Browsing by Subject "Sequencing"
Now showing 1 - 7 of 7
Results Per Page
Sort Options
Item Beyond the cycle: investigating the sequencing, binding affinity, and utility of aptamers selected with CE-SELEX.(2023-02) Brinza, NicholasAptamers are single stranded DNA or RNA sequences that bind with high affinity to a target molecule. The discovery of sequences that can bind to a target of interest starts with a mixture of random sequences which are refined through repeated incubation with the target followed by amplification of binding sequences in a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers produced by multiple SELEX rounds can assist bioanalytical scientific research through detecting molecules of interest in biosensors and have the potential to improve drug delivery to specific cells in humans. However, developing aptamers capable of useful application is difficult due to multiple unique challenges. Quantifying the ability for a pool of aptamers produced by SELEX to bind to a target has no single perfect method. In the field of aptamer research, scientists employ a variety of binding affinity tests which contain specific advantages and disadvantages. Reported values for binding affinity vary between different methods. When a pool does show evidence of most sequences binding, identifying the best sequences in a mixture of more than thousands of unique base pair arrangements provides a separate challenge. Finally, even after finding a sequence with evidence of strong binding to a desired target, there remains uncertainty if the aptamer will bind to the target as well in a real-world application as it does in a carefully controlled lab environment. This study examines aptamers at three distinct stages of development for separate capillary electrophoresis SELEX (CE-SELEX) experiments. For an aptamer already selected against transferrin receptor 1 (TfR1) with high affinity, a combination of flow cytometry and confocal microscopy provided evidence the aptamer did not significantly internalize into cancerous liver cells used to model human drug delivery. A separate aptamer pool, previously developed over 4 rounds of selection against low-density lipoprotein receptor (LDLR), is analyzed with preexisting bioinformatic tools as well as custom Python code on next generation sequencing (NGS) data, revealing a primer dimer sequence from polymerase chain reaction (PCR) which impacted the selection of aptamer length candidates but remained at a lower frequency than one possible aptamer candidate. After performing 4 rounds of CE-SELEX against mouse leptin, testing the binding affinity of rounds 1-3 with CE showed weak evidence of binding in early rounds which was lost in later rounds after correcting for an earlier false positive result. Documenting CE problems and optimization, even for an unsuccessful aptamer selection, may help improve future CE research, while detailing the experimental set up, discovery, and correction of the false positive result is important to help prevent future misleading conclusions.Item Cd4 T Cell Activation And Ox40 Agonist Immunotherapy In Tuberculosis(2023-09) Gress, AbigailAfter Mycobacterium tuberculosis (Mtb) infection millions of effector T cells traffic to the lungs, but relatively few find antigen and become activated. We used an antigen receptor reporter mouse (Nur77-GFP) infected with Mtb to distinguish recently activated CD4 T cells from others in the lungs. Recently activated Nur77-GFPHI cells more often expressed protective markers and were enriched for expanded TCR clonotypes. Nur77-GFPHI cells differentially expressed co-stimulatory genes including Tnfrsf4/OX40 and were functionally more protective than Nur77-GFPLO. Nur77-GFPLO cells more often expressed markers of terminal exhaustion, cytotoxicity, and the trafficking receptor S1pr5, associated with vascular localization. A short course of immunotherapy with an agonist monoclonal antibody targeting OX40+ cells during early infection transiently expanded CD4 T cell numbers and shifted their phenotype towards parenchymal protective cells, which decreased the lung bacterial burden and extended host survival, offering an additive benefit to antibiotics. We have newly identified OX40 as a conserved marker of recently activated CD4 T cells at the infection site and a target for immunotherapy in tuberculosis.Item Characterization of clinical cases associated with porcine circovirus type 3: genetic analysis, frequency of detection and association with histologic lesions(2022-06) Yang, ZhenPorcine circovirus type 3 (PCV3) is a recently discovered porcine circovirus species, firstly identified in swine herds from the