, this protein modification may constitute a novel target for antifungal agents.