Biochemistry, Biophysics, and Structural Biology Commons^™

Personalizable Metabolic Models To Enable Immune Characterization And Drug Discovery, Joshua Loecker

Department of Biochemistry: Dissertations, Theses, and Student Research

Cellular metabolism is reshaped by disease and physiological state, and genome-scale metabolic networks (GSMNs) offer a systematic way to interrogate this biology and identify therapeutic targets. Building a GSMN from expression data, however, requires a sequence of stages including read processing, normalization and gene-activity classification, multi-omic integration, model reconstruction, simulation, and biological interpretation. These are typically handled by disconnected tools spanning multiple programming languages, file formats, and databases. The resulting reliance on heterogeneous interfaces and manual steps threatens reproducibility. This work develops an ecosystem of computational tools that carry an analysis from raw sequencing reads to mechanistic biological interpretation. First, …

Effect Of Novel Antimicrobial Compounds On Fungal And Bacterial Pathogens, Megan Wilderman

Honors Program: Senior Projects (Public)

Antimicrobial resistance is a growing concern in medicine¹ leading to a need for the development of novel compounds. Two compounds were tested in this study, 2-hydroxyethylhydrazine (HEH) and para-nitrophenyl-isocyanide (NPIC). HEH targets phosphatidylcholine (PC) production², and NPIC targets mitochondrial function³. Choline (Cho) is a compound that bypasses the inhibition of HEH through the Kennedy pathway.² Two-fold serial dilutions were conducted in 96-well plates to test the sensitivity of different bacteria to HEH and fungi to HEH and NPIC in different media types. A choline condition was also included in the HEH dilutions to test …

Interview With Orca Award Winner David Hage, Unl Chemistry; Drug-Protein Interactions, Mark Griep, David Hage

UNL ORCA Interview Series

Prof. David Hage was awarded the ORCA in 2018. He was born in La Crosse, Wisconsin, and earned a bachelor’s in chemistry at the University of Wisconsin in La Crosse. Next, he earned a doctorate from Iowa State University and did postdoctoral work at the Mayo Clinic in Rochester, Minnesota. In 1989, he joined UNL as an assistant professor of chemistry and quickly rose through the ranks. He has won many awards and, for the past 13 years, has been the James Hewet University Professor of Chemistry. Allow me to share some remarkable facts about Dave before we get into …

Molecular Pathway Dysregulation In Chronic Liver Disease: Metabolic And Mechanotransductive Drivers Of Hepatic Pathophysiology, Samantha Harvat

Department of Chemical and Biomolecular Engineering: Dissertations, Theses, and Student Research

Metabolic dysfunction–associated steatotic liver disease (MASLD) represents a rapidly growing cause of chronic liver injury, yet the molecular interactions linking metabolic stress, inflammation, fibrosis, and hepatocellular carcinoma (HCC) remain incompletely defined. This thesis integrates transcriptomic analyses across multiple GEO datasets to identify coordinated metabolic, inflammatory, and mechanotransductive pathways that collectively drive disease progression.

Analysis of human liver biopsy datasets (GSE126848 and GSE89632) revealed consistent suppression of glycolysis, β-oxidation, and oxidative phosphorylation across MASLD and MASH samples, accompanied by modest compensatory TCA cycle activation. These alterations reflect impaired metabolic flexibility, mitochondrial stress, and increased ROS susceptibility. Concurrently, cytokine-network enrichment demonstrated activation …

Comparative Analysis Of Citrate Synthase Activity In Skeletal Muscle, Liver, And Adipose Tissue In Beef Cattle, Abigail Webb

Honors Program: Senior Projects (Public)

Mitochondrial efficiency plays a central role in determining how cattle partition nutrients toward muscle growth or adipose deposition, yet little is known about tissue-specific differences in mitochondrial density among tissues or among beef cattle. This study aimed to quantify mitochondrial density in muscle, liver, and adipose tissue samples from beef cattle and evaluate its potential relationship with metabolic efficiency. Tissue samples of semitendinosus muscle (ST), liver, and subcutaneous adipose (SC) were collected, homogenized, and analyzed for citrate synthase (CS) activity using a fluorometric assay as a proxy for mitochondrial density. Preliminary analyses revealed that the semitendinosus muscle displayed the highest …

Interview With Orca Award Winner Donald Weeks; Unl Biochemistry; Biotechnology, Mark Griep, Donald Weeks

