Biological Sciences electronic theses and disserations (MU)
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The items in this collection are the theses and dissertations written by students of the Division of Biological Sciences. Some items may be viewed only by members of the University of Missouri System and/or University of Missouri-Columbia. Click on one of the browse buttons above for a complete listing of the works.
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Item Characterization of Argonaute-associated proteins in meiotic silencing by unpaired DNA(University of Missouri--Columbia, 2025) Sy, Victor Thanh; Shiu, Patrick K.T.[EMBARGOED UNTIL 08/01/2026] Neurospora crassa, a filamentous fungus, employs a post-transcriptional gene silencing (PTGS) mechanism known as meiotic silencing by unpaired DNA (MSUD) to protect its genome during meiosis. This process, which relies on RNA interference (RNAi)-related proteins, targets and silences genes lacking a homologous pairing partner during prophase I. While some of these proteins function within the nucleus or shuttle between the nucleus and cytoplasm, many are localized in the perinuclear region, where MSUD activity is highly concentrated. MSUD targets genetic parasites like transposable elements and viruses. When such a parasite generates an unpaired DNA segment during homologous pairing, an RNA signal is transcribed and exported from the nucleus. In the perinuclear region, this aberrant RNA is processed by an RNAi protein complex into short-interfering RNAs (siRNAs), which then guide the suppressor of meiotic silencing-2 (SMS-2) Argonaute to degrade homologous mRNAs. In Neurospora, the suppressor of ascus dominance-9 (SAD-9) protein is homologous to the DEAD-box helicases Belle and Vasa in Drosophila. DEADbox helicases are known to recruit Argonaute proteins in certain organisms. Deletion mutants of sad-9 are highly impaired in meiotic silencing. Localization experiment shows that SAD-9 functions to recruit the SMS-2 Argonaute to the perinuclear region during MSUD. While the absence of SAD-9 does not appear to impact vegetative growth and development, it severely impairs sexual reproduction. The heat shock protein 70 (HSP70) family is a highly conserved group of chaperone proteins found in virtually all organisms. Within the cell, HSP70 performs diverse functions, including protein folding, responding to stress, and facilitating protein translocation to organelles. HSP70 has also been implicated in RNAi processes. In Drosophila, an HSP70 protein has been shown to interact with Argonaute, altering its conformation to enable the loading of siRNAs. Several HSP-70 proteins exist in Neurospora, and HSP70-1 mediates meiotic silencing. HPS70-1 interacts with the SMS-2 Argonaute and could act as its molecular chaperone. Crosses lacking HSP70-1 exhibit severe fertility defects, although growth and asexual sporulation are only mildly affected. In fission yeast, two Argonaute-binding (ARB) proteins mediate the loading of siRNAs onto Argonaute, which subsequently uses them to search for homologous mRNAs. The homologs in Neurospora, ARB1 and ARB2, also play a role in silencing. Deletion mutants of arb1 and arb2 act as semi-dominant suppressors of MSUD. While having a diffused localization in the cytoplasm, ARB1 and ARB2 preferentially accumulate outside of the nuclear envelope. The two ARB proteins interact with each other and with the SMS-2 Argonaute, supporting the notion that they help deliver siRNAs to the latter. In spite of their importance in sexual development, ARB1 and ARB2 do not appear to contribute to vegetative growth.Item Evading early transgene silencing in plants : design and molecular factors(University of Missouri--Columbia, 2025) Edwards, Seth Alexander; Slotkin, R. Keith[EMBARGOED UNTIL 08/01/2026] Transgenes are fragments of DNA that are foreign to the genome that have been artificially integrated. Inserting foreign DNA into plants is the basis behind the success of modern molecular crop improvement. Though transgenesis often improves plant fitness through beneficial trait delivery, (such as herbicide and disease resistance) and delivering the reagents necessary for gene editing, plant cells regularly silence transgenes which hampers the speed of crop improvement progress. In this work, I addressed both design and molecular factors of transgenes that are important in early transgene silencing. Transgene silencing has been historically investigated well after the process was triggered. Therefore, I investigated methods of early activation of transgene silencing, with the goal of avoiding early activation of transgene silencing. In Chapter 2, I used silencing-prone transgenes to understand the importance of the quality of integration. Transgenes are typically integrated into the genome in a multi-copy, often fragmented manner. I found that high quality integrations can outweigh poorly designed transgenes, even over several