Browsing by Author "Gentry-Weeks, Claudia, committee member"

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Open Access
A kinetic model development of the M13 bacteriophage life cycle
(Colorado State University. Libraries, 2014) Smeal, Steven William, author; Fisk, Nick, advisor; Prasad, Ashok, advisor; Gentry-Weeks, Claudia, committee member
A kinetic model which can simulate the M13 bacteriophage (a virus which only infects bacteria) life-cycle was created through a set of ordinary differential equations. The M13 bacteriophage is a filamentous phage with a circular single-stranded DNA genome. The kinetic model was developed by converting the biology into ordinary differential equations through careful studying of the existing literature describing the M13 life cycle. Most of the differential equations follow simple mass-action kinetics but some have an additional function, called the Hill Function, to account for special scenarios. Whenever possible, the rate constants associated with each ordinary differential equation were based off of experimentally determined constants. The literature describing M13 viral infection did not provide all of the rate constants necessary for our model. The parameters which were not experimentally determined through literature were estimated in the model based on what is known about the process. At present, no experiments were performed by our lab to verify the model or expand on the information available in the literature. However, the M13 phage model has improved the understanding of phage biology and makes some suggestions about the unknown factors that are most important to quantitatively understanding phage biology. The kinetic model is genetically structured and simulates all well-known and major features of viral phage infection beginning when the first viral ssDNA has entered the cytoplasm and ends right before the cell is ready to divide. The model includes DNA replication, transcription, translation, mRNA processing and degradation, viral protein P2 and viral DNA interaction, viral protein P5 and viral single-strand DNA (ssDNA) interaction, P5 and mRNA interactions, and the assembly of new phage. Additionally, the model has implemented an interaction of P2 and P10, which has not been directly verified through experiments, to account for the negative effect P10 has on DNA replication. The interaction of the host cell and virus infection was not explicitly modeled, but a subset of cellular resources were set aside for phage reproduction based on experimental estimates of the metabolic burden of phage infection. Specifically, limited amount of host resources RNA polymerase, DNA polymerase 3, and ribosomes were allocated to phage reproduction. All other host resources such as nucleotides and amino acids were assumed to be in abundance and did not limit phage replication. The model was verified by comparing the output of the model to a set of existing experimental results in literature. The model reproduced both the experimentally measured levels of phage proteins and mRNA, and the timing and dynamics of virus production for the first cell cycle after infection. All of the unknown parameters were based off the model results at the end of the first cell cycle. When the model was extended to account for phage production through multiple cell divisions, the model predicts the cell has the ability to cure itself from the infection in 7 - 8 cell cycles, which we found literature supporting our results after we made the conclusions. Once the model was created we studied how host resources, RNA polymerase and ribosomes, were distributed during the infection process. We were also able to replicate an experiment describing the effects that the viral DNA binding protein P5 had on the translation of five other viral proteins in-silico. The role of P5 inhibition in the viral life-cycle is unclear and our in-depth analysis of P5 function has revealed a possible explanation of how P5 translational inhibition could be an evolutionary advantage. Additionally, we proposed a mechanism which has not been strongly suggested to exist in literature. We are anticipating the development of the model will aide in the progress of phage display on filamentous phage and we believe the current model can be easily amendable to account for other phage like phages such as Ike filamentous phage. We discuss further additions and modifications to the model that will allow more exact treatment of early events in the phage life-cycle and more explicit coupling of phage life-cycle and host biology.
Open Access
A surface protease of Lyme disease bacteria degrades host extracellular matrix components and induces inflammatory cytokines in vitro
(Colorado State University. Libraries, 2012) Russell, Theresa Michelle Tidd, author; Bamburg, James R., advisor; Johnson, Barbara J. B., advisor; Luger, Karolin, committee member; Cohen, Robert E., committee member; Gentry-Weeks, Claudia, committee member
For nearly two decades, the paradigm in Lyme disease research has been that Borrelia burgdorferi does not produce proteases capable of damaging host molecules. Lyme disease has been considered, therefore, to be the consequence of an exuberant inflammatory response to infecting bacteria. This prevailing concept, however, has created a conundrum for the field. The bacterial burden in infected tissue is low, but the degree of inflammation is remarkable and seemingly out of proportion to this burden. The studies described in this dissertation provide evidence that, contrary to current thinking, B. burgdorferi does possess a protease that degrades numerous molecules of the host extracellular matrix (ECM). In addition to destabilization of the ECM which would be expected to benefit the organism, characterization of this proteolytic activity demonstrates that ECM fragments are produced that are known to be pro-inflammatory. These bioactive fragments may amplify the inflammatory processes triggered by the presence of the bacteria itself. When this hypothesis was tested directly by exposing chondrocytes to the borrelial protease in vitro, inflammatory cytokines and chemokines that are hallmarks of Lyme disease were induced. The studies herein suggest a new model for the pathogenesis of Lyme disease and offer an explanation for the paradox of debilitating inflammatory disease in the presence of few infecting organisms. Lastly, in contrast to current serology-based Lyme disease diagnostic tests, the activity of this protease in vitro may generate diagnostic biomarkers enabling detection of active B. burgdorferi infection.
