Theses and Dissertations at Montana State University (MSU)
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Item Mechanisms of RNA-targeting CRISPR systems and their applications for RNA editing(Montana State University - Bozeman, College of Agriculture, 2022) Nichols, Joseph Edward; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.Genetic modification studies are central to understanding gene function and are the bedrock of molecular biology. The development of novel, CRISPR-based technologies for genome engineering in the last decade has revolutionized nearly every field of biology by simplifying the process of editing DNA genomes. In contrast, there are currently no comparable tools for editing RNA. Our goal is to develop facile CRISPR-based RNA editing methods that will transform our understanding of RNA metabolism, viruses and the repair pathways that govern RNA biology. I didn't initially come to MSU intending to study SARS-CoV-2, but the growing importance of this topic, combined with unanticipated intersections with my interest in CRISPRs, ultimately lead to several projects in this area. While participating in genomic surveillance, we identified a naturally occurring deletion within ORF7a, a viral accessory protein. We determined that this deletion results in the loss of function of ORF7a, limiting the virus' ability to evade host interferon responses, and reduced viral fitness. My focus then moved to Type-III CRISPR systems. While CRISPR has become synonymous with genome engineering, these systems naturally evolved in prokaryotes as an adaptive immune system against bacteriophages. Type-III CRISPR systems are unique, as they are one of two groups of CRISPR systems to target RNA rather than DNA. To develop type III systems for editing RNA, we designed and purified a series of type III complexes and showed that these systems function as programable nucleases. We then adapted a method for targeted RNA repair in vitro following cleavage and demonstrate that this approach results in edited RNA. In addition to cleaving the RNA target, target recognition by type III CRISPR systems also activates a polymerase domain that generates signaling molecules that activate ancillary CRISPR nucleases. Working with several members of the team, I set out to determine substrate preferences for each ancillary nuclease in Thermus thermophilus. We expected that activating these immune components would result in dramatic changes in bacterial growth kinetics. However, my experiments failed to identify a reliable phenotype, suggesting that this expression system is not a faithful representation of Type-III immunity.Item Intersection of SARS-CoV-2 and CRISPR-CAS defense systems(Montana State University - Bozeman, College of Agriculture, 2021) Wiegand, Tanner Roy; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.Viral predators exploit cellular resources in all domains of life. To defend against these genetic invaders, bacteria and archaea have evolved adaptive immune systems comprised of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins. In this dissertation, I investigate the biological mechanisms and biotechnological applications of CRISPR-Cas systems. The sequences that interspace the eponymous repeats of CRISPR loci are derived from mobile genetic elements, including bacteriophages (i.e., viruses that infect bacteria). When the locus is transcribed into CRISPR-RNA, these spacer sequences guide nucleases to RNA or DNA molecules with complementary sequences, resulting in degradation of the target nucleic acid. While recent work has illuminated many details of CRISPR-RNA-guided surveillance and target interference, the process of new sequence adaptation remains more mysterious. Initially, the goal of this research was to understand how new spacer sequences are acquired and integrated at CRISPR loci. High throughput sequencing of spacers acquired in in vivo adaptation assays revealed that some spacer sequences are reproducibly acquired in the I-F CRISPR system of Pseudomonas aeruginosa, and that the I-F CRISPR-guided surveillance complex enhances the efficiency of new spacer acquisition. We then used bioinformatic and in vitro acquisition assays to show that adaptation in many systems is dependent on the presence and phasing of sequence motifs in the transcriptional leaders of CRISPR loci. Collectively, these results expand our understanding of how CRISPR-Cas systems adapt to new