Thesis (doctoral)--Ludwig Maximilians Universität zu München, 1979.

Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1988.

In order to understand glycoprotein biosynthesis and processing, we have studied the glycosylation and intracellular assembly kinetics of murine IgM which are expressed functionally only at specific stages of B-cell differentiation by the corresponding tumor cell lines. We have shown that the majority of carbohydrate chains on intracellular IgM contain predominantly Man₈GlcNac₂ rate limiting step in the carbohydrate processing is the transport from the RER to the Golgi apparatus. We made comparisons of carbohydrate structures on secretory and membrane-bound u chains produced by different cell lines. Our results show that the carbohydrates on WEHI231 membrane-bound IgM are less processed, and the processing at individual glycosylation sites is different for IgMs produced by plasmacytoma (MOPC104E) and hybridoma (MPC11xW279.2) cell lines. In addition, we also show that the glycosylation and processing are dramatically altered by lowering the glucose concentration in the cell culture medium. These results are a beginning for our understanding of the influence of the polypeptide on the final glycosylation patterns of a glycoprotein, and the genetic and environmental control over the carbohydrate processing during intracellular transport. The kinetic studies on IgM synthesis and maturation in WEHI231 as well as WEHI279.1/12 cells have led to the conclusion that membrane bound IgM and soluble IgM are segregated and processed individually even in the same cell. These differences appear to lead to the changes in carbohydrate/processing for membrane-bound and soluble IgM.

Many protein-linked oligosaccharides are believed to impart biological specificities to the molecules. The knowledge of detailed structural characteristics of oligosaccharides is essential for understanding their functions. In order to develop methodology for characterization of oligosaccharides of glycoproteins, and to compare glycosylation patterns of different immunoglobulins, oligosaccharides of IgM from two cell lines, MOPC 104E and PC 700, were analyzed. Homogeneous preparations of glycopeptides carrying individual glycosylation sites of the heavy chain were obtained from the two IgM's. The oligosaccharides of these glycopeptides were prepared by hydrazinolysis, and fractionated by HPLC under conditions that resolve oligosaccharides by charge and size, and by affinity chromatography on Concavalin A-Sepharose. Structures of some of these oligosaccharides were determined by 400 MHz NMR spectroscopy. HPLC fractionation by charge resolved oligosaccharides with zero, one, two, and three sialic acids. As indicated by HPLC analyses, oligosaccharides at all the glycosylation sites of both the IgM's were highly heterogeneous. A comparative study on oligosaccharides prepared by peptide-N-glycosidase F digestion of glycopeptides showed a similar degree of heterogeneity. Therefore, it was concluded that the observed heterogeneity of oligosaccharides was not an artefact caused by hydrazinolysis. Major differences between the glycosylation patterns of the two IgM's were evident from analyses of the oligosaccharides by both chromatographic techniques and NMR spectroscopy. MOPC IgM contained a high proportion of sialylated oligosaccharides when compared to PC IgM. Furthermore, the major oligosaccharide structures of MOPC IgM were of triantennary type whereas PC IgM contained biantennary oligosaccharides as its major species. In both the IgM's, a decreased trend of oligosaccharide processing was observed from the N-terminus to the C-terminus.

We have chosen the murine immunoglobulin M (IgM) as system to study glycoprotein biosynthesis and carbohydrate processing. Secreted IgM heavy chain (m) has five glycosylation sites which location and structures have been determined. m chain variable region (VH) is involved in antigen binding, while the constant region (CH) is responsible for the effector functions in which the carbohydrate plays an important role. We have determined the carbohydrate structures present at each glycosylation site of IgM produced by a hybridoma cell line (PC 700) and its derived mutants and compared them to IgM from myeloma cell MOPC 104E. PC 700 mutants secrete altered IgM. The alterations include: deletion of one or more constant domains (mutants: 128, 313, and 562) and m chain hyperglycosylation (mutants 21 and 38). Gene analysis indicated that deletions can arise from two different mechanisms. One of these involve a major gene change (mutant 128), while others come from base point mutations (mutants 313 and 562). Cells 21 and 38 did not appear to have m gene insertions. Determination of purified single glycosylation site structures show that PC 700 m chain is processed only to biantennary. Heavy chain protein fragmentation and carbohydrate studies indicate that mutants 21 and 38 alterations are due to an increase in oligosaccharide processing and reduction of unprocessed structures. There is a trend of processing going from PC 700 < 21 < 38. In addition, our results show how growth cell conditions can affect the carbohydrate processing without altering the determinants of m chain oligosaccharide structures. Studies on the IgM molecule illustrate the need for precisely define structure-function relationships. This would allow the selection of the best antibodies for studies such as those involved in immunotherapy.

Thesis (M.Sc.) -- Dept. of Microbiology, University of Adelaide, 1973.

Thesis (Ph. D.)--Ohio State University, 2008. / Non-Latin script record

x, 156 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1988

Madariaga virus (MADV) has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June 2017, a fatal MADV infection was confirmed in a community of Darien Province. We conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. In addition, by applying a catalytic, force-of-infection (FOI) statistical model to two serosurveys from Darien Province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. In 2017, MADV and VEEV IgM seroprevalences were 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%, VEEV: 16.8%, Una virus (UNAV): 16.0%, and Mayaro virus: 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households, raising questions about its vectors and enzootic transmission cycles. Insomnia was associated withMADVand VEEV infections, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in Aruza and Mercadeo, respectively. The lack of additional neurological cases suggests that severe MADV and VEEV infections occur only rarely. Our results indicate that over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased-potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially in some locations of eastern Panama. / National Institute for Health Research / Revisión por pares

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