Open Access
Bacterial culture components activating colorimetric transition in polydiacetylene nanofiber composites
(Colorado State University. Libraries, 2020) Bhattacharjee, Abhishek, author; Li, Yan Vivian, advisor; Gentry-Weeks, Claudia, committee member; Diddi, Sonali, committee member
Polydiacetylene (PDA) demonstrates colorimetric transition behaviors due to conformational changes in π conjugated backbone of PDA macromolecules at external stimuli of bacteria, suggesting potential applications in biosensors. However, the bacterial culture components activating colorimetric transition in PDAs are still undetermined due to the complexity of the bacterial system. In this study, PU-PDA nanofiber composite was prepared via electrospinning and tested with components from Escherichia coli (E. coli) culture including supernatant fluid, cell pellet, and extracellular polymeric substances (EPS). When PU-PDA nanofiber was tested with supernatant fluid, it changed color from blue to red. In contrast, bacterial cell pellets could not induce a color change, suggesting the color-changing substances (CCS) are not cell-associated, rather can be found in the spent media (supernatant fluid) generated by E. coli during its growth phase. Intense color change in the nanofiber by the autoclaved supernatant fluid indicated that the CCS may not be a protein, DNA, or RNA since they denature in high heat and pressure from the autoclaving process. With an increase in storage time of the supernatant fluid, the color-changing rate was reduced significantly, suggesting a degradation in CCS with time. Free EPS from the supernatant fluid could induce a color change in the nanofibers, which confirmed that EPS contains the CCS. No significant changes were found in the morphology of PU-PDA nanofibers before and after the exposure of E. coli culture components. Critical bacterial concentration (CBC) was found approximately 9 × 108 CFU/ml, suggesting the efficiency of the PU-PDA nanofiber composite to be used as a biosensor. Additionally, solvatochromism of the nanofiber composite was investigated using organic solvents commonly used in extracting bacterial culture components. The results from this study provided a guideline for using PU-PDA nanofiber composite as a biosensor in point-of-care applications.
Open Access
Development and implementation of novel drug delivery system for transdermal materials
(Colorado State University. Libraries, 2024) Sun, Yu, author; Li, Yan Vivian, advisor; Liu, Jiangguo, advisor; Bailey, Travis, committee member; Gentry-Weeks, Claudia, committee member
There is a constant need for developing transdermal dressing materials with advanced properties such as infection monitoring and wound closure facilitation for effective chronic wound treatments. On the other than, the advancement in drug delivery systems has created innovation of precise targeting of treatment, nontoxicity, cell access, controlled release profiles, treatment variations, and antibiotics activity conservation, which offers a great opportunity in developing novel wound dressing materials. Recently, the application of nanomaterials, especially nanoparticles, in drug delivery systems has shown great potential in effective wound treatment. In this proposal, three projects are focused on developing nanostructured scaffolds loaded with antibiotic agents for novel wound dressing applications. First, the antibiotic encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles will be integrated into monolithic nanofiber scaffolds that can be tested in transdermal materials for antibacterial properties. Second, the antibiotics loaded PLGA nanoparticles incorporated monolithic nanofiber scaffolds will be developed into core-shell fibrous scaffolds to provide a drug delivery system with controlled release of antibiotics. Integration of the nanoparticles into nanofibers includes monolithic and core-shell structures to provide controlled release of antibiotic agents. The mechanisms of controlled release are investigated via experimental and computational methods. Finite difference methods and machine learning are used for developing mathematical models capable of numerically quantifying antibiotic release rates, which provides a theoretical understanding of the release process in the nanostructure scaffolds. The work provides implication of utilizing PLGA nanoparticles in scaffolds to develop effective transdermal materials. Additionally, the computational models would provide tools to understand the mechanism of controlled release process, which may assist in the design of the nanoparticles as well as the nanostructured scaffolds.
