Doctor of Philosophy, The Ohio State University, 2018, Chemical Engineering

The development of platform technologies can dramatically impact the speed and ease with which new biological processes/products can be optimized and brought to market. Platform technologies offer researchers predictable, generalizable procedures for achieving a pre-defined outcome. This dissertation describes the development of two platforms for i) engineering RNA-based tools for gene regulation and ii) engineering self-cleaving intein affinity purification tags with more tightly-controlled cleavage kinetics for recombinant protein purification. Bacterial trans- acting small regulatory RNAs (sRNAs) have great potential for applications in the field of metabolic engineering, due to their modular nature and the relative ease with which they may be engineered for novel regulatory function at the level of mRNA translation control. Furthermore, these sRNAs act at their target mRNAs through relatively simple base-pairing interactions, and (in many cases) have been demonstrated to be portable from microbe to microbe, while also providing the benefit of tuning gene expression of multiple genes for optimization of metabolic pathway flux. Chapter 2 describes the development of a genetic system for engineering novel, multi-acting sRNA regulators derived from the E. coli-native DsrA sRNA. Chapter 3 establishes thorough design rules for these engineered sRNAs to ensure robust regulation of the target gene, and describes a design basis that provides these semi-synthetic sRNAs with unprecedented specificity for their target mRNAs. Chapter 4 describes the validation and use of a previously-established, yeast-surface display-based protein evolution platform for the engineering of self-cleaving intein affinity tags with applications in bioseparations. Affinity tag technology greatly simplifies the process of purifying diverse recombinantly-expressed proteins, but tag removal remains a non-trivial barrier to implementation of affinity capture for protein purification at scale. Affin (open full item for complete abstract)

Committee: David Wood (Advisor); Jeffrey Chalmers (Committee Member); Andre Palmer (Committee Member) Subjects: Chemical Engineering

Doctor of Philosophy, The Ohio State University, 2024, Chemistry

Regulation of gene expression is essential in life. The transcription and translation of genes are modulated and responsive to cellular cues. One common theme for transcription and translation control in bacteria is through modulation of the structure of messenger RNAs (mRNAs) in response to cellular cues such as the concentration of ions, metabolites or change in temperature. These changes in the mRNAs can in turn affect transcription and translation. Similar mechanisms have also been shown to exist in eukaryotic systems such as fungi and plants. This modulation process by mRNA can operate both in both protein-dependent and protein-independent manner. Exploiting these gene regulatory processes in the laboratory is of great value because it can provide new insights into the development of novel antibiotics, new tools for genome engineering and new platforms for synthetic biology. In this work, we investigated two gene expression regulation systems involving RNA and protein: the SMK box riboswitch and the trp RNA binding attenuation protein (TRAP) to gain insights into the structural and dynamics that contribute to gene regulation. The SMK box riboswitch is a cis-acting non-coding RNA that was discovered in members of the Lactobacillales family of Gram-positive bacteria. It modulates translation of metK gene in response to the cellular concentration of S-adenosylmethionine (SAM). Previously, X-ray crystallographic analysis of the SMK aptamer bound to SAM established the structural basis of ligand-mediated repression. Based on results from NMR, titration calorimetry and chemical structure probing experiments, in the absence of SAM, SMK is proposed to be in equilibrium between an alternative fold (ISO) that has no obvious ligand-binding site, and a ligand-binding-competent state (PRIMED). To understand how the SMK box riboswitch functions, we need to know how it undergoes conformational fluctuations between alternative structures. We first used stopped-flow fluorescen (open full item for complete abstract)

Committee: Mark Foster (Advisor); Rafael Brüschweiler (Committee Member); Venkat Gopalan (Committee Member); Tina Henkin (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2024, Chemistry

