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 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 Identification and Characterization of Two Novel Sleep Promoting Neurons Located in the Ventral Nerve Cord of Drosophila Melanogaster(2023) Jones, Joseph David; Dissel, StephaneSleep is a phenomenon that is ubiquitous in the animal kingdom and can be found in nearly every evolutionary niche that has come about. Humans cannot live without it, and it is required for optimal physical and mental health. Despite its importance, we have yet to discover the reason or reasons for which animals require sleep, making the study of sleep a rich vein of inquiry. Sleep is regulated by the circadian clock and the sleep homeostat, which primarily reside in the brain and communicate via complex neurocircuitry. Though difficult to study in mammalian systems, Drosophila melanogaster, the fruit fly, has become a tractable model for this kind of study. With its high degree of homology to mammals and unparalleled genetic toolbox, it is an invaluable asset in studying the neurobiological and molecular aspects of many behaviors, including sleep. In the Drosophila sleep field, the 23E10-GAL4 driver is the most widely used tool to modulate sleep. 23E10-GAL4 expresses in the dorsal Fan-Shaped Body (dFB), a brain region whose associated neurons are believed to be a major sleep regulating center. However, because the dFB contains a group of 31 neurons and the 23E10-GAL4 driver expresses in many other neurons of the fly's nervous system, it is difficult to link individual neurons to observed sleep phenotypes when manipulating 23E10-GAL4 neurons. To identify which neurons in the 23E10-GAL4 driver modulate sleep, we undertook a Split-GAL4 approach to genetically dissect out the individual neurons expressed in the 23E10-GAL4 driver. We screened 22 Split-GAL4 lines based on 23E10-GAL4 and identified a pair of neurons in the ventral nerve cord, the equivalent of the fly's spinal cord, that modulate sleep when manipulated. Through immunohistochemical experiments and RNAi knockdown, we found that these cells use acetylcholine as their primary neurotransmitter. Furthermore, we found that the number of dFB neurons contained in a Split-GAL4 line did not indicate whether it promoted sleep. Therefore, this dissertation has both identified a novel pair of sleep regulating neurons in the fly and has also raised new questions concerning past and future studies done using the 23E10-GAL4 driver in Drosophila sleep research.Item Functional Analysis of TET-Family 5-Methylcytosine Dioxygenases in Non-CpG DNA Demethylation(2023) Dey, Aninda Sundar; Mukherji, MridulOne of the most widely studied epigenetic marks in DNA is 5-methylcytosine (5mC). Research has indicated that in the mammalian genome around 5-6% of all cytosines are methylated (5mC), with a strong preference for the CpG dinucleotide context. In addition, several recent studies have uncovered extensive C5 cytosine methylation (5mC) at non-CpG (5mCpH, where H = A/C/T) sites. 5mC plays an important function in tissue-specific gene expression, human development, X-chromosome inactivation, genomic imprinting, and as a biomarker of ageing. The dynamic regulation of 5mC epigenetics is also essential for various stages of pluripotency, differentiation, and development. In humans, demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation of dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair (TDG/BER). To study the functional analysis of TET-family 5-methylcytosine dioxygenases in non-CpG DNA demethylation, efficient purification of enzymatically active untagged human TET2 and His-Tagged mouse TET1 using cation exchange chromatography and affinity chromatography was established. In addition, using the positive and negative mode of the mass spectrometer we have developed an improved and highly sensitive positive/negative ion-switching-based liquid chromatography-tandem mass spectrometry (LC–MS/MS) method that can separate and quantify modified cytosine bases produced by TET-family 5-methylcytosine dioxygenases. The substrate specificity of the TET enzymes has been explored in the CpG context, but little is known about the enzyme responsible for the active demethylation of 5mCpH sites. Initial studies have reported that the human TET2 can rapidly oxidize 5mCpG sites, but oxidation of both the 5mCpA and 5mCpC sites was negligible. Using the established high-sensitive method, we demonstrated that the human TET2 (including mouse TET1) can oxidize 5mCpH sites in double-stranded DNA in-vitro. In addition, we have demonstrated the substrate specificity of the human TET2/mouse TET1 dioxygenase and the role of DNA flanking sequences in the demethylation of methylated CpG and CpA sites in DNA. Our results showed that human TET2/mouse TET1 does have DNA sequence preference like other DNA modifying enzymes. Our findings also indicated that TET-mediated mCpA oxidation has a higher flanking sequence preference compared to mCpG oxidation. These results may help elucidate the novel role of TET2-mediated mCpG and mCpA demethylation in the genome. And finally, in the last set of experiments, the activity of ten different TET2 clinical mutations was studied. Over the last decade, hundreds of frame-shift, nonsense, and missense mutations in the TET2 gene have been identified in patients. TET2 is one of the most frequently mutated genes in myelodysplastic syndromes (MDS), MDS-myeloproliferative neoplasms (MDS-MPNs) and acute myeloid leukemia derived from MDS and MDS-MPN (sAML). However, our understanding of biochemical abnormalities associated with TET2 mutations is very limited because all the study has been focused on the CpG context. In this project, the activity of ten different TET2 clinical mutations was studied in both the CpG and CpA contexts for a comparative analysis to better understand the biochemical abnormalities associated with TET2 mutation.