Open Access
Development and thermal characterization of polydiacetylene (PDA) nanofiber composites for smart wound dressing applications
(Colorado State University. Libraries, 2016) Alam, A K M Mashud, author; Li, Yan Vivian, advisor; Park, Juyeon, committee member; Gentry-Weeks, Claudia, committee member
Conventional methods of identification of microbiological pathogens infection in wound have many challenges such as the need for specialized instruments and trained personnel, and the long detection time. There is a critical need for an innovative method that is simple, accurate, sensitive, reliable, and rapid in pathogen detection practices. Wound dressings containing PDA nanofibers could be used as a diagnostic tool for the detection of onsite bacterial infection. By early wound infection diagnosis, the smart wound dressing would allow physicians to start timely treatment which would reduce hospitalization time and patient suffering. PDAs are of great interest in the development of chromatic sensors due to their unique optical property of undergoing a chromatic transition from blue to red upon external stimuli. 10,12-Pentacosadiynoic acid (PCDA) and poly (ethylene oxide) (PEO) were used in this study to develop fiber composites via an electrospinning method at various mass ratios of PEO to PCDA, solution concentrations, and injection speeds. High mass ratios of PEO to PCDA, low polymer concentrations, and low injection speed promoted fine fibers with smooth surfaces. The colorimetric transition of the fibers was investigated by heating the fibers at temperatures ranging from 25 °C to 120 °C. A color switch from blue to red was observed when the fibers were treated at temperatures higher than 60 °C. The color transition was more sensitive in the fibers made with a low mass ratio of PEO to PCDA due to the high fraction of PDA in the fibers. The large diameter fibers also promoted the color switch due to the high reflectance area in the fibers. All of the fibers were analyzed using Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) and compared before and after the color change occurred. The colorimetric transitional mechanism is proposed to occur due to conformational changes in the PDA macromolecules.
Open Access
Macrophage receptor-mediated recognition, response and treatment of Leishmania major infection
(Colorado State University. Libraries, 2011) Nelson, Keith G., author; Mason, Gary, advisor; Gentry-Weeks, Claudia, committee member; Zeidner, Nordin, committee member; Bamburg, James, committee member
We investigate the role of macrophage receptors in the recognition of Leishmania major and response to the parasite, focusing on complement receptors (CR). CR1 and CR3 are the main complement receptors on murine macrophages, recognizing specific parasite surface antigens and parasites opsonized by complement component 3 (C3). Utilizing C3-deficient mice, we show a clear role for complement in enhancing parasite infectivity, and recognition of and response to L. major by the murine host. In our in vitro experiments blocking CR1 and CR3, there is a clear effect of the route of parasite recognition and entry into the macrophage on parasite phagocytosis and cytokine response to infection by L. major, although Th1/Th2 bias is unaffected by blocking complement receptors. Using parasite strains lacking in the two most common surface molecules of L. major (LPG and gp63), we show a strong role for gp63 in the interaction with C3 and complement receptors, particularly CR3. Lastly, we demonstrate the utility of targeting anti-leishmanial therapeutics in infected hosts to the macrophage through another macrophage receptor, the scavenger receptor.
Open Access
Odor encoder: computational design of a novel allosteric enzyme activation system for providing enhanced olfactory abilities to trained odor detecting sentinel animals
(Colorado State University. Libraries, 2022) Scroggins, Michael, author; Snow, Christopher, advisor; Peebles, Christie, advisor; Gentry-Weeks, Claudia, committee member
From the perfume of a flower, to the aroma of a favorite food, to what for bioengineers is the all-to-familiar smell of E. coli, olfactory senses play in important role in how animals interact with the world around them. An offensive odor can inform us that an object is unsafe to eat or be around, a familiar scent can recall memories of events from decades in our past, and even our natural body odors can affect our mating selection preferences. Yet there are many chemicals, both natural and synthetic, for which we do not possess the ability for olfactory detection. An everyday example of this is the natural gas that we use in our homes and which is naturally odorless, but which is commonly spiked with the odorant tert-butyl mercaptan (TBM) to provide the characteristic sulfuric smell we associate with natural gas. Because of this added odorant we can rapidly detect a leaking gas via the smell of the TBM and address the situation as needed to ensure the safety of ourselves and our community. Unfortunately, there are some hazardous and odorless chemicals which we cannot simply spike with an odorant molecule, and for these situations it would be ideal to have alternative options for facilitating a rapid olfactory detection. Therein lies the goals of the Odor Encoder project; to create enhanced olfactory abilities via a conditionally activated enzyme which produces a smellable product in the presence of a target odorless molecule. The approach to achieving this goal was creation of a genetically modified bacterial organism which could be engineered for conditional expression of an odorant producing enzyme in-situ within the nasal microbiome of trained odor detecting animals. The odorant producing enzyme chosen for this purpose was salicylic acid methyltransferase, a.k.a SAMT, which produces the characteristic odorant molecule methyl salicylate via methylation of salicylic acid. The probiotic E. coli strain Nissle 1917 was selected as the bacterial organism for inoculation of the nasal microbiome, and an expression plasmid was created which could produce both salicylic acid and methyl salicylate from endogenously produced metabolites via dual expression of SAMT along with a salicylate synthase enzyme known as irp9. Conditional production of methyl salicylate was achieved via two methods. The first method involved conditional enzyme expression via use of a riboswitch specific to the small molecule theophylline. The second method involved conditional enzyme activity via constitutive expression of a crippled form of SAMT which may potentially have its enzymatic activity restored via theophylline induced allosteric activation. The allosteric rescue method utilized computational design methods to design novel theophylline-specific allosteric cavities in SAMT, and theophylline induced allosteric reactivation of enzyme activity will be investigated via production and screening of the computationally designed enzyme library.
