Browsing by Author "Kennan, Alan, advisor"
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Item Open Access Developments in automated electrochemical biosensors to improve point of care diagnostics(Colorado State University. Libraries, 2022) Schenkel, Melissa, author; Kennan, Alan, advisor; Henry, Charles, committee member; Snow, Christopher, committee member; Ross, Eric, committee memberThe onset of the COVID-19 pandemic brought public attention to the pre-existing need for developments in diagnostics, especially at the point of care. While traditional techniques, like PCR, can be highly sensitive and specific, they are also time consuming, expensive, and require trained personnel in a laboratory setting and expensive equipment. The need for point of care diagnostic options was made evident in early 2020 when laboratories could not keep up with the high demand for COVID-19 testing. Lateral flow assays (LFAs) like home pregnancy tests offer a platform that is inexpensive, easy to use, and can produce results rapidly at the point of care. Unfortunately, LFAs usually exhibit poor sensitivity and limits of detection compared to traditional techniques. Electrochemical biosensors can provide a diagnostic platform that is quick, cost effective, accurate, highly sensitive, and quantitative. While electrochemical biosensors incorporated in lateral flow devices have improved sensitivity, they typically require complex fabrication techniques, and the nitrocellulose platform can limit electrochemical performance. The Henry group has recently reported a new class of capillary-driven fluidic devices using alternating layers of patterned polyethylene terephthalate (PET) films and double-sided adhesives (DSA) that can control flow for sequential delivery of reagents. This work presents recent developments in automated electrochemical biosensors to improve point of care diagnostics. The incorporation of electrochemical biosensors with the aforementioned novel fluidic devices provides a diagnostic platform that has the potential to achieve the sensitivity and selectivity rivaling that of traditional techniques while maintaining the ease of use of an LFA. Chapter 2 of this dissertation first presents an electrochemical immunosensor for detection of SARS-CoV-2 N-protein. This sensor was then adapted and optimized for compatibility in a fluidic device. This included optimizing ease of functionalization with manufacturing-friendly techniques, exploring different buffers for assay steps, and optimizing assay components Ultimately, these studies led to automated, concentration-dependent detection of SARS-CoV-2 N-protein upon a single sample addition step. Chapter 3 of this dissertation presents a novel device design that improved flow rates, decreased device malfunctions, and incorporated commercial electrodes. This device was developed for measurements of C-reactive protein, a common biomarker of inflammation. Utilizing gold electrodes has the potential for more sensitive detection compared to carbon electrodes and aptamers as biological recognition elements provides many advantages as well. While work on this project is still underway, the results presented herein demonstrate the ability of this novel diagnostic device to be adapted for various analytes. Future work includes continued assay and device optimization, with the intent for multiplexed detection of multiple analytes. Overall, the work presented here provides a novel platform for point of care diagnostics and demonstrates its application to two different analytes.Item Open Access Engineering and evolving helical proteins that improve in vivo stability and inhibit entry of Enfuvirtide-resistant HIV-1(Colorado State University. Libraries, 2019) Walker, Susanne N., author; Kennan, Alan, advisor; Yao, Tingting, committee member; McNally, Andrew, committee member; Paton, Robert, committee memberMethods for the stabilization of well-defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Enfuvirtide is a 36-residue chemically synthesized helical peptide that targets the viral gp41 protein and inhibits viral membrane fusion. Enfuvirtide-resistant HIV, however, has been prolific, and this peptide therapy requires daily injection due to proteolytic degradation. In this dissertation I have developed a method for stabilizing helical peptide therapeutics termed helix-grafted display proteins. These consist of the HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these earlier protein biologics inhibit HIV-1 entry with modest and variable potencies (IC50 190 nM - >1 μM). After optimization of the scaffold and the helix, our designer peptide therapeutic potently inhibited HIV-1 entry in a live-virus assay (IC50 1.9-12.4 nM). Sequence optimization of solvent-exposed helical residues using yeast display as a screening method led to improved biologics with enhanced protein expression in Escherichia coli (E. coli, a common bio-expression host), with no appreciable change in viral membrane fusion suppression. Optimized proteins suppress the viral entry of a clinically-relevant double mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide-resistant. Protein fusions engineered for