MOORE, COURTNEY MARIE (The University of Arizona., 2016)
A goal of diabetes therapeutics is to deliver agents that target insulin secreting pancreaticβ-cells with high specificity while leaving other cells unaffected.Linking two ligands for two different receptors promotes binding to cells expressing the complementary receptors, thereby increasing the specificity for the target cells. Both Glucagon LikePeptide 1 (GLP-1) and Cholecystokinin (CCK) receptors are expressed on the surface of pancreatic β-cells. A ligand composed of GLP-1 linked to CCK-6 was produced and evaluated for its effect on β-cell insulin secretion. A combination of unlinked monomers activates insulin secretion at 100 nM in the absence of stimulatory glucose, an effect similar to CCK alone. However, the GLP-1/CCK-6 dimer potentiated glucose stimulated insulin secretin (GSIS) at low concentrations with higher sensitivity than the combined monomers.These findings suggest that the GLP-1 effect on GSIS(potentiation) is retained in the bivalent ligand, but the CCK effect may be attenuated or shifted in efficacy. The observed enhanced sensitivity to potentiate GSIS indicates that the binding affinity of the GLP-1/CCK-6 is increased via bivalent interactions as expected. Since bivalent binding requires the expression of both complementary receptors, this also suggests that the GLP-1/CCK-6 may have enhanced specificity for β-cells.
Schnoebelen, Carly Lynn (The University of Arizona., 2013)
In general chemistry classes nationwide, peer-led team learning (PLTL) has proven to be an effective teaching method. The general chemistry class in this study, Chemical Thinking at the University of Arizona, uses a modified form of PLTL where preceptor-led sessions supplement lectures to help facilitate student learning. Yet aside from the individual experiences of students and preceptors, it is not well known how preceptors interact with students to help them learn in this model. To investigate this, we video-recorded preceptors interacting with students during weekly review sessions. We characterized many of the teaching strategies used by preceptors and identified an evolution in the use of these strategies as a preceptor gained experience. This evolution supports current research in learning progressions, which suggests that conceptual understanding develops over time. It is important for preceptors to possess a strong conceptual understanding of chemistry as well as teaching strategies specific to the course content, and our research indicates that both of these develop through experience. The results of this study are being used to implement a training program aimed at accelerating the development of teaching skills for new preceptors.
The translation of messenger RNA (mRNA) into proteins is a central process of all cells. The initiation of this process is highly orchestrated, involving many different players, such as initiation factors, helicases and the ribosome components. Ded1 is an RNA helicase and member of the DEAD-box RNA helicase family. This protein plays key roles in resolving 5' UTR secondary structures and recruiting ribosomal components. While Ded1’s role in translational initiation is well-studied, its role in translational repression during stress is unclear. Previous work has displayed that Ded1 physically interacts with the eukaryotic initiation factor 4G (eIF4G) in yeast, a protein serving as the nucleation point for the pre-initiation complex (PIC). Here, we report the creation of eIF4G1 mutants deleting the RNA-2 and RNA-3 domains of this protein, which have been reported binding sites for Ded1. Deletion of both domains failed to phenocopy ded1 mutants that displayed resistance to rapamycin, a drug that causes translational repression. We also show that eIF4G1 co-immunoprecipitates with Ded1 after deletion of these regions, but their interaction is weakened. Further, treatment with RNAse A indicates that the interaction between these two proteins is not RNA-mediated but is instead protein-protein binding. This suggests that eIF4G1's RNA-2 and RNA-3 domains are not the sole binding regions for Ded1 but are involved. Our results indicate that these mutations do not cause defects in translational repression and that the in vivo interaction of these two proteins may be more complex than the in vitro interactions observed.
Multivalent ligands serve as a
promising diabetes treatment option for
delivery of therapeutic agents directly to
pancreatic ?-cells for restoration of function.
