Theses and Dissertations at Montana State University (MSU)
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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 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 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.