serum-stability also potently inhibit HIV-1 entry.Item Open Access Exploration of protein engineering methods towards biomaterials, therapeutic protein scaffolds, and localization detection within mammalian cells(Colorado State University. Libraries, 2019) Bjerke, Jennifer N., author; Kennan, Alan, advisor; Snow, Christopher, committee member; Williams, Robert M., committee member; Nyborg, Jennifer, committee memberProteins are large biomolecules entangled with complex chemistry that uniquely control the processes, function and efficiency of everyday life. These macromolecules are the final product of the central dogma, and as in any synthesis, the proper reactants must combine in order to produce the correct products. As technologies develop to understand their production and resulting structure, sequence and activities, they have increasingly become a bulk chemical platform in which nearly any desired application can be engineered- ranging from materials to therapeutics. The first half of this dissertation will discuss the potential to break down the central dogma in order to create new biological materials through incorporation of novel building blocks, known as amino acids. The second half will focus solely on the design and analysis of engineered proteins to create scaffolds for biological therapeutics that have the capability to bind almost any disease relevant target. The second chapter of this dissertation describes the synthesis of novel non-canonical amino acids that add new chemistries to any protein of interest. Using amber codon suppression, and careful engineering of tRNA synthetase and cognant tRNA's, these new amino acids can be site selectively incorporated. To start, derivatives of phenylalanine are selected since this machinery has been successfully implemented for many new amino acids. The synthesis of bithiophene explores the utilization of phase transfer catalysis and transition metal cross- coupling to deliver a racemic mixture of the non-canonical amino acid with a bithiophene moiety. The third chapter of this dissertation will discuss the engineering potential of nanobodies, a monomeric protein responsible for all binding interactions, isolated from the variable heavy chain of camelid antibodies. We had previously reported a cationic resurfacing strategy that endowed mammalian cell penetration in three nanobody scaffolds. We have since explored and refined this strategy, along with endowing the capability to bind new targets. In the end we found that, when fused to sfGFP, a polyarginine version (PolyR-NB) increased internalization over the original cationic GFP nanobody (CatNB), but the original CatNB scaffold was more adaptable to extensive mutagenesis. Therefore, the PolyR nanobody scaffold can be utilized to aid the internalization of therapeutic cargo proteins that would otherwise not be able to cross the lipid bilayer, and the CatNB scaffold can become a therapeutic itself, modified to bind any intracellular target of disease relevant proteins. To further prove the later point, the complimentary determining regions (CDRs) of a separate nanobody, the BC2 nanobody, were grafted upon the CatNB framework, keeping all resurfacing and structure intact. Remarkably, the CatNB-BC2 CDR nanobody was able to maintain binding to its wild type partner. The final chapter will showcase the efforts towards development of a facile luminescent assay that detects protein delivery to the cytosol of cells. Adapted from the NanoBit assay developed by Promega to identify protein-protein interactions, our assay utilizes the split nanoluciferase technology to produce a luminescent signal once an exogenous protein has recombined with its other half in the cytosol. This system, in theory, could be applied to identify endosomal release of virtually any therapeutically relevant protein.Item Open Access Preparation and investigation of unnatural polar sidechains in designed coiled-coils(Colorado State University. Libraries, 2008) Diss, Maria L., author; Kennan, Alan, advisorProgrammed self-assembly of discrete molecular species to form complex aggregates provides the opportunity to both refine and exploit current knowledge of molecular recognition patterns. Self-assembly events dominate the precise control and understanding of macromolecular structure, placing a premium on development and discovery of molecular recognition motifs. One such motif is the alpha helical coiled coil. A key natural strategy for controlling specific assembly is the burial of polar side chains (particularly asparagines) at central hydrophobic core positions. Alignment of the polar side chains opposite each other, rather than opposite hydrophobic alternatives, drives formation of the intended complex. Utilizing this polar contact, we sought to control coiled-coil formation through the use of unnatural polar side chains. These unnatural side chains included various chain length arginine derivatives and the corresponding urea analog, citrulline, along with its chain length variants. Synthetic methodology compatible with solid phase peptide synthesis was developed to form the desired functionalized primary amine side chain. Guanidinylation occurred