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- Genre: Academic theses
Description
Surface plasmon resonance (SPR) has emerged as a popular technique for elucidating subtle signals from biological events in a label-free, high throughput environment. The efficacy of conventional SPR sensors, whose signals are mass-sensitive, diminishes rapidly with the size of the observed target molecules. The following work advances the current SPR sensor paradigm for the purpose of small molecule detection. The detection limits of two orthogonal components of SPR measurement are targeted: speed and sensitivity. In the context of this report, speed refers to the dynamic range of measured kinetic rate constants, while sensitivity refers to the target molecule mass limitation of conventional SPR measurement. A simple device for high-speed microfluidic delivery of liquid samples to a sensor surface is presented to address the temporal limitations of conventional SPR measurement. The time scale of buffer/sample switching is on the order of milliseconds, thereby minimizing the opportunity for sample plug dispersion. The high rates of mass transport to and from the central microfluidic sensing region allow for SPR-based kinetic analysis of binding events with dissociation rate constants (kd) up to 130 s-1. The required sample volume is only 1 μL, allowing for minimal sample consumption during high-speed kinetic binding measurement. Charge-based detection of small molecules is demonstrated by plasmonic-based electrochemical impedance microscopy (P-EIM). The dependence of surface plasmon resonance (SPR) on surface charge density is used to detect small molecules (60-120 Da) printed on a dextran-modified sensor surface. The SPR response to an applied ac potential is a function of the surface charge density. This optical signal is comprised of a dc and an ac component, and is measured with high spatial resolution. The amplitude and phase of local surface impedance is provided by the ac component. The phase signal of the small molecules is a function of their charge status, which is manipulated by the pH of a solution. This technique is used to detect and distinguish small molecules based on their charge status, thereby circumventing the mass limitation (~100 Da) of conventional SPR measurement.
ContributorsMacGriff, Christopher Assiff (Author) / Tao, Nongjian (Thesis advisor) / Wang, Shaopeng (Committee member) / LaBaer, Joshua (Committee member) / Chae, Junseok (Committee member) / Arizona State University (