Master of Science, The Ohio State University, 2017, Biomedical Engineering

Low back pain is a widespread debilitating disorder of significant socio-economic importance and intervertebral disc (IVD) degeneration has been implicated in its pathogenesis. Despite its high prevalence the underlying causes of LBP and IVD degeneration are not well understood, contributing to the difficulty in identifying relevant treatment strategies that specifically target this disease. Recent work in musculoskeletal degenerative diseases such as rheumatoid arthritis and osteoarthritis have revealed a critical role for immune cells, specifically mast cells in their pathophysiology, eluding to a potential role for these cells in the pathogenesis of IVD degeneration. This study sought to characterize the presence and role of mast cells within the IVD, focusing on chemo-attractants (CCL2/MCP-1 and stem cell factor) which may recruitment mast cells to the IVD and mast cell-IVD cell interactions using immunohistochemistry and 3D in-vitro culture methods. Mast cells were upregulated in painful human intervertebral disc tissue and were able to induce an inflammatory, catabolic and pro-angiogenic phenotype in bovine nucleus pulposus and cartilage endplate cells at the gene level. Bovine annulus fibrosus cells in particular from the healthy IVD however, demonstrated a protective role against key inflammatory (IL-1ß and TNFa) and pro-angiogenic (VEGFA) genes expressed by mast cells, and mitigated neo-angiogenesis formation in vitro. In conclusion, mast cells can infiltrate and elicit a degenerate phenotype in IVD cells, enhancing key disease processes in the degenerate IVD and making them a potential target for low back pain therapeutics.

Committee: Devina Purmessur (Advisor); Christopher Breuer (Committee Member); Safdar Khan (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, Case Western Reserve University, 0, Chemistry

The imbalance between the production of reactive oxygen/nitrogen species (ROS/RNS) and their consumption by antioxidants leads to excess free radicals and peroxides, which can attack various components of cell or the entire organism. Polyunsaturated fatty acids (PUFAs) are especially vulnerable to free radical mediated oxidation. The resulting oxidative degradation of PUFAs gives reactive bifunctional aldehyde such as 4-hydroxy-2-nonenal (4-HNE) as well as polyoxygenated products with the full carbon chain of the fatty acid such as epoxyketooctadecenoic acids (EKODEs). Many of lipid peroxidation (LPO) products are highly electrophilic and can subsequently react with biological nucleophiles including protein side chains and DNA bases, consequently leading to enzyme inactivation and gene mutation. A number of metabolic fates of LPO products have been discovered and are critical for counteracting the oxidative damage. In addition, parallel catabolic pathways of 4-HNE were identified while the mechanism was not fully elucidated yet. The first goal of my work was to generate a better understanding of the transformations involved in the catabolic pathways of 4-HNE. An LC-MS/MS based assay was developed to probe the presence of 4-hydroxy-acyl-CoA kinase activity, which generates 4-phosphoacyl-CoA (4-P-acyl-CoA) as a key intermediate during the isomerization of 4-hydroxy-acyl-CoA to 3-hydroxy-acyl-CoA. This assay was used to guide the purification of the kinase from liver tissues. A number of purification techniques including ammonium sulfate precipitation, fast protein liquid chromatography (FPLC) and SDS-PAGE were utilized. Protein sequencing of the resulting protein fractions gave five kinase candidates. Sedoheptulose kinase, the most relevant candidate with oxidative stress was tested for 4-hydroxy-acyl-CoA kinase activity. In addition to the catabolic fates of LPO products, we also investigated the chemical nature and biological consequences of the protein modifi (open full item for complete abstract)

Committee: Gregory Tochtrop (Advisor) Subjects: Biochemistry; Chemistry; Organic Chemistry

Doctor of Philosophy, The Ohio State University, 2015, Microbiology

Atrazine is one of the most widely used herbicides in the world. It is primarily used in the production of corn in the United States. Although it may marginally increase crop yields, atrazine is also an endocrine disruptor in non-target organisms. Its moderate solubility in water allows for atrazine to contaminate surface and ground waters far removed from the point of application to soil. Although atrazine can be degraded abiotically, its primary mode of attenuation in natural environments is through bacterial degradation. Full mineralization of atrazine to CO2, H2O, Cl- and NH4+ has been demonstrated in Pseudomonas ADP, which contains the complete suite of atz atrazine catabolic genes. The overall hypothesis of this study is that the microorganisms and catabolic pathways reported in the literature do not universally account for the atrazine biodegradation observed in different natural environments. Furthermore, it is hypothesized that in situ pre-enrichment methods yield atrazine degraders uncultivable by classical laboratory enrichment, including anaerobic bacteria. The discovery of atrazine catabolic genes other than those in the atz pathway and the demonstrated involvement of consortia of bacteria in atrazine biodegradation suggest that the full diversity of environmental atrazine biodegradation has yet to be elucidated. In order to further elucidate the bacteria and genes responsible for atrazine biodegradation in different environments, locations with different exposures to atrazine were chosen for study. Among them were a farm location that was the site of multiple pesticide spill events throughout its history and a constructed wetland that receives river water containing agricultural runoff. Samples from these sites were assessed for atrazine biomineralization via biometer studies. In addition to traditional environmental sampling, these sites were also sampled with Bio-Sep beads to allow for in situ pre-enrichment for atrazine-degrading microbes. (open full item for complete abstract)

Committee: Olli Tuovinen PhD (Advisor); Michael Boehm PhD (Committee Member); Charles Daniels PhD (Committee Member); Michael Ibba PhD (Committee Member) Subjects: Agricultural Chemicals; Biology; Environmental Science; Microbiology; Molecular Biology