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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 NMR hydrophilic metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium antimicrobials in Escherichia coli and Bacillus cereus exposed to DABCO and mannose functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2021) Aries, Michelle Lynne; Chairperson, Graduate Committee: Mary J. Cloninger; This is a manuscript style paper that includes co-authored chapters.Novel antibiotics developed using a new scaffold are needed to combat the rising tide of antibiotic resistant bacteria. Multivalent antibiotics are a relatively new approach that have the potential to greatly increase the efficacy of antibiotics while making it difficult for bacteria to develop resistance. Dendrimers are an attractive framework for the multivalent presentation of antibacterial moieties. Quaternary ammonium compounds (QAC) are a positively charged class of membrane disruptors that are attracted to the large negative charge on phospholipid membranes. Nuclear magnetic resonance (NMR) metabolomics is a quantitative method used for comparison of metabolic profiles of wild type and mutated bacterial samples, enabling the study of bacterial response to antimicrobials. Proton (1 H) NMR hydrophilic metabolomics was used to study gram-negative and gram-positive bacteria upon exposure to 1,4-diazabicyclo-2,2,2-octane (DABCO) with a 16-carbon chain tethered onto a mannose functionalized poly(amidoamine) (PAMAM) dendrimer (denoted as DABCOMD), a membrane disrupting multivalent QAC. Stock Escherichia coli (E. coli) (denoted as wild type) and DABCOMD mutated E. coli (denoted as mutants) were collected in the mid log and stationary phases. The same procedures were used for Bacillus cereus (B. cereus) as for E. coli samples (denoted as unchallenged), except that a DABCOMD challenged sample set was added (denoted as challenged). The challenged sample set procedures were identical to the unchallenged, except DABCOMD was included at 33 % of the MIC value in the growth media for growth curve acquisition and sample collection. The greatest differences observed between the metabolic profiles of the wild type and mutated E. coli samples and between the challenged and unchallenged B. cereus samples were in energy-associated metabolites and membrane-related pathways. The mutants in all sample types were associated with higher levels of spent energy molecules (including AMP and NAD+) and peptidoglycan related compounds (including N-acetylglucosamine). Overall, more changes were observed for B. cereus (gram-positive), especially in challenged mutant B. cereus samples, than for E. coli (gram-negative) samples. Since DABCOMD is a positively charged multivalent membrane disruptor, both B. cereus and E. coli mutated to garner protection by altering their peptidoglycan layer composition, which is energetically costly.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 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 synthesis of N-acetyllactosamine functionalized dendrimers, and the functionalization of silica surfaces using tunable dendrons and beta-cyclodextrins(Montana State University - Bozeman, College of Letters & Science, 2017) Ennist, Jessica Helen; Chairperson, Graduate Committee: Mary J. Cloninger; Mary J. Cloninger was a co-author of the article, 'The synthesis of N-acetyllactosamine functionalized dendrimers and their role in galectin-3 mediated cancer cellular aggregation studies' which is contained within this thesis.; Eric A. Gobrogge, Kristian H. Schlick, Robert A. Walker and Mary J. Cloninger were co-authors of the article, 'Cyclodextrin-functionalized chromatographic materials tailored for reversible adsorbtion' in the journal 'ACS applied materials and interfaces' which is contained within this thesis.Galectin-3 is beta-galactoside binding protein which is found in many healthy cells. In cancer, the galectin-3/tumor-associated Thomsen-Friedenreich antigen (TF antigen) interaction has been implicated in heterotypic and homotypic cellular adhesion and apoptotic signaling pathways. However, a stronger mechanistic understanding of the role of galectin-3 in these processes is needed. N-acetyllactosamine (LacNAc) is a non-native ligand for galectin-3 which binds with comparable affinity to the TF antigen and therefore an important ligand to study galectin-3 mediated processes. To study galectin-3 mediated homotypic cellular aggregation, four generations of polyamidoamine (PAMAM) dendrimers were functionalized with N-acetyllactosamine using a four-step chemoenzymatic route. The enzymatic step controlled the regiochemistry of the galactose addition to N-acetylglucosamine functionalized dendrimers using a recombinant beta-1,4-Galactosyltransferase-/UDP-4'-Gal Epimerase Fusion Protein (lgtB-galE). Homotypic cellular aggregation, which is promoted by the presence of galectin-3 as it binds to glycosides at the cell surface, was studied using HT-1080 fibrosarcoma, A549 lung, and DU-145 prostate cancer cell lines. In the presence of small LacNAc functionalized PAMAM dendrimers, galectin-3 induced cancer cellular aggregation was inhibited. However, the larger glycodendrimers induced homotypic cellular aggregation. Additionally, novel poly(aryl ether) dendronized silica surfaces designed for reversible adsorbtion of targeted analytes were synthesized, and characterization using X-ray Photoelectron Spectroscopy (XPS) was performed. Using a Cu(I) mediated cycloaddition 'click' reaction, beta-cyclodextrin was appended to dendronized surfaces via triazole formation and also to a non-dendronized surface for comparison purposes. First generation G(1) dendrons have more than 6 times greater capacity to adsorb targeted analytes than slides functionalized with