threats. Following the emergence of SARS-CoV-2, and the ensuing international COVID-19 pandemic, my research goals pivoted to developing methods to track the spread of this coronavirus and to understanding how it was evolving. Long read genomic sequencing was used to determine the likely evolutionary origin of SARS-CoV-2 samples isolated from wastewater and human patients. This work led to the identification of isolates with large genomic deletions and shows that while these mutations cause a replication defect in the virus, similar mutations have appeared multiple times, independently in the evolution of SARS-CoV-2. Finally, we show that type III CRISPR-Cas systems can be repurposed for molecular detection of SARS-CoV-2 and investigate how these new diagnostic platforms can be improved.Item The synthesis of N-acetyllactosamine functionalized dendrimers, and the functionalization of silica surfaces using tunable dendrons and beta-cyclodextrins(Montana State University - Bozeman, College of Letters & Science, 2017) Ennist, Jessica Helen; Chairperson, Graduate Committee: Mary J. Cloninger; Mary J. Cloninger was a co-author of the article, 'The synthesis of N-acetyllactosamine functionalized dendrimers and their role in galectin-3 mediated cancer cellular aggregation studies' which is contained within this thesis.; Eric A. Gobrogge, Kristian H. Schlick, Robert A. Walker and Mary J. Cloninger were co-authors of the article, 'Cyclodextrin-functionalized chromatographic materials tailored for reversible adsorbtion' in the journal 'ACS applied materials and interfaces' which is contained within this thesis.Galectin-3 is beta-galactoside binding protein which is found in many healthy cells. In cancer, the galectin-3/tumor-associated Thomsen-Friedenreich antigen (TF antigen) interaction has been implicated in heterotypic and homotypic cellular adhesion and apoptotic signaling pathways. However, a stronger mechanistic understanding of the role of galectin-3 in these processes is needed. N-acetyllactosamine (LacNAc) is a non-native ligand for galectin-3 which binds with comparable affinity to the TF antigen and therefore an important ligand to study galectin-3 mediated processes. To study galectin-3 mediated homotypic cellular aggregation, four generations of polyamidoamine (PAMAM) dendrimers were functionalized with N-acetyllactosamine using a four-step chemoenzymatic route. The enzymatic step controlled the regiochemistry of the galactose addition to N-acetylglucosamine functionalized dendrimers using a recombinant beta-1,4-Galactosyltransferase-/UDP-4'-Gal Epimerase Fusion Protein (lgtB-galE). Homotypic cellular aggregation, which is promoted by the presence of galectin-3 as it binds to glycosides at the cell surface, was studied using HT-1080 fibrosarcoma, A549 lung, and DU-145 prostate cancer cell lines. In the presence of small LacNAc functionalized PAMAM dendrimers, galectin-3 induced cancer cellular aggregation was inhibited. However, the larger glycodendrimers induced homotypic cellular aggregation. Additionally, novel poly(aryl ether) dendronized silica surfaces designed for reversible adsorbtion of targeted analytes were synthesized, and characterization using X-ray Photoelectron Spectroscopy (XPS) was performed. Using a Cu(I) mediated cycloaddition 'click' reaction, beta-cyclodextrin was appended to dendronized surfaces via triazole formation and also to a non-dendronized surface for comparison purposes. First generation G(1) dendrons have more than 6 times greater capacity to adsorb targeted analytes than slides functionalized with monomeric beta-cyclodextrin and are 2 times greater than slides functionalized with larger generation dendrons. This study reported beta-cyclodextrin functionalized surfaces can undergo a triggered release of the adsorbent, but otherwise retained the targeted analyte through multiple aqueous washes. Therefore, a new generation of G(1) dendronized surfaces capable of reversible adsorption were developed by heterogeneously appending sulfonic acid/pyridine end-groups. Auger Electron Spectroscopy (AES) was used to quantify the ratio of groups installed. Furthermore, G(1) dendronized surfaces were functionalized homogenously with sulfonic acid and pyridine for comparison and with chiral amino acids for chiral recognition studies.Item The synthesis and study of TF antigen functionalized dendrimers and dendrimer end group characterization and indium(III) promoted