Browsing by Author "Geiss, Brian, committee member"
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Item Open Access Allostery of the flavivirus NS3 helicase and bacterial IGPS studied with molecular dynamics simulations(Colorado State University. Libraries, 2020) Davidson, Russell Bruce, author; McCullagh, Martin, advisor; Bernstein, Elliot, committee member; Barisas, George, committee member; Geiss, Brian, committee memberAllostery is a biochemical phenomenon where the binding of a molecule at one site in a biological macromolecule (e.g. a protein) results in a perturbation of activity or function at another distinct active site in the macromolecule's structure. Allosteric mechanisms are seen throughout biology and play important functions during cell signaling, enzyme activation, and metabolism regulation as well as genome transcription and replication processes. Biochemical studies have identified allosteric effects for numerous proteins, yet our understanding of the molecular mechanisms underlying allostery is still lacking. Molecular-level insights obtained from all-atom molecular dynamics simulations can drive our understanding and further experimentation on the allosteric mechanisms at play in a protein. This dissertation reports three such studies of allostery using molecular dynamics simulations in conjunction with other methods. Specifically, the first chapter introduces allostery and how computational simulation of proteins can provide insight into the mechanisms of allosteric enzymes. The second and third chapters are foundational studies of the flavivirus non-structural 3 (NS3) helicase. This enzyme hydrolyzes nucleoside triphosphate molecules to power the translocation of the enzyme along single-stranded RNA as well as the unwinding of double-stranded RNA; both the hydrolysis and helicase functions (translocation and unwinding) have allosteric mechanisms where the hydrolysis active site's ligand affects the protein-RNA interactions and bound RNA enhances the hydrolysis activity. Specifically, a bound RNA oligomer is seen to affect the behavior and positioning of waters within the hydrolysis active site, which is hypothesized to originate, in part, from the RNA-dependent conformational states of the RNA-binding loop. Additionally, the substrate states of the NTP hydrolysis reaction cycle are seen to affect protein-RNA interactions, which is hypothesized to drive unidirectional translocation of the enzyme along the RNA polymer. Finally, chapter four introduces a novel method to study the biophysical coupling between two active sites in a protein. The short-ranged residue-residue interactions within the protein's three dimensional structure are used to identify paths that connect the two active sites. This method is used to highlight the paths and residue-residue interactions that are important to the allosteric enhancement observed for the Thermatoga maritima imidazole glycerol phosphate synthase (IGPS) protein. Results from this new quantitative analysis have provided novel insights into the allosteric paths of IGPS. For both the NS3 and IGPS proteins, results presented in this dissertation have highlighted structural regions that may be targeted for small-molecule inhibition or mutagenesis studies. Towards this end, the future studies of both allosteric proteins as well as broader impacts of the presented research are discussed in the final chapter.Item Open Access Assessing and understanding the generation and function of RNA decay intermediates in non-insect borne flaviviruses(Colorado State University. Libraries, 2019) Mundell, Cary T., author; Wilusz, Jeffrey, advisor; Geiss, Brian, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee memberCellular gene expression is an intricate process regulated on many levels that allows the cell to react correctly to stimuli or to maintain homeostasis. RNA viruses must act to preferentially drive production of their own messenger RNAs (mRNAs) and proteins in order to successfully replicate and ensure continued infection. Due to the necessity for RNA viruses to remain in the cytoplasm, regulatory factors that affect host mRNAs likely also affect the transcripts of RNA viruses. RNA decay represents a major pathway of regulation for mRNAs. A multitude of RNA viruses possess unique mechanisms that act to prevent the decay of viral transcripts and allow for successful translation. Members of the viral family Flaviviridae are positive sense, single-stranded RNA viruses that do not possess a poly(A) tail. Therefore, it is highly likely that these transcripts would be marked as deadenylated and shuttled down one of the RNA decay pathways that exist in the cell. Interestingly, members of the genera Flavivirus of the family Flaviviridae possess a conserved structured 3' untranslated region (UTR) that acts to interfere with the decay processes of the major cytoplasmic cellular 5'-3' decay enzyme XRN1. In addition, members of the generas Hepacivirus, Hepatitis C Virus (HCV) and Pestivirus, Bovine Viral Diarrhea Virus (BVDV), possess XRN1 stalling elements within their 5' UTRs. These stalling