Theses and Dissertations at Montana State University (MSU)
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Item The synthesis and characterization of fluorescently labeled, lactose-functionalized poly(amidoamine) (PAMAM) dendrimers(Montana State University - Bozeman, College of Letters & Science, 2024) Frometa, Magalee Rose; Chairperson, Graduate Committee: Mary J. CloningerCellular uptake of lactose-functionalized poly(amidoamine) dendrimers (PAMAM) has yet to be fully understood and deeply studied. Before sufficient cellular uptake studies can be made, optimization of the synthesis of the lactoside, and the coupling and purification of dye-tagged lactose-functionalized PAMAM had to be completed, as reported here. The synthesis of the requisite lactoside derivative for dendrimer functionalization was optimized. The coupling of the dye, Alexa Fluor 647, to the lactoside-functionalized PAMAM was performed in the presence of a sodium acetate buffer and utilized size separation methods to ensure purity. The structures of the lactoside derivatives and of lactose functionalized PAMAMs were confirmed via nuclear magnetic resonance (NMR) spectroscopy. The purity and degree of labeling (DOL) of the dye labeled, lactose-functionalized PAMAMs were determined with UV-vis. Results show high success of yield and purity resulting from the optimized procedure described in this study.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 Multivalently presented carbohydrates can be used as drug delivery vehicles and to study protein carbohydrate interactions(Montana State University - Bozeman, College of Letters & Science, 2018) VanKoten, Harrison Wesley; Chairperson, Graduate Committee: Mary J. Cloninger; Wendy M. Dlakic, Robert Engel and Mary J. Cloninger were co-authors of the article, 'Synthesis and biological activity of highly cationic dendrimer antibiotics' in the journal 'Molecular pharmaceutics' which is contained within this thesis.; Rebecca Moore, Coleen Murphy and Mary J. Cloninger were co-authors of the article, 'Probing the LEC-1 and LEC-10 oxidative stress pathway in Caenorhabditis elegans using GALBeta1-4FUC dendrimers' which is contained within this thesis.Dendrimers in general excel as drug delivery vehicles since there are many different ways they can be assembled and different ways to tailor them to the system being studied. Glycodendrimers are generally nontoxic and can be further developed to meet the needs of what is being studied. For instance, in the studies below, a quaternity ammonium compound (QAC) has been attached to a glycodendrimer to determine the antimicrobial activity of a multivalently presented QAC in studies of minimum inhibitory concentration (MIC), biofilm prevention, and bacterial resistance. Results include comparable MICs to those of established antibiotics, prevention of biofilm formation but not disruption of an established biofilm, and establishment of multivalency as a strategy to counteract bacterial resistance. Another heterogeneously functionalized dendrimer was synthesized to study drug release characteristics of a prodrug attached to a cleavable substrate. In these studies, the upregulation of several proteins during cancer progression was taken advantage of including; MMP-2, -7, -9, and galectin-3. Glycodendrimers are tools used to study protein carbohydrate interactions. Study of galectins and their corresponding Beta-galactosides have illuminated their role in several essential biological processes. Multivalency plays a crucial role in many protein-carbohydrate interactions. Galectins are known to interact multivalently with various ligands. Although the role of galectins in this process is not yet fully understood, galectins have been proposed to serve as protective proteins during periods of high oxidative stress. We describe the synthesis of GalBeta1-4Fuc functionalized poly(amidoamine) (PAMAM) dendrimers in order to test C. elegans' response to high oxidative stress. In order to test the function of GalBeta1-4Fuc in vivo, C. elegans were treated with RNAi to knockdown lec-1 or lec-10, and then treated with glycodendrimer and exposed to oxidative stress. C. elegans that were pre-treated with the glycodendrimers were less susceptible to oxidative stress than untreated controls. The glycodendrimers mainly appeared within the digestive tract of the worms, and uptake into the vulva and proximal gonads could also be observed in some instances. This study indicates that multivalently presented GalBeta1-4Fuc can protect C. elegans from oxidative stress by binding to galectins.Item The carbohydrate associated with myrosinase(Montana State University - Bozeman, College of Letters & Science, 1962) Huotari, FrancesItem Carbohydrate chemistry : synthetic and structural investigation of the phytotoxins found in Helminthosporium sacchari, and Rhynochosporium secalis(Montana State University - Bozeman, College of Letters & Science, 1980) Beier, Ross CarltonItem The use of In(OTf)3 as a Lewis Acid in carbohydrate chemistry, and exploration in silicon tethered