United States in the summer of 2015 via next generation sequencing (NGS). The objective of this thesis is to characterize clinical cases associated with PCV3, including investigating the genetic variability of PCV3 in the US shortly after its discovery, describing PCV3 detection frequency in veterinary diagnostic samples and investigating its association with clinical signs and histologic lesions.In Chapter 1, the current literature on PCV3 was reviewed. In Chapter 2, the genetic variability of PCV3 was investigated. We sequenced the whole PCV3 genome from seven samples obtained from swine herds in Iowa, Minnesota and North Carolina. Except for one pair of sequences that were from the same system, the DNA sequences of the genomes had 0.25-1.37% nucleotide differences at the whole genome level, 0.45-0.79% in open reading frame 1 (ORF1) and 0.31-2.4% in the ORF2. This was consistent with the genetic variability of PCV3 previously described in the US and worldwide. In a phylogenetic analysis, our PCV3 genomes along with other US PCV3 sequences clustered in multiple subgroups containing also global PCV3 references, indicating that the US sequences did not appear to have a recent common ancestor and therefore were most likely not the result of a recent introduction from other countries. In Chapter 3, the frequency of PCV3 and its association with clinical signs and lesions was investigated. We found PCR assay results were positive for PCV3 for 27% samples, 35% submissions, 38% sites and 81% states in a database of 730 diagnostic swine cases containing 2,177 samples. PCV3 was detected in multiple specimen types and in pigs of all ages and clinical presentations. Of 58 types of histologic lesions evaluated, we found that PCV3 detection was associated with myocarditis, cardiac vasculitis, and interstitial pneumonia in growing pigs. A high PCV3 detection rate was observed in aborted fetuses. In conclusion, results from this thesis indicate there was less than 2% genetic variation in US PCV3 genomes sequenced in the early years after its discovery, and US PCV3 clustered into different clades of global PCV3 references, suggesting that PCV3 was not a recent introduction into the US swine population. We also demonstrated that PCV3 was widespread in US swine herds. PCV3 detection was associated with myocarditis, cardiac vasculitis, and interstitial pneumonia in growing pigs, and PCV3 could be a cause of fetal death.Item Detection, diagnostics, and characterization of virus-like organisms and conformational disease-like proteins in plants(2016-10) Bratsch, Sara AnnThe projects in this thesis all investigated virus-like organisms in agronomically important plants. Chapter one consists of reports describing viruses identified in new hosts or locations. Tobacco rattle virus (TRV) was identified in symptomatic Phryma leptostachya L, a native perennial, from plants in an uncultivated habitat and suggests that TRV may be endemic to North America. Since TRV is the causal organism of corky ringspot of potato this study raises the possibility that native perennial plants could serve as a potential reservoir to cause disease in potato. Canna yellow mottle virus was isolated for the first time from symptomatic Canna indica in Kenya. Cut flowers are a major agronomic crop of Kenya and growers should plant only virus indexed plants to limit losses from virus infection. Orchid fleck virus was confirmed by microscopy and sequence analysis for the first time in the United States in Phalaenopsis hybrida. Asymptomatic P. hybrida tested by one step reverse transcription polymerase chain reaction (RT-PCR) did not yield the expected product while a two step RT-PCR, creating cDNA first, yielded the expected product. This indicates the one step RT-PCR diagnostic test can yield false negatives for asymptomatic plants. Chapter two describes the production of polyclonal antibodies for the detection of Orchid fleck virus (OFV). OFV is a mite transmitted virus and has been reported word wide. The previous project identified a need for a reliable, inexpensive method to detect OFV in plants use for propagation, breeding, conservatories, or virus indexing projects. Polyclonal antibodies were produced in rabbits against Escherichia coli expressed OFV phosphoprotein and matrix protein. The resulting antiserums were assayed in PTA-ELISA and DAS-ELISA. OFV phosphoprotein antisera in PTA-ELISA readily differentiated between healthy and OFV infected orchid (Phalaenopsis hybrida) tissue. OFV matrix antisera in PTA-ELISA detected bacterially-expressed protein but did not differentiate between healthy or OFV infected tissue. The OFV phosphoprotein antiserum