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 Cancer processes probed by multivalency: investigations with galectin-3 and lactose functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2019) Fricke, Mackenzie Sue; Chairperson, Graduate Committee: Mary J. Cloninger; Samuel P. Bernhard was an author and Willy Totten, Katarina Achazi, Paul Hillman, Rainer Haag and Mary J. Cloninger were 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 thesis.; Kyle Tweedy and Mary J. Cloninger were co-authors of the article, 'Lactose functionalized dendrimers impact galectin-3 mediated cancer cell migration in vitro' submitted to the journal 'ACS chemical biology' which is contained within this thesis.Cancer has become a prevalent disease that is the second leading cause of death in the United States. Various cancers have been identified as either over or under expressing a sugar binding protein: galectin-3. The target of this research is to investigate cancerous events that are impacted by galectin-3 and mediate these events through the use of a multivalent binding partner to galectin-3. This binding partner is lactose functionalized PAMAM dendrimers. Apoptosis has been reported as another phenomenon that galectin-3 impacts. By using a reporter assay, viability, cytotoxicity and apoptosis were observed for cancer cell line A549 in the presence of exogenously added galectin-3 and/or lactose functionalized dendrimers. It was found that exogenous galectin-3 and glycodendrimers did not have any significant impact on these cell viability tests. Therefore, glycodendrimers can be used to probe multivalent effects without threat of toxicity. Metastasis was investigated through a modified in vitro scratch assay. By monitoring the migration of cancer cells, it was found that exogenously added galectin-3 retarded cell migration. When glycodendrimers were included, migration was partially restored. This revealed the implications of exogenous galectin-3 regarding the metastatic potential of carcinomas. When the implications of the domains of galectin-3 were investigated, it was found that the truncated galectin-3 containing only the carbohydrate recognition domain (CRD) was unable to replicate the same effects observed in full length galectin-3. Immunofluorescence microscopy was used to locate the multivalent binding partner and galectin-3 in the assay. While endocytosis of galectin-3 was observed, no colocalization with the multivalent binding partner was observed intracellularly, supporting the hypothesis of an extracellular interaction mediating the results. Multivalent interactions between glycodendrimers and galectin-3 impacted cellular migration. Angiogenesis revealed that exogenous galectin-3 induced neovascularization. Glycodendrimers impacted galectin-3 mediated angiogenesis. Glycodendrimers alone could elicit effects either enhancing or negating angiogenesis depending on the dendrimer generation. Fluorescent tags revealed glycodendrimer accumulation on or inside the cells and galectin-3 on the surface of cell groups. Overall, these studies show that glycodendrimers can interact multivalently and affect cellular processes.Item Glycodendrimers : tools to study multivalent galectin-1 interactions(Montana State University - Bozeman, College of Letters & Science, 2015) Cousin, Jonathan Martin; Chairperson, Graduate Committee: Mary J. CloningerGalectin-1 is a carbohydrate binding protein that mediates cancer processes through multivalent interactions with glycoproteins expressed on the surface of cancer cells and in the extracellular matrix. A series of multivalent PAMAM dendrimers were functionalized with lactose and applied to the study and mediation of multivalent galectin-1 interactions. An ELISA was designed to study the interaction of galectin-1 with surface immobilized glycodendrimers. The results of the ELISAs indicate that galectin-1 binds well to the multivalent framework. DLS and fluorescence microscopy were used to study that interaction of galectin-1 with glycodendrimers in solution. These solution-based assays indicate that the glycodendrimers nucleate galectin-1 into nanoparticles. The ability of the glycodendrimers to organize the galectin-1 into biologically active arrays was investigated in cellular assays. A homotypic cellular aggregation assay using DU145 human prostate carcinoma cells, which express a putative galectin-1 ligand (Mucin1), was designed to study the influence of multivalent glycodendrimers on cellular aggregation/tumor formation. All generations of glycodendrimers were observed to inhibit cellular aggregation by diverting the galectin-1 from its native role in cellular cross-linking of cancer cells. To