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The MS-818 compound is used in the proliferation process of neuronal cells and many biological activities that accompany this process such as astrocyte differentiation, inhibition of neuronal apoptosis, and fraction repairs. We do know the effects of this compound, but the mechanism of action remained uncertain until now. To determine the pathway of this compound, NT2 cells were cultured and lysed to isolate the proteins. Affinity Chromatography was performed in order to immobilize the MS-818 compound to a Hi-Trap NHS column. The NT2 protein sample was injected through the column and eluted with a MS-818 concentrated, high salt content elution buffer. SDS-PAGE was then performed to isolate the proteins that bound to MS-818. The gel was visualized using Coomassie Blue. The results indicate that there are two proteins associated in the mechanism of this compound. A standard protein marker ranging from 10 kDa to 250 kDa was used to compare the bands. The findings indicate that one of the protein bands is slightly less than 250kDa and the other is between 50-75 kDa. When the proteins are confirmed by mass spectrometry sequencing, this will help to promote this compound as a drug candidate.

atfish production is the largest aquaculture industry in the United States and infectious agents are responsible for 45% of all economic losses. Ictalurid herpesvirus 1 or Channel catfish virus (CCV) has a great economic impact on channel catfish aquaculture; yet it also has the potential for becoming a highly efficient vaccine vector eliciting long-lived immune responses against itself and, as a recombinant, other important catfish pathogens (bacteria, myxosporean, and fungi). However, little is known about CCV’s genome, its gene functions or genetic interactions with its host. Better understanding of CCV biology and pathogenesis could enable more rational vaccine design and other control strategies for CCV. My thesis is that “systems biology” can enable much more rapid understanding of CCV biology and pathogenesis. To test this thesis I needed to first more fully annotate the CCV genome, then construct a rapid system for generating CCV mutants and recombinants for systems biology research and then apply these tools in a systems biology experiment. I experimentally annotated the CCV proteome by proteogenomic mapping followed by real-time PCR and confirmed the expression of 37 of the 76 previously predicted ORFs (25 for the first time) as well as 17 novel ORFs. I next constructed two different infectious clones of CCV: one as three overlapping bacterial artificial chromosomes (BACs) and the other as a full length CCV BAC. These CCV BACs facilitate CCV mutant and recombinant production and I regenerated a genotypically wild-type and an attenuated virus. To further simplify CCV mutant production, I next adapted the CCV infectious clone for lambda phage crossover recombination cloning to enable sequence transfer into a specific CCV locus by a simple one-step in-vitro reaction. Finally, I used the CCV infectious clone, in combination with affinity purification, to identify interacting partners of the CCV zinc RING finger proteins ORF9, ORF11 and ORF12 to provide insight into the topology of one presumptive CCV-channel catfish molecular interaction network module. The work in this dissertation supports my thesis and the CCV BAC tools were patented; together these provide tools to facilitate and accelerate the development and testing of better CCV vaccines.

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Despite the growing use of hybrid catfish (Ictalurus furcatus × I. punctatus) in aquaculture, few studies have investigated the comparative effects of temperature on physiological performance with other cultured catfish. Therefore, growth, metabolism, and blood-oxygen affinity of blue (I. furcatus), channel (I. punctatus), and hybrid catfish were compared at moderate (23°C) and high (33°C) water temperatures. Channel catfish had the greatest growth and were in better condition compared to blue and hybrid catfish. Hybrid catfish standard metabolic rates did not differ from blue or channel catfish, while hybrid catfish had higher maximum metabolic rates, larger metabolic scopes, and better swimming performance than blue and channel catfish. Blue and hybrid catfish had a higher affinity to bind oxygen than channel catfish, with hybrid catfish generally having larger Bohr effects than blue and channel catfish. These results indicate hybrid catfish likely achieve superior performance due to larger metabolic scopes and Bohr effects.

This thesis describes the use of functional nucleic acid (FNA) superstructures entrapped within monolithic macroporous sol–gel-derived silica for solid-phase flow-based sensing of small molecules and macromolecular proteins. The work described herein overcomes a long-standing issue with entrapment of biomolecule into sol–gel-derived materials; the mesoporous pore morphology required to retain entrapped biomolecules prevents detection of large analytes as these can’t access the entrapped species. It is shown that large DNA superstructures can be produced through rolling circle amplification of a functional nucleic acid, resulting in concatemeric FNA species with dameters of several microns. Such species can be entrapped within macroporous sol-gel derived materials with micron-sized pores with minimal leaching, thus allowing for detection of a wide range of molecules, including biomolecules. Optimal materials for entrapment of FNA superstructures was achieved using a high-throughput material screening method, which minimized biomolecule leaching while maintaining FNA activity. Using an optimized material, concatemeric aptamer superstructures were entrapped within macroporous monolithic columns for flow-based detection of small molecules and proteins, extending the range of analytes that can be analyzed using biohybrid monolithic columns. Preliminary studies on the formation and properties of a DNAzyme superstructure for detection of E. coli detection were also performed, which provided valuable information on factors that must be controlled to allow reproducible fluorescence-based detection of E. coli using the crude intracellular matrix as the target. / Thesis / Master of Science (MSc)