can be used by in PTA-ELISA to reliably detect OFV. Chapter three describes the molecular and biological characterization of a new Nepovirus causing a leaf mottling disease in Petunia hybrida. The sequence of the majority of the genome was determined by next generation sequencing and the sequence of remainder of the genome was obtained using a 5’ RACE amplification and RT-PCR using poly-A tail and virus specific primers. Due to phylogenetic relationship and sufficient genome dissimilarity to characterized viruses I propose the name of Petunia Chlorotic Mottle Virus for a new Nepovirus. The fourth chapter describes characterization of filamentous virus-like particles in members of the Asteraceae plant family including sunflower, chrysanthemum, coneflower, gerbera daisy, and zinnia. The filaments were 7-10 nanometers in diameter and could exceed 3,000 nm in length. The N-terminal sequences of the major proteins associated with purified filaments from several species were nearly identical and shared homology with the kunitz soybean trypsin inhibitor (KTI) family of proteins. CID MS/MS sequencing of the major proteins of purified sunflower filaments also shared homology with a KTI sequence. A Western blot using antiserum prepared against recombinant sunflower KTI protein labeled the observable protein bands from sunflower filaments. Filaments composed of a major protein of KTI have been found across the Asteraceae family but have not been observed in dandelion, thistle, or lettuce.Item Evaluating the information content of human microbiomes(2022-03) Hillmann, BenjaminMicrobes vastly outnumber all other organisms on earth and are integral to many aspects of the ecological fitness of the earth’s soils, oceans, animals, and plants. Unfortunately, most of the microbes in these communities cannot be cultured, so to observe these communities’ biological functions, we must study their DNA. After a researcher sequences a microbial community, they utilize informatics methods to correlate the taxonomic and functional profiles to their traits of interest. However, these methods assume that the underlying taxonomic and functional profiling are accurate. If procedures are developed to identify the profiles of a community more accurately, the increased precision will enable higher power testing of hypotheses and detection of these communities’ causal roles. We propose novel, accurate, and data-efficient methods for taxonomic and functional profiles in shotgun metagenomic datasets.Item Evaluating the Utility and Causative Genetics of Soybean Mutants(2016-03) Campbell, BenjaminSoybean (Glycine max (L.) Merr.) is the second most widely planted crop in the United States by acreage, but yet its genetic resources, mapping methodologies, and breeding improvements lag behind those of other major crop species. In the 20th century, soybean researchers gathered a wealth of natural soybean genetic diversity in the forms of soybean’s wild relative G. soja, soybean landraces, soybean elite lines, and spontaneous mutants. Starting in that same century, researchers began inducing soybean mutations through chemical or irradiation mutagenesis to generate new phenotypes. In the 21st century, these mutagenesis efforts have expanded and have been coupled with new genomics tools to enhance soybean functional genomics. These new mutagenesis efforts and genomics tools will be discussed in chapter one. One of the challenges facing soybean is the difficulties in gene mapping, cloning, and validation. A major focus of this dissertation is the adaptation of new genomics tools and mapping methodologies to soybean in order to facilitate the identification of causative mutants in soybean. Chapter two demonstrates a more classical approach to gene mapping and soybean whole plant transformation to identify the causative loci for three spontaneous chlorophyll deficient mutants. In contrast, chapter three utilizes a combination of new genomics approaches to map and clone a fast neutron induced mutant and validates the result using both a second mutant allele from a historic soybean mutant and transformation of an Arabidopsis mutant. Chapter four builds off of the results of chapter three in leveraging the genomic mapping approach to clone a spontaneous canopy architecture mutant. Several unexpected results and conclusions are reported in the following chapters. Chapter two provides evidence to challenge the widely held idea of gene redundancy in soybean provides an effective buffer against mutations. Additionally, to our knowledge, the research of chapter two reports the first instance of identical mutations affecting two different paralogs resulting