Master of Science, The Ohio State University, 2009, Chemical Engineering

Cell assays for high-throughput screening (HTS) of potential drug candidates are important tools in the process of drug discovery. Most cellular assays are currently based on 2-D monolayer cell cultures, but 3-D cell cultures could better mimic the in vivo characteristics of actual organism tissues. Unfortunately, assays using 3-D culture models usually require significant manual manipulation and are therefore not suitable for HTS. Research under Dr. Shang-Tian Yang has resulted in a functioning system for high-throughput 3-D cellular assays using engineered cells to express enhanced green fluorescence protein (EGFP) quantifiable through fluorometry. System improvement to allow rapid assessment of cellular events, such as specific gene expression or cell cycle progress is limited by the long persistence of the current reporter protein in the cells. In this study a new fluorescence reporting cell line was established using a destabilized EGFP (d4EGFP) expressed in Chinese hamster ovary (CHO) cells. Correlating fluorescence with cell number for the d4EGFP cell line in 2-D assays indicated that d4EGFP expression may be too low for use in high-throughput cell number reporting. The fluorescence and cell number correlation in 3-D assays indicated better performance could be achieved in 3-D but the fluorescence was sensitive to duration between sampling, possibly due to oxygen transfer limitation, hindering reliable use for cell number reporting. Response to such factors could still serve a purpose for culture condition monitoring, and could be applied in culture development and optimization.

Committee: Shang-Tian Yang PhD (Advisor); Jeffrey Chalmers PhD (Committee Member) Subjects: Cellular Biology; Chemical Engineering

Bachelor of Arts, Wittenberg University, 2019, Biochemistry/Molecular Biology

Trypanosoma brucei, a parasitic protozoan, is the etiologic agent of African Sleeping Sickness (Trypanosomiasis), a disease that threatens the lives of thousands in sub-Saharan Africa, with a 100% mortality rate in untreated cases. Transfer RNA (tRNA) is an adaptor molecule which acts as the intermediary between nucleotide and amino acid sequences, linking the information encoded in the genome to protein synthesis. Naturally occurring nucleotide modifications of tRNA are critical for translation; and therefore, essential for the viability of the organism. RNA editing involves the chemical modification of one nucleotide to another; in T. brucei, some tRNAs undergo essential editing events from adenosine to inosine. The heterodimeric enzymes, called adeonsine deaminases acting on tRNA (ADAT2/3), catalyzes the deamination of the first position of the anticodon in tRNA. Inosine in the wobble base position allows a single tRNA to decode multiple codons, implicating ADAT2/3 as a potential anti-parasitic therapeutic target. This enzyme consists of two structurally unique subunits, ADAT2 and ADAT3, both of which bind Zn2+. Given its essentiality in parasitic viability, it is important to understand the enzyme's structure and function. Since the structure of ADAT2/3 has been difficult to elucidate, a Tripartite Split-Green Fluorescent Protein (GFP) Association Assay was used to determine the correct orientation of each subunit in vitro. This assay uses GFP split into three fragments, which fluoresce when they are all in close proximity to one another. By strategically tagging the termini of the enzyme with the two small linker fragments, and incubating with the larger reporter, the correct orientation of the ADAT2/3 heterodimer can be determined on the basis of fluorescence of a reassembled GFP. To validate the results of the assay, a general cross linking experiment, followed by mass spectrometry, should also be conducted. Ultimately, elucidation of ADAT2/3 structure is imp (open full item for complete abstract)

Committee: Margaret Goodman (Advisor); Michelle McWhorter (Committee Member); Daniel Marous (Committee Member) Subjects: Biochemistry; Microbiology; Molecular Biology; Parasitology

Doctor of Philosophy, The Ohio State University, 2012, Chemical and Biomolecular Engineering

Drug screening is a long and costly process confronted with low productivity and challenges in using animals. 3-D cell-based high-throughput platforms have been developed to improve drug-screening efficiency and minimize animal testing. However, online monitoring of cell proliferation, pH, and dissolved oxygen (DO) has been a challenge in 3-D cell-based assays. In this work, a 40 micro-well plate bioreactor (40-MBR) system was developed as a high-throughput platform for 3-D cell cultures. This 40-MBR has similar dimensions of a 384-well plate (384-MWP) can provide real-time and noninvasive monitoring of cell proliferation, pH, and DO in 3-D microenvironments. A colon cancer cell line expressing enhanced green fluorescent protein (EGFP) under the control of a constitutive CMV promoter was tested with two potential cancer drugs using the 40-MBR and 384-MWP. Compared to the 384-MWP, the 40-MBR gave more reliable and highly reproducible growth kinetic data with reduced experimental errors. This study demonstrated the potential application of the 40-MBR as a high-throughput platform for screening potential cancer drugs and evaluating their cytotoxic effects in the early-stage drug discovery. Cytotoxicity and embryotoxicity of chemicals were also investigated in the 40-MBR using EGFP-expressing embryonic stem cells (ESCs). Embryonic stem cell test (EST) has been used as an in vitro model for assessing embryotoxicity. However, the current EST can only provide end-point data and cannot predict embryotoxicity of chemicals affecting organs such as bone. In this study, a novel high-throughput embryotoxicity assay was developed using EGFP-expressing ESCs under the control of a survivin promoter. Survivin expression is closely associated with embryo development and cell differentiation. For control, ESCs expressing EGFP under the control of a CMV promoter were used to monitor cytotoxicity of chemicals. Using survivin as a diagnostic marker for predicting embryotoxicity was fi (open full item for complete abstract)

Committee: Shang-Tian Yang (Advisor); Jeffery Chalmers (Committee Member); Andre Palmer (Committee Member) Subjects: Chemical Engineering