UNL ORCA Interview Series

Emeritus Professor Donald Paul Weeks was awarded the ORCA in 2014, a year before he retired. He grew up in a small farm south of Terre Haute, Indiana. In his youth, he participated in 4-H programs that must have sparked an interest in science because he earned his bachelor's degree from Purdue University in 1963. Purdue is about 90 miles north of Terre Haute. Don's doctoral work was at the University of Illinois in Champaign-Urbana for a project about protein synthesis during maize germination. Next, he did postdoctoral work at the Fox Chase Cancer Center in Philadelphia, where his research …

Functional Genomics Of Phosphatidylcholine Biosynthesis In Saccharomyces Cerevisiae, Amanda Marie Maliva

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

Phosphatidylcholine (PtdCho) is a prominent structural building block of cell and organelle membranes in animal, plant, and fungal cells and some bacteria. Due to its abundant nature in the plasma membrane and intracellular organelle membranes in eukaryotes, the biosynthesis of PtdCho must remain regulated to preserve the correct membrane lipid composition. Dysregulations in PtdCho metabolism can lead to mitochondrial dysfunction, cell death, and the development of diseases including cancer, atherosclerosis, and hepatic diseases. Knowledge of PtdCho has remained limited to the genes involved in its three biosynthetic pathways (Kennedy (K), methylation (M), and acyltransferase (A)), despite the growing realization of …

Aptamer Selection And Development Of Electrochemical Aptamer-Based Sensors For Neuropeptide Y, Evelyn Adela Carreto Guevara

Department of Chemistry: Dissertations, Theses, and Student Research

In this thesis, we report the results of four aptamers obtained from an aptamer selection process called the systematic evolution of ligands by exponential enrichment (SELEX) with affinity to Neuropeptide Y (NPY). NPY is a highly abundant peptide transmitter with widespread distribution throughout the body which affects various physiological functions. These functions include energy homeostasis, appetite regulation, stress response, and anxiety regulation. Dynamic monitoring of NPY levels is crucial for better understanding behaviors in individuals with neurological disorders associated with NPY. Aptamers are synthetic oligonucleotides obtained through SELEX which have a binding affinity to a target of interest when in …

Biocomputing Approach To Modeling And Modulating Calcium Signaling, Sehee Sun

School of Computing: Dissertations, Theses, and Student Research

Biocomputing is an emerging field that seeks to perform computational tasks using biological substrates and processes. Unlike conventional computing systems based on silicon hardware, biocomputing leverages the parallelism, energy efficiency, and complex dynamics of living systems. Among various cellular mechanisms, calcium (Ca2+) signaling stands out as a central regulator of diverse biological functions, offering a promising basis for programmable logic and control in living cells.

This thesis introduces a novel framework for modeling and modulating Ca2+ dynamics using biologically inspired Boolean logic circuits. Specifically, we propose the Ca2+ Boolean Logic (CaBL) model, in which Ca2+ fluxes and interactions are abstracted …

Novel Chloroviruses With Unique Glycans On Their Major Capsid Proteins, Cayden J. Homolka

Honors Program: Senior Projects (Public)

Chloroviruses, of the family Phycodnaviridae, are large, dsDNA viruses with a genome of 290 to 410 kb. These virus genomes are so large that they are classified as “giant viruses”. Within this genome, up to 16 tRNAs and 330 to 450 proteins are encoded. Among the massive amounts of genes and proteins are unique carbohydrate synthesis/metabolism genes, which are responsible for the glycosylation of chloroviruses major capsid proteins. In most viruses, these proteins are glycosylated by the host enzymes located in the endoplasmic reticulum. Instead, Chloroviruses encode their own glycosylation machinery; essentially decorating themselves. Furthermore, these chlorovirus glycans are unlike …

Implications Of Type Iv Pilus Retraction For Dna Uptake By Acinetobacter Baumannii, Yafan Yu

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

Horizontal gene transfer (HGT) via natural competence allows bacteria to incorporate extracellular DNA (eDNA) into their genomes, facilitating genetic diversity and adaptation. In Acinetobacter species, natural competence depends on DNA binding and uptake mediated by type IV pili (T4P). T4P are dynamic extracellular filaments composed primarily of the major pilin subunit PilA, along with a few minor subunits. T4P drive a range of cellular functions including twitching motility, biofilm formation, and DNA uptake, with some functions dependent on pilus retraction and others not. However, how Acinetobacter T4P bind DNA and how T4P/eDNA interactions impact biofilm formation remain unclear. Here, I …

Screening For Chloroplast Inner Envelope-Thylakoid Membrane Contact Site Proteins, Evan W. Labrant

