Doctor of Philosophy, The Ohio State University, 2023, Chemical Engineering

Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e., erythrocruorins) derived from annelid sources. Therefore, the goal of Chapter 3 was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins. The long-term storage stability and portability of hemoglobin (Hb)-based oxygen carriers are important design criteria in the development of these therapeutics for use in emergency medicine in austere environments. Lyophilization or storing proteins in a freeze-dried form is known to increase storage lifetime and reduce overall weight. In Chapter 4, we (open full item for complete abstract)

Committee: Andre Palmer (Advisor); Jeffrey Chalmers (Committee Member); Dana McTigue (Committee Member); Eduardo Reategui (Committee Member); David Wood (Committee Member) Subjects: Biochemistry; Chemical Engineering

PhD, University of Cincinnati, 2020, Engineering and Applied Science: Electrical Engineering

The objective of this research work is to develop a functional lab-on-chip (LOC) and a smartphone compatible point-of-care-testing (POCT) platform to quantitatively determine the concentration of a target biomarker in biological fluids for disease diagnostics in resource-limited environment. Majority of deaths related to infectious diseases occur in resource-poor countries having limited access to clinical laboratory facilities and trained personnel. Developing reliable diagnostic tests to be used at the point-of-care can result in earlier disease diagnosis, improved patient treatment, and more efficient outbreak prevention. The realization of an ideal POCT system largely relies on the development of cheap and disposable micro?uidic devices that can be easily integrated to low power electronics with a user-friendly interface.In this research, for the first time, a new microchannel capillary flow assay (MCFA) LOC for high-sensitive chemiluminescence ELISA using on-chip reagent lyophilization has been developed and applied for the detection of malarial biomarker Plasmodium Falciparum Histidine Rich Protein 2 (PfHRP2). This work also reports the design and development of a smartphone based POCT analyzer for detecting chemiluminescent signals from preloaded MCFA LOC. MCFA LOCs were designed for single-step sample loading and capillary liquid transport to initiate chemiluminescence sandwich ELISA in the reaction chambers. A methodology towards lyophilization of chemiluminescent substrate while restoring the substrate functionality in artificial serum was successfully established which paves the way towards developing a sample-to-answer type POCT platform . On sample addition, the functionally designed microchannels with adequately hydrophilic surfaces pull the sample towards the dried reagents and reconstitutes them.The designed microfluidic chip controls sequential arrival of reconstituted reagents in the reaction chamber producing the desired chemil (open full item for complete abstract)

Committee: Chong Ahn Ph.D. (Committee Chair); Marc Cahay Ph.D. (Committee Member); Leyla Esfandiari Ph.D. (Committee Member); Jungyoup Han Ph.D. PMP (Committee Member); Rashmi Jha Ph.D. (Committee Member) Subjects: Electrical Engineering

Master of Science, Miami University, 2019, Chemistry and Biochemistry

Metabolic profiling of cell lines and tissue extracts involves sample processing that includes a drying step prior to re-dissolving in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, around forty metabolites were identified to have statistically significant differences in abundance in re-dissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in lyophilized samples, for example, n-methyl-α-aminoisobutyric acid and ethanol, whereas some metabolites were only present in speed-vacuum dried samples, for example, trimethylamine and n-acetylneuraminic acid. This research demonstrates that the choice of drying technique used during preparation of samples of human cell lines or tissue extracts can significantly influence the observed metabolome making it important to carefully consider the selection of a drying method prior to preparation of such samples for metabolic profiling.

Committee: Michael Kennedy PhD (Advisor); Rick Page PhD (Committee Chair); Michael Crowder PhD (Committee Member); Neil Danielson PhD (Committee Member) Subjects: Chemistry

Doctor of Philosophy, Case Western Reserve University, 2015, Macromolecular Science and Engineering

Aerogels are an interesting class of materials that possess many exotic and extreme properties. These properties are developed as the gel network is produced from solution. As the gel develops, it builds a hierarchical structure, possessing architectures at different size scales through molecular and macro-scale interactions. Once the solvent is removed, and the resultant aerogel is produced, the hierarchical nature of the material produces many desirable properties including: extremely high porosities (greater than 90% pore volume)[1], extremely low thermal conductivities (10-30 mW/m-k)[1], very low densities (as low as 0.002 g/cm3)[2], low refractive indices (as low as 1.01),[3] low dielectric constants (between 1.0 and 1.5),[4] high surface areas,[5,6] and the slowest speed of sound through a solid material. The first chapter of this thesis deals with the structure/property relationships of polymer/clay aerogels interfused with uniformly distributed air bubbles were examined. Through the incorporation of a polyelectrolyte in a montmorillonite (MMT) clay solution, the viscosity was systematically changed by the addition of ions with different charges. The bubbles were achieved via high speed mixing and were stabilized through the use of the surfactant sodium dodecyl sulfate (SDS). As the charge of the ion increased from +1 (Na+ ions) to +2 (Ca2+ ions) to finally +3 (Al3+ ions), the modulus of the resultant aerogels increased. The foamed polymer/clay aerogels showed a reduction in thermal conductivity while retaining similar mechanical properties to unfoamed polymer/clay aerogels. The most promising composition was one which contained 5% MMT clay/5% poly(vinyl alcohol)/0.5% xanthum gum/0.5% SDS/0.2% Al2(SO4)3·6(H2O) possessing a density of 0.083 g/cm3, an average modulus of 3.0 MPa, and a thermal conductivity of 41 mW/m·K. The second project investigated the feasibility of incorporating ground recycled polyurethane (PU) foam into clay/polymer aerog (open full item for complete abstract)

