▼ An inflammatory neurodegenerative disease, Multiple Sclerosis (MS), affects the central nervous system and results in a progressive physical and cognitive disability in the patients. Hemoglobin expression in neurons has been found to be increased in neurons in postmortem MS brain tissue compared to controls. The role of hemoglobin in neurons is unknown but evidence suggests it may be involved in neuronal respiration and may provide protection from the damaging effects of free radicals and reactive oxygen species. The ability to modulate hemoglobin expression in neurons may be beneficial in diseases such as MS where mitochondrial dysfunction and oxidative damage are involved in disease pathology. Both transformed neural cell lines and primary cultured neurons were tested to see if they expressed hemoglobin. Neuroblastoma cells (SHSY-5Y) and neura-2a cells were treated with hydrogen peroxide (H2O2). Then primary neurons were treated with L-methionine (L-met), trichostatin A (TSA), hydrogen peroxide (H2O2), and erythropoietin (EPO). We confirmed that hemoglobin is expressed in primary cultured neurons and the expression of hemoglobin could be modulated in this culture system. Advisors/Committee Members: Freeman, Ernest.

▼ For years, it was thought that sphingolipids were only structural elements in the plasma membrane found in cells but it has recently been found that sphingolipids are active participants in many different cell processes. Lipid rafts, a type of domain, are enriched with sphingolipids and cholesterol. Lipid rafts are important in the mechanisms of disease because their specific physical properties allow for more efficient facilitation of certain cell signaling processes to occur. An important sphingolipid is ceramide-1-phosphate (Cer-1-P), which is an inhibitor of the phosphoinositide-3-kinase (PI3K) pathway and induces cell proliferation. Beyond proliferation, the binding of Ca2+ to the phosphate headgroup of Cer-1-P is vital to facilitating inflammatory responses and Ca2+ likely plays an important role in phase properties of Cer-1-P. The mechanisms of how Cer-1-P controls different processes are currently being researched, but more information is needed about its phase behavior in model membranes with cholesterol and Ca2+. My experiments centered on investigating the monolayer morphology and phase behavior of Cer-1-P with these two constituents. My findings show that Ca2+ strongly affects the Cer-1-P monolayer phase behavior and morphology by inducing the formation of solid phase Cer-1-P domains. Furthermore, the phase behavior of Cer-1-P is also greatly affected by the presence or absence of Ca2+. Finally, my results demonstrate that cholesterol transforms expanded Cer-1-P phases to more condensed phases. Advisors/Committee Members: Gericke, Dr. Arne.

▼ Targeted drug delivery via nanoparticles is an emerging field that has great potential in the improvement of cancer treatment. The goal of this project is to develop a multi-component drug delivery system that targets and selectively delivers the chemotherapeutic drug, Dox, to cancerous cell lines that over express the cell surface glycoprotein CD44, a receptor for hyaluronic acid. 10nm gold nanoparticles (GNPs) were used as the delivery vehicle for a thiolated derivative of Doxorubicin referred to as MPDOX. In order to target the tumorigenic cell lines that over expressed CD44 receptors, reducing end thiolated hyaluronic acid was used as the targeting ligand. After the individual facets of the nanoconjugate system were synthesized, the optimal conditions for stable nanoconjugate assembly were determined. The stability of the nanoconjugate system was then assessed with a salt stability assay that determined the level of protection hyaluronic acid provided for the nanosystem in the presence of a salt solution. The presence of both the targeting ligand and drug on the resulting nanoconjugates was then demonstrated by UV absorbance to confirm the formation of the full 3-part nanoconjugate system. After the composition of the nanoconjugates was determined, MTS assays were performed in order to generate dose dependent cell viability curves and IC50 values for treatment of two ovarian cancer cell lines (A2008, C-13) with either the free drug or the nanoconjugate system at various time points. The IC50 values were then compared to determine the type of treatment that was most efficient. When compared to the free drug, equal levels of cytotoxicity were observed in the cancerous cells. However, it was noted that a decrease in cytotoxicity in the non-targeted cells was not observed. It can be concluded that future work is needed to enhance of efficacy of the nanosystem to target cancerous cell lines over non-cancerous cells. Confocal studies were then performed to visualize the uptake of Doxorubicin and MPDOX in cellular compartments. The findings from this project may provide new insights that can be helpful in future cancer tissue specific targeted drug delivery. Advisors/Committee Members: Basu, Soumitra.

▼ Circadian rhythm is one of the most fundamental regulators of physiology and behavior. The transcription-translation feed-back loop of clock genes control metabolic activity by regulating the transcription of key proteins and enzymes. The transcription proteins BMAL1 and CLOCK are two of the proteins that are involved in translation-transcription feed-back loop. They dimerize and bind to promoter regions and positively regulate the expression of other clock genes. The expressed clock proteins in turn can negatively regulate the action of CLOCK and BMAL1 and/or regulate the expression of other metabolic proteins. Because BMAL1 is an integral part of the circadian rhythm and its roles in whole-body metabolism, the modification of its expression can disrupt muscle metabolism. The overexpression of BMAL1 in skeletal muscles may have a broader impact on whole-body metabolic regulation. Advisors/Committee Members: Novak, Colleen.

▼ Reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitrite (NO2-) are small reactive molecules present naturally in biological systems. Elevated levels of ROS and RNS (oxidative / nitrosative stress) are associated with inflammatory diseases and cancer. Vitamin B12, known as cobalamin (Cbl), participates in two B12-dependent enzyme reactions in mammals that are inactivated during oxidative / nitrosative stress. Cob(I)alamin (Cbl(I)), an intermediate in the B12-dependent methionine synthase reaction and a biosynthetic precursor of the B12 coenzymes, is readily oxidized to cob(II)alamin (Cbl(II)).The kinetics of the reaction between Cbl(I) and NO2- have been investigated and a reaction mechanism proposed. Advisors/Committee Members: Brasch, Nicola.