glycosylation(Montana State University - Bozeman, College of Letters & Science, 2017) Mattson, Amanda Lynn; Chairperson, Graduate Committee: Mary J. Cloninger; Anna K. Michel and Mary J. Cloninger were co-authors of the article, 'Using indium(III) as a promoter for glycosylation' in the journal 'Carbohydrate research' which is contained within this thesis.Polyamidoamine (PAMAM) dendrimers were functionalized with the Thomsen-Friedenreich (TF) antigen to study the multivalent effects on the galectin-3 mediated homotypic aggregation of A549 cells. TF antigen functionalized dendrimers of generations 2, 3, 4, and 6 were found to induce cellular aggregation. This is in contrast with previously observed results using lactose functionalized dendrimers, in which lactose functionalized generation 2 dendrimers were able to inhibit cellular aggregation. Additionally, TF antigen functionalized generation 6 dendrimers did not induce cellular aggregation as effectively as lactose functionalized generation 6 dendrimers. These preliminary results suggest that when compared to lactose functionalized dendrimers, the stronger galectin-3 binding affinity for TF antigen dendrimers may allow for more galectin-3 recruitment, creating aggregates with less freedom to rearrange into an optimized conformation. This suggests the reversibility of the binding event is important for effective protein interactions. The synthesis of TF antigen was achieved using indium triflate catalyzed glycosylation reactions. The development of indium(III) as a glycosylation promoter involved the analysis of indium bromide, indium chloride, and indium triflate for use in glycosylation reactions with a variety of alcohol acceptors. In(OTf) 3 mediated glycosylations of acetonide protected mannosides afforded exclusively alpha products in high yields. Acetylated mannosides gave moderate yields of exclusively alpha products using InBr 3 and InCl 3. Benzylated galactosides gave moderate yields of alpha, beta product mixtures using In(OTf) 3, with beta products formation being favored. Indium(III) was also used to synthesize alpha-1-2-dimannoside and alpha-galactose-1-2-mannoside in high yield. Additionally, spin labelled PAMAM dendrimers were preliminarily studied via electron paramagnetic resonance (EPR) to analyze the spatial arrangement of end groups. Dendrimers were functionalized with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) covalently tethered as a dimer. At low percent loading, a strong effect arising from the dimeric spin-labeled end groups was observed. EPR spectra of dendrimers bearing a higher loading of the dimeric spin-labeled end groups indicated that the end group arrangement approached a random distribution at approximately 40 to 50 percent loading. This suggests that covalently clustered pairs of end groups are significantly different from randomly distributed end groups on PAMAM dendrimers at low loading and become equivalent to randomly functionalized dendrimers around 50% functionalization.Item The effect of a natural covS mutation on virulence factor expression and innate immune evasion in a hypervirulent strain of group A streptococcus(Montana State University - Bozeman, College of Agriculture, 2013) Stetzner, Zachary William; Chairperson, Graduate Committee: Benfang LeiGroup A Streptococcus (GAS) is a highly versatile pathogen that is able to colonize multiple locations on the body, resulting in numerous diseases such as mild pharyngitis and the potentially lethal necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The high mortality rates associated with severe invasive diseases are particularly concerning. GAS strains isolated from patients with severe invasive infections frequently display hypervirulence, but the basis for this hypervirulence is not fully understood. The objective of this project is to elucidate the underlying mechanism behind this hypervirulent phenotype in a serotype M3 STSS isolate, MGAS315. A comparative study of MGAS315 and a serotype M28 puerperal sepsis isolate, MGAS6180, revealed that MGAS315 has a substantially higher capacity to invade soft tissue and inhibit neutrophil recruitment than MGAS6180 in a murine model of subcutaneous infection. Deletion of the platelet-activating factor (PAF) acetylhydrolase gene sse reduced MGAS315 skin invasion and innate immune evasion. These results cannot be explained by the proposal that the hypervirulence of MGAS315 is