Hepatitis C Virus (HCV) is a bloodborne pathogen that infects approximately 3 million people in the United States and 140 million people worldwide. Once infected, only 15-25% of patients are able to clear the virus from their systems without treatment, leaving 75-85% of affected individuals with a chronic, life-long infection. Chronic HCV is often asymptomatic until decades after infection, so many patients are unaware of the need for treatment until damage has already reached advanced stages. Long-term HCV infection can lead to several serious diseases, including chronic hepatitis, liver cirrhosis, and liver cancer. In the United States, chronic HCV infection is the leading cause for liver transplants. As a RNA virus, mutations in the HCV genome are relatively common. Currently, there are 6 genotypes and at least 50 subtypes of the virus, which can affect response both to pharmaceutical treatment and to the host innate immune response.
When HCV infects a cell, the cell fights the infection by turning on the expression of antiviral genes, such as interferon-beta (IFNb). Once IFNb is produced, it is secreted from the cell and in turn activates expression of interferon-stimulated genes (ISGs) in the same cell and surrounding cells, thereby triggering the host innate immune response. HCV produces proteins that are capable of blocking IFNb. Without IFNb, the host is unable to fight off the HCV infection, which allows the infection to become chronic. Our lab has shown that the HCV non-structural protein NS5A inhibits Sendai Virus (SV)-induced IFNb gene expression, and is also vital to viral replication.
This study focuses on two mutant forms of HCV NS5A. NS5A 10A is the K2040 mutant with a lysine deletion, and has been shown to result in increased levels of viral replication. NS5A H27 is the L2198S mutant with a lysine to serine substitution, and has been shown to result in decreased levels of viral replication. We hypothesize that the differences in levels of replication between the two mutants is due to differential inhibition of SV-induced IFNb gene expression. Cells expressing NS5A 10A should have lower levels of antiviral gene expression, while expression of NS5A H27 should lead to higher levels of antiviral gene expression. RT-PCR and q-RT-PCR was performed on HEK 293 cells in order to measure differences in gene expression of IFNb and ISGs MX1, OAS1, and TRIM14 in the presence or absence of Sendai Virus and NS5A. GAPDH was used as an endogenous control, as GAPDH levels are unaffected by viral infection. Cells were infected using Sendai Virus in order to trigger the IFNb antiviral pathway, and were transfected with the different mutant forms of NS5A.

Oxidative stress in the brain is a known contributor to the development of neurodegenerative diseases, including Alzheimer’s. The focus of this project is to target the amyloid-β plaque formations and reactive oxygen species (ROS) derived from misregulated metal-ions that lead to disease-causing oxidative stress. The present investigation is measuring the antioxidant reactivity of the new molecule L4. L4 contains two radical scavenging pyridol groups along with a metal-binding nitrogen rich ligand system. It was hypothesized that increasing the number of pyridol groups in our small molecule library would increase the radical scavenging activity, which in turn may provide cells protection from oxidative stress. The radical scavenging ability of L4 was quantified using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay and this was compared to other radical scavenging small molecules to evaluate the effect of the additional radical scavenging group on the antioxidant activity. The interaction of L4 with redox active metal-ions such as copper(II) was also evaluated to show the molecule’s ability to target misregulated metal-ions in diseased tissues.

Monika Hailey
SRS 2017
Neilson Group
Synthesis of Silicon-Nitrogen Polymer Precursors
The Neilson research group focuses on developing synthetic routes to new organic-inorganic hybrid polymers. Specifically, one class of potential polymers contain silicon-nitrogen bonds, alternating with organic spacer groups along the polymer backbone. These two elements were chosen in order to obtain a system whose stability is similar to that of organic (carbon-based) polymers. Organic polymers are very stable and can be found in everyday life. In addition, silicon-oxygen polymers are used in several commercial applications. Silicon-nitrogen polymers could possibly serve as precursors to other new polymeric and/or solid state materials.
Experiments were conducted to produce a variety of small molecule precursors to the new silicon-nitrogen polymer system. Seven silicon-nitrogen small molecules were synthesized, in fairly good yield, and characterized using 1H NMR spectroscopy. When attempting to purify some of these small molecules, there was some thermal decomposition, possibly leading to the desired polymer. Future experiments will investigate the synthetic potential of these new compounds.

Molecularly imprinted polymers (MIPs) are advantageous to chemists both in their ability to drive the equilibrium of a reaction toward a desired product and in chromatography. In this project we focused on the use of MIPs in a chromatographic sense to selectively isolate menthyl-(hydroxymethyl)-phenyl phosphinate in the SP form from a mixture of both diastereoisomers. Both R and S configurations are made in equal proportions but the yield from isolation and crystallization of each pure diastereoisomer is low. Production of a polymer containing pockets specific to the configuration of one diastereoisomer enables an easier method to isolate one diastereoisomer through absorption by the polymer and subsequent release. The potential for MIPs for these P-stereogenic compounds lies in the increase yield of pure crystals and therefor decreased cost of production.

This project was aimed to prepare stable isosteric analogs of S-adenosylmethione (SAM) whose sulfur atom is replaced by a nitrogen atom and to evaluate these analogs for the SAM riboswitch-binding activities and antibacterial activities. In bacteria, SAM binds to the SAM riboswitch, which regulates the biosynthesis of methionine and cysteine, two amino acids essential for survival. Therefore, synthetic molecules that bind to SAM riboswitches have the potential to kill bacterial cells.
Three different classes of SAM riboswitches exist in bacteria (SAM I, II, and III). Each class of SAM riboswitch gene under control of T7 promoter was prepared by the overlapping extension polymerase chain reaction of synthetic oligonucleotides. Each SAM riboswitch gene was successfully cloned into the pUC19 plasmid and verified by DNA sequencing. A high concentration of each SAM riboswitch DNA was prepared by PCR and further converted to the corresponding SAM riboswitch RNA molecules by in vitro transcription using T7 RNA polymerase. All three classes of SAM riboswitches will be tested for binding to the synthesized SAM analogs.