Proteins are fundamental macromolecules in biology, serving as the building blocks of cells and tissues, while also playing crucial roles as enzymes, structural components, signaling molecules, and transporters, thus governing various essential biological processes. These versatile molecules contribute significantly to the maintenance, regulation, and functionality of living organisms, embodying the molecular machinery that drives and sustains life. The secondary structure of a protein, formed by local folding patterns like alpha helices and beta sheets, significantly influences its stability by establishing a backbone conformation. This structural arrangement not only determines the protein's stability but also plays a critical role in dictating its activity, as it forms the basis for the protein's specific shape, which is crucial for interactions with other molecules and functional roles within biological processes. Protein engineering techniques allow the modification of amino acid sequences to probe how alterations impact the stability and activity of a protein, providing insights into the importance of specific secondary structures. By selectively modifying or designing secondary structures, such as helices or sheets, protein engineers can assess their contributions to stability and activity, enabling the fine-tuning of protein properties for various applications in biotechnology, medicine, and beyond. In chapter one we reviewed literature to find out the importance of protein loop on stability and activity of proteins. We also focused on studies Rop, the model protein that we used in this dissertation. In chapter two we focused on probing the loop of the four-helix bundle protein Rop with LDAD sequence, exploring its impact on stability, activity, and structure through the creation of four libraries: NNK4, NNK5, R55Q NNK4, and R55Q NNK5. Our results revealed that contrary to the typical expectation longer loops destabilize proteins, in Rop, two 5-amino ac (open full item for complete abstract)

Committee: Thomas Magliery Dr. (Advisor); Rafael Brüschweiler Dr. (Committee Member); Marcos Sotomayor Dr. (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Chemistry

Doctor of Philosophy, Miami University, 2023, Chemistry and Biochemistry

Proteins can misfold in a crowded cellular environment due to physiological, pathological, and environmental stress. The fate of such misfolded proteins to either refolding by chaperones or degradation by the proteasome is determined by a protein-protein complex comprising the molecular chaperone Hsp70 and the E3 ubiquitin ligase CHIP. The decision to shunt proteins to the refolding or degradative pathways is known as the triage decision, and sufficient mechanistic details are still lacking. We hypothesized that the ensemble of different folding states of a single misfolded protein could be represented by a library of model proteins, each with a varying degree of thermodynamic stability. We termed the novel library of proteins we devised as “Folding Sensors”. Structure-guided protein engineering, computational modeling, biophysical techniques, and structural biology were utilized in this process. According to thermal denaturation data, the spectrum of folding sensors showed varying degrees of quantitative thermal stability at a given temperature. The crystal structure of the most stable folding sensor closely resembled the structure of the TPR domain, which served as the initial basis for the folding sensors, while also forming a disulfide bond as expected. We further speculate that Hsp70 recognized this variant as a natively folded state protein due to its' low affinity towards Hsp70. We further discovered that Hsp70 tightly binds to the moderately stable folding sensors. We further observed that CHIP differentially ubiquitinated substrates based on the thermal stability and the folding state. The process was chaperone dependent. The folding sensor mimicking a natively folded protein was ubiquitinated the least while the variants with more unfolding showed more ubiquitination. Altogether we developed folding sensors with varying thermodynamic stability to serve as a model to study chaperone-mediated triage by the Hsp70/CHIP. This preliminary study provided insights (open full item for complete abstract)

Committee: Richard Page (Advisor); Carole Dabney-Smith (Committee Chair); Timothy Wilson (Committee Member); Dominik Konkolewicz (Committee Member); Ann Hagerman (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Chemistry

Doctor of Philosophy, The Ohio State University, 2022, Chemistry

Proteins, the basic building blocks of life, are the most varied class of molecules and are created by the countless combinations of the 20 classical amino acids. The unique factors that determine a protein's three-dimensional characteristics and activities are its distinct amino acid sequences. While it is critical that we construct a comprehensive model to understand and anticipate the impacts of amino acids and changes in protein sequences, our present knowledge is context-sensitive and confined to just a few attributes over which we have very little control. Our categorization of these molecules is based on the structural homology of proteins, which is a result of natural evolution. The knowledge obtained from analyzing these protein "families" has enabled us to give certain positions and amino acid identities the weight they deserve in determining various biophysical features such as thermodynamic stability, solubility, catalysis, and binding. This dissertation focuses on an exploration of the protein folding problems and evaluates the information that we have learned in terms of utilizing protein molecules in immunotherapeutics. I introduce two major projects that I have conducted which include one based on the model protein Rop focusing on the question of protein folding and the myths of the hydrophobic core, and one leaning towards the applied side where we designed and produced Notch DLL1 based constructs that can function as potential signaling pathway activator for cancer treatments. Predicting the impact of mutation on protein folding is significantly hampered by the inability to precisely interpret the link between protein sequence and structural stability. Using rigorous high-throughput approaches, we studied this intricate connection in order to directly evaluate assumptions developed by earlier de novo design research and acquire a more comprehensive knowledge of protein folding and structure. Through combinatorial repackaging of the hydrophobi (open full item for complete abstract)