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

Most life on Earth is dependent upon the capture of solar energy and subsequent fixation of carbon. In members of the Archaeplastida lineage these biological processes take place in semi-autonomous organelles called chloroplasts. Chloroplasts are bounded by a double membrane envelope and contain the photosynthetic membrane system, thylakoid membranes, where light capture takes place. While a great deal is known about how thylakoid lipids are synthesized and protein complexes therein are properly maintained, very little is known regarding the processes by which lipids are transported to thylakoids which lack de novo glycerolipid synthesis. We have worked to identify candidate proteins …

Sodium And Potassium In Health And Cellular Function, Jordyn Branz

Honors Program: Senior Projects (Public)

Sodium (Na⁺) and potassium (K⁺) are essential electrolytes that regulate numerous physiological processes. These include cell function, nerve transmission, and muscle contraction. The Na+/K+-ATPase plays a critical role in maintaining the balance of these ions by actively transporting three sodium ions out of the cell and two potassium ions into the cell against their concentration gradients. This process is vital for maintaining the resting membrane potential and supporting the function of excitable cells. The Na+/K+-ATPase consists of two main subunits, with the alpha-subunit being primarily responsible for ion transport activity. Isoforms alpha1, alpha2, and alpha3 are tissue-specific and contribute to …

Next-Generation Biomedical Implants And Stimuli Responsive Soft Optical Arrays Via Surface Functionalized Elastomeric Materials, John M. Kapitan

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

The demands of emerging technologies, such as soft robotics, stretchable electronics, and next generation biomedical implants, has led to the development of diverse polymeric and hybrid organic/inorganic materials specifically tuned to meet these challenges. These materials typically have low Young’s moduli and are inert with high chemical stability. However, this has led to materials that are static in nature, lacking any meaningful way of responding to changes in their environment and adapting to these changes. This has driven a shift towards the design, manufacturing, and characterization of various stimuli responsive surfaces, relying on precise control of surface chemical coatings and …

Dental Disparities: An Analysis Of Dental Caries Risk Factors Promoting Disparities Between Rural And Urban Communities In Nebraska, Nick Lounsbery

Honors Program: Senior Projects (Public)

The prevalence of dental caries in the United States remains a public health concern, as it is one of the most common chronic conditions across people of all ages. Research shows that preventative measures including brushing, fluoridated water, and regular checkups are effective strategies to mitigate tooth decay. However, there appears to be a divide between rural and urban populations, as the presence of dental cavities is significantly greater in remote areas in the State of Nebraska. Research methods used in this analysis included analyzing accredited academic literature to illustrate how cavities form from a biochemical perspective. Research methods also …

Induced Mutagenesis In West Nile Virus Non-Structural Protein 3 With A Discussion Of The Mutation's Effects On Viral Properties, Caden Connealy

Honors Program: Senior Projects (Public)

West Nile Virus (WNV), a member of the Flaviviridae family and introduced to the Western hemisphere in 1999, presents a unique and emerging threat to human health. Infection in humans is often mild but can occasionally result in severe neurological impairment and even death. West Nile Virus non-structural protein 3 (NS3) plays an important role in the process of viral genome amplification. More specifically, the C-terminal domain of the protein folds into a helicase enzyme, which is responsible for the “unwinding” of double-stranded RNA intermediates and RNA secondary structure on the WNV genome. This is a vitally important step in …

Domain Communication In Bifunctional Proline Utilization A From Sinorhizobium Meliloti, Emma Bitterman

Honors Program: Senior Projects (Public)

The reduction potential of flavin bound to Sinorhizobium meliloti proline utilization A (SmPutA) is beneficial in providing vital information on enzyme activity and the active site environment. Flavin is crucial in biological metabolism such as aerobic respiration and other important processes like oxygen activation that are catalyzed by flavin-dependent enzymes. (4). In this study, we seek to determine whether the redox potential of SmPutA-bound flavin can be used to characterize potential long-distance communication and analyze the possibilities of inactivation reversibility. SmPutA is a large bifunctional enzyme that catalyzes the overall oxidation of proline to glutamate via two catalytic domains (1). …

Applications Of Archaeal Antioxidant Coenzyme M As An Agricultural Biostimulant, Jeremy Hunter Brown

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

As the demand to meet global food security goals continue to increase, the need to increase efficiency and productivity of agricultural output also rises. This calls for innovative and cost-effective methods for improving plant growth and yield. Here we explore how antioxidants, molecules that inhibit oxidation and are necessary for redox based signaling and redox homeostasis, can be utilized as chemical biostimulants. Specifically, we investigated Coenzyme M, an antioxidant from archaea that holds potential economic advantages over other antioxidants. Chapter 1 provides a comprehensive review of antioxidants and how exogenously application of them affects plant growth and biology, highlighting the …

