Cell Biology and Biophysics Electronic Theses and Dissertations (UMKC)
Permanent URI for this collection
The items in this collection are the theses and dissertations written by students of the Division of Cell Biology and Biophysics. 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.
Browse
Recent Submissions
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 Pregnancy outcomes in females with hidradenitis suppurativa(2024) Walsh, David Paige, Jr.; Wyckoff, Gerald J.; Wang, Shizhen, (Jeff); Lee, Yugyung, 1960-; Monaghan-Nichols, PaulaA pregnancy datamart was constructed from Cerner Health Facts EMR data using pypreg, a python package developed by the author based on a validated pregnancy identification algorithm by Keran Moll. The demographics of the pregnancy datamart were consistent with the demographics of Health Facts overall. Various measures were compared against national data for the United States including rates of cesarean section, 42-day maternal mortality, severe maternal morbidity, preeclampsia, fetal growth restriction, gestational diabetes, and gestational hypertension. These measures were compared using Chi-square and Breslow-Day. No evidence suggested the rates observed in the datamart were substantially different from national figures. Thus, providing a degree of validity to the data. During construction of the datamart, several data quality problems were identified in Health Facts and were addressed. The quality issues included patient identifiers that are defined to be unique being linked to other unique patient identifiers, patient encounters with more than one date of admission, patient encounters being listed at more than one facility, and patient age not changing appropriately over time. A small percentage of the total encounters and patients were recommended to be removed from the general database to avoid unforeseen problems due to their inclusion in research. With the pregnancy datamart established, pregnancy outcomes were studied between the populations with and without hidradenitis suppurativa. Several covariates were selected based on association with inflammatory imbalance or association with condition for use in a multinomial logistic regression. This model type was selected to model all outcomes simultaneously to avoid the problem of multiple testing. Additionally, two models were considered. The first model treated each pregnancy as an independent event, while the second model only looked at the first record pregnancy for each patient. The odds ratios for negative pregnancy outcomes were elevated in both model types. When considering model predictions on counterfactual data and the estimated marginal means, several negative outcomes showed significant increases in risk.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 Discovery of IKBKE and CD24 siRNAs for the treatment of triple-negative breast cancer(2023) Liu, Yanli, 1988-; Cheng, Kun (Professor); Mohan, Ryan D.Triple-negative breast cancer (TNBC) represents approximately 10-20% of all newly diagnosed breast cancers and is classified as a subtype with the absence of ER, PR, and HER2 expression. TNBC displays high aggressiveness, a tendency to metastasize, a poor prognosis, and a low survival rate. Unlike other breast cancer subtypes, TNBC has limited treatment options due to the lack of targeted therapies. Therefore, there is a significant need to develop new therapies for TNBC. IKBKE, also known as IKKε or IKKi, is a member of the IKK (IκB kinase) family and exhibits high expression levels in various cancers. IKBKE functions as an oncogene in breast cancer and is overexpressed in approximately 30% of breast carcinomas. IKBKE is shown to be aberrantly amplified in TNBC and associated with proliferation, migration, and survival in TNBC cells. Breast cancer also expresses high levels of CD24, which is a heavily glycosylated glycosylphosphatidylinositol (GPI)-anchored surface protein and plays an important role in tumor growth, invasion, and metastasis. Moreover, overexpression of CD24 in tumors is associated with resistance to therapies. Small interfering RNA (siRNA) can specifically knockdown the expression of target genes. It represents a promising tool for cancer therapy as it can silence aberrant genes that are essential for the progression of cancer cells. However, successful siRNA cancer therapy relies on the development of safe and effective RNA delivery systems because naked siRNA is unstable and has limited cellular uptake. Numerous delivery carriers have been investigated to increase the stability and improve the cellular uptake of siRNAs. This dissertation focuses on two primary research objectives. The first objective centered on discovering siRNAs targeting IKBKE and CD24 for the treatment of TNBC. The second research objective aims to utilize a recently discovered anti-PD-L1 human domain antibody as an immune checkpoint inhibitor for cancer immunotherapy. In Chapter 1, we briefly introduced the background of the research, the statement of the problems, and research objectives. In Chapter 2, we reviewed potential treatment options for TNBC and provided a concise introduction to cancer immunotherapy, specifically focusing on PD-1/PD-L1 immune checkpoint inhibitors. In Chapter 3, we utilized a cholesterol peptide-based delivery system to condense IKBKE siRNA to form nanocomplexes for the treatment of TNBC. The stability, cellular uptake, and penetration capability of the cholesterol peptide/siRNA (CCP/siRNA) nanocomplex was significantly increased. Importantly, the CCP/siRNA nanocomplex significantly inhibited tumor growth in an orthotopic TNBC mouse model. These data suggest that IKBKE siRNA could be a promising therapeutic strategy for TNBC. In Chapter 4, we designed four CD24 siRNAs for the treatment of TNBC by targeting different regions of human CD24 mRNA. The results showed the pre-designed siRNA reduced the CD24 expression at both mRNA and protein levels in TNBC cells. CD24 siRNA efficiently inhibited the proliferation, migration, and invasion of TNBC cells. Moreover, silencing of CD24 induced tumor cell apoptosis and cell cycle arrest. Further, we evaluated the association of CD24 expression with doxorubicin resistance. We found that both CD24 mRNA and protein levels were upregulated in doxorubicin-treated MDA-MB-231 cells and CD24 siRNA sensitized MDA-MB-231 cells to doxorubicin which was reflected by a decreased IC50 value. Overall, targeting CD24 with siRNAs may be a promising therapeutic target for TNBC or other cancers with overexpressed CD24. In Chapter 5, we presented the work on the discovery of anti-PD-L1 human domain antibodies (dAbs) using the phage display technique for cancer immunotherapy. In this study, seven anti-PD-L1 domain antibodies were discovered. Among them, the CLV3 dAb showed the highest binding affinity to human and mouse PD-L1 proteins with KD values of 137.5 nM and 266.8 nM, respectively. The CLV3 dAb also exhibited potent binding affinity to PD-L1 overexpressing DU145 cells. CLV3 inhibited PBMC apoptosis in a co-culture system and showed better tumor penetration compared to antibody. The CLV3 dAb significantly inhibited tumor growth and increased the survival of CT26 tumor-bearing mice. The CLV3 dAb is therefore a promising PD-L1 inhibitor that can be used for cancer immunotherapy.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.