Committee: Thomas Magliery (Advisor) Subjects: Biochemistry; Biophysics

Doctor of Philosophy, The Ohio State University, 2022, Chemistry

Predicting the effect of a mutation in a protein on its function is still a complex unsolved problem. Analysis of a large number of protein mutants based on their activity, affinity and stability is an excellent method to understand this sequence-function relationship. Alanine scanning attempts it rationally, where the individual mutant is cloned, purified, and assayed to evaluate the consequence of a mutation. Large protein libraries coupled with a functional screen use a more comprehensive way to tackle this problem by saving time and costs of cloning and purifying individual variants. Here we have employed both approaches for two different protein systems to understand their sequence-function relationship. Since library studies are easier to perform on model proteins with a robust structure, we used Rop for our research. It is a small homodimer that controls the replication of the ColE1 plasmid by enhancing the interaction of RNAs that initiate plasmid replication. To probe the sequence fitness landscape of Rop and understand its binding interface at the molecular level, we developed a deep mutational scanning approach and verified it using a model experiment. We generated sixty-two individual point libraries spanning its whole sequence and enriched them using a growth-based assay. We used three environmentally different positions, i.e., core, loop, and surface and validated the functionality of the approach. We also identified unique functional variants at surface and core positions. The comprehensive sequence-function map showed that helix H1/H1' is more important for binding than H2/H2'. We found that layers 5,6 and 7 of the hydrophobic core are less tolerant to mutations, positions 57-63 are highly tolerant, and the stretch of positions from 10-15 is entirely intolerant to mutations. We also identified interesting multiple-point mutants with strong selection and thermal stability from high throughput thermal scanning, which need further analysis. Since Rop (open full item for complete abstract)

Committee: Karin Musier-Forsyth (Committee Member); Thomas Magliery (Advisor) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2022, Biochemistry Program, Ohio State

Antibodies are important tools in scientific research and medicine due to their ability to bind a wide variety of targets with high specificity. The minimal binding fragment of human antibodies, the single-chain FV (scFV) fragment, is engineered by genetically fusing the two binding domains of an antibody together and spacing them with an artificial linker. The length of the linker has been demonstrated to change a host of biophysical characteristics such as binding affinity, stability, and oligomeric state. However, the effect that the linker composition has on these properties is not well understood as previous comprehensive linker studies have been primarily completed with limited variations in linker composition. In chapter 2 we constructed and purified scFV constructs with varying lengths and four different compositions, (EAAAK)n, (DDAKK)n, Pn, An, and compared them to the commonly used (GGGGS)n linker. Constructs were characterized by differential scanning fluorimetry, gel filtration, and protease assays to determine thermal stability, oligomeric state, and proteolytic stability, respectively. We show that the majority oligomeric state when the interdomain linker is 15 amino acids or fewer is dimer. This suggests that despite the popular use of 15 amino acid long linkers in scFV construction, the linker should be at least 20 amino acids in length when monomeric scFVs are desired. Additionally, scFV dimers with higher stability and homogeneity can be generated with linkers that have higher conformational rigidity. Overall, our results contribute to greater understanding of how the design of the linker composition and length impacts the of the biophysical properties of scFVs. In chapter 3 we explore the incorporation of functional peptides into the scFV interdomain linker. We hypothesized that the artificial interdomain linker region would be a favorable place to insert a functional cell penetrating peptide (CPP) with minimal negative effects on the biop (open full item for complete abstract)

Committee: Tom Magliery (Advisor); Mike Tweedle (Committee Member); Ross Dalbey (Committee Member); Mark Foster (Committee Member) Subjects: Biochemistry

Master of Science (MS), Ohio University, 2022, Chemistry and Biochemistry (Arts and Sciences)