Chilling- And Dark-Regulated Photoprotection In Miscanthus, An Economically Important C4 Grass, Jared Haupt, Katarzyna Glowacka

Department of Biochemistry: Faculty Publications

Tolerance of chilling dictates the geographical distribution, establishment, and productivity of C4 crops. Chilling reduces enzyme rate, limiting the sink for the absorbed light energy leading to the need for quick energy dissipation via non-photochemical quenching (NPQ). Here, we characterize NPQ upon chilling in three Miscanthus accessions representing diverse chilling tolerance in C4 grasses. High chilling tolerant accessions accumulate substantial amounts of zeaxanthin during chilling nights in both field and growth chamber settings. Chilling-induced zeaxanthin accumulation in the dark enhances rate of NPQ induction by 66% in the following morning. Based on our data, the emerging ways for the unique …

Analysis Of Natural Structures And Chemical Mapping Data Reveals Local Stability Compensation In Rna, Robert L. Cornwell-Arquitt, Riley Nigh, Michael T. Hathaway, Joseph D. Yesselman, David A. Hendrix

Department of Biochemistry: Faculty Publications

RNA molecules adopt complex structures that perform essential biological functions across all forms of life, making them promising candidates for therapeutic applications. However, our ability to design new RNA structures remains limited by an incomplete understanding of their folding principles. While global metrics such as the minimum free energy are widely used, they are at odds with naturally occurring structures and incompatible with established design rules. Here, we introduce local stability compensation (LSC), a principle that RNA folding is governed by the local balance between destabilizing loops and their stabilizing adjacent stems, challenging the focus on global energetic optimization. Analysis …

Characterization Of The Final Enzyme In The Coenzyme M Biosynthesis Pathway In Methanosarcina, Connor John Hines

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

Methanogenesis is known to be one of the oldest forms of energy generation on our planet. Methanogens rely on this method of energy conservation and have evolved to metabolize a variety of substrates, including the waste products of other organisms. This feature has made methanogens beneficial to both wastewater treatment and landfill-to-energy projects. Methane itself is a greenhouse gas twenty times more potent than carbon dioxide, viewed as a particularly influential contributor to global greenhouse emissions. Climate change was known to be on the horizon throughout the 20^th century but has made itself known vocally during this 21^{st …}

Student Comprehension Of Biochemistry In A Flipped Classroom Format, Ed Harris, Evan A. Schroder, Teryn J. Berks

Department of Biochemistry: Faculty Publications

Concept-heavy courses such as Biochemistry in life and physical science curricula are challenging for many college-aged students. It is easy for students to disengage in a lecture and not learn the subject matter while in class. To improve student learning and participation, we employed a flipped format for the first half of the course and compared learning outcomes and attitudes with the traditional lecture in the second half of the course. The experimental course was an upper-level biochemistry course taken primarily by juniors (49%), seniors (40%), and some graduate students (10%) at the college level. Our results indicate that lower-performing …

A Data-Driven Discovery System For Studying Extracellular Microrna Sorting And Rna-Protein Interactions, Sasan Azizian

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

Interactions between microRNAs (miRNAs) and RNA-binding proteins (RBPs) are pivotal in miRNA-mediated sorting, yet the molecular mechanisms underlying these interactions remain largely understudied. Few miRNA-binding proteins have been verified, typically requiring extensive laboratory work. This study introduces DeepMiRBP, a novel hybrid deep learning model designed to predict microRNA-binding proteins. The model integrates Bidirectional Long Short-Term Memory (Bi-LSTM) networks with attention mechanisms, transfer learning, and cosine similarity to offer a robust computational approach for inferring miRNA-protein interactions.

DeepMiRBP is implemented through two distinct architectures. The first architecture employs a Y-shaped model that uses Bi-LSTM networks and transfer learning to extract contextual …

Role Of The Malate Dehydrogenase-Citrate Synthase Metabolon In Metabolic Flux Regulation, Joy John Omini

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

The TCA cycle enzymes- malate dehydrogenase (MDH) and citrate synthase (CS)- interact to transfer oxaloacetate from the active site of MDH to the active site of CS, through an electropositive channel. The MDH-CS metabolon is highly conserved across different domains of life, and it has been associated with many advantages. Some of these advantages include protecting oxaloacetate from competing enzymes like aspartate aminotransferase, allowing the thermodynamically unfavorable MDH forward reaction to occur and increasing the local concentration of oxaloacetate within the active site of CS. These advantages and conservativeness of the MDH-CS metabolon indicate that it is highly important to …

