Doctor of Engineering, Cleveland State University, 2018, Washkewicz College of Engineering

Hepatocellular carcinoma (HCC) is an invasive and aggressive cancer of the liver that arises due to chronic cirrhosis. Research into understanding HCC has focused on two-dimensional (2D) and three-dimensional (3D) technologies to simulate the liver microenvironment and use animal models to model how HCC affects the rest of the body. 3D hydrogel models are desired because they can mimic the transport behavior observed in vivo by structurally mimicking the extracellular matrix (ECM) without the ethical concerns of animal models. However, hydrogels can be toxic to cells and require optimal procedures for appropriate handling. In this study, we created 3D models of liver diseases on high-throughput platforms. First, we optimized hydrogel attachment on micropillar chips by coating them with 0.01 w/v % PMA-OD in ethanol. Next, we optimized the protocol for encapsulation of viable Hep3B cells PuraMatrix peptide hydrogel, using a higher seeding density (6 * 106 cells/mL) and two post-print media washes. Then, we established the ability to transduce adenoviruses in situ in encapsulated cells and successfully demonstrated their dose-response behavior towards six compounds. In the second part, we scaled up to using the microwell chip platform and optimized the polymerization of oxidized methacrylated alginate (OMA) for Hep3B encapsulation. First, we plasma-treated microwell chips for 15 minutes at high RF to minimize bubbles. Then, we optimized micro-scale photopolymerization conditions at 45 % methacrylated OMA (OMA-45) and 2 w/v % OMA with 0.05 w/v % PI and reflective background under either low intensity, long duration (2.5 mW/cm2 for 2 minutes) or high intensity, short duration (4.0 mW/cm2, 30 seconds) light by testing cell viability at these conditions. Third, we used these OMA conditions to develop a high-throughput, real-time 3D cell migration assay on a newly engineered 384-pillar plate with sidewalls. We first developed a set of a protocols where out-of-focus cells ar (open full item for complete abstract)

Committee: Moo-Yeal Lee Ph.D. (Committee Chair); Joah Belovich Ph.D. (Committee Member); Nolan Holland Ph.D. (Committee Member); Chandra Kothapalli Ph.D. (Committee Member); Xue-Long Sun Ph.D. (Committee Chair) Subjects: Biomedical Engineering; Biomedical Research; Chemical Engineering; Materials Science; Nanoscience

Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology

MicroRNA-122 (miR-122) is a liver-specific microRNA that maintains liver homeostasis by regulating lipid metabolism, cell differentiation and viral infections in vertebrates. In clinics, hepatocellular carcinoma (HCC) patients with low miR-122 levels are usually associated with poor prognosis, implying miR-122 functions as a tumor suppressor. This notion is supported by the phenotypes of miR-122 genetic knockout (KO) mouse that develops spontaneous hepatitis, steatosis, fibrosis and HCC with age. Although several studies have shown the importance of miR-122 in maintaining liver homeostasis, the mechanisms by which loss of miR-122 contributes to these liver pathological processes remains largely unknown. In the current study, we explored the role of miR-122 in regulating liver inflammation and fibrosis by combining molecular, biochemical, and bioinformatic analysis. We demonstrated that the increased expression of the chemokine CCL2 in the liver is one of the causes of liver inflammation upon deprivation of miR-122. Blocking CCL2 using specific neutralizing antibody (CCL2 nab) ameliorates liver inflammation and tumorigenesis through decreasing the population of CD11b+/Gr1+ cells and their corresponding downstream pathways such as the IL-6-Stat3-cMYC axis and TNF-alpha-NF-kappaB axis. Along with the shrinking tumors in the CCL2 nab treated liver, CCL2 nab also activated natural killer (NK) cells and increased their cytotoxicity toward tumor cells. Besides its robust role in inhibiting liver inflammation and HCC tumors, miR-122 also has a strong anti-fibrosis function. Analyzing database that contained both clinical and expression profiles of liver cirrhosis patients revealed downregulation of miR-122 in the cirrhotic liver tissues compared to normal livers. Ectopic expression of miR-122 in LX-2, an immortalized human hepatic stellate cell (HSC) cell line, reduced cell proliferation and fibrotic gene expressions. Moreover, co-culture of miR-122 expressed HCC cells with (open full item for complete abstract)

Committee: Kalpana Ghoshal Ph.D (Advisor); samson Jacob Ph.D (Committee Member); Jianhua Yu Ph.D (Committee Member); David Brigstock Ph.D (Committee Member) Subjects: Biology; Molecular Biology; Oncology

Master of Sciences (Engineering), Case Western Reserve University, 2017, Biomedical Engineering

Patient-derived xenografts (PDX) are living ex situ tumor models that aid clinicians in selecting potent therapies for cancer patients. Unfortunately, a liver PDX is difficult to develop due to poor liver tumor engraftment in mice hosts. Tissue engineering studies have suggested that beneficial factors may exist in extracellular matrix (ECM) that can enhance tumor viability after transplant. We conducted comparative analysis of three published decellularization protocols for efficient cell removal. ECM samples produced by Freeze-thaw with Triton X-100 (TX-100), Sodium Dodecyl Sulfate (SDS) with TX-100, and TX-100 alone, were analyzed with nuclear labeling and structural analysis. SDS with TX- 100 was efficient and caused minimal alterations to the matrix structure. Additionally, we demonstrated with developed assays that liver cell lines can respond to isolated decellularized matrix and mimic in vivo liver activity. The work completed provides confidence to study the effects of decellularized liver matrix on patient-derived liver tumors.