Apoptosis is a form of controlled cell death that occurs as a normal part of an organism's growth and development. Apoptosis is often initiated as a result of external or internal stresses that the cell cannot overcome and thus becomes committed to die. Defects in a cell's apoptosis machinery can result in uncontrolled cell division, leading to cancer or auto-immune disorders. Our work focuses on studying the interactions between pro- apoptotic (pro-death) and anti-apoptotic (pro-survival) proteins involved in the intrinsic apoptosis pathway. Specifically, we are interested in developing molecules that inhibit anti-apoptotic BCL2 family proteins in order to facilitate cell death in diseases that result from dysregulated apoptosis mechanisms. The primary area of my research involves studying the interaction between the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2. Bax interacts with Bcl-2 via its helical BH3 domain, causing Bax to remain inactivated. In times of cellular stress, activated Bax becomes unbound from Bcl-2 and initiates the release of cytochrome c from mitochondria, leading to cell death via apoptosis. Our lab recently developed a series of molecules based on the small protein scyllatoxin (ScTx) as inhibitors of the Bax:Bcl-2 interaction. Notably, synthetic ScTx can be modified to target anti-apoptotic Bcl-2 proteins by replacing natural amino acids within the ScTx helix with Bax BH3 residues that are important for Bcl-2 recognition. 4 My studies have included establishing screens to co-crystalize Bcl-2 or Bcl-2 (G145A) (a mutant of Bcl-2 that resists interactions with other BCL2 BH3 domains) with ScTx-based BH3 domain mimetics or truncated Bax BH3 domains. In addition, we have conducted isothermal titration calorimetry (ITC) studies with ScTx-Bax structural variants in complex with Bcl-2. These studies have helped to elucidate structural requirements and energetic properties for targeting anti-apoptotic Bcl-2 with ScTx-Bax BH3 (open full item for complete abstract)

Committee: Justin Holub (Advisor); Scott Hooper (Committee Member); Katherine Cimatu (Committee Member); Norbert Sträter (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, Case Western Reserve University, 2022, Chemical Engineering

Since the 1970s, elastin-like polypeptides (ELPs) have been extensively characterized for their reversible phase transition in solutions. ELPs are soluble in aqueous solution at temperatures below their transition temperature (Tt) and undergo phase transition when the temperature is raised above Tt, at which ELPs are insoluble. While applications of surface-grafted ELPs continue to rise, there is limited knowledge of whether the phase transition behavior of surface-grafted ELPs follows that of the free ELPs. The motivation of this research study is to understand the surface phenomena of ELPs to support the development of ELP-based electrodes. The thesis pioneers the study of molecular arrangement of surface-grafted ELPs on confined spaces of metal electrodes. The discovery of grafting conditions presented in this thesis provides a path to consistent fabrication of ELP-based electrodes. The demonstrated applications of surface-grafted ELPs provide insight into their physical properties and phase transition behavior.

Committee: Julie Renner (Advisor); Michael Hore (Committee Member); Chung-Chiun Liu (Committee Member); Donald Feke (Committee Member) Subjects: Biophysics; Chemical Engineering; Higher Education

Doctor of Philosophy, The Ohio State University, 2021, Chemical Engineering

The purification of recombinant proteins is critical to fundamental research and pharmaceutical development. In clinical manufacturing, biopharmaceuticals must be highly purified to meet stringent safety guidelines, and thus require a downstream purification process tailored specifically to each drug product. Paradoxically though, discovery-phase pharmaceutical research depends on screening diverse arrays of candidate molecules, making product-specific purification schemes impractical. This creates an inherent bottleneck in the transition from drug discovery to clinical evaluation to production-scale manufacturing. As a result, platform purification technologies like affinity chromatography – which enable highly specific isolation of proteins yet require minimal product-specific process optimization – have become a critical driver of pharmaceutical development. Affinity capture is performed using a stationary-phase substrate functionalized for direct affinity to a specific peptide or protein domain. In the absence of a known, convenient, naturally occurring affinity domain within a given protein of interest, an ‘affinity tag' is recombinantly appended to the purification target to facilitate selective isolation of the desired product. Introducing an affinity tag is a fairly trivial modification with modern cloning and heterologous expression techniques, and this approach is widely practiced at bench scale due to its inherent versatility; affinity tag function is generally agnostic to the protein that the tag is fused with, thus, a single affinity tag platform can purify a variety of hypothetical products. Despite this utility, affinity tags have a few fundamental limitations that are particularly problematic for pharmaceutical production. Tags may alter structure or inhibit function of their fusion partners, and many tags are immunogenic and thus cannot be administered in fusion to a therapeutic. In response to these problems, several proteolytic techniques ha (open full item for complete abstract)

Committee: David Wood (Advisor); Andre Palmer (Committee Member); Jeffrey Chalmers (Committee Member) Subjects: Biochemistry; Chemical Engineering; Molecular Biology; Pharmaceuticals

