As a basic building block in many biological molecules, the element nitrogen (N) is essential for life. Dinitrogen (N2) is abundant in Earth’s atmosphere, but this form is biologically unavailable. To be biologically available, N2 must undergo a reduction reaction to the fixed form, ammonia (NH3). The industrial Haber-Bosch process, which accounts for approximately 50% of the worlds fixed nitrogen, uses energy from fossil fuels to achieve high pressures and temperatures to catalyze the reaction. The energy used by Haber-Bosch accounts for approximately 2% of the world’s annual supply. The remainder of fixed nitrogen is produced biologically by nitrogen fixing microorganisms (diazotrophs), utilizing nitrogenase enzymes. Nitrogenase enzymes catalyze the reduction at ambient temperature and pressure, deriving the necessary energy from the energy rich molecule adenosine triphosphate (ATP). The focus of this dissertation is enriching our understanding of how nitrogenase enzymes can catalyze this crucial reaction under ambient conditions. This, of course, leads to a better understanding of biological nitrogen fixation, but also reveals the strategies that nature has evolved which can inform on the development of cleaner more efficient industrial processes.

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CRISPR (clustered regularly interspaced short palindromic repeats)-guided nucleases such as Cas9 remain atop the most exciting biological systems in gene editing and therapeutic potential. The modern adaptation of the Streptococcus pyogenes (Spy) Cas9 machinery for directed DNA editing relies on two major components; a Cas9 protein, and an RNA guide. This study aims at modification of the guide RNA to probe structural requirements and modification tolerance while studying the biochemical properties of the ribonucleoprotein complex. We find that the SpyCas9-RNP is not only capable of heavy substitutions and modifications in the guide, but specific properties can be tuned by careful and directed modifications. DNA substitutions in the 3’ end of the guide RNA improves cleavage activity and efficiency in vitro. Various RNA chemistries such as 2’-fluoro and other RNA mimics maintain biochemical activity but can be used to improve stability against nucleases. Additionally, tethering of the tracrRNA and crRNA together into a sgRNA was found to affect the fundamental properties of the Cas9 protein, improving activity, but reducing target binding affinity and cleavage rate. Directed modification of the guide RNA can be used to exploit certain biochemical properties for effective therapeutic applications.

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Cysteine dioxygenase (CDO) is a non-heme iron enzyme that can be found in mammalian tissue. It is mainly localized in the liver but is also present in the brain, kidney, and adipose tissue. CDO converts cysteine to cysteine sulfinic acid, which is the first step in cysteine metabolism in the human body. CDO contains a novel cofactor located near the metal binding site that is present in another enzyme, galactose oxidase, where it is essential for redox function. This suggests that the linkage may play an important role in CDO as well. The cofactor consists of Y157 and C93. Mutation of the C93S causes a drop in activity to 57.1% and a mutation of the Y157F causes a drop to 8.1%. The metal center was studied using XAS revealing that the addition of cysteamine, an activator of CDO, changes the conformation of the binding site significantly. CDO differs from the rest of the cupin super family in that it does not contain a 2-his-1-carboxylate binding motif but rather the carboxylate is replaced with another histidine. A mutation of one of the binding residues, H140D, caused the enzyme to be non-active. Also the mechanism of the CDO was studied by conducting activity assays with various inhibitors and activators that yielded contradicting results with previously published work.

Strength and tone of the pelvic floor muscles are thought to play an important role in pelvic floor disorders, and are key outcomes monitored in pelvic floor rehabilitation, yet the standard approach to measuring these outcomes is through subjective assessment using manual palpation. While intravaginal dynamometers (IVD) have been designed to measure these characteristics in research settings, most are not sufficient to withstand the rigors of clinical use. This work presents the bench validation and subsequent updated design of a new mechanical constant speed mechanism for future inclusion in an IVD. Opening speeds of the original mechanism were validated with and without external loading using video analysis. The bench validation showed that the speed of arm opening was lower than the ranges specified for clinical use and was influenced by external loading. The mechanism was updated, and the bench test was repeated. The updated mechanism was found to provide output speeds that are within the ranges required clinically and were minimally impacted by external loading. The next step of this work is to reduce the size of the mechanism and improve output speeds to allow it to be assembled into a clinical prototype IVD.

This thesis presents research on the feasibility of applying compliant-ortho-planar springs (COPS) to rotational applications. The primary motivation of this research is the application of COPS to a continuously variable transmission (CVT). The design space limitations, loading conditions, stresses, stress concentrations, and limitations of current design tools, such as pseudo-rigid-body models (PRBM) for COPS, are discussed. A new 3D PRBM is presented along with a discussion on the possible applications of such to a rotating COPS. Stress stiffening and lateral stability are two major phenomena occurring in a rotating COPS. Both phenomena are a direct result of the inertial loads a COPS would be subjected to in a rotational environment. The results show how stress stiffening and lateral buckling in the legs are influenced by design parameters. Conclusions and recommendations for further research are recommended.

Indiana University-Purdue University Indianapolis (IUPUI) / Complex biological systems are composed of many regulatory components, which can be measured with the advent of genomics technology. Each molecular assay is normally designed to interrogate one aspect of the cell state. However, a comprehensive understanding of the regulatory mechanism requires characterization from multiple levels such as genome, epigenome, and transcriptome. Integration of multi-omics data is urgently needed for understanding the global regulatory mechanism of gene expression. In recent years, single-cell technology offers unprecedented resolution for a deeper characterization of cellular diversity and states. High-quality single-cell suspensions from tissue biopsies are required for single-cell sequencing experiments. Tissue biopsies need to be processed as soon as being collected to avoid gene expression changes and RNA degradation. Although cryopreservation is a feasible solution to preserve freshly isolated samples, its effect on transcriptome profiles still needs to be investigated. Investigation of multi-omics data at the single-cell level can provide new insights into the biological process. In addition to the common method of integrating multi-omics data, it is also capable of simultaneously profiling the transcriptome and epigenome at single-cell resolution, enhancing the power of discovering new gene regulatory interactions. In this dissertation, we integrated bulk RNA-seq with ATAC-seq and several additional assays and revealed the complex mechanisms of ER–E2 interaction with nucleosomes. A comparison analysis was conducted for comparing fresh and frozen multiple myeloma single-cell RNA sequencing data and concluded that cryopreservation is a feasible protocol for preserving cells. We also analyzed the single-cell multiome data for mesenchymal stem cells. With the unified landscape from simultaneously profiling gene expression and chromatin accessibility, we discovered distinct osteogenic differentiation potential of mesenchymal stem cells and different associations with bone disease-related traits. We gained a deeper insight into the underlying gene regulatory mechanisms with this frontier single-cell mutliome sequencing technique.