Candida Albicans Farnesol Synthesis And Secretion, Daniel J. Gutzmann

School of Biological Sciences: Dissertations, Theses, and Student Research

Candida albicans is a polymorphic fungus and opportunistic commensal found primarily in the gastrointestinal and skin of healthy individuals. Several barriers prevent C. albicans from causing disease including a healthy immune system and microbiome. When these barriers become comprised, C. albicans can transition to a pathogen and disseminate through the intestinal mucosa leading to life-threatening bloodstream and invasive infections with mortality rates of up to 64%. Morphogenic plasticity is key to this transition and impacts virulence, adaptation to different host environments, and evasion of host immune responses. One regulator of morphogenesis is farnesol. Farnesol is a secondary metabolite of …

Developing A Comprehensive Genome-Scale Metabolic Model For The Arabidopsis Thaliana Root System, Lohani Esterhuizen

Department of Chemical and Biomolecular Engineering: Dissertations, Theses, and Student Research

Arabidopsis thaliana (Arabidopsis) is the most well-established model plant to date. Being the first plant to have its genome mapped, studies on Arabidopsis have provided insurmountable insights into the physiological and biochemical nature of plants. Methods that allow us to computationally study the metabolism of organisms include the use of genome-scale metabolic models (GEMs). Despite its popularity, no GEM currently maps the metabolic activity in the root system of Arabidopsis, which is the first organ to face and respond to stress conditions in the soil. This work aims to develop and implement a comprehensive GEM of the Arabidopsis root system …

Exploring Chilling Stress And Recovery Dynamics In C4 Perennial Grass Of Miscanthus Sinensis, Karolina Sobańska, Monika Mokrzycka, Martyna Przewoźnik, Tomasz Pniewski, Katarzyna Glowacka

Department of Biochemistry: Faculty Publications

The increasing cultivation of perennial C4 grass known as Miscanthus spp. for biomass production holds promise as a sustainable source of renewable energy. Unlike the sterile triploid hybrid of M. × giganteus, which cannot reproduce through seeds, M. sinensis possesses attributes that could potentially address these limitations by effectively establishing itself through seed propagation. This study aimed to investigate how 18 genotypes of M. sinensis respond to chilling stress and subsequent recovery. Various traits were measured, including growth and biomass yield, the rate of leaf elongation, and a variety of chlorophyll fluorescence parameters, as well as chlorophyll …

Structural Analysis Of Dj-1 Glyoxalase Activity By Mix-And-Inject Serial Synchrotron Crystallography, Coleman Dolamore

Department of Biochemistry: Dissertations, Theses, and Student Research

Observing enzyme structures while they are catalyzing a reaction has been a major goal of both enzymology and structural biology for decades. With the advent of time-resolved serial X-ray crystallography using X-ray free electron lasers (XFELs) and synchrotron sources, enzyme reactions can be monitored in real time in crystallo. Here, we use two different approaches to study related enzymes involved in methylglyoxal detoxification. For human DJ-1, we used pink beam mix-and-inject serial crystallography (MISC) at the Advanced Photon Source (BioCARS 14-ID) to probe the controversial mechanism of DJ-1’s action on methylglyoxal by using serial Laue diffraction. The high flux …

Identifying The Relative Abundance Of Cell-Cell Signaling Peptides During Mammalian Hibernation And Anesthetic Administration By Label-Free Peptidomics Analysis, Somayeh Mousavi

Dissertations and Doctoral Documents, University of Nebraska-Lincoln, 2023–

Cell-cell signaling molecules known as neuropeptide and peptide hormones, distributed in both the central nervous system (CNS) and the peripheral nervous system (PNS), regulate a variety of physiological processes including body temperature, feeding, circadian rhythm, reproduction, energy homeostasis, and more. While a number of active neuropeptides structures with physiological functions have been discovered, advances in peptidomic mass spectrometry are enabling researchers to uncover novel, previously unknown, active neuropeptide structures with potential for bioactivities. Using liquid chromatography coupled with tandem mass spectrometry (MS/MS), it is possible to identify a wide range of neuropeptides in single experiments without preselecting specific peptides. Mass …

Structural And Biophysical Insights Into Targeting Of Claudin-4 By A Synthetic Antibody Fragment, Satchal K. Erramilli, Pawel K. Dominik, Chinemerem P. Ogbu, Anthony A. Kossiakoff, Alex J. J.

Department of Biochemistry: Faculty Publications

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes …