Molecular Biology and Biochemistry Electronic Theses and Dissertations (UMKC)
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The items in this collection are the theses and dissertations written by students of the Division of Molecular Biology and Biochemistry. Some items may be viewed only by members of the University of Missouri System and/or University of Missouri-Kansas City. Click on one of the browse buttons above for a complete listing of the works.
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Item Virus-host interactions mediated by pea enation mosaic virus 2 biomolecular condensates(2024) Brown, Shelby L.; May, Jared P.; Bame, Karen J. (Karen Joyce); Gaddis, Monica Louise, 1955-The concept of phase separation applied to biological systems has been rapidly building momentum and interest. Phase separation is the conversion of a single-phase solution into two distinct phases: a dilute phase and a concentrated droplet phase. When applied to cells, the term droplet refers to membraneless organelles or condensates that concentrate biomolecules like proteins and RNA. Viruses can interfere with host condensates, like the nucleolus or stress granules, as well as generate condensates to facilitate viral processes. Our research suggests that electrostatic interactions in the intrinsically disordered region of p26, a movement protein encoded by the +sense RNA plant virus Pea enation mosaic virus 2 (PEMV2), drive p26 phase separation to form viral condensates. We demonstrate co-localization of p26 with host proteins, specifically fibrillarin and G3BP1, into condensates during virus infection and subsequently illustrate the importance of G3BP1 phase separation in the plant anti-viral response. These findings outline a key role for p26 phase separation in the coordination of virus-host interactions, viral ribonucleoprotein (vRNP) formation, and systemic virus movement. This work explores how host- and virus-induced phase separation impacts virus-host interactions to promote or restrict a virus infection.Viral condensate research has predominantly centered around the formation of membraneless replication factories by negative sense viruses. However, the function and composition of cytoplasmic condensates formed by positive sense RNA viruses, which utilize membrane-associated replication factories, has been largely uninvestigated. Mass spectrometry revealed that p26 condensates were enriched with ribosomal proteins and fibrillarin, a host rRNA methyltransferase hijacked by PEMV2 to support virus movement. Our data shows that p26 expression represses global translation >40% in plants. In corroboration, polysome profiling exposed significant defects in monosome formation for p26-overexpression and virus-infected samples, whereas infection with a mutant virus lacking p26 partially rescued monosome formation. Our findings suggest that p26 binds rRNA with a high affinity, yet there was no significant alteration in rRNA abundance, processing, or 2’-O-methylation. Therefore, we propose that p26-mediated sequestration of fibrillarin, mRNA, rRNA, and ribosomal proteins into condensates may serve as a switch to repress translation in favor of virus trafficking, a process incompatible with active translation.Item Exploiting the Power of Sleep to Rescue Long-Term Memory Defects in a Drosophila Memory Mutant(2024) Holder, Brandon Lee; Dissel, StephaneSleep is a behavior indispensable for life that is evolutionarily conserved throughout the animal kingdom, signifying its importance. Investigations into the function(s) of sleep reveal a vast array of processes influenced and bolstered by sleep, from the cellular level to whole-body functioning. Memory is another behavior indispensable for life that is also conserved throughout the animal kingdom. Short-term and long-term memory work collectively to generate associations that can inform future decision making and influence the survival of organisms. Sleep and memory have been shown to interact in a reciprocal relationship, either supportively or destructively, indicating that these behaviors are not mutually exclusive. Drosophila melanogaster has emerged as a powerful model organism to study sleep, memory, and their interaction. Sharing significant genetic homology in both physiological function and disease states, in addition to its formidable genetic toolbox, the fruit fly has provided ingresses into probing the nebulous realm of sleep- and memoryrelated processes. Previous studies have shown that pharmacological sleep induction with gaboxadol both prior to and following training can restore courtship long-term memory in the Drosophila memory mutant rutabaga. To identify the sleep-related center responsible for long-term memory restoration, we employed CsChrimson optogenetic activation using sleep-center specific Split-GAL4 lines to recapitulate memory restoration. All three sleep centers examined, the dorsal fan-shaped body, ventral nerve cord – sleep promoting, and ventral fan-shaped body, restored courtship long-term memory only when activated both prior to and following training. We found that pre-training activation restored the ability of memory mutants to form short-term memory in a sleep-independent manner, while posttraining activation restored consolidation into long-term memory in a sleep-dependent manner. We also found significant connectivity between these three sleep-related centers and the established courtship long-term memory circuit, and that the ventral fan-shaped body is indispensable for long-term memory restoration, regardless of sleep center activated. While attempting to extend this interaction into another memory paradigm, we identified several opportunities for optimization within an established appetitive visual memory assay. By addressing sexually dimorphic, color combination, and video-recording elements, we present an optimized, sex-specific paradigm that generates significant learning in fewer replications and numbers of flies.Item Regulation of the development and regeneration of the zebrafish posterior lateral line(2024) Bell, Jon Michael; McGraw, Hillary FayeMechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Damage to hair cells can arise from multiple factors such as high decibel and prolonged sound exposure, genetic lesions, age, illnesses, and ototoxic medications including aminoglycoside antibiotics and some chemotherapy drugs. Aquatic vertebrates such as zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line (LL) system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, zebrafish lateral line hair cells can robustly regenerate following damage throughout the life of the animal. Our work focuses on investigating the cellular and molecular mechanisms that drive development and regeneration of the zebrafish LL to make inferences about mammalian hair cells and drive medical interventions and therapies for conditions affecting hearing and balance. We used the zebrafish lateral line to research two different mutations effects on lateral line development. Foxg1 is a transcription factor in mammals that functions to promote normal development of multiple tissues including the inner ear. Murine studies demonstrate Foxg1 function is necessary for inner ear hair cell development and proper cochlear morphology. Foxg1 interacts with many critical pathways and cellular processes of hair cell development and homeostasis. YEATS domain-containing 2 (YEATS2), functions as part of an epigenetic protein complex acting as a reader of post-translational histone modifications. As a subunit of the histone acetyltransferase (HAT) Ada-two-A-containing (ATAC) protein complex, YEATS2 has been minimally investigated outside of cancer research where YEATS2 transcription affects collective cell migration and proliferation. Work investigating ATAC complex proteins in murine development models show early embryonic lethality, but YEATS2 function has not been determined. The necessity for Foxg1 in the mammalian inner ear, and function of YEATS2 during cancer collective cell migration and proliferation make both ideal targets of investigation. In foxg1aᵃ²⁶⁶ mutant zebrafish larvae, lateral line development is significantly delayed, proliferation is decreased, and fewer average hair cells form per neuromast compared to heterozygous siblings. Following regeneration, foxg1aᵃ²⁶⁶ zebrafish show reduced proliferation and differentiation into hair cells. Coinciding with the reduction in average hair cell numbers we observed a reduction in central localized cells labeled with α-Isl1. In yeats2ⁿˡ²⁴ mutant embryos we see truncated migration of the posterior lateral line primordium, but no changes in proliferation. To better characterize the function and effect YEATS2 mutations have on LL development we generated a CRISPR/Cas9 mutant line, yeats2ᴬᵀᴳ, that recapitulates the yeats2ⁿˡ²⁴ phenotype. These reduced cell numbers in the LL suggest that Foxg1a function is critical for the proper development and regeneration of support cells and hair cells. We also demonstrate a novel developmental role for YEATS2 in collective cell migration of the zebrafish posterior lateral line primordium. Our work provides novel data for Foxg1a and YEATS2 in zebrafish and shows both genes are potential therapeutic targets for development and regeneration of human inner ear hair cells.Item The Clinical Associations and Physiological Mechanisms Linking Bone and Cardiac Health(2024) Vallejo, Julian Alfredo; Wacker, Michael; Johnson, Mark L. (Mark Louis)Osteoporosis and cardiovascular disease represent two significant clinical burdens worldwide, which have been found to be clinically intertwined. The skeleton is highly sensitive to mechanical stimuli with osteocytes/osteoblasts sensing mechanical strain and activating bone remodeling. Bone is multifaceted exhibiting