due to the acquisition of additional prophage-encoding virulence factors. SsE is negatively regulated by the two-component regulatory system CovR/S, and MGAS315 has a CovS G457V mutation compared with the covS gene of the serotype M1 isolate MGAS2221. We hypothesize that the CovSG457V mutation is responsible for the hypervirulence of MGAS315. To test this hypothesis, the mutated covS gene in MGAS315 was replaced with wild-type covS, resulting in MGAS315wtcovS. The mRNA levels of CovR/S-controlled capsule synthase gene hasA and the IL-8 peptidase gene spyCEP in MGAS315wtcovS were 24% and 3% of those in MGAS315, respectively. Repairing the covS mutation in MGAS315 also reduced the PAF acetylhydrolase activity in the culture supernatant and SsE production as measured by western blotting analysis. These results indicate that the CovSG457V mutation enhanced the expression of CovR/S-controlled virulence factors. More importantly, repairing the CovS G457V mutation attenuated the innate immune evasion, skin evasion, and virulence during infection. Collectively, this work demonstrates the CovS G457V mutation increases virulence factor expression and enhances innate immune evasion, thereby contributing to the hypervirulence of MGAS315.Item The immunology of spontaneous cure in the Nippostrongylus brasiliensis-mouse system(Montana State University - Bozeman, College of Agriculture, 1980) Benjamin, William HiramItem Analysis of the mechanisms of immune expulsion from mice of Hymenolepis diminuta and Hymenolepis nana(Montana State University - Bozeman, College of Agriculture, 1976) Isaak, Dale DarwinItem Hemagglutinin from Acrididae (Grasshopper) : preparation and properties(Montana State University - Bozeman, College of Letters & Science, 1984) Stebbins, Mark RichardItem Type-2 diabetes and innate immunity : new connections revealed by multi-dimensional fractionation of blood plasma prior to proteomic analysis(Montana State University - Bozeman, College of Letters & Science, 2010) Laffoon, Scott Bradley; Chairperson, Graduate Committee: Edward DratzWe compared levels of protein isoforms in human blood plasma from patients with newly diagnosed and untreated type-2 diabetes (T2DM) with non-diabetic controls in samples obtained from US NIH. We immunodepleted fourteen of the most abundant proteins from pooled plasma samples and separated the depleted samples into six fractions by reverse-phase liquid chromatography at 80°C. Proteins from these fractions were labeled with new high quantum yield, hydrophilic and spectrally resolved fluorescent detection dyes developed at MSU and resolved on large-format (24cm x 20cm) two-dimensional gels. By fluorescence analysis of 2D gels, using >1.4 fold change and p<0.05 acceptance criteria, we have identified five T2DM associated proteins and isoforms, including: two isoforms of zinc-alpha glycoprotein (ZAG), one isoform of serum amyloid A-1 (SAA-1) preprotein, one isoform of cysteine-rich secreted protein-3 (CRISP-3), one isoform of haptoglobin, and an A1-apolipoprotein fragment. Complement factor H related-5 (CFHR-5) is the likely identification of a sixth protein found significantly down in T2DM. Changes in the plasma levels of CRISP-3 and CFHR-5 strengthen the hypothesis that T2DM is a disease involving innate immunity. Three of these proteins are known to specifically bind to the transport protein, human serum albumin (HSA). Also, CRISP-3 is a specific and high-affinity ligand of alpha 1 beta glycoprotein, which is an HSA binder. To investigate HSA binding properties, we quantitatively measured the binding of a dye probe by HSA at neutral pH. These measurements revealed that HAS binding of the probe correlates with several metabolic parameters of central importance to the diagnosis of T2DM, including fasting plasma glucose (FPG). Therefore, this assay may reveal altered properties of HSA that could be developed for the clinical assessment of individuals' metabolic status. We sought modifications of HSA or altered cargo of HSA that may cause the difference in binding. 1D gels of plasma proteins reacted with maleimide dye showed no changed levels of the oxidation state of HSA's lone thiol, Cys-34. However, 1D blots of plasma proteins reacted with the oxidative carbonyl probe, hydrazide-biotin conjugate, and probed with luminol reactive HRP-neutravidin showed a surprising anti-correlation of HSA oxidation with hemoglobin A1c, an indicator of glycemic control.