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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 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.