Master of Science, The Ohio State University, 2021, Chemical Engineering

Bridging the gap between protein purification platforms at the laboratory scale and at the industrial scale remains a challenge, as there is no “one size fits all” technology that works for every protein. To date, only technology based on Protein A has managed to enter mainstream usage both in the lab and in industry, but it only works for monoclonal antibody and Fc fusions. Affinity chromatography can be used to purify most proteins, but a major downside of this is that they require the use of affinity tags – proteins purified with these tags must undergo further treatment to remove the tags, which is impractical at the industrial scale. Research into intein-based chromatography offers a possibility to get around this: engineered inteins can be used as a “self-cleaving” affinity tag that leaves a tagless target protein after elution. However, the rate at which inteins cleave themselves off a protein varies wildly depending on the target protein. This work aims to reduce that dependency by introducing a mutation known to reduce the intein's dependence on the +2 C-extein residue in the natural splicing version of the Npu intein into the engineered cleaving version to determine if it can also reduce its dependency on the +2 residue. The experiments performed in this work did not detect a significant difference in variance, however. In addition, it also appeared to slow down the overall cleavage kinetics, suggesting that the intein cleavage reaction is subject to different controls than the splicing reaction.

Committee: Andre Palmer (Committee Member); David Wood (Advisor) Subjects: Chemical Engineering

PHD, Kent State University, 2021, College of Arts and Sciences / School of Biomedical Sciences

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by the accumulation of beta-amyloid plaques and neurofibrillary hyperphosphorylated tau tangles. According to the most recent report from the U.S. Centers for Disease Control and Prevention, Alzheimer's disease is the 6th leading cause of death in the United States and the number of cases as increased almost 150% in the last 20 years in the United States. The disease starts as a subtle change in memory that progresses into noticeable cognitive decline, often referred to as mild cognitive impairment, and progresses into severe and pervasive memory loss. One of the hallmarks of Alzheimer's disease is the accumulation of the misfolded protein product of the amyloid precursor protein, beta-amyloid. Beta-amyloid has been long considered and widely supported by genetics and biochemical observation to promote Alzheimer's disease pathology. One of the earliest and long-supported theories, the amyloid hypothesis, posits beta-amyloid accumulation as having a central role in the pathogenesis of Alzheimer's disease as a promoter of tau seeding and inflammatory signaling which causes immune dysfunction. Beta-amyloid therapeutics represent the majority of the clinical trial candidates for Alzheimer's disease therapeutics. Recent clinical failures of anti-beta-amyloid trials, including Aducanumab and Gantenerumab, have raised concerns for the ability to manage beta-amyloid accumulation. The current pipeline of drugs targeting both beta-amyloid and tau are ineffective; therefore warranting alternative therapeutic options. A possible alternative non-pharmacological option for targeting beta-amyloid plaque aggregation is using energy to disrupt large beta-amyloid plaques into smaller fragments that may be cleared by microglia, the innate immune cells of the brain. One manner in which we can generate sufficient energy in a minimal to non-invasive safe manner is to use an alternating magnetic (open full item for complete abstract)

Committee: Min-Ho Kim Ph.D. (Advisor); Fayez Safadi Ph.D. (Advisor); Colleen Novak Ph.D. (Committee Chair); Gary Koski Ph.D. (Committee Member); Songping Huang Ph.D. (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Nanoscience; Nanotechnology; Neurobiology; Neurology; Neurosciences

Master of Science (MS), Ohio University, 2020, Biomedical Engineering (Engineering and Technology)

