Yager, Eric Theses Advised
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Item Native Transcriptome Profiling of Virus-Infected Chinese Hamster Ovary Cells(Albany College of Pharmacy and Health Sciences Theses, 2021-12) Sher, Melissa '21Chinese Hamster Ovary (CHO) cells, used extensively to produce therapeutic recombinant proteins, may become infected with viruses without apparent impact to cell metabolism or morphology. As contamination is currently detected using lengthy culture-based methods, (Pharmacopeia U. S., USP Viral Safety of Biotechnology Products Derived from Cell Lines of Human or Animal Origin ) (Pharmacopeia U. S., Chapter Virology Test Methods, Current Version) contamination may spread before an infection is discovered, (RL, 1998) thereby squandering resources and impacting the amount of therapeutics available to patients. A molecular diagnostic method quantifying genes known to be differentially expressed (DEGs) during viral infection has the potential to facilitate early identification of viral contaminants. Unfortunately, the transcriptional landscape of CHO cells during infection is not well documented, and the proposed diagnostic method cannot be designed without thorough characterization of healthy and infected CHO transcriptomes. The present study characterized the transcriptome of CHO cells (healthy and infected with a common viral contaminant, Minute Virus of Mice, (MVM) (RL, 1998) (Jacoby RO, 2003)) whose mechanism of infection is known to impact gene expression (Adeyemi RO P. D., 2014) (Majumder K, 2017) using native RNA nanopore sequencing (Oxford Nanopore Technologies). Native transcriptome analysis of virus-infected CHO cells elucidated a panel of detectable genes which were differentially expressed during viral infection. Differential expression of these biomarkers could not be confirmed by an orthogonal RT-PCR. However, alternative uses of native transcriptome sequencing are promising for development of a rapid, non-specific molecular diagnostic method to monitor the health of CHO cell cultures.Item Determining the Role of Cellular Glycosphingolipid Biosynthesis on Influenza Virus Infectivity(Albany College of Pharmacy and Health Sciences Theses, 2018-08) Williams, Clare '18Influenza A virus (IAV) is able to mutate rapidly and has therefore developed resistance to available antivirals that target viral proteins. Glycosphingolipids (GSLs) are present in all eukaryotic cells and have been known to play critical roles during the lifecycle of various enveloped viruses. The enzyme glucosylceramide synthetase (GCS) is a key enzyme intermediate during GSL biosynthesis and could play a role in the generation of influenza virus particles. Various stages of the IAV lifecycle were analyzed in the absence of GCS; fluorescence microscopy was used to look at viral binding, gene expression and protein synthesis of IAV proteins was analyzed, and infectious virus particles were counted through the use of TCID50 and plaque assays. Host factor gene expression was also analyzed over the course of infection. It was found that GCS plays a role in the initial binding of IAV to the surface of host cells. This may lead to a decrease in IAV gene expression and protein production, and ultimately the amount of infectious virus released. It was also found that IAV upregulates GCS expression during infection. These findings can be used to further the understanding of how IAV interacts with GSLs and potentially lead to the development of a vaccine or antiviral treatment for influenza virus infection.