in nearly identical phenotypes. Chapter three demonstrates that array comparative genomic hybridization technology and whole genome sequencing of mutant and wild-type bulks can be effectively combined to map and clone a fast neutron mutant from a small F2 population. The chapter also provides an example of the high complexity of mutations that can result from fast neutron irradiation. Chapter four describes the mapping and characterizing a short petiole mutant. The research identifies that the short petiole trait (lps1) is due to a three base-pair in frame insertion in an uncharacterized gene. It was found that the mutation decreases petiole length primarily by decreased cell length and that the short petiole trait could be agronomically beneficial through improved harvest index. The results from chapter four suggest that there is the capacity to improve soybean’s productivity and agronomics through modifications to canopy architecture, as has been demonstrated in other major crop species. The fifth and final chapter discusses potential future directions for soybean genomics research. New population designs with improved efficiency are described. Additionally, suggestions are made for how to utilize current technologies to improve next generation population designs.Item Transcriptional disruptions and functional correlates in a human induced pluripotent stem cell – derived motor neuron model of Spinocerebellar ataxia type 1(2022-08) Sheeler, CarrieIt is hypothesized that degeneration of the motor neurons (MNs) in the brain stem and spinal cord contributes to premature lethality in spinocerebellar ataxia type 1 (SCA1) by affecting the strength of swallowing and respiratory drive. While we can recreate some aspects of MN pathology in mouse models, loss of MNs has only been seen in SCA1 patient populations. This, in addition to other species differences that exist between rodents and humans, accentuates the need for translational human models of disease which can be used to uncover therapeutically relevant cellular and molecular mechanisms underlying dysfunction in vulnerable cell types. To investigate potential mechanisms underlying SCA1 pathology in human MNs, I developed a human induced pluripotent stem cell (iPSC)-derived MN model of SCA1. This is, to our knowledge, the first cell-type specific iPSC-derived model made to study SCA1. Previous work in mouse models has demonstrated both that ATXN1 regulates cellular transcription (Ingram et al 2016, Rousseaux et al 2018, Friedrich et al 2019) and that its entry into the nuclei of cells is important for the longevity of mouse models of SCA1 (Handler et al 2022). Thus, I predicted that the presence of mutant ATXN1 in hiPSC-derived MNs would drive measurable transcriptomic changes in SCA1 samples compared to unaffected controls. Furthermore, transcriptomic disruptions might indicate functional pathways of interest for future studies into the lethality of SCA1. I performed bulk RNA sequencing on MN-enriched cultures to assess underlying transcriptional changes that may be affecting SCA1 human MNs, and subsequently identified disruptions in key cellular processes including extracellular matrix (ECM) regulation, calcium ion binding, and mitotic cell cycle regulation. Proper regulation of ECM is key to many aspects of neuronal development and function, including extension of neurites, proper placement and presentation of receptors and ion channels, cell-cell communication, synapse formation, and intracellular transport. As such, I predicted that broad changes in ECM regulation might result in measurable changes in some of these capabilities in SCA1 MN enriched cultures. Neurite outgrowth was measured in motor neuron progenitors (pMNs) and immature motor neurons (iMNs). I determined that SCA1 samples do not exhibit remarkable disruptions in neurite outgrowth at either timepoint. Assessment of spontaneous calcium activity exhibited a similar degree of physiological maturity between SCA1 and unaffected control MN enriched cultures. Additionally, measurement of miniature excitatory postsynaptic currents (mEPSCs) demonstrated formation of synapses but no functional differences in SCA1 samples compared to controls. However, glutamate evoked calcium activity demonstrated a reduced amplitude of calcium response in SCA1 cultures. This occurred despite any measurable transcriptomic changes in glutamate receptor expression. This indicates potential underlying disruptions in receptor activity and calcium dynamics in SCA1 MN-enriched cultures and provides a potential avenue of interest for future work investigating disruptions in communication in the SCA1 spinal cord.