Open Access
The analysis of Burkholderia pseudomallei virulence and efficacy of potential therapeutics
(Colorado State University. Libraries, 2011) Propst, Katie L., author; Schweizer, Herbert, advisor; Dow, Steven, advisor; Gentry-Weeks, Claudia, committee member; Goehring, Lutz, committee member
Burkholderia pseudomallei is the causative agent of the disease melioidosis and is classified as a category B Select Agent. There are currently many challenges associated with both the study of this pathogen and its treatment in the clinical setting. Prior to these studies, there was no attenuated B. pseudomallei strain available that was exempt of Select Agent regulations and approved for study outside of biosafety level 3 (BSL-3) containment, and consequently basic research on this pathogen was largely hindered. The first purpose of these studies was to extensively characterize the attenuation of two B. pseudomallei mutant strains using melioidosis animal models. The two mutants constructed were Bp82 and Bp190, Δ purM derivatives deficient in adenine and thiamine biosynthesis. These mutants were found to be fully attenuated in immune competent and immune deficient mouse and hamster melioidosis models. Bp82 is currently exempt of all Select Agent regulations and can be safely handled in the BSL-2 setting, greatly accelerating research on this priority pathogen. Since basic research on B. pseudomallei was not common in the Western world until its Select Agent classification, much is still unknown regarding the bacterial factors contributing to its virulence. A second purpose of this research was to determine whether resistance-nodulation-cell division (RND) efflux systems and iron acquisition siderophores impact the virulence of B. pseudomallei in a pneumonic murine melioidosis model. This was examined using a clinical isolate naturally devoid of a characterized efflux system and the gene cluster for malleobactin siderophore synthesis, and by the construction of isogenetic mutants. The two characterized B. pseudomallei efflux pumps, AmrAB-OprA and BpeAB-OprB, were both found to be completely dispensable during in vivo murine infection. The removal of one or both of these systems did not reduce lethality of the mutant strains. Unlike that observed with similar bacterial pathogens, the lethality of B. pseudomallei was also not reduced upon the removal of either the malleobactin or pyochelin siderophores. This finding indicates B. pseudomallei is likely capable of utilizing alternative systems for iron acquisition within the host. In addition to the challenges associated with the study of this pathogen, there are also many clinical challenges associated with melioidosis, providing a basis for the final two purposes of this research. One particular challenge is the high frequency of patient relapse, even after appropriate prolonged antibiotic therapy. A third purpose of this research was to determine whether traditional antibiotic therapy could be augmented by the co-administration of immunotherapy. Cationic liposome-DNA complexes (CLDC), which are potent activators of the innate immune system, were found to synergistically reduce intracellular B. pseudomallei concentrations in macrophages in vitro when combined with the antibiotic ceftazidime. In addition, this combination therapy also significantly increased mouse survival during both acute and chronic melioidosis. A similar enhancement to ceftazidime therapy was observed with recombinant IFN-γ, illustrating the potential of immunotherapy to improve clinical outcome and decrease patient relapse. The lack of an effective approved vaccine for human use is another substantial clinical challenge associated with melioidosis and its prevention. The final purpose of these studies was to develop an effective mucosal vaccine, offering both short-term protection from acute pneumonic disease and long-term protection from disseminated chronic melioidosis. CLDC was identified as a highly effective mucosal adjuvant within complexed to heat-killed B. pseudomallei, and this adjuvant offered moderate protection from acute disease when combined with Burkholderia protein subunits. The longest-term protection from lethal challenge in our murine model, lasting beyond 100 days, was elicited by the fully attenuated live Bp82 strain. Since this strain is both fully attenuated and exempt of Select Agent regulations, it has great potential clinically for high-risk persons as an effective live vaccine strain.