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Item When a lectin binds a sugar, and other sweet tales(Montana State University - Bozeman, College of Letters & Science, 2019) Bernhard, Samuel Pruitt; Chairperson, Graduate Committee: Mary J. Cloninger; Mackenzie S. Fricke was an author and Katharina Achazi, Paul Hillman, Willy Totten, Rainer Haag and Mary J. Cloningerwere co-authors of the article, 'The toxicity, uptake, and impact on galectin-3 mediated apoptosis of lactose functionalized dendrimers' submitted to the journal 'Biomolecules Special Issue: Moving Forward with Dendrimers' which is contained within this dissertation.The current state of chemotherapy and cancer treatment leaves much to be desired. Treatment is generally non-specific and relies on high dosage to achieve therapeutically relevant concentrations at target sites. Glycopolymer-drug conjugates, featuring targeting molecules and therapeutic prodrug on a water-soluble polymeric scaffold, offer a solution to these contemporary problems. Here, the complexity of glycopolymer design is explored through the lens of a biologically significant carbohydrate-binding receptor. In particular, galectin-3 is a complex Beta-galactoside binding lectin that experiences altered expression in many cancer pathologies and is implicated in metastasis, angiogenesis and poor overall prognosis. Galectin-3 mediates undesired cancer promoting processes through carbohydrate binding and oligomerization. A more complete understanding of the role galectin-3 plays in cancer progression will guide development of methods in the therapeutic intervention of these processes. In the interest of understanding galectin-3 and using it as a targeted receptor, its binding characteristics have been assessed through fluorescence lifetime and dynamic light scattering measurements. Employment of carbohydrates and glycopolymers including mannose, lactose, and lactose functionalized poly(amidoamine) (PAMAM) dendrimers, dendritic polyglycerols (dPG), and linear polymers (LP) provided insight into the carbohydrate binding avidity of galectin-3 and its propensity to oligomerize or form micron scale aggregates. A relationship between scaffold size and receptor recruitment was observed, which sheds light into multivalent binding motifs initiated by these glycopolymers and establishes a threshold for minimum requisite lactose functionality on lactose functionalized dendritic polyglycerols. In vitro cell based glycopolymer studies with AlexaFluor 647 and lactose functionalized PAMAM dendrimers revealed size-dependent uptake and demonstrated that accumulation occurs within the lysosome. Cellular aggregation experiments revealed that lactose functionalized LPs and dPGs influence galectin-3 mediated homotypic cellular aggregation and, in fact, augment this aggregation through receptor recruitment and cross-linking. The results reported here have provided a more fundamental understanding of galectin-3 binding interactions and have laid the groundwork for optimized glycopolymer-drug conjugate design.Item Chromatographic, spectroscopic and microscopic analyses reveal the impact of iron oxides and electron shuttles on the degradation pathway of 2,4,6- trinitrotoluene (TNT) by a fermenting bacterium(Montana State University - Bozeman, College of Agriculture, 2003) Borch, Thomas; Chairperson, Graduate Committee: William P. Inskeep and Robin Gerlach (co-chair)Contamination of surface and subsurface environments with explosives such as 2,4,6-trinitrotoluene (TNT) is a worldwide problem. The fate and analysis of TNT were investigated in numerous artificially contaminated model systems. We developed a unique high performance liquid chromatography gradient elution method for the analysis of commonly observed TNT metabolites and EPA explosives. Column temperature was identified as the key parameter for optimal separation. Iron (hydr)oxides play an important role in the reduction, sorption and fate of TNT in soil and sediment. Consequently, characterization of the nature and properties of natural and synthetic Fe (hydr)oxides is important for determining reaction mechanisms and surface-associated chemical processes. This work thus summarizes the potential applicability of imaging and spectroscopic techniques for eliciting chemical and physical properties of iron (hydr)oxides. TNT is persistent in soils due to its low redox potential and sorption. Batch and column studies revealed some of the first results on TNT desorption behavior in two well-defined model soil systems. Biosurfactants were found to be the most promising technique for enhanced TNT desorption. Batch studies with a Cellulomonas sp. in the presence of ferrihydrite and the electron shuttle anthraquinone-2,6-disulfonate (AQDS) were conducted to reveal biotic and abiotic mechanisms contributing to the degradation of TNT. Strain ES6 was found to reduce TNT and ferrihydrite with enhanced reduction in the presence of AQDS. Ferrihydrite stimulated the formation of more reduced TNT metabolites such as 2,4-diamino-6-nitrotoluene. Interestingly, a completely different degradation pathway was observed in AQDS-amended iron-free cell suspensions, showing a rapid transformation of TNT to 2,4-dihydroxylamino-6-nitrotoluene, which transformed into unidentified polar products. The influence of iron phases (i.e. hematite, magnetite, and ferrihydrite) and secondary Fe mineral formation on the degradation of TNT was also evaluated. The initial reduction of TNT was fastest in the presence of hematite; however, the further reduction of hydroxylamino-dinitrotoluenes was fastest, in the presence of magnetite and ferrihydrite (no AQDS). The impact of AQDS was predominant in the presence of hematite resulting in the formation of 2,4,6-triaminotoluene. Ferrihydrite underwent reductive dissolution with the formation of secondary hematite. The enhanced TNT reduction in ferfihydrite-amended systems was therefore most likely due to redox-active Fe(II) rather than secondary Fe phases.Item Continuous chromatography using time-varying eluant flow(Montana State University - Bozeman, College of Engineering, 1989) Swanson, Mark DavidItem An investigation of the effects