Committee: David Schiraldi Ph.D. (Advisor); Mary Ann Meador Ph.D. (Advisor); Gary Wnek Ph.D. (Committee Member); Eric Baer Ph.D. (Committee Member) Subjects: Aerospace Materials; Automotive Materials; Chemistry; Engineering; Experiments; Inorganic Chemistry; Materials Science; Organic Chemistry; Polymer Chemistry; Polymers

MS, University of Cincinnati, 2014, Engineering and Applied Science: Environmental Engineering

Surface water contains natural organic matter (NOM) that reacts with disinfectants creating disinfection byproducts (DBPs), some of which are USEPA regulated contaminants. Characterizing NOM can provide insight with respect to DBP formation and water treatment process adaptation to climate change as the nature of NOM varies. This study collected NOM from the Ohio River over 15 months (April 2010 to July 2011) in order to assess seasonal variability in NOM characteristics. The NOM was characterized using fluorescence spectroscopy, UV254, TOC, high performance liquid chromatography – size exclusion chromatography (HPLC-SEC), and elemental analysis. NOM was concentrated, freeze-dried (lyophilized), and validated with the source NOM creating a standardized lyophilized NOM that may be used in water treatment process evaluations investigating utility adaptation to seasonal changes. Additionally, NOM was concentrated at multiple concentration factors, lyophilized, and reconstituted allowing for the determination of optimal NOM concentration and reconstitution conditions. The NOM was characterized using UV254, TOC, HPLC-SEC, fluorescence spectroscopy, and DBP formation. Raw Ohio River water NOM was concentrated in the following order: ultrafiltration (UF), cation ion exchange, reverse osmosis (RO), sulfate removal, and lyophilization. Lyophilization allows for long-term storage of NOM while providing the ability to reconstitute at various NOM concentrations compared to liquid material with a short shelf-life. Lyophilized NOM was used for elemental analysis while UF effluent, concentrate, and reconstituted lyophilized NOM were employed for all other analyses. A single RO concentration factor (150X) was used during the 15-month study while 50X, 100X, 150X, 200X, and 250X were used to determine the optimal RO concentration factor versus reconstitution factor. Parallel factor ii analysis (PARAFAC) determined the locations of principle components within fluorescence excitatio (open full item for complete abstract)

Committee: Dominic Boccelli Ph.D. (Committee Chair); Jonathan Pressman Ph.D. (Committee Member); Margaret Kupferle Ph.D. P.E. (Committee Member) Subjects: Environmental Engineering

Doctor of Philosophy (PhD), Ohio University, 1997, Chemical Engineering (Engineering)

Human growth hormone (hGH) is a polypeptide with 191 amino acids. Previously, it was found that an hGH analog with a single amino acid substitution acted as an hGH antagonist. Previously a genetically engineered anchorage-dependent mouse L cell line was created that produced and secreted the hGH antagonist (hGHG120R) at a level of 10 mg/1 in culture media supplemented with 3-5% NuSerum IV. The multistep downstream process that was developed for the purification of hGHG120R consisted of cell clarification, salt precipitation, membrane ultrafiltration, size exclusion chromatography (SEC), reverse phase high performance liquid chromatography (RP-HPLC), phase separation and lyophilization. The total operating time for the purification protocol was approximately 120 hours and the average overall recovery was 51%. The objective of this work is to produce a system suitable for the large-scale production of hGHG120R, by increasing the specific production rate and versatility of the culture system, and at the same time easing the burden of the purification process, in terms of both, time and efficiency. A proper combination of genetic elements, cell line, and media formulation is employed. We have used dihydrofolate reductase mutant (DHFR) Chinese hamster ovary (CHO) cells, stably transfected with an expression vector driven by the relatively strong human cytomegalovirus immediate-early gene-regulatory region, to express high levels of hGHG120R. Protein expression levels of 4-5 mg/l were obtained from stably established CHO cells. The hGHG120R tested to be biologically active. These cells were then adapted to grow in suspension in CHO-S-SFM (serum-free media). High cell densities, typically, ~1.0 – 2.0 × 106 cells/ml were obtained in spinner flask cultures. Partial purification of hGHG120R from CHO cell cultured media using the previously established protocol, revealed that the level of impurities in SFM was significantly lower than the serum- supplemented DMEM. This suggest (open full item for complete abstract)

Committee: Darin Ridgway (Advisor) Subjects: Engineering, Chemical