in one step through the use of a di-2-Cl-Z protected pyrazole derivative. Urea formation also proceeded in one step through the use of a preformed p-methoxybenzyl-p-nitrophenyl carbamate. Deprotection to give the desired functional group occurred through standard cleavage conditions. With the establishment of the synthetic methodology, numerous peptides were synthesized incorporating these new polar groups into the hydrophobic core. Additionally, asparagine, aspartic acid, and glutamic acid were used as core residues. Heterodimeric mixtures of these sequences with guanidine, urea, amide and carboxylic acid binding partners form a large number of reasonably stable coiled coils (Tm ≥ 60 °C), allowing for application-specific tuning. A number of four-component selective recognition systems are also presented, in which two distinct heterodimers form from an input of four different peptides. More impressively, examples of six-component systems are demonstrated. Control mixtures establish subtle structural requirements for successful recognition. Having demonstrated successful recognition motifs within a dimeric system, a trimeric system has been investigated. Once again, these polar contacts allow for heterotrimerization. However, the relatively narrow stability range of these trimers does not allow for successful exchange experiments (Tm = 43 to 59 °C).Item Open Access Protein engineering strategy for the stabilization of HIV-1 α-helical peptides(Colorado State University. Libraries, 2019) Tennyson, Rachel Lee, author; Kennan, Alan, advisor; Ackerson, Christopher, committee member; Snow, Christopher, committee member; Gustafson, Daniel, committee memberMany disease-relevant protein-protein interactions (PPIs) contain an alpha helix and helical binding cleft at their interface. Disruption of these interactions with helical peptide mimics is a validated therapeutic strategy. However, short peptides typically do not fold into stable helices, which significantly lowers their in vivo stability. Researches have reported methods for helical peptide stabilization but, these approaches rely on laborious, and often expensive, chemical synthesis and purification. The research I have preformed aims to stabilize disease-relevant helices through protein engineering. In contrast to chemically constrained helical peptides, a protein can be expressed in a cellular system on a much larger scale. Recently, we reported a new strategy termed "helix-grafted display" that overcomes the traditional hurdles of helical mimics and applied it to the challenge of suppressing HIV entry. Our helix grafted proteins, potently inhibits formation of the extracellular PPI involving C-peptide helix, and HIV gp41 N-peptide trimer, as tested in HIV CD4+ cells. Further optimization of the helical sequence by yeast display yielded new proteins that suppress HIV-1 entry and express substantially better in E. coli. Furthermore, fusion proteins designed to improve the serum stability of these helix grafted proteins have been made that potently suppress HIV-1 entry. Collectively, I report a potential cocktail of evolved HIV-1 entry inhibitors that are functional against an Enfuvirtide-resistant strain and are designed for serum stabilities that rival current monoclonal antibody drugs.Item Open Access Studies concerning platinum-catalyzed 1,6-enyne cycloisomerizations: a unified synthetic approach to the Gelsemium alkaloids(Colorado State University. Libraries, 2015) Newcomb, Eric Thomas, author; Kennan, Alan, advisor; Ferreira, Eric, committee member; Prieto, Amy, committee member; McNaughton, Brian, committee member; Akkina, Ramesh, committee memberThe development and application of transition metal-catalyzed enyne cyclization reactions is an ever growing and active area of research in modern organic synthesis. One prolific class of catalysts studied in this broad arena is that of pi-acidic metal complexes. Through further understanding of the fundamental processes of these alkynophilic metal catalysts, we are able to test new transformations in more complex settings. Presented herein are our contributions to the understanding and further implementation of Pt-catalyzed alkyne activation chemistries. In particular, we have developed a chirality transfer protocol to synthesize highly enantioenriched O-tethered cyclopropane-containing compounds. The substrate scope for this process is broad, and the overall transformation is highly stereospecific. Additionally, we further refined a purported mechanistic pathway and extended this chemistry in a number of additional systems. Furthermore, we explored the use of this cycloisomerization chemistry in our synthetic approach to the Gelsemium alkaloids. Specifically, the development of a Pt-catalyzed tandem cycloisomerization/[3,3]-sigmatropic rearrangement allowed us to build a motif shared among a large number of the alkaloids. Following successful implementation of this reaction, we then studied the use of additional late-stage cyclizations to synthesize gelesenicine. Our final two steps, a highly efficient hypervalent iodine-mediated cyclization followed by an iminyl radical cyclization, provided the natural