Linking multiple ligands for different
receptors promotes binding to cells that
express the receptor combination
complimentary to the ligands, thus achieving
specificity for the target cell. Three
receptors that are specific for pancreatic ?-
cells are glucagon-like peptide-1 (GLP-1),
cholecystokinin (CCK), and ?2-adrenergic
receptors. For this study, a dimeric ligand
composed of GLP-1 and CCK6 and a
trimeric ligand composed of GLP-1, CCK6,
and yohimbine (Yhm), an ?2-AR
antagonist, were designed and evaluated for
their ability to effect insulin secretion. It
was found that the GLP-1/CCK6 dimer
potentiates GSIS at lower concentrations but
its maximal response is lower than that of
the combination of monomers. Overall,
these effects of the dimeric ligand compared
to its constituent monomers were not large
indicating that it would not provide high
specificity and affinity for pancreatic ?-
cells. The GLP-1/CCK6/Yhm trimer
significantly increased insulin secretion at
concentrations >1 nM while the monomers
had a negligible effect until >50 nM. The
trimer also exhibited a 2-site activity curve
with EC50 values of 6.24 nM (0.1 nM to 25
nM) and 83 nM (10nM to 250 nM). These
results indicate the trimeric ligand is a
favorable candidate for an effective ?-cell
Rounseville, Benjamin James (The University of Arizona., 2018)
To better study the role of a specific kinase and bypass the well-known challenge of
selectively targeting kinases, we designed a ligand-gated split-protein system where two halves
of a kinase are reassembled via the addition of a Chemical Inducer of Dimerization (CID) with
concomitant activation of the enzyme. This was done by splitting the catalytic domain of
tyrosine kinase Src into two inactive halves, and linking each to a copy of E. coli dihydrofolate
reductase (eDHFR). Trimethoprim (TMP) is a tight and selective ligand for eDHFR, and in the
presence of a di-TMP ligand, the previously mentioned Src-eDHFR halves dimerize, achieving
reassembly into a functional kinase with measureable activity.
To demonstrate the use of our Src-eDHFR system in cellulo, we expressed the splitkinase
in mammalian cells and measured overall phosphorylation via western blotting. We have
shown that the activity of our Src-eDHFR system is dependent on the presence of the di-TMP
ligand. In addition, we have shown that two well identified mutations (K298M and T341M)
behave the same way in our split system as they do in the native, unaltered protein. Overall, our
eDHFR-Src system seems able to replicate the function of Src in cellulo, giving future
researchers a useful tool to elucidate the role Src plays in different cellular processes.
Nguyen, Celina Tien (The University of Arizona., 2017)
A mosquito-borne virus endemic to the tropics and subtropics, dengue (DENV) is the most common arbovirus in the world. Among other obstacles, an incomplete understanding of specific host-virus protein-protein interactions (PPI) challenges the development of vaccines and antiviral therapies for DENV. Previous studies on DENV PPI depend on genetic modifications, which can be tedious and time-consuming to accomplish. Mixing the principles of chemical biology and virology, the work in this thesis aims to take advantage of chemical modifications on DENV to pull down DENV-host PPI. First, this thesis will present the chemical modification of the DENV E glycoprotein via the metabolic incorporation of an unnatural sugar. This modification seemed to produce a morphology change in the viral structure, rendering it an impractical chemical modification. Then, this thesis will introduce an attempt to use bioorthogonal chemical modifications to DENV to pull down an established DENV PPI with an antibody as a proof-of-concept experiment to demonstrate the possibility of using bioorthogonal chemistry for identifying DENV PPI. Currently, this experiment is stalled by the presentation of background labelling. In the future, we hope to optimize the pull-down proof-of-concept and later use the same protocol to identifying PPI in DENV-infected mosquitoes.
McLeod, Kaitlyn Renee (The University of Arizona., 2015)
The melanocortin system is comprised of 5 homeostatic G-protein coupled receptors, which regulate diverse physiological functions including skin pigmentation, erectile dysfunction, and feeding behavior. Melanocortin peptides have been applied to MC4Rs to control feeding behavior and MC1Rs to control skin pigmentation and melanoma progression. Orexin receptors, like MC4R, are expressed in the hypothalamus and affect feeding behavior, suggesting a complex relationship between orexin and melanocortin signaling. Competitive ligand binding and cAMP accumulation assays showed no stimulation of intracellular melanocortin signaling by orexin peptides, making direct cross talk between these receptors unlikely. Melanocortin peptides have also been applied to the treatment of melanoma, a cancer plagued by poor diagnosis and prognosis. Previous experimentation showed Rad-tagged NDP-α-MSH selectively binding melanoma tumors in vivo; repetitive treatments with Rho-MT-II, a non-specific melanocortin agonist, resulted in melanoma tumor shrinkage¹. In vitro, MTT colorimetric assays showed greater cell death when A375 melanoma cells were treated with an MC1R antagonist rather than an agonist. However, induction of hypotonic stress and apoptosis through simultaneous treatment with BzATP and MC1R agonist resulted an increased cell death. These results mark an important step towards developing targeted melanoma treatments.
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