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Item Development and characterization of a novel isothermal DNA amplification reaction(Montana State University - Bozeman, College of Engineering, 2021) Ozay, Burcu; Chairperson, Graduate Committee: Scott McCalla; This is a manuscript style paper that includes co-authored chapters.Isothermal nucleic acid amplification chemistries are gaining popularity as nucleic acid detection tools that can replace the current gold standard methods, PCR and its derivatives, with their simplicity, speed and applicability to point-of-care applications. In this work, we have developed and characterized a novel isothermal amplification chemistry, ultrasensitive DNA amplification reaction (UDAR). UDAR differs from similar chemistries with its unique, biphasic response with a high-gain output that can be captured with a cell-phone camera. The switch-like, nonlinear characteristics provide a definitive on/off signal for potential use in applications such as molecular diagnostics and DNA circuits. Tunability of the reaction was explored and the relationship between thermodynamic properties of the reaction templates and the reaction output was established. Limitations on fluorescent staining of reaction components by two popular commercial nucleic acid stains, SYBR Green II and SYBR Gold, were determined for a more accurate evaluation of the reaction output and reaction product analysis. A mathematical model of the reaction output was built and outputs from three different UDAR templates were successfully simulated. This model revealed important information on reaction pathways and helped identify the impact of individual reaction events. A comprehensive literature review of enhancement strategies for isothermal amplification reactions was conducted to serve as a guide to improve and modify these reactions according to different needs and applications. Lastly, UDAR was applied to microRNA detection, which are putative biomarkers for diseases such as cancer, malaria, and traumatic brain injury. Five different miRNAs were successfully detected by UDAR, down to 10 fM concentration. UDAR-based miRNA quantification is possible, with linear calibration curves provided between 10fM and 1 nM. This work has significant contributions to the growing field of isothermal nucleic acid amplification based-molecular detection systems by introducing a unique isothermal amplification chemistry, establishing design and manipulation techniques, and guiding improvement efforts of these technologies.Item Improving transport in hydrogels for 3D bioprinting applications(Montana State University - Bozeman, College of Engineering, 2021) Abbasi, Reha; Chairperson, Graduate Committee: James Wilking; Aaron D. Benjamin was an author and Madison Owens, Robert J. Olsen, Danica J. Walsh, Thomas B. LeFevre and James N. Wilking were co-authors of the article, 'Light-based 3D printing of hydrogels with high-resolution channels' in the journal 'Biomedical physics & engineering express' which is contained within this dissertation.; Thomas B. LeFevre was an author and Aaron D. Benjamin, Isaak J. Thornton, and James N. Wilking were co-authors of the article, 'Coupling fluid flow to hydrogel fluidic devices with reversible "pop-it" connections' in the journal 'Lab on a chip' which is contained within this dissertation.; Zahra Mahdieh was an author and Galip Yiyen, Robert A. Walker and James N. Wilking were co-authors of the article, 'Light-based 3D bioprinting of hydrogels containing colloidal calcium peroxide' submitted to the journal 'Bioprinting' which is contained within this dissertation.Hydrogels are soft, water-based gels with widespread applications in medicine, tissue engineering, and biotechnology. Many of these applications require structuring hydrogels in three-dimensional space. Light-based 3D printers offer exquisite spatial control; however, technologies for light-based 3D-printing of hydrogels remain limited. This is mainly caused by poor material transportation through the hydrogel. For example, limited transport of oxygen and other nutrients through 3D printed tissue constructs containing living cells leads to low cell viability. Here, we describe three experimental research studies focused on improving material transport in 3D-printed hydrogels. In the first part of this thesis, we describe a generalizable method for light-based 3D printing of hydrogels containing open, well-defined, submillimeter-scale channels with any orientation. These submillimeter channels allow for bulk liquid flow through the hydrogel to improve nutrient and oxygen transport. In the second part of this thesis, we describe a simple, reversible, plug-based connector designed to couple tubing to a hydrogel-based fluidic device to allow for pressurized liquid flow through the system. The resulting connection can withstand liquid pressures significantly greater than traditional, connector-free approaches, enabling long-term flow through 3D-printed hydrogels. In the third part of this thesis, we characterize the printability of photopolymerizable resins containing particles that slowly dissolve to release oxygen and thereby improve cell viability. The light-based 3D bioprinting technologies we describe in this thesis will improve material transport through 3D printed hydrogels and enable a wide variety of applications in 3D bioprinting and hydrogel fluidics.Item The