sites block the action of the exonuclease and generate decay intermediates. The generation of these decay intermediates represses XRN1 activity in the infected cell. Herein we demonstrate a new method for studying RNA decay through the use of XRN1-resistant RNAs (xrRNAs). In this method we utilize the well characterized xrRNA of Dengue Virus Type 2 (DENV2) as a readout to study the decay rates of relatively large RNA constructs. We show that not only is utilizing an xrRNA an effective method for confirming XRN1-mediated decay, but that the accumulation of the readout xrRNA can be utilized to understand changes in the decay kinetics of RNA substrates. We further utilize this method to demonstrate a lack of XRN1 stalling elements within the poliovirus internal ribosomal entry site (IRES) element. We provide evidence that the stalling of XRN1 in the 5' UTR of BVDV is dependent on both the presence of the entire IRES structure and the presence of a stem loop 5' to the IRES element through the analysis of a series of truncations. Finally, we demonstrate one possible role for the HCV and BVDV decay intermediates as the truncated IRES element maintains translatability in an in vitro system. Collectively, these data better define the structural requirements for the novel XRN1 stalling elements located in the 5' UTR of non-insect borne members of the Flaviviridae as well as the potential function of the decay intermediates.Item Open Access Automated sample preparation using adaptive digital microfluidics for lab-on-chip devices(Colorado State University. Libraries, 2018) Grant, Nicholas, author; Chen, Thomas W., advisor; Chong, Edwin, committee member; Geiss, Brian, committee memberThere have been many technological advances in the medical industry over the years giving doctors and researchers more information than ever before. Technology has allowed more sensitive and accurate sensors and has also driven the size of many sensor devices smaller while increasing sensitivity. However, while many aspects of technology have seen improvements, the sample preparation of biological tests has seen lagging development. The sample preparation stage is defined here as the extracting of required features from a given sample for the purpose of measurement. A simple example of this is the solid phase extraction of DNA from a blood sample to detect blood borne pathogens. While this process is common in laboratories, and has even been automated by large and expensive equipment, it is a difficult process to mimic in lab-on-chip (LoC) devices. Nucleic Acid isolation requires common bench top equipment such as pipettes, vortexers, and centrifuges. Current lab based methods also use relatively large amounts of reagents to perform the extraction adding to the cost of each test. There has been a lot of research improving sensing techniques proposed for Lab on Chip devices, but many sensing methods still require a sample preparation stage to extract desired features. Without a complimentary LoC sample preparation system, the diversity of LoC device remains limited. The results presented in this thesis demonstrate the general principle of digital microfluidic device and the use of such device in a small hand-held platform capable of performing many sample preparation tasks automatically, such as the extraction and isolation of DNA. Liquids are transported using a technique called Eletro-wetting on Dielectric (EWOD) and controlled via a programmable microprocessor. The programmable nature of the device allows it to be configured for a variety of tests for different industries. The device also requires a fraction of the liquids lab based methods use, which greatly reduces the cost per test. The results of this thesis show a promising step forward to more capable LoC devices.Item Open Access Design, fabrication and testing of an electrically controlled microfluidic capillary microvalve based on hydrophobicity(Colorado State University. Libraries, 2019) Kulkarni, Gitesh S., author; Chen, Thomas W., advisor; Chong, Edwin K. P., committee member; Geiss, Brian, committee memberMicrofluidics is a promising disciple that combines "micro" amount of fluid handling in "micro" sized channels and has found applications in diverse fields such as biotechnology and environmental monitoring. Combination of microfluidics with digital electronics technology has spurred creation of Lab-on-a-Chip (LOC) devices that are field-deployable and bought to market in the last few decades. In these devices, positioning/transportation of liquids has remained a critical issue. A sample of fluid needs to be acquired from a specimen reservoir and moved to a different reservoir location for analysis. Inexpensive, reliable and straightforward methods to do this transportation makes such instruments low-cost and robust for use in the field for a variety of purposes. Current ways to do fluid movement require high electric field and hence requiring the use of high voltages (thousands of volts), making the device bulkier. Another approach to use a pneumatic pump for droplet movement is also detrimental in making