reactions(Montana State University - Bozeman, College of Letters & Science, 2008) Bizier, Nicholas Paul; Chairperson, Graduate Committee: Mary J. CloningerThe feasibility of using Indium (III) triflate as a Lewis Acid for a number of different carbohydrate reactions was explored. First, the use of In(OTf)3 as a catalyst in the acylation of a number of carbohydrates was explored. The utility and compatibility of the In(OTf)3 reaction conditions with a number of protecting groups on the sugars were examined by 1H NMR analysis. In the second part of the carbohydrate methodology, the feasibility of using In(OTf)3 to promote glycosylation reactions using a number of glycosyl donors was evaluated. These studies were conducted using 1H NMR, 13CNMR, and Micro-ToF mass spectrometry. In both cases, In(OTf)3 was found to be a viable if highly acidic Lewis acid for these reactions. Silicon tether methodology was explored with the goal of obtaining 1,3 and 1,2-amino alcohols. The parameters for this reaction were explored after precursors and 1,3-amino alcohols were synthesized. These studies were conducted using 1H NMR, 13C NMR, IR spectroscopy, and Micro-ToF mass spectrometry. While some success was achieved, this route is unlikely to be widely adopted due to the multi-step syntheses that are required to obtain viable reaction precursors.Item Glycodendrimer mediation of Galectin-3 induced cancer cell aggregation(Montana State University - Bozeman, College of Letters & Science, 2010) Sprenger, Julie Jeannine; Chairperson, Graduate Committee: Mary J. CloningerGalectin-3 is a cell surface protein that plays an important role in tumor aggregation, tumor progression and metastasis via its interaction with carbohydrates in the biological system. A synthetic, carbohydrate-functionalized, multivalent framework is ideal to study biological protein/carbohydrate interactions. In this research, dendrimers are used as a platform for the display of carbohydrates to study multivalent galectin-3/carbohydrate interactions as they pertain to tumor aggregation. The hypothesis is that the addition of carbohydrate functionalized dendrimers will mimic natural galectin-3 glycoconjugate ligands, affecting the aggregating behavior of neoplastic cells. A galectin-3 inhibitor may prove to be an effective cancer therapeutic agent. Results from homotypic aggregation assays show a change in aggregation with the addition of particular carbohydrate-functionalized dendrimers. Two of the glycodendrimers significantly inhibit tumor cell aggregation.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.Item Evaluating protein-carbohydrate interactions induced by multivalent carbohydrate-functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2010) Schlick, Kristian Henri; Chairperson, Graduate Committee: Mary J. CloningerUnderstanding protein-carbohydrate interactions is essential for elucidating biological pathways and cellular mechanisms but is often difficult due to the prevalence of multivalent interactions. A better understanding of the basic behavior of protein-carbohydrate interactions is critical for controlling cellular proliferation and recognition processes for novel therapeutic methods to be successful. Many procedures that exist for evaluating protein-carbohydrate interactions are often limited to monovalent interactions or small polymers. Given that many cellular processes, such as those attributed to the immune system, are enhanced multivalently or are aggregation-driven, there is a need to reveal the behavior and basic requirements for multivalent binding and aggregation. Evaluating these interactions on large, multivalent scaffolds such as synthetically controllable dendrimers provides an important tool towards accurately determining the role of glycosylation in biological systems. Here, different approaches to measure the interactions of proteins with glycodendrimers are described, ranging from simple qualitative assays to novel quantitative methods of assessment. Quantitative methods such as Isothermal Titration Calorimetry and Surface Plasmon Resonance are severely limited when used with multivalent systems, and do not provide as accurate results as monovalent systems. When dealing with multivalent systems, inhibition assays often provide more reproducible results. Through these experiments, it has become increasingly apparent that aggregates play a significant role in multivalent systems, and current methods to evaluate these interactions leave much room for improvement. Assay design is important both for basic identification and understanding of any interaction, especially higher-order interactions involving multivalency. Endgroup patterning and presentation was explored to determine their role in multivalent affinity enhancements. Using a novel fluorescence lifetime method, glycodendrimer-mediated aggregation was successfully characterized. The work here evaluates the effectiveness of assays used for carbohydrate interaction, translated to a multivalent scaffold, with special consideration to large-order aggregates.