Committee: Samuel Senyo PhD (Advisor); Eben Alsberg PhD (Committee Member); Analisa DiFeo PhD (Committee Member); Anirban Sen Gupta PhD (Committee Member) Subjects: Biomedical Engineering

Master of Science, The Ohio State University, 2016, Biomedical Sciences

Sorafenib therapy has been shown to have only a small clinical benefit for liver cancer patients. There is an urgent needed to develop new therapeutic strategies for the treatment of advanced stage HCC. In this report, we screened several repurposed therapeutics in order to identify synergistic drug combinations. We demonstrate that the combination of 2-deoxy-glucose and sorafenib drastically inhibit HCC cell viability in Hep3B, Huh7 and sorafenib resistant Huh7 cells. Cell cycle analysis revealed that this therapeutic combination induced complete G0/G1 arrested HCC cells. Our studies suggest that this cell-cycle arrest is due to the depletion of cellular ATP. Overall, this report provides strong evidence for the clinical potential of sorafenib + 2-deoxyglucose combination therapy.

Committee: Samson Jacob PhD (Advisor); Kalpana Ghoshal PhD (Committee Member) Subjects: Medicine; Molecular Biology; Therapy

Master of Science, The Ohio State University, 2012, Electrical and Computer Engineering

Wind turbine has been popular in the area of renewable energy source. Wind turbine has shown the biggest growth in the past 10 years compared to other renewable sources. Permanent Magnet Synchronous Generator (PMSG) used as wind turbine generator. PMSG is suitable for the application due to its high efficiency, high torque-to-size ratio, and low maintenance requirement. The intermittent characteristic of wind requires a wind turbine to have good control system. This thesis discusses the Field Oriented Control (FOC) and Hysteresis Current Control (HCC) technique used to control the generator-side and grid-side respectively. In order to perform these controls, good understanding of the PMSG and electrical grid are required. In addition, reference frame theory and Space Vector Modulation are explained as supplement theories used for the Field Oriented Control. A MATLAB/Simulink simulation has been developed to simulate the control algorithms.

Committee: Ali Keyhani PhD (Advisor); Mahesh Illindala PhD (Committee Member) Subjects: Alternative Energy; Energy; Engineering

Doctor of Philosophy, The Ohio State University, 2012, Molecular, Cellular and Developmental Biology

MicroRNAs are conserved, small (20-25 nucleotide) noncoding RNAs that negatively regulate expression of mRNAs at the post-transcriptional level. MicroRNA signature is altered in different disease states including cancer and some microRNAs act as oncogenes or tumor suppressors. To identify microRNAs that may play a causal role in hepatocarcinogenesis we used an animal model in which C57BL/6 mice fed choline deficient and amino acid defined (CDAA) diet develop nonalcoholic steatohepatitis (NASH)-induced hepatocarcinogenesis after 70 weeks. Microarray analysis identified 30 hepatic microRNAs that are significantly (P<.01) altered in mice fed CDAA diet for 6, 18, 32 and 65 weeks compared to those fed choline sufficient and amino acid defined diet (CSAA). Real-time RT-PCR analysis demonstrated upregulation of oncogenic miR-155, miR-181b, miR-221/222 and miR-21 and downregulation of the most abundant liver specific miR-122 at early stages of hepatocarcinogenesis. Western blot analysis showed reduced expression of hepatic PTEN, a target of miR-21, and C/EBPβ, a target of miR-155, in these mice at early stages. DNA binding activity of NF-κB that transactivates miR-155 gene was significantly (P=0.002) elevated in the liver nuclear extract of mice fed CDAA diet. Further, the expression of miR-155, as measured by in situ hybridization and real-time RT- PCR, correlated with diet-induced histopathological changes in the liver. Ectopic expression of miR-155 promoted growth of hepatocellular carcinoma (HCC) cells whereas its depletion inhibited cell growth. Notably, miR-155 was significantly (P=0.0004) upregulated in primary human HCCs with concomitant decrease (P=0.02) in C/EBPβ level compared to matching liver tissues. The expression of tissue inhibitor of metalloprotease 3 (TIMP3), a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic transforming growth factor β (TGFβ) and its downstream media (open full item for complete abstract)

Committee: Samson Jacob PhD (Advisor); Kalpana Ghoshal PhD (Advisor); Said Sif PhD (Committee Member); Thomas Schmittgen PhD (Committee Member) Subjects: Biomedical Research; Molecular Biology

Master of Science, The Ohio State University, 2013, Pathology

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. There are a limited number of therapeutic options currently available to delay the advancement of HCC, and the chance of survival decreases as the disease progresses. DNA methylation of certain tumor suppressor genes and hypomethylation of oncogenes have been shown to initiate HCC. 5-hydroxymethylcytosine (5-hmC), recently identified sixth base of the genome, can cause demethylation by its conversion to cytosine through reactions catalyzed by the TET enzymes. Research on the role of this novel modification in the liver and HCC is needed before it can be therapeutically targeted. To this end, we planned to perform systematic analysis of the role of 5-hmC in liver biology and its aberrations in HCC. In the present study, DNA from primary human HCC and matching benign livers were used to compare alterations in 5-hmC level, if any, in tumors. Mouse models of HCC were also used to further validate the results obtained from human specimens. We observed remarkable decrease in global 5-hmC content in HCCs of both human and rodent origin using multiple techniques that include dot blot, immunohistochemistry, and LC/MS analysis. Furthermore, specific regions of EGFR, H19, and 7SL loci also exhibited reduced 5-hmC levels in the tumor samples. Since aberrations in methylation can cause cancer including HCC, it would be important to identify differentially hydroxymethylated genes in the liver and the consequence of their differential hydroxymethylation in HCC.

Committee: Kalpana Ghoshal (Advisor); Samson Jacob (Committee Member) Subjects: Biology; Genetics; Molecular Biology; Oncology