generations. This approach of identification is time-consuming and laborious, so in Chapter 3 and 4 I investigated the pathway of silencing to prevent transgenes from transitioning from early silencing to late-stage silencing. I identified both highly important and non-important factors by way of gene editing technologies. Together these findings demonstrate approaches to mitigate the likelihood of transgene silencing. Transgene silencing requires transcription, but to this point, it has been unclear what aspects of transcription are relevant for silencing. In Chapter 6, I found that two portions of a transgene promoter can independently trigger transgene silencing. In transgenes with the same predicted transcript, through a combination of machine learning and novel long-read RNA sequencing in Chapter 7, I found that the promoter can change the types of transcripts that are produced. And in Chapter 5, I determined that other than the standard mRNA polymerase RNA Polymerase II, RNA Polymerase III transcripts can also be recognized in the initiation of silencing. These transcriptional findings provide the potential roles of the promoter for importance in the initiation of silencing. Overall, this research advances the understanding of transgene silencing. From genetic considerations to early transcriptional identifiers, this work contributes to new hypotheses of the molecular progression of transgene silencing as well as a deeper understanding of evading silencing to promote quicker plant improvement processes. My thesis tells the future generation of plant genome engineers to focus on the quality of the integration event and the promoter of the transgene transcript.Item Identification of small molecule inhibitors targeting Cache Valley virus replication and establishment of reverse genetics for swine mammalian orthoreovirus(University of Missouri--Columbia, 2025) Khalid, Muhammad Afnan; Ma, Wenjun[EMBARGOED UNTIL 08/01/2026] Bunyaviruses are important zoonotic pathogens, and many of them are highly pathogenic and cause severe diseases with high mortality in infected humans. Cache Valley virus (CVV), an emerging bunyavirus, is an arthropod-borne, tri-segmented, enveloped RNA virus of veterinary and public health significance. CVV affects many mammalian species, most commonly causing central nervous system defects and abortions in sheep and goats, and a few human cases with neurological illness have been reported from different parts of the USA. Currently, no commercial vaccines and antivirals are available for human and animal use. To develop an antiviral with broad-spectrum activity against different bunyaviruses, our group has performed high-throughput screening of more than twenty-six thousand small molecules predicted to penetrate the blood-brain barrier and identified 239 small molecules with antiviral activity against the Rift Valley Fever Virus (RVFV) MP-12 vaccine strain expressing Ranielle Luciferase. Thirty-five small molecules were selected for further investigation. In this study, we screened these thirty-five small molecules against the CVV human isolate using the gold standard plaque reduction assay and identified 11 compounds that showed effective inhibition of CVV replication. To explore the mechanism of identified compounds that inhibit CVV replication, we established a minigenome reporter assay and tested them. Results showed that three compounds significantly reduced luciferase expression compared to positive (Ribavirin) and mock controls, indicating that these compounds potentially target viral polymerase RdRp of CVV, thereby inhibiting viral replication. One compound has been demonstrated to effectively inhibit two other bunyaviruses, RVFV and Andes virus, by our group. Our results indicate that this compound could be a good antiviral candidate with broad-spectrum activity against different bunyaviruses. We will further test its efficacy against other bunyaviruses and optimize it in order to develop safe and effective broad-spectrum antivirals against emerging bunyaviruses that threaten human and animal health. Mammalian Orthoreovirus (MRV), a member of the Reoviridae family, infects various mammalian species, including humans, and is important for veterinary and public health. Due to its segmented genome, it can reassort among different strains when they infect the same host, resulting in novel and virulent strains. Our group has isolated reassortant MRVs from US bats and swine, which can infect human, swine, and canine cells without prior adaptation. Our pig studies reveal that both swine MRV1/Porcine/USA/2018 and bat MRV1/40/Bat/USA/2018 viruses are able to infect and replicate in pigs, but they show different tissue tropism. The swine isolate was dominantly detected in the gastrointestinal tract of infected pigs, while the bat isolate was restricted to the respiratory tract of infected animals. We hypothesize that the different tissue tropism of both swine and bat MRVs