monomeric beta-cyclodextrin and are 2 times greater than slides functionalized with larger generation dendrons. This study reported beta-cyclodextrin functionalized surfaces can undergo a triggered release of the adsorbent, but otherwise retained the targeted analyte through multiple aqueous washes. Therefore, a new generation of G(1) dendronized surfaces capable of reversible adsorption were developed by heterogeneously appending sulfonic acid/pyridine end-groups. Auger Electron Spectroscopy (AES) was used to quantify the ratio of groups installed. Furthermore, G(1) dendronized surfaces were functionalized homogenously with sulfonic acid and pyridine for comparison and with chiral amino acids for chiral recognition studies.Item The synthesis and study of TF antigen functionalized dendrimers and dendrimer end group characterization and indium(III) promoted glycosylation(Montana State University - Bozeman, College of Letters & Science, 2017) Mattson, Amanda Lynn; Chairperson, Graduate Committee: Mary J. Cloninger; Anna K. Michel and Mary J. Cloninger were co-authors of the article, 'Using indium(III) as a promoter for glycosylation' in the journal 'Carbohydrate research' which is contained within this thesis.Polyamidoamine (PAMAM) dendrimers were functionalized with the Thomsen-Friedenreich (TF) antigen to study the multivalent effects on the galectin-3 mediated homotypic aggregation of A549 cells. TF antigen functionalized dendrimers of generations 2, 3, 4, and 6 were found to induce cellular aggregation. This is in contrast with previously observed results using lactose functionalized dendrimers, in which lactose functionalized generation 2 dendrimers were able to inhibit cellular aggregation. Additionally, TF antigen functionalized generation 6 dendrimers did not induce cellular aggregation as effectively as lactose functionalized generation 6 dendrimers. These preliminary results suggest that when compared to lactose functionalized dendrimers, the stronger galectin-3 binding affinity for TF antigen dendrimers may allow for more galectin-3 recruitment, creating aggregates with less freedom to rearrange into an optimized conformation. This suggests the reversibility of the binding event is important for effective protein interactions. The synthesis of TF antigen was achieved using indium triflate catalyzed glycosylation reactions. The development of indium(III) as a glycosylation promoter involved the analysis of indium bromide, indium chloride, and indium triflate for use in glycosylation reactions with a variety of alcohol acceptors. In(OTf) 3 mediated glycosylations of acetonide protected mannosides afforded exclusively alpha products in high yields. Acetylated mannosides gave moderate yields of exclusively alpha products using InBr 3 and InCl 3. Benzylated galactosides gave moderate yields of alpha, beta product mixtures using In(OTf) 3, with beta products formation being favored. Indium(III) was also used to synthesize alpha-1-2-dimannoside and alpha-galactose-1-2-mannoside in high yield. Additionally, spin labelled PAMAM dendrimers were preliminarily studied via electron paramagnetic resonance (EPR) to analyze the spatial arrangement of end groups. Dendrimers were functionalized with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) covalently tethered as a dimer. At low percent loading, a strong effect arising from the dimeric spin-labeled end groups was observed. EPR spectra of dendrimers bearing a higher loading of the dimeric spin-labeled end groups indicated that the end group arrangement approached a random distribution at approximately 40 to 50 percent loading. This suggests that covalently clustered pairs of end groups are significantly different from randomly distributed end groups on PAMAM dendrimers at low loading and become equivalent to randomly functionalized dendrimers around 50% functionalization.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 Effect of monomeric binding affinity on scaffold mediated protein aggregation(Montana State University - Bozeman, College of Letters & Science, 2015) Goodman, Candace Kay; Chairperson, Graduate Committee: Mary J. CloningerThe intermolecular interactions that occur in a system determine the degree and duration of the contact. They govern processes from signaling and recognition to aggregation and tumor formation. The ability to control and affect intermolecular processes requires an understanding of the assembly process and factors modulating the assembly, such as the strength of individual interactions (binding affinity) and the number of interactions between molecules (valency). Functionalized PAMAM dendrimers were used as nucleating scaffolds to study the significance of intermolecular interactions on aggregate assembly. Dendrimers functionalized with biotin, lactose and mannose units spontaneously aggregated when added to the appropriate protein binding partner (streptavidin, galectin-3, and Concanavalin A, respectively). Aggregates were characterized to provide insight regarding the effects of binding affinity, protein valency and concentration on the average diameter, regularity (polydispersity) and kinetics of aggregate formation. A number of tools were used in this investigation, including dynamic light scattering (DLS), fluorescence microscopy (FM) and fluorescence lifetime spectroscopy (FLS). FLS instrumentation was reconfigured to enable high thoughput formats. A discussion of the validation and re-design of the FLS instrumentation is included.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 Design and synthesis of new nonlinear optical chromophores incorporating diphenylphosphino groups and their incorporation into electroactive dendrimers(Montana State University - Bozeman, College of Letters & Science, 2000) Madrigal, Luis Gustavo