development of hybrid biomaterials using the virus-like particle (VLP) from bacteriophage P22(Montana State University - Bozeman, College of Letters & Science, 2016) Edwards, Ethan James; Chairperson, Graduate Committee: Trevor Douglas; Rajarshi Roychoudhury, Benjamin Schwarz, Paul Jordan, John Lisher and Trevor Douglas were co-authors of the article, 'Co-localization of catalysts within a protein cage leads to efficient photochemical NADH and/or hydrogen production' which is contained within this thesis.; Dissertation contains several articles of which Ethan James Edwards is not the main author.A broad range of bio-composite materials have been developed through inspiration from biology. In particular, natural systems that confine, co-localize and protect their contents has inspired the design and synthesis of the P22 virus-like particle (VLP) to effect a suite of biomaterials. These materials were realized by taking advantage of the native protein architecture of P22 as an initiation point and platform for material synthesis. Introducing a reactive cysteine on the P22 coat protein provided an initiation point for polymer synthesis. Atom transfer radical polymerization (ATRP) was initiated creating a polymer framework on the interior of the P22 VLP. Using this polymerization technique (ATRP) a photocatalytic crosslinker was successfully incorporated for reduction of methyl viologen. Next, a manganese porphyrin imaging agent was loaded creating a T 1-enhanced MRI contrast agent, as an alternative to the highly toxic Gadolinium currently used. Inspired by photosynthetic machinery, the P22-xAEMA system was labeled with a co-catalyst system, creating a co-localized photocatalytic nanoparticle capable of photochemically producing NADH/hydrogen. The production was controlled by labeling density of catalysts resulting in a tunable biomaterial. The design of a complex bio-hybrid material was developed by combining both synthetic and genetic approaches. Coupling the enzyme Alcohol Dehydrogenase D from Pyrococcus furiosis with a small molecule catalyst led to a coupled catalytic system between a synthetic catalyst and biologically derived enzyme. The P22 VLP system was studied by atomic force microscopy (AFM) and cryoelectron microscopy (cryo-EM) unraveling its biophysical properties and providing insights for further material design. 2D-crystal arrays were formed from a variety of P22-protein biomaterials, for the development of functional P22 arrays. Lastly, the P22 VLP was monitored by charge detection mass spectrometry, giving insight into the stability of the scaffolding protein. These studies show the versatility of this system for both material synthesis and fundamental biochemical understandings. Overall, the work here continues to progress and push the boundaries of protein cage nanoparticles as platforms for material synthesis. The development of hybrid biomaterials from VLPs serve to improve our basic understandings of the natural systems they are derived from and provide additional design principles for improved complex biohybrid materials.Item Factors affecting the implementation of agriscience programs in Montana(Montana State University - Bozeman, 1992) Cavey, Brian M.; Chairperson, Graduate Committee: C. Van ShelhamerItem Nox protein expression, purification and structure analysis(Montana State University - Bozeman, College of Agriculture, 2004) Baniulis, Danas; Chairperson, Graduate Committee: Algirdas J. JesaitisFlavocytochrome b558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the NADPH oxidase. Six homologues of gp91-phox, the large subunit of Cytb, have been identified (Nox family). Understanding of the structure and function of the Nox proteins is limited. To distinguish solvent-accessible and membrane or conformation sequestered regions on native structure of gp91-phox, a number of proteolytic enzyme cleavage products on the lipid reconstituted protein were identified using mass spectrometry, in this study. Affinity-purified rabbit anti-peptide antibodies binding to intact neutrophils suggested extracellular localization of gp91-phox regions, however, results using control CGD-cells suggested that these antibodies may cross-react with an unusual non-gp91-phox species in the normal and CGD-derived plasma membranes. Further, a monoclonal antibody CL5 epitope was mapped to the region 135-DPYSVALSELGDR on the gp91-phox, the prototype for the Nox family proteins. Epitopes of previously described mAb 54.1 and CL5 in gp91-phox align with Nox family proteins with high degree of identity and the use of these two monoclonal antibodies as immunoprobes for Nox family proteins was evaluated. Ab 54.1 was found to be specifically reactive with homologous Nox protein fragments expressed in E. coli. Nox3 protein expressed in HEK293H cells was also detected by 54.1, but not by CL5. Nox1 expression in stably transfected NIH 3T3 was examined using the antibodies, but no detectable binding to Nox1 was observed in immunoblotting assays and by flow-cytometry analysis. The antibodies were also used to probe for presence of potential truncated forms of gp91-phox expressed in chronic granulomatous disease (CGD) affected neutrophils with premature termination of gp91-phox synthesis. Analysis did not detect any smaller size protein fragments by immunoblotting. In addition, two other proteins were found to be crossreactive with 54.1 and CL5, they were identified