of acid solutions of vanadium in column chromatography(Montana State University - Bozeman, College of Letters & Science, 1954) Van Vorous, TheodoreItem Chromatographic separation of the high molecular weight straight chain fatty acids(Montana State University - Bozeman, College of Letters & Science, 1955) Shellenberger, Thomas E.Item Purification and characterization of a lytic factor having phospholipase A 2 like activity in Trichomonas vaginalis(Montana State University - Bozeman, College of Agriculture, 2003) Lubick, Kirk James; Chairperson, Graduate Committee: Donald Burgess.Item Surface substrates for high performance laser desorption ionization mass spectrometry and their interfacing to chromatography(Montana State University - Bozeman, College of Letters & Science, 1999) Han, MeiItem Using chromatographic and mass spectrometry tools to probe albumin and its cargos : in search of understanding type II diabetes(Montana State University - Bozeman, College of Letters & Science, 2011) Bowden, Jared Newell; Chairperson, Graduate Committee: Edward DratzWe measured molecules carried as cargos on the abundant blood protein human serum albumin (1) in patients with newly diagnosed, untreated type II diabetes (T2D) compared to healthy controls (HC). The HSA cargos measured included lipids, minerals, peptides, and metabolites. Differences in these cargos associated with T2D were measured, using chromatography and mass spectrometry, seeking to identify biological markers that may enhance early diagnosis of T2D. An extrinsic fluorescent probe of binding sites on HSA, ANS, revealed that there were distinct differences in loading of hydrophobic cargo between HC and systemic lupus erythematosus, T2D, and Lyme disease plasma samples. A decrease in mineral levels on HSA was also measured in T2D plasma compared to healthy control plasma, using ICP-MS. Zinc ions showed the largest changes and were reduced three fold in T2D. The hydrophobic cargo of HSA revealed a decrease in HSA-associated fatty acids in T2D, measured by GCMS using negative chemical ionization. In this same GCMS study new classes of glycine-containing compounds bound to HSA were found to be increased by two fold in T2D in the hydrophobic extract of HSA. A metabolomic study using RP-uHPLC QTOF MS in both positive and negative ionization modes examined differences in the hydrophobic extract of whole plasma in T2D compared to healthy controls. Increased levels of branched chain amino acids were found in T2D compared to HC. Decreased levels of phosphatidylcholines, phosphatidylethanol amines, and vitamin D3 metabolites were found in T2D compared to HC. The results suggests that the HSA cargo in T2D, SLE, and other disease states, may provide new diagnostic markers and lead to deeper understanding of the mechanisms of disease in humans.Item Monitoring protien cage nanopaticle morphology for applications in medicines and materials(Montana State University - Bozeman, College of Letters & Science, 2011) Johnson, Benjamin Lawrence; Chairperson, Graduate Committee: Trevor DouglasProtein cage nanoparticles are naturally occurring proteins found in all domains of life. The breadth of structural knowledge and the ability to modify protein cage nanoparticles both chemically and genetically set them apart for use as platforms for biomedical templates and materials synthesis. The work described herein focuses on the use of protein cage nanoparticles as a protective agent from a suite of viral pathogens. Protein cage nanoparticles exist in many different morphological forms both within a specific particle and between particles. It is essential to characterize these different states in order to engineer a protein cage nano particle for biomedical and materials synthesis. Described here is an expanded protocol for determining the morphological state with the bacteriophage P22 capsid. Using multiple techniques including multi angle light scattering, analytical ultra centrifugation, agarose gel electrophoresis and transmission electron microscopy these states are described and characterized. P22 exits in four different morphological states: the procapsid, empty shell, expanded shell and so-called "wiffleball". Also characterized in the work is the small heat shock protein from Methanococcus jannaschii, which exists in two morphological states. One of the states being the assembled 12 nm cage structure and the other state being a disassembled cage structure that is most commonly described at elevated temperatures. The characterization of these structures can aid in the understanding the mechanism of formation for the immunological phenomena induced bronchial associated lymphoid tissue.Item Mannose/tempo functionalized pamam dendrimers : their relative locations and components of affinity towards Concanavalin A(Montana State University - Bozeman, College of Letters & Science, 2004) Samuelson, Lynn Elizabeth; Chairperson, Graduate Committee: Mary J. Cloninger.Surface functionalized dendrimers are being used for several applications including the study of protein-carbohydrate interactions. Mannose-functionalized dendrimers with varying concentrations of saccharides on the dendrimer surface were synthesized. Spin labels (2,2,6,6-tetramethylpiperidine N-oxide) were incorporated onto the dendrimer's surface as well. Linebroadening effects in the EPR spectra of these compounds allowed us to determine the distance between spin labels (and thus between carbohydrates). The mannose-spin labeled functionalized dendrimers were further studied to determine effects of the spin label in hemagglutination inhibition assays. Affinity chromatography was employed to separate any mixture of compounds based on their affinity towards Concanavalin A, a mannose specific protein. The spin label on these compounds was used to study the relative conformations of the different compounds obtained from the affinity column. Synthesis of glucosamine funtionalized dendrimers was undertaken unsuccessfully. Had the synthesis been a success, TEMPO residues would have been attached to the amino sugar. EPR studies would have been used to determine the relative locations of the TEMPO labeled carbohydrates directly.