The increasing applications of nanoparticles in biomedical research and consumer products has raised concerns regarding their potential toxicity. Although the effects of nanoparticles on human health and environment are still under investigation, previous studies have revealed significant evidence of nanoparticle toxicity to cells. With the cell plasma membrane being the first entity that first interacts with nanoparticles upon possible exposure, numerous studies have focused on nanoparticle interactions with cell plasma membrane. Nanoparticles entering a biological fluid are covered by proteins which adsorb to the particle surface, commonly called the nanoparticle protein corona. Protein corona changes nanoparticle characteristics, thereby altering their biological behavior. A better understanding of how the corona regulates nanoparticle-membrane interactions can help the design of the biomedical applications of nanoparticles. This study seeks to investigate the role of the corona from human serum on nanoparticle-induced cell membrane damage. Plain silica nanoparticles, known for disrupting the lipid bilayer of the cell membrane, were used as nanoparticle models. The effects of pristine nanoparticles are recorded after interactions with model membranes and red blood cells and compared to results from nanoparticles with the protein corona on their surface. The results show that while the applied silica nanoparticles are disruptive to the cell membrane in their pristine form, the damage to the cell membrane is significantly reduced after the formation of the protein corona. Furthermore, the specific proteins adsorbed on the nanoparticle surface play an important role in the ability of the particles to induce membrane damage with the examination of a number of abundant proteins in the corona, it is concluded that one of the main reasons behind these interactions is the surface coverage of the nanoparticles by proteins.

Committee: Amir Farnoud (Advisor) Subjects: Biomedical Engineering

Master of Science, The Ohio State University, 2020, Chemistry

Rop protein is a four-bundle dimeric protein that assists RNAI to form a kissing complex with RNAII. By engagement in the kissing complex, the DNA polymerase will fail to replicate from the ColE1 origin and consequently, the copy number of the ColE1 plasmids will be downregulated. Rop's monomer is a helix-turn-helix structure with a tight turn containing a Schellman motif. We have studied the stability and activity of the Rop protein with different mutations in the loop section of the protein, residues 29-32, Leu-Asp-Ala-Asp (LDAD) in wild-type. The Arg55 residue appears to make an ionic contact with Asp32 in the wild-type. We engineered four variants, LDAG, R55Q_LDAN, LGGAD, and R55Q_LGDAD and compared their stability and activity with AV_Rop, as the native Rop with LDAD sequence in its loop region. To measure their stability, first, all these four variants were cloned into a T7 expression vector, and after expression and purification, their thermal stability was measured by CD thermal scanning. The result showed that the variant R55Q_LGDAD was the most stable variant. LGGAD and LDAG were more stable than wild-type one but less than R55Q_LGDAD. Finally, the variant R55Q_LDAN was the least stable variant. To measure the activity of the variants, all the variants were cloned into p15a vector and using green fluorescent protein (GFP) expression from a ColE1 plasmid, their activity was measured. We also ran a 1 ns MD simulation to validate our simulation system for future longer runs. We have plan to calculate RMSD, RMSF, DSSP, SASA, HB, Ramachandran plot, and free energy surface of all variants under longer runs.

Committee: Thomas Magliery (Advisor); Rafael Brüschweiler (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy, University of Akron, 2019, Chemical Engineering

In the study of this Ph.D. dissertation, two research topics related to zwitterionic materials have been investigated. Even though the applications have different objectives, the unique properties that existed in zwitterionic materials have, including charge property and antifouling property, have been used in both of research. The first topic is the development of zwitterionic-peptides gene delivery system. The gene delivery system, with high efficacy, low toxicity, long blood circulation time and targeting the specific cancer cell, is investigated. The second topic is the functionalization of protein therapeutics with zwitterionic polymers. The protein therapeutics with better solubility, stability, and activity is developed. The non-viral gene delivery system is under research due to their low toxicity, low immunogenic and large DNA loading size in gene therapy. Peptides gene delivery system is reported with the cationic charge and buffering effect which overcomes the barrier and delivery DNA into the nucleus. In our group, the economic dextran-peptide hybrid gene delivery system was developed with high transfection efficiency and low toxicity. The first topic of my research was expanded as a continuous work under the same research interest. The effect of the design of peptides length, zwitterionic group and targeting group was studied for the optimization objects on achieving low toxicity, transfection efficiency and blood circulation time, which was summarized into three research projects under this topic. The system was adjusted by the peptides length for toxicity and economic purpose. The system was functionalized with the zwitterionic group for improved stability, enhanced endosomal escape and longer blood circulation time. The system was also conjugating with targeting ligand for targeting gene delivery. It was found that the shorter length of peptides will not provide enough charge to form stable micelle with report DNA. The zwitterionic functionalized (open full item for complete abstract)

Committee: Gang Cheng (Advisor); Jie Zheng (Advisor); George Chase (Committee Member); Lingyun Liu (Committee Member); Ge Zhang (Committee Member); Coleen Pugh (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering; Polymers