further probe multivalent interactions in cancer, the glycodendrimers were applied to a tube formation assay to study galectin-1 angiogenic processes. Galectin-1 was observed to accelerate neovascularization, and the impact of the galectin-1 was mildly inhibited by the glycodendrimers.Item Glycodendrimer mediation of galectin-3 cancer processes and indium(III) as a glycosylation promoter(Montana State University - Bozeman, College of Letters & Science, 2014) Michel, Anna Kaczmarek; Chairperson, Graduate Committee: Mary J. CloningerGalectin-3 is a carbohydrate-binding protein that is found inside and at the surface of most healthy cells, where it plays a role in cellular differentiation, proliferation, and death. Galectin-3 is also overexpressed in many forms of cancer, where it interacts with beta-galactosides on the surface of the transmembrane protein Mucin 1 (MUC1), which is also upregulated in many cancers. The interaction between MUC1 and galectin-3 leads to enhanced tumor formation, invasion, and metastasis. Glycodendrimers were synthesized and used as tools to probe the galectin- 3/MUC1 mediated cancer cellular aggregation. Cellular aggregation assays were designed and performed using three different cancer cell lines. Results indicate that cellular aggregation can either be inhibited or intensified depending on the size of the dendrimer and on the number of carbohydrate endgroups present. Confocal micrographs of cancer cells confirm that the pattern of galectin-3 on the outside of the cell is altered in the presence of glycodendrimers. These finding indicate that glycodendrimers mediate cellular aggregation properties, giving insight into the mechanism of action. The results also establish glycodendrimers as potential targeting agents and prodrug delivery systems when considering cancer drug design. A dendrimer bearing a matrix metalloproteinase (MMP) substrate was synthesized to explore dendritic potential as drug delivery systems. Results show that the substrate can be cleaved from the dendrimer when incubated with cancer cells known to overexpress MMP, indicating a potential for glycodendrimers as a prodrug carrier. A novel method for glycosylation was also explored. Indium(III) provides many advantages over standard glycosylation promoters (such as BF 3 x OEt 2) in that it is not air-or water-sensitive, and there is no need for additional purification of In(III) before use. Carbohydrate donors with a variety of protecting groups were tested against a range of glycosyl acceptors, including a carbohydrate. Results show that using In(III) as a glycosylation promoter gives comparable yields in less time when compared to a common glycosylation promoter.Item 67 kDa metastasis-associated laminin binding protein : substrate recognition, catalytic activity and homology modeling(Montana State University - Bozeman, College of Letters & Science, 2002) Kazmin, Dmitri AlexandrovichItem Heterologous expression of laminin peptide 11 on a virus particle surface for use in malignant tumor cell targeting(Montana State University - Bozeman, College of Letters & Science, 2000) Arnold, Thomas DarmodyItem Novel compounds inhibiting HIV infection, breast cancer metastasis, and bacterial growth(Montana State University - Bozeman, College of Letters & Science, 2012) Shepard, Joyce Brewer; Chairperson, Graduate Committee: Martin Teintze; Royce A. Wilkinson, Jean R. Starkey and Martin Teintze were co-authors of the article, 'Novel compounds containing multiple guanide groups that inhibit breast cancer metastases' in the journal 'International journal for cancer research' which is contained within this thesis.; Royce A. Wilkinson was a main author, and Cassidy Cooper, Sarah K. Walton, Amanda R. Radke, Robert L. Watkins, Thomas J. Wright, Elizabeth Erikson, Mohamed E. Labib, Jovanka M. Voyich, and Martin Teintze were co-authors of the article, 'Broad-spectrum antibacterial activity in novel compounds containing multiple phenylguanide or biguanide groups' in the journal 'Journal of antimicrobial chemotherapy' which is contained within this thesis.We synthesized novel guanide, biguanide, phenylguanide, and naphthyguanide derived compounds on linear, branched, and dendrimer backbones that are effective inhibitors of HIV infection, breast cancer metastasis, and bacterial growth. HIV utilizes CXCR4 as a co-receptor for cellular entry. Blocking CXCR4 inhibits infection with X4 strains of the virus. Initial competition assays demonstrated that some of the phenylguanide compounds bound to CXCR4 with high affinity. The derivatives with high CXCR4 affinity inhibited X4 viral infection, but did not inhibit R5 or X4R5 viruses. Importantly, many cancers overexpress