Theses and Dissertations at Montana State University (MSU)
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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 Systems for studying the non-ubiquitous functions of the TATA-binding protein(Montana State University - Bozeman, College of Agriculture, 2003) Tucker, Tammy Alice; Chairperson, Graduate Committee: Edward E. Schmidt.Item Alpha-galactoside-binding proteins from plant membranes : distribution, function, and relation to helminthosporoside-binding proteins of sugarcane(Montana State University - Bozeman, College of Agriculture, 1978) Kenfield, Douglas SherwoodItem Identification and characterization of novel protein-protein interactions with the basal transcription factor, TATA-binding protein(Montana State University - Bozeman, College of Agriculture, 2006) Prigge, Justin Robert; Chairperson, Graduate Committee: Ed Schmidt.Recruitment of the TATA-Binding Protein, TBP, to the promoter is a critical, rate-limiting step that drives the initial phase of nearly all gene transcription events. Because of this, many transcriptional regulators target TBP, either to localize TBP at the promoter, or to relay signals between other promoter-bound protein complexes and the basal transcription machinery. Studies described herein were designed to identify novel protein-protein interactions with TBP. To do this, we screened mid-gestational pregnancy-associated cDNA prey libraries using two different yeast two-hybrid systems. Screens in both systems revealed both known and novel TBP interactors. B'-Related Factor 1 and Transcription Factor II A were identified in screens that used full-length TBP as bait. These proteins are known to interact with TBP and thus validated our system. In addition to known interactors, novel interactions with both the N-terminal (TBP-N) and C-terminal (TBP-C) domains of TBP were identified. Coactivator- Associated Arginine Methyltransferase 1 (CARM1), Pax Transactivation Domain- Interacting Protein (PTIP), and Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Factor (LITAF) all interacted with TBP-N. Proteins that interacted with TBP-C were Huntingtin-Associated Protein 1 (HAP1), four members of the Protein Inhibitor of Activated STAT (PIAS) family of transcriptional regulatory proteins, and Zinc Finger Protein 523 (ZFP523). The TBP interaction domains on PIAS1 and HAP1 were mapped to further define each interaction. Mapping studies revealed that TBP interacts with a single region on PIAS1, and with two separate regions on HAP1. We also show that TBP co-precipitates with PIAS1, PIAS3, HAP1, and PTIP. In conclusion, our studies, in agreement with previous published data suggest that TBP interacts with many classes of regulatory proteins, including transcriptional activators, repressors, and individual components of the transcriptional co-regulatory complexes. They also support the hypothesis that the TBP N-terminus is a protein interaction module and may provide clues to the function of the vertebrate-specific N terminus of TBP.Item Loss of the murine TATA-binding protein N terminus leads to placental labyrinth defects but not maternal adaptive immune responses(Montana State University - Bozeman, College of Agriculture, 2007) Sealey, Amy Lynn; Chairperson, Graduate Committee: Edward E. Schmidt.The TATA-binding protein (TBP) is a component of the basal transcription machinery. The TBP C-terminal core is conserved in all eukaryotes, but the N terminal region is shared between vertebrates. To study this, we generated a mouse line lacking 111 of the 135 amino acids of the vertebrate-specific sequence. The mutant tbp allele was designated tbp?N, and the majority of homozygous mutants, tbp?N/?N, died at midgestation due to defects in the placenta. Previous studies in our laboratory showed that tbp?N/?N fetuses survived the midgestational crisis if the mother was severely immunocompromised or if mutant fetuses were supplied tbp+/+ placentas. From these data, we hypothesized that the TBP N terminus regulates placental activity that is required for tolerizing the maternal immune system to the conceptus. Recent histological analysis of embryonic day 8.5 (E8.5) to E12.5 placentas in immune wildtype mothers revealed that the tbp?N/?N placental vascular region, or labyrinth, does not develop.Item Molecular and systemic functions of the vertebrate-specific TATA-binding protein N terminus(Montana State University - Bozeman, College of Agriculture, 2009) Lucas, Olivier; Chairperson, Graduate Committee: Edward E. Schmidt; Michael W. White (co-chair)The invertebrate/vertebrate transition and associated innovations can be regarded as a major event in evolution. Recent molecular progresses invite to an analysis of the events leading to the apparition of vertebrates and the underlying embellishment in gene regulation. In eukaryotes, the TATA-Binding Protein (TBP) has a central role in transcription initiation of most genes. TBP is comprised of a highly conserved DNA binding domain and, in vertebrates, it also contains a novel region: the N-terminal TBP protein coding sequence. The role of the TBP-N is largely unknown, but previous studies suggest that it is important for fetal survival. Most animals lacking the TBP-N (tbp Delta N/Delta N) die before weaning. The goal of the present work was to establish a deeper knowledge of the vertebrate-specific TBP-N. It was hypothesized that TBP-N could be involved in protein-protein interactions and that the high degree of similarity of TBP-N protein sequences in different species could correlate with similar functions. To test those hypotheses, two independent approaches were taken: (1) Protein-protein interactions involving the TBP-N via unbiased screens were characterized. (2) The mouse TBP-N was replaced by a similar and homologous TBP-N, in vivo, through homologous recombination. The TBP-N-replacement mutation was characterized through pathway analyses, bioinformatics, and whole-animal physiology. Screens for proteins interacting with the TBP-N of hagfish (hf), a basal vertebrate, uncovered hfPitxA. The Pitx family of transcription factors are proteins important in vertebrate development. The mouse paralogs of hfPitxA, Pitx1 and Pitx2, were found to interact with the mouse TBP-N. Moreover, the interaction appeared functional as it regulated the expression of nppa, a known target gene of Pitx2. In vivo replacement of the mouse TBP-N with the similar hfTBP-N did not affect the survival. Gene expression analysis indicated that lipid metabolism pathways were affected in animals lacking the TBP-N or when the hfTBP-N was present. Further analyses pointed toward a potential defect in insulin response and an abnormal hepatic fat storage. The data presented here argues in favor of an important role for TBP-N in vertebrate-specific gene regulation. More specifically, it is likely involved in heart development and in regulation of lipid metabolism.