Doctor of Philosophy, University of Akron, 2019, Biomedical Engineering

Spinal cord injury (SCI) results in permanent motor and sensory deficits, primarily caused by localized cell death. Treatment strategies which focus on guiding cell behavior (exogenous or endogenous) are attractive. Neural stem cells (NSCs) can contribute to recovery indirectly (secreted neurotrophic factors) or directly (by differentiating into functional cell types). Their efficacy is also clearly enhanced with an active biomaterial carrier to keep them within the site of injury and guide their behavior. Here, a biomaterial-based approach for treating SCI was investigated, consisting of NSCs seeded within a biomimetic scaffold made from methacrylamide chitosan (MAC). The scaffold contained tethered, biotinylated recombinant growth factors to specify the lineage of the encapsulated NSCs. This scaffold showed promising tissue-level improvements but failed to restore locomotor function. Next, a new approach for covalently immobilizing azide-tagged recombinant proteins was developed. This approach was tested on interferon-gamma (IFN-gamma, which induces neuronal differentiation from NSCs) and found to enable immobilization to multiple materials while retaining its bioactivity. A new, open-source gait analysis technique was then adapted to include SCI-specific parameters. This technique was tested on a treatment which is known to be effective, intracellular sigma peptide (ISP, which reduces inhibitory cues from the microenvironment) and found to sensitively measure benefits. The NSC-seeded scaffolds were tested again, with two major improvements: they were primed in subcutaneous tissue prior to transplantation into the spinal cord and ISP was co-administered. This approach was based on a finding that NSC-seeded scaffolds with immobilized IFN-gamma increased the expression of developmental markers after subcutaneous maturation. Ultimately, subcutaneous maturation with ISP was found to improve function. The tissue-level analysis suggested that this was due to an indirec (open full item for complete abstract)

Committee: Nic Leipzig PhD (Committee Chair); Rebecca Willits PhD (Committee Member); Hossein Tavana PhD (Committee Member); Bi-Min Zhang Newby PhD (Committee Member); Adam Smith PhD (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, The Ohio State University, 2019, Biochemistry

Each year approximately 3 million people are exposed to toxic organophosphorus compounds (OPs) such as pesticides and nerve agents. These chemicals target the parasympathetic nervous system causing a variety of horrific symptoms and eventually respiratory failure, seizures and death. The current treatment for OPs is a drug cocktail consisting of atropine and pralidoxime (2-PAM) given in coordination with diazepam. However these drugs have not been shown to prevent the long term, more severe, side effects of OP poisoning. Therefore the search for additional therapeutics is underway. Bioscavengers have shown significant promise as potential therapeutic treatments; however, a strong lead molecule has yet to be optimized for therapeutic use. Initially, butyrylcholinesterase (BChE) was identified as a strong candidate. However the economics of its purification and subsequent use have proven extremely prohibitive. However, enzymatic bioscavengers show significant promise as potential therapeutics. Specifically, human Paraoxonase-1 (PON1) has been shown to provide protection form relatively high doses of certain G-type nerve agents and pesticides. However it is perhaps even more difficult to isolate than BChE. As a result, many studies have sought to identify a recombinant enzyme which still retains the ability to hydrolyze organophosphates and can also be easily purified from a bacterial system. The resulting series of variants yielded a particularly interesting enzyme, the G3C9 variant of PON1. Since its generation, numerousiii structural and mechanistic studies have been undertaken in order to gain a better understanding of PON1's hydrolytic mechanism. However these studies typically were inconclusive, as to date several mechanisms have been proposed for its catalytic activity, all of which have their merits and shortcomings. This ambiguity makes engineering PON1 for improved phosphotriesterase activity significantly difficult, ultimately limitin (open full item for complete abstract)

Committee: Thomas Magliery Ph.D. (Advisor); Jane Jackman Ph.D. (Committee Member); Christopher Hadad Ph.D. (Committee Member); Ross Dalbey Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2018, Biochemistry