endocrine/paracrine functions and connections to the central nervous system through which it modulates a number of tissues including itself. During osteoporosis, bone cellular network integrity and the anabolic response to loading is compromised. Thus, the clinical associations of cardiovascular and skeletal health may be explained by crosstalk via bone-humoral and/or bone-neural mechanisms. To investigate the links among heart and bone health clinically, the impact of osteoporosis on cardiovascular disease patient outcomes was examined utilizing data from Cerner Health Facts. Next, cellular and animal studies were undertaken to determine the effects of bone cell-secreted molecules and in vivo bone mechanical loading on cardiac physiology. The Health Facts analysis revealed a significant association of history of osteoporosis with prolonged length of hospital stay, elevated NT-proBNP levels and greater mortality in patients admitted for cardiovascular disease. Men with a history of osteoporosis displayed worse overall outcomes for cardiovascular disease compared to women including remaining hospitalized for 1.02 days longer and having 2.13-fold higher mortality. In vitro studies showed that administration of conditioned media from bone cells exposed to fluid flow shear stress enhanced cardiomyocyte cell viability during CoCl2 treatment (p<0.05) and hypoxia/reoxygenation (p<0.05). In vivo, tibia mechanical loading in adult mice acutely and transiently lowered heart rate (p<0.01) and enhanced heart rate variability (p<0.01), which was mediated by neuronal afferents in the hindlimb and downregulated sympathetic nervous system tone. This cardiac response to loading was largely diminished by middle age. Daily tibia loading over three weeks in adult mice resulted in significantly lower resting heart rate (p<0.05) and higher heart rate variability (p<0.05) compared to non-loaded mice. These findings suggest that endocrine/neural pathways associated with bone mechanical loading may regulate cardiac physiology and link bone and cardiovascular health. Investigation of bone-heart crosstalk networks could aid in identifying novel therapeutic targets to improve standard of treatment for musculoskeletal and cardiovascular diseases.Item Methamphetamine, interleukin-6 and perineuronal nets in synaptic plasticity(2024) Essel, Leslie Brian; Wyckoff, Gerald; Bame, Karen J. (Karen Joyce)The proper functioning of the central nervous system (CNS) is dependent on the strength and integrity of neurons and their synaptic connections. Neuronal synapses allow for the flow of information that enhances cellular communication. The plasticity of these synapses essentially drives activity-dependent learning and memory. This plasticity may be enhanced and/or injured by factors including xenobiotics and components of the neural extracellular matrix (ECM). This study evaluated the effect of modulating methamphetamine (METH) action on learning and memory, the effect of METH-induced metabolites on neuronal structure and the biochemical and structural analysis of the lecticans and hyaluronan (HA) in perineuronal nets (PNNs), which are essential neural ECM components. METH is a highly addictive CNS stimulant which acts by enhancing catecholamine neurotransmission, causing euphoric, addictive, withdrawal, and neurotoxic effects. METH is clinically used in the treatment of attention-deficit/hyperactivity disorder (ADHD), narcolepsy and treatment-resistant obesity. This is however limited by the neurotoxicity effect of METH which results from oxidative stress and lipid peroxidation mechanisms. Interleukin-6 (IL6) is a pleiotropic cytokine that is a mediator of immune and inflammatory processes. IL6 is known to mediate METH-induced neurotoxicity. We used mice models to test a combination of a clinical dose of METH and IL6 deficiency on learning and memory. We showed METH-improved short-term memory with IL6 deficiency, suggesting a beneficial therapeutic effect of this combination. Next, we employed untargeted metabolomics using high-resolution liquid chromatography-mass spectrometry (LCMS) to identify differentially expressed METH metabolites. Metabolites associated with the neuronal cell membrane were identified for continued METH use, whereas a metabolite involved in cellular energetics was identified in METH withdrawal. These metabolites may hold potential therapeutic and diagnostic purposes. Finally, we solved the crystal structures of the G1 domains of two lecticans brevican and versican and used biochemical methods to characterize the valency of complexes and the kinetics of the binding affinities of lecticans and HA. The crystal structure, multimerization and relative binding affinities of the lecticans for HA suggest a basis for PNN formation. Taken together, we have shown how the synaptic plasticity may be modified and identified compounds with diagnostic and therapeutic potential.