as GRP58 and gelsolin, respectively, two universally expressed cytosolic proteins with regulated association with the plasma membrane. Finally, to help in ongoing structural biology efforts, a recombinant human Cytb expressing PLB-985 cell line was used to develop process of large-scale production of the protein for application in structural biology experiments.Item Photosynthetic and glycoalkaloid responses of potato (Solanum tuberosum L.) to Colorado potato beetle (Leptinotarsa decemlineata Say) defoliation(Montana State University - Bozeman, College of Agriculture, 2006) Dinkins, Courtney Louise Pariera; Chairperson, Graduate Committee: Robert K. D. Peterson.Photosynthetic and glycoalkaloid responses of potatoes (Solanum tuberosum L.) to varying levels of Colorado potato beetle (Leptinotarsa decemlineata Say) and manual defoliation were measured on 'Cal Red' and 'Russet Burbank' plants. No alteration in photosynthesis was observed on the remaining tissue of an injured leaf for Cal Red and Russet Burbank leaves defoliated by larval Colorado potato beetles nor for Russet Burbank leaves defoliated by adult Colorado potato beetles. No significant differences were observed between actual Colorado potato beetle and manual defoliation for both varieties. In both of the whole-plant defoliation studies, defoliation level consistently did not result in increased or decreased gas exchange parameters of individual leaves compared to undefoliated controls. There was no evidence of delayed leaf senescence in defoliated treatments. Plants defoliated by Colorado potato beetles had a significantly greater production of glycoalkaloids than in control and in manually defoliated plants for both skin and inner tissue.Item Mycofumigation with Muscodor albus : effects on Verticillium wilt and black dot root rot of potato, effects on Glomus intraradices and ectomycorrhizal fungi, and M. albus proliferation in soil(Montana State University - Bozeman, College of Agriculture, 2008) Grimme, Eva; Chairperson, Graduate Committee: Barry J. Jacobsen.Muscodor albus Worapong, Strobel & Hess, isolate CZ-620 (MA) is an endophytic fungus that produces volatile organic compounds (VOCs) and non-volatile antimicrobial compounds. The use of these VOCs to inhibit or kill a wide range of microorganisms is termed mycofumigation. This dissertation focuses on parameters of MA mycofumigation including: production and bioactivity of previously un-described water-soluble antimicrobial compounds produced by MA; distribution of antimicrobial compounds from a MA point source in three soil types as measured by effects on Verticillium dahliae and Colletotrichum coccodes; control of V. dahliae and C. coccodes on potato; the ability of MA to colonize soil; and the effects of mycofumigation on ectomycorrhizal fungi (EMF) in vitro and on the colonization of onion roots by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. The bioactivity of water-soluble compounds produced in potato dextrose broth was significantly increased as measured in growth reduction of C. coccodes, V. dahliae, and Rhizoctonia solani. No reduction was observed for Aphanomyces cochlioides and Pythium ultimum. Antimicrobial compounds from a MA colonized barley point source reduced V. dahliae and C. coccodes populations in soils by 60-100% at distances up to 9 cm from the inoculation source depending on soil type. Mortality rate ranging from 70-100% was observed within a 3 cm radius from the inoculation source. In both field and greenhouse trials, MA colonized barley formulation reduced Verticillium wilt and black dot root rot severity and reduced populations of both pathogens in potato tissue as measured by real-time quantitative PCR and serial dilution. Planting directly into mycofumigated soil previously infested with V. dahliae or C. coccodes resulted in equal control of the pathogens when compared to a one-week mycofumigation period prior to planting. After six weeks of incubation MA did not colonize sterile soil further than 0.5 cm away from a MA inoculation point. In vitro experiments showed that most of the tested EMF were inhibited in the presence of MA VOCs, but were able to resume growth when removed from VOCs. Incorporating MA into soil had no negative but supportive effect on onion root colonization by the AM fungus G. intraradices.Item Effects of organic acids and heavy metals on the biomining bacterium : Acidithiobacillus caldus strain BC13(Montana State University - Bozeman, College of Engineering, 2010) Aston, John Earl; Chairperson, Graduate Committee: Brent M. Peyton; William Apel (co-chair)Acidithiobacillus caldus is an important microorganism to biomining and acid-mine formation. However, its degree of characterization is not commensurate to its significance in such systems. Specifically, studies enumerating effects of organic acids and metals on this microorganism are limited. The work presented in this dissertation improves understanding of At. caldus with respect to interactions with these compounds. All experiments discussed in this dissertation used At. caldus strain BC13. The organic acids; pyruvate, acetate, 2-ketoglutarate, succinate, fumarate, malate, and oxaloacetate were each toxic to At. caldus strain BC13. Depending on the organic acid tested, concentrations between 250 and 5,000 uM completely inhibited the growth of At. caldus strain BC13 (chapter two). Subsequent experiments, reported in chapter