Insulin is a peptide hormone that is the primary regulator of glucose homeostasis in vertebrates. Insulin is secreted by the endocrine pancreas in response to increased interstitial glucose levels; insulin initiates the uptake of glucose by peripheral tissues. Since its first use in 1921, insulin has been the primary treatment for the metabolic condition known as Type I Diabetes Mellitus (T1DM; caused by the absolute lack of insulin), and a component in the treatment of Type II Diabetes Mellitus (T2DM; caused by the relative lack of insulin in relation to peripheral insulin resistance). The ubiquitous clinical use of insulin has led to investigation of its structure-function relationships. Such studies have uncovered the rich evolutionary history of insulin and its usefulness as a model molecule. Indeed, the study of insulin has revealed a number of concepts of protein structure/function relationships and the in vitro and biosynthesis of proteins. This dissertation is a continuation of the nearly century-old field of insulin biochemistry. The first section of the thesis examines the evolutionary constraints responsible for the conservation of several structural features within the insulin B chain as they relate to the biosynthesis, stability, and biological activity of the hormone. Concepts expounded in these studies may be generalized to the evolution and folding xvii process of globular proteins as a class. Furthermore, such studies may be used to inform the design of therapeutic insulin analogs as exemplified in the second part of the dissertation. This section demonstrates how conserved structural features of insulin may be exploited and modified to produce favorable therapeutic effects even if such modifications would be unfavorable in the context of vertebrate physiology. This approach underscores the importance and usefulness of a multidisciplinary approach to the study of insulin both as a model molecule and as a therapeutic agent.

Committee: Michael Weiss MD, PhD (Advisor); Paul Carey PhD (Committee Chair); Faramarz Ismail-Beigi MD, PhD (Committee Member); George Dubyak PhD (Committee Member) Subjects: Biochemistry; Biophysics; Endocrinology; Medicine

Master of Science, Miami University, 2018, Cell, Molecular and Structural Biology (CMSB)

Enzymes, while incredibly complex, are limited in functionality to their twenty canonical amino acids. To shortcut this limitation, nature has evolved to utilize post translational modification (PTM) of protein structure to regulate cellular functions. Whether this be as essential cofactors or acting as regulators of enzyme activity, controlling cellular processes by PTMs are absolutely essential to the molecular-level events that coordinate and sustain life. Within a massive population of possible binding sites, enzymes responsible for PTMs manage to find their precise target through highly selective recognition sites. LplA has been shown to be a convenient tool for attaching small molecule precursors to its recognition site. The aim of this study is to modify the enzyme Lipoic acid ligase A (LplA) to effectively carve a tunnel through the backside of the enzyme. By carving a tunnel through the ligase, this work aims to broaden the substrate compatibility range of LplA for post translational modification of target proteins. Additionally in this study, crystal structures of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) will be solved and analyzed for evidence of S-nitrosylation. It is the goal of this study to gain a better understanding of how this PTM regulates heme binding to GAPDH.

Committee: Richard Page (Advisor); Michael Robinson (Committee Chair); Yoshi Tomoyasu (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, The Ohio State University, 2018, Chemical Engineering

Recombinant therapeutic proteins changed the world over 30 years ago when insulin, the first therapeutic protein, was approved. Since then, over 200 therapeutic proteins have been approved to treat a wide range of diseases from diabetes to immune disorders. Currently, there is no universal platform that can be used to purify any given target protein in a quick and inexpensive manner. The self-cleaving split intein tag technology remedies this issue by creating a universal platform that can purify any traceless, tagless target protein rapidly and economically. Chapter 2 discusses the combination of the split intein purification strategy with cell-free protein synthesis (CFPS) systems to reduce the time it takes to produce therapeutic proteins. With the cell-free systems, proteins can be produced in hours compared to days or even weeks. The combination of CFPS and the split intein tag technology has been utilized in the creation of a device to produce biologics on demand. The BioMOD device aims to produce a single-dose of any therapeutic protein within 24 hours, specifically with a military application in mind. Chapter 3 discusses the use of magnetic beads to mediate the split intein purification. Combining the split intein and magnetic beads creates a more efficient purification process that requires less buffer and set-up time. Four target proteins are used to demonstrate the applicability of the system. Chapter 4 discusses the regeneration of a commercially available resin that has been used to covalently immobilize the N fragment of the split intein using a thioester bond. Due to the commercially available resin having a high price point and the lengthy amount of time it takes to immobilize the N fragment, regeneration of the resin was necessary. A panel of buffers was screened to find the best regeneration buffer. Using the best buffer, a life cycle analysis was done using 20 regeneration cycles to show the resin could be regenerated multiple times. The develo (open full item for complete abstract)

Committee: David Wood (Advisor); Jeffrey Chalmers (Committee Member); Andre Palmer (Committee Member) Subjects: Chemical Engineering