product. Additionally, the synthesis was highly efficient--14 steps--without the use of protecting groups.Item Open Access The application of new methodology to complex molecule synthesis: studies toward the synthesis of pordamacrine A and liphagal(Colorado State University. Libraries, 2015) Seizert, Curtis A., author; Kennan, Alan, advisor; Ferreira, Eric, committee member; Chen, Eugene, committee member; Prieto, Amy, committee member; Hansen, Jeffrey, committee memberThe coevolution of organic synthesis and methodology has contributed greatly to the growth of both fields. This has been enabled by the invention of new methods during the prosecution of a synthesis in order to solve an unforeseen problem as well as by the novel application of independently developed methods to complex synthetic settings. Our own studies have encompassed both of these strategies, and we present their results herein. Our initial efforts consisted of synthetic studies towards the complex hexacyclic alkaloid pordamacrine A. This molecule presented many difficulties, and we were forced develop and employ new methods in its synthesis. Ultimately, these studies were stymied by the difficulty of forming the central carbocyclic ring system of this molecule. Among the methods used in the synthesis of pordamacrine A was a variant of a previously reported boron promoted Ireland-Claisen rearrangement. This rearrangement has been reported in very few papers in the literature, and many details of the reaction were undisclosed at the outset of ourstudies. We report here our investigations of the scope and stereochemical features of this rearrangement. Finally, methods based on the use of Pt carbenoids have formed a central element in our group's research focus. We apply here the use of this intermediate to the synthesis of liphagal, a complex tetracyclic compound. Our explorations of Pt-catalyzed cycloaddition reactions based on Pt carbenoids in this study have shed valuable light on the scope of this method. Though our studies culminated in a formal synthesis of an epimer of the natural product, we expect that future work towards liphagal will be able to use this methodology to make the correct diastereomer of liphagal, potentially in enantioenriched form.Item Open Access Understanding the role of prion-like domains in ribonucleoprotein granule dynamics(Colorado State University. Libraries, 2019) Boncella, Amy Elizabeth, author; Ross, Eric, advisor; Kennan, Alan, advisor; Peersen, Olve, committee member; Ackerson, Chris, committee memberRibonucleoprotein (RNP) granules are membraneless organelles, comprised of RNA-binding proteins and RNA, that are integrally related with the cellular stress response. Stress granules and processing bodies (p-bodies) are the two primary types of RNP granules that reversibly assemble upon stress. Interestingly, many of the proteins that localize to stress granules and p-bodies contain aggregation-prone prion-like domains (PrLDs). Furthermore, mutations in the PrLDs of a number of stress granule-associated proteins have been linked to various neurodegenerative diseases, leading to the idea that aggregation-promoting mutations in these PrLDs cause stress granule persistence. Altogether, these finding suggest an important role for these domains in the dynamics of these assemblies. In order to gain a greater understanding of how PrLDs contribute to RNP granule biology, I have taken two different approaches. The first was to investigate how aggregation-promoting mutations affect stress granule and p-body dynamics. I introduced various aggregation-promoting mutations into the PrLDs of different stress granule and p-body proteins and assessed the ability of these granules to disassemble, hypothesizing that these mutations would cause RNP granule persistence, as is observed in disease. Interestingly, despite successfully increasing the aggregation propensity of these PrLDs, stress granules and p-bodies do not persist and can efficiently disassemble after stress relief. Given that aggregation-promoting mutations in PrLDs of RNP granule proteins fail to cause granule persistence, I took a second, less targeted approach towards understanding the roles of these domains in RNP granules. I focused on investigating how PrLDs are recruited to RNP granules by screening a set of PrLDs for ability to assemble into foci upon stress. Interestingly, many PrLDs are sufficient to assemble into foci upon various stresses, with robust recruitment to stress granules upon heat shock. Furthermore, several compositional biases are observed among PrLDs that are and are not sufficient to assemble upon stress. Using these biases, we have developed a reasonably accurate composition-based predictor of PrLD recruitment into heat shock-induced stress granules, which has been further validated using rational mutation strategies. This predictor is reasonably successful at predicting whether a PrLD will assemble into stress granules upon stress. Additionally, scrambling of PrLD sequences does not disrupt recruitment to stress granules. Together, these results suggest that PrLD localization to stress granules is based on composition rather than primary sequence.