three, showed that At. caldus strain BC13 used pyruvate as a sole carbon source. Chapter four discusses the toxicities of the heavy metals; lead, zinc, and copper to At. caldus strain BC13. Lead was by far the most toxic metal tested, with an observed minimum inhibitory concentration of 7.5 mM. Conversely, zinc and copper had minimum inhibitory concentrations of 75 and 250 mM, respectively. The sorption of lead, zinc, and copper was also studied, and is discussed in chapter 5. Between pH 5.5 and 7.0, zinc and copper sorbed to At. caldus strain BC13 with similar capacity and affinity as that observed to other acidithiobacilli, however at pH 2.0, significant sorption of zinc and copper to viable cells was observed, whereas previous work did not report sorption of zinc or copper to viable acidithiobacilli cells below pH 3.0. Chapter six reports efforts to qualify changes in protein expression of At. caldus strain BC13 when exposed to organic acids or heavy metals. Matrix assisted laser desorption ionization mass spectrometry and one-dimensional gel electrophoresis qualified the up-regulation of an integral membrane protein with a molecular weight of approximately 25 kDa. Efforts to identify up-regulated proteins were not successful, but any proteins that are regulated in response to organic acids or heavy metals in biomining microorganisms would likely be of commercial application.Item Disease control and plant defense pathways induced by Bacillus mojavensis isolate 203-7 and Bacillus mycoides isolate BmJ(Montana State University - Bozeman, College of Agriculture, 2008) Neher, Oliver Thomas; Chairperson, Graduate Committee: Barry J. Jacobsen.The objective of this study was to investigate the plant defense pathways induced by Bacillus mojavensis isolate 203-7 (203-7) and B. mycoides isolate BmJ (BmJ) and to test their ability to control fungal pathogens on tomato and cucumber by means of systemic acquired resistance (SAR). An Arabidopsis thaliana mutant - Botrytis cinerea pathosystem was used to investigate plant defense pathways activated by 203-7 and BmJ. A. thaliana wild type (Col-0), ein2-1, jar1-1, NahG, ndr1-1/npr1-2, and npr1-1 mutants were induced by application of bacilli, distilled water, or chemical inducers. Both bacilli reduced disease severity on wild type and NahG mutants, but provided no reduction on jar1-1, indicating that induction was salicylic acid (SA) independent but jasmonic acid (JA) dependent. 203-7 induced plants had lower disease severity on npr1-1 and ein1-2 mutants but were equivalent to buffer controls on jar1-1 mutants. BmJ did not decrease disease severity on npr1-1, jar1 or ein2-1 mutants. Enzyme assays confirmed the induction of chitinase, beta-1,3-glucanase, and superoxide dismutase by 203-7 and BmJ. These results demonstrate that induction by 203-7 is JA dependent and NPR1 independent. BmJ is SA independent but NPR1 and JA/ethylene dependent. Bacilli were tested for their ability to control B. cinerea grey mold on hydroponically grown greenhouse tomatoes and Glomerella cingulata var. orbiculare on cucumber by means of SAR. Weekly foliar applications of bacilli were able to significantly (a=0.05) reduce the severity of grey mold leaf lesions and to reduce the area under the disease progress curve (AUDPC) calculated for seven Botrytis stem canker ratings. Chitinase, beta-1,3-glucanase, and SOD activity of apoplastic fluids were not significantly (a=0.05) increased by the treatments. Bacilli reduced total and live spore production of G. orbiculare per mm 2 of lesion and increased beta-1,3-glucanase activity of cucumber apoplastic fluids. Applications of BmJ compared to fungicides for the control of anthracnose in cucumber (var. 'General Lee') and cantaloupe (var. 'Athena') were evaluated in 2004 and 2005 field experiments. BmJ applied seven days before inoculation provided disease reduction in cucumber and cantaloupe statistically equal to the fungicide treatments.Item Risk assessment of plant-based pharmaceuticals and biologics(Montana State University - Bozeman, College of Agriculture, 2006) Shama, Leslie Marie; Chairperson, Graduate Committee: Robert K. D. Peterson.Biotechnology is evolving to produce pharmaceutical proteins in plants. Plantbased pharmaceutical production creates concerns of exposure in an open environment and contamination of the food supply. Consequently, quantitative human health and ecological risk assessments were conducted for aprotinin, gastric lipase, and LT-B expressed in maize. A comparative, qualitative risk assessment was conducted for conventionally derived and plant-cell derived Newcastle disease virus vaccine. Effect and exposure scenarios were modeled for each quantitative risk assessment and each scenario was based on a tiered approach in which inadvertent exposure through ingestion were examined to determine a risk characterization of the plant-based pharmaceuticals. The qualitative Newcastle disease virus vaccine risk assessment characterized risks based on the potential exposures to the poultry being vaccinated, to humans administering the vaccine, and to non-target birds. For the human-health risk assessment, the dietary exposure evaluation model (DEEM) was used to estimate the inadvertent dietary intake of the pharmaceutical proteins in food. The ecological risk assessment used Monte Carlo simulations to evaluate the exposure of each protein in maize for four receptor species.