ORCID

0000-0002-4328-5988

Graduation Date

12-2021

Document Type

Master's Thesis

Degree

Master of Science

Program

Biological Science

Program Director

Meredith Protas, PhD

First Reader

Roger Lawrence, PhD

Second Reader

Ian Barr, PhD

Abstract

Abstract 1: Glycosylation plays an important role in facilitating viral transduction by acting as preliminary cell surface receptors. For this reason, the structural determinants in glycans that dictate viral tissue tropism need to be extensively studied to improve the efficacy of gene therapy vectors in basic research and eventually the clinic. Elucidating the dependencies for viral transduction initiation and understanding how these structural nuances of glycans initiate virion specific tropic effects is paramount when considering how to use vectors to improve clinical outcomes for patients suffering from illnesses with few treatment options. The goal of this project was to use MALDI-TOF-MS to provide baseline N-glycan profiles of the cell lines and tissues used to test gene therapy vectors. In doing so these profiles will be valuable to the field by clarifying what structural determinants may influence viral tropism. It was discovered Neu5Ac sialic acid content differs qualitatively amongst the seven cell lines analyzed. These differences may play into why some cell lines such as CHO-K1 and COS-7 can transduce more preferentially with some AAV serotypes like AAV5. In addition, sialic acid differences were also assessed in three tissue types used in transduction assays.

Abstract 2: After injury to the CNS, reactive astrocytes form a protective extracellular matrix to isolate damaged tissue. These astrocytes influence the surrounding tissue by upregulating the production of proteoglycans containing chondroitin sulfate. Due to the new cellular environment, chondroitin sulfate (CS) glycosaminoglycan chains are upregulated with predominately 4-O-sulated sulfation patterns. These sulfation patterns are known to inhibit axonal guidance, and ultimately neuronal regeneration. While the inhibitory effect of CS is well known, the mechanism by which these specific sulfation patterns may interact with receptors also known to have inhibitory effects on neuro-regeneration such as protein tyrosine phosphatase σ is unknown. To characterize these interactions reductive amination was used to immobilize these CS chains onto solid beads. Chondroitin sulfate was isolated from the organs of an ARSB null mouse model which lacks the N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB) which is involved in the degradation of glycosaminoglycans (GAGs). Disruption of arylsulfatase B leads to the production of CS chains with 4-O-sulfated non-reducing ends exclusively. Key findings indicate that purified GAG chains retain their ligand specificity after being covalently immobilized onto solid supports, and that these systems can be utilized to characterize the relationship between inhibitory forms of CS and protein tyrosine phosphatase σ.

Comments

This M.S. thesis contains two parts. The first being: Expanding the understanding of viral tropism by characterizing the N-glycomic profiles of transducible cell lines and tissue types, and the second is, characterizing functional receptors that mediate the inhibitory relationship between 4-O-sulfated chondroitin sulfate and neurons. These two sections are to be read separately and independent from one another. Thank you for reading.

Available for download on Monday, December 02, 2024

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