LoC devices portable due to sizes of associated electronics and electrical parts. This thesis presents the design of a microfluidic valve using capillary action, hydrophobicity, and low voltages (several volts). The use of low voltages brings the "micro" realm to the digital electronics part of LOC. It could lead to better portability, low-power operation of LOC devices, and consequently more adoption in field applications. The design process is based on practical considerations found during experimentation. This method was tested, and results are presented for various biochemical mediums, including KCl, PBS, GMOPS, Cell culture and FBS.Item Open Access Flavivirus control of lipid metabolism: implications for virion formation, function and pathogenesis(Colorado State University. Libraries, 2018) Gullberg, Rebekah, author; Perera, Rushika, advisor; Crick, Dean, committee member; Di Pietro, Santiago, committee member; Geiss, Brian, committee member; Wilusz, Jeff, committee memberDengue viruses (DENV) are the most aggressive arthropod-born viruses worldwide with no currently available antivirals. There is a clear need to understand host viral interactions that can be exploited for therapeutic options. DENV are members of the Flaviviridae family with a positive sense single-stranded RNA genome surrounded by a virally encoded capsid protein, a host cell derived lipid envelope and an icosahedral shell of virally encoded glycoproteins. Its genome is replicated in virally–induced invaginations in the endoplasmic reticulum of the host cell that consistently develop in a time-dependent manner. These invaginations display a highly curved architecture and seem to increase the membrane contact sites within the ER and its vicinity. Functionally, these membranes condense the replication machinery, provide a scaffold to coordinate replication, and hide the viral double stranded RNA intermediate from the host cellular defenses. It has been shown that fatty acid synthesis is increased during infection to provide substrates for this membrane expansion. To identify further changes to cellular metabolism, we have profiled the metabolome of DENV serotype 2 (DENV2) infected Human Hepatoma cells (Huh7) cells at key time-points in virus replication. We have found time-dependent changes in cellular essential fatty acid metabolism. Furthermore, we have interrogated a library of siRNAs directed at the unsaturated fatty acid biosynthesis pathway to determine key enzymes involved in viral replication. We have identified that stearoyl Co-A desaturase 1 (SCD1), the rate-limiting enzyme responsible for converting stearic to oleic acid, is critical for viral replication, maturation and infectious particle formation. Finally, we have profiled the serum metabolome of acute-phase patients with dengue diseases, chikungunya virus infection, or an unknown febrile illness to identify metabolic changes with potential use as prognostic biomarkers. Hypothesis: Since dengue viruses are enveloped viruses, lipid metabolites in the human host are a critical resource hijacked by these viruses for their replicative advantage. Important metabolites will be altered during infection in a time dependent manner and can be quantified and correlated directly to their role in viral genome replication and infectious particle assembly and release. These metabolic changes could also be identified in human bio-fluids and could function as early biomarkers of disease manifestation.Item Open Access Modeling conformational heterogeneity in biomolecules(Colorado State University. Libraries, 2023) Klem, Heidi, author; Paton, Robert, advisor; McCullagh, Martin, advisor; Levinger, Nancy, committee member; Kennan, Alan, committee member; Geiss, Brian, committee memberRegulation of biocatalytic cascades is essential for biological processes but has yet to be exploited in real-world applications. Allostery is a prime example, where binding of an effector molecule alters function in a remote location of the same biomolecule. V-type allostery is especially fascinating, as the reaction rate can be either increased or decreased in response to effector binding. Determining how conformational changes affect the reaction rate is challenging due to the disparity of timescales between the underlying molecular processes. Experimental methods, such as X-ray crystallography, can help to capture large-scale conformational change. However, the resulting structures are not guaranteed to correspond to the biophysical state relevant to the research questions being addressed. Structural changes that occur during the chemical reaction are particularly elusive to this approach. To understand the connection between conformational change and catalytic consequence, a description of the reaction mechanism and relevant configurations is needed. Quantum mechanical (QM) methods can be used to propose enzyme reaction mechanisms by modeling femtosecond motions of forming and breaking bonds. Large-scale conformational changes take place over much longer timescales that cannot be simulated at the QM level, therefore requiring classical simulation