CXCR4, including breast cancer. CXCR4 activation leads to cellular chemotaxis, angiogenesis, and cell survival, all of which promote cancer survival and proliferation. Compounds with high CXCR4 affinity were evaluated for inhibition of breast cancer metastasis. In vitro toxicity of all the derivatives was determined, followed by in vitro migration inhibition. Three derivatives with the best selectivity indexes for CXCR4 were examined in an in vivo lung colony metastasis assay. Spermidine trisphenylguanide (SI = 1785) was evaluated at 50 micrometers, 200 micrometers, and 300 micrometers and showed increasing inhibition of lung metastases (P = 0.34, 0.3, 0.02, respectively). Spermidine bis-2-naphthylguanide (SI = 1230) and spermine tris-2-naphthylguanide (SI = 191) were evaluated at 100 micrometers and showed significant reduction of lung metastases (P = 0.1 and 0.04, respectively). The topical antiseptic biguanide chlorhexidine, is structurally similar to our derivatives. So, the derivatives were tested for antimicrobial activity against drug susceptible and resistant pathogenic Enterococcus, Staphylococcus, Acinetobacter, and Pseudomonas strains. THAM trisphenylguanide, DNT2300 biguanide and phenylguanide, and DNT2200 phenylguanide demonstrated broad spectrum bacteriocidal activity similar to chlorhexidine. Preliminary in vivo studies on mice treated with THAM trisphenylguanide either immediately after methicillin resistant Staphylococcus aureus (MRSA) infection or an hour post MRSA infection showed significant reductions in bacterial burden in the intraperitoneal cavity, heart and kidney in the immediate treatment group and slight reductions of bacterial burden in the one hour treatment group. This data shows the potential for treatment of MRSA infections with the tested compounds. Thus, different subsets of the novel guanide compounds discussed here can inhibit HIV infection, breast cancer metastasis, and bacterial growth.Item Structural investigations of the cancer-associated laminin binding protein and Nos L : a novel copper binding protein(Montana State University - Bozeman, College of Letters & Science, 2005) Taubner, Lara Marie; Chairperson, Graduate Committee: Valerie CopieThis thesis consists of two distinct projects, one on the metastasis-associated laminin binding protein and the other on the putative copper chaperone NosL, both related by the common aim of investigation of the relationship between protein structure and function using nuclear magnetic resonance techniques. In the first part of this dissertation, the role that the metastasis-associated laminin binding protein or LBP plays in the spread and development of cancer was investigated. Functional domains of LBP were delineated by limited proteolysis, overexpressed, and then assayed for their ability to bind to the previously identified in vivo binding partner laminin. These assays demonstrated that, at least under the conditions used in this assay, binding to laminin was localized to domain 137-230, a region that encompasses a previously identified binding site known as Peptide G. This protein, like the full-length recombinant laminin binding protein, aggregated under conditions used for nuclear magnetic resonance experiments and therefore could not be analyzed with this technique. Contrary to previous findings on a synthetic peptide corresponding to residues 205-229, this sequence within the context of the 200-295 construct demonstrated no laminin binding activity. Furthermore, the peptide lacked the predicted alpha-helical content and tertiary structure as ascertained by nuclear magnetic resonance and by circular dichroism spectroscopy. A potential role for the disorder exhibited by this region of LBP is proposed, and suggests possible new functions for the laminin binding protein in angiogenesis. NosL, the subject of the second part of this thesis, is a highly conserved copper(I) binding lipoprotein encoded by the nitrous oxide reductase (nos) gene cluster of denitrifying bacteria. To identify functional features and structural homologues of this protein, the structure of apo NosL Was solved using nuclear magnetic resonance techniques. The high-resolution structure of NosL consists of one four-strand antiparallel beta sheet, one three strand antiparallel beta sheet and two alpha-helices organized in a twisted butterfly-like fold that is structurally homologous to MerB, an alkyl mercury lyase. Chemical perturbation mapping performed on the copper(I)=protein defined regions of NosL potentially involved in copper binding, and thus allowed preliminary identification of the copper-binding ligand Met 109.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.