is due to the S1 segment-encoded surface sigma-1 protein, which is the major attachment protein and binds to the host receptor. To test our hypothesis, I attempted to establish a reverse genetics system for the novel porcine MRV1/Porcine/USA/2018 isolate and then rescue a recombinant swine MRV with the S1 segment from the bat MRV1/40/Bat/USA/2018 isolate. I constructed ten plasmids containing ten gene segments from the porcine MRV under the T7 promoter with conserved sequences at both the 5' and 3' UTRs for each gene and confirmed them by sequencing. The swine MRV was not successfully rescued despite multiple attempts and modifications. It is expected that the failure to rescue the porcine MRV can be resolved in the future. Additionally, to understand the seroprevalence of MRVs in pigs, we have established an in-house ELISA assay to detect MRV-specific antibodies. In this ELISA, we coated the whole virus (MRV1/Porcine/USA/2018) as an antigen and screened pig serum samples collected from different parts of the USA. I have screened 405 samples and found that 240 were positive for MRV, with a 60 percent positive rate. In the future, these MRV-positive serum samples will be further characterized by the HI assay. This result indicates a high seroprevalence in the US swine population, suggesting that MRV could pose threats to animals when co-infected with other pathogens.Item Molecular surveillance of mammalian orthoreovirus in bat and swine populations(University of Missouri--Columbia, 2025) Gupta, Dipali; Ma, Wenjun[EMBARGOED UNTIL 08/01/2026] Mammalian orthoreovirus (MRV) affects a wide range of mammalian species, including humans, bats, and domestic and wild animals. Genetic reassortment among different strains of MRVs increases potential for the emergence of variants that can further expand their host range beyond what is presently known. Evidence of interspecies transmission raises a zoonotic concern, as MRV has previously been reported to cause respiratory, gastrointestinal, and neurological disturbances in infected hosts including humans. Although bats and pigs have been recognized as reservoir and mixing hosts for many agents of viral zoonoses, not much information has been documented about the infection of MRVs in bat and pig populations to date. Herein, we screened 565 bats, and 362 lung and intestinal tissue homogenates of swine samples collected in the USA in 2022 and 2024, respectively, to determine the prevalence of MRVs in bats and swine using a real-time reverse-transcription polymerase chain reaction assay. Results showed that all bats were negative for MRVs, whereas two swine tissue homogenates; one from lung and one from intestine samples were positive for MRV. These results suggest that MRV's has shown tropism for both respiratory and gastrointestinal organs. However, attempts to isolate the virus in MDCK cells failed despite high viral genome copy numbers. Although our results suggest low MRV prevalence in both bat and pig populations, it is difficult to draw conclusions about MRV prevalence in both species due to our study limitations. Further studies are needed to understand MRV prevalence in bats and swine in the USA.Item Comparison of sedated respiratory-gated computed tomography (CT) to anesthetized inspiratory : expiratory breath-hold CT in dogs(University of Missouri--Columbia, 2025) Navarro-LaBoy, Iliana Maria; Vientós-Plotts, Aida I.[EMBARGOED UNTIL 08/01/2026] Dogs experiencing respiratory compromise typically have multiple respiratory disorders that can be underestimated or overlooked by thoracic radiography. The anesthetized, ventilator-assisted inspiratory: expiratory breath-hold (I:E BH) CT technique provides comprehensive evaluation of respiratory diseases of a diagnostic quality that is superior to images obtained without breath-holds. Sedated respiratory-gated (RG) CT has the potential to provide high-quality diagnostic images, avoid the potential risk of general anesthesia, and minimize motion artifact seen on sedated CT scans without respiratory gating. It was hypothesized that sedated RG-CT would generate images of comparable quality to anesthetized I:E-BH CT. Sedated RG-CT followed by I:E-BH CT images were prospectively acquired in fifty client-owned dogs with respiratory clinical signs. Thoracic CT scans from both techniques were assessed to 1) compare the presence or absence of the four major CT lung patterns and their sub-patterns, 2) provide a severity score for pulmonary parenchymal disease and a quantification score of the normal lung parenchyma, 3) identify bronchomalacia (defined as segmental and subsegmental airway collapse resulting in airflow limitation) and compare scoring between the scans. This study showed no significant difference between the two groups of images for the presence of the four major CT lung patterns and sub-patterns (p >0.05 for all), severity scores of parenchymal disease or normal lung, or the diagnosis of bronchomalacia.