techniques. This dissertation focuses on the challenges posed by conformational change in the field of computational biocatalysis. The first chapter examines the prevalence of conformational change in enzymes, its relationship to catalysis, and the difficulties it presents. The second chapter looks at the influence of active site structural features on reaction rates in the allosteric enzyme IGPS using QM approaches and energy decomposition schemes. The third chapter covers the development of methods that use molecular dynamics (MD) simulations to analyze relevant structural states from simulation data and identify long-range communication pathways in biomolecules. The fourth chapter presents a Python code, enzyASM, that automates the generation of QM-based truncated active site models and discusses ongoing developments that will aid reproducibility and standardization in this field of research. The fifth and final chapter summarizes the implications of this Thesis work in computational biocatalysis and envisions how remaining challenges can be addressed to maximize potential to solve real-world problems.Item Open Access SARS-CoV-2 viral RNA biology and its impact on the infected cell(Colorado State University. Libraries, 2023) Altina, Noelia H., author; Wilusz, Jeffrey, advisor; Argueso, Lucas, committee member; Geiss, Brian, committee member; Perera, Rushika, committee member; Yao, Tingting, committee memberThe fine-tuning of the replication and transcription of RNA viruses often requires the interaction of viral RNAs with cellular RNA binding proteins. This project addresses fundamental knowledge gaps on the molecular mechanisms that underlie SARS-CoV-2 gene expression, regulation, and viral RNA-host interactions. After infection, SARS-CoV-2 generates a large set of sub-genomic mRNAs, each containing an identical ~70 base 'leader' region in their 5'UTR (from position 14 to position 75 in RefSeq NC_045512) and a 229 base region at the 3'UTR (from position 29,675 to position 29,903 in RefSeq NC_045512) generated by discontinuous transcription. The accumulation of a considerable amount of these leader/3'UTR regions during the infection represents a possible sink for cellular RNA binding proteins. We demonstrated that PTBP1, a cellular protein involved in the regulation of alternative splicing, binds to the SARS-CoV-2 leader region. SARS-CoV-2 infection critically impacted the splicing of several cellular pre-mRNAs that are normally regulated by PTBP1. Mechanistically, we suggest that SARS-CoV-2 sequesters and influences the re-localization of shuttling splicing factors like PTBP1 from the nucleus to the cytoplasm resulting in significant effects on the host cell splicing machinery leading to changes in cellular mRNA splicing patterns during SARS-CoV-2 infection. Given the current extensive interest in epigenetic methylations of both cellular and viral RNAs, our study also explored the role of post-transcriptional RNA modifications on viral mRNAs. We demonstrated that SARS-CoV-2 can usurp the cellular enzyme, namely PCIF1, to place the m6Am modification at the cap proximal position in its mRNAs. This double methylation is usually found on all host mRNAs that initiate with an adenosine residue, and thus SARS-CoV-2 likely installs this modification on its mRNAs to avoid host immune recognition. Interestingly, we also discovered that capping and m6Am modification are tightly regulated throughout the infection. The highest levels of these 5' end RNA modifications were observed at 12 hours post infection, correlating with the full establishment of viral gene expression in infected cells. These findings indicate that 5' end modification of SARS-CoV-2 transcripts is not simply a default process but rather undergoes unanticipated regulation throughout the infection. Collectively, the data presented provide not only new insights into the complex interactions that SARS-CoV-2 has with the RNA biology of the cell during infection, but also identify attractive potential targets for developing novel anti-coronavirus drugs to treat future emerging coronavirus diseases.Item Open Access The application of carbon composite electrodes for the analysis of environmental and biological pathogens(Colorado State University. Libraries, 2023) McMahon, Catherine J., author; Henry, Charles S., advisor; Prieto, Amy L., committee member; Farmer, Delphine, committee member; Geiss, Brian, committee memberFast, reliable, and accurate detection of heavy metals is crucial in preventing adverse health effects. Heavy metal contamination comes from various human anthropological endeavors, and can leach into water, food, and consumer products such as cosmetics. Electrochemical detection of heavy metals has become a popular alternative to traditional analysis, using highly sensitive spectroscopic techniques. Carbon composite electrodes have been used for electrochemical sensors due to their chemical inertness, large potential window, and resistance to fouling. However, they can often suffer from poor electrocatalytic behavior, resulting in the need for extensive surface modifications. Moreover, traditional carbon composite electrodes have been limited in their pattern-ability and difficultly in fabrication. Thermoplastic electrodes were developed in 2017 to address these needs and are further discussed and characterized in this dissertation for applications towards heavy metal analysis. Overall, this dissertation seeks to use carbon composite electrodes to improve detection efforts for both environmental pollutants (i.e heavy metals) and biological analytes. Chapter 2 introduces the use of stencil-printed carbon electrodes (SPCEs) for the analysis of heavy metals in cosmetic samples from Nepal, Ghana, and Uganda. The approach utilizes a previously developed method and adapts it, expanding its utility. The goal of the work is to develop a method that is capable of screening for heavy metal pollutants outside of traditional laboratory settings. An alternative sample extraction approach is detailed as well as the development of a laboratory standard for heavy metal analysis in cosmetics. In addition to the electrochemical analysis, extensive analysis using inductively coupled plasma optical emission spectroscopy is conducted on the cosmetics samples, to better understand the Pb contamination and matrix complexity of the samples. Chapter 3 focuses on the use of TPEs for the detection of heavy metals. Six formulations of TPEs, with different graphites and polymer binders, are characterized to better understand how the unique surface properties impact the analysis of heavy metals. The detection of Pb is used as a proof-of-concept model. The results illustrate that both the polymer and graphite can have intensive impact on the application of TPEs. Of the various formulations tested, polystyrene and polymethyl methacrylate show promise in detecting heavy metals within relevant ranges. Chapter 4 pivots from heavy metal analysis and investigates the use of SPCEs for the detection of SARS-CoV-2 nucleocapsid protein. With the onset of the COVID-19 pandemic in 2020, my research focus pivoted to address the need to develop reliable, accurate, and fast point-of-care diagnostics for SARS-CoV-2 to help manage the spread of the virus. SPCEs are modified based on an ELISA (enzyme-linked immunosorbent assay) for the electrochemical detection of the N-protein. The assay developed sets the framework for a potential POC diagnostic, while meeting the industry need for fewer false negatives and lower limits of detection. In summary, this dissertation seeks to implement and expand the utility of different kinds of carbon composite electrodes for the detection of heavy metals and biological analytes. The work described in this dissertation sets the framework for improving upon carbon-based electrochemical sensors for environmental and biological sensors. This work provides materials, methods, and fundamental characterization of carbon composite electrodes, and how different surface treatments and modifications can expand their utility in electrochemically sensing applications.Item Open Access Zika virus noncoding sfRNA sequesters viral restriction factors involved in RNA splicing and nucleic acid editing(Colorado State University. Libraries, 2019) Ontiveros Valles, Jesús Gustavo, author; Wilusz, Jeffrey, advisor; Geiss, Brian, committee member; Chen, Chaoping, committee memberZIka virus (ZIKV) is a single-stranded positive sense RNA flavivirus that is transmitted primarily by Aedes aegypti. To date, all vector-borne flaviviruses are known to generate stable subgenomic flavivirus RNAs (sfRNA), due to the stalling of the major cytoplasmic 5'-3' exoribonuclease XRN1 at a knot-like three helix junction structure located in viral 3' untranslated regions (UTRs). Formation of sfRNAs not only stalls XRN1, but also represses its function. sfRNA decay intermediates accumulate to high levels in infected cells and studies with other flaviviruses have implicated sfRNAs in cytopathology. Our objective was to characterize the function of ZIKV sfRNAs to gain insight into ZIKV pathogenesis. Specifically, we identified host proteins that interact with ZIKV sfRNA and have begun to evaluate their role in cytopathology and pathogenesis. RNA pull-down experiments revealed that PHAX and SF3B1, critically important RNA splicing factors involved in nuclear-cytoplasmic shuttling, bind sfRNA. Additionally, the cytidine deaminase APOBEC3C was found to bind ZIKV sfRNA. Knockdown and subsequent overexpression of these RNA Binding Proteins (RBPs) identified the nucleic acid deaminase APOBEC3C and the splicing-associated factor PHAX as negative viral restriction factors whose activity may be suppressed and/or altered by sfRNA interaction during ZIKV infection. sfRNA interactions with the splicing factors also resulted in the accumulation of aberrantly spliced transcripts, possibly due to sequestration of the host cell proteins. Thus, in addition to targeting XRN1, sfRNAs appear to interact with a set of RBPs to disrupt cellular mRNA decay regulation as well as other RNA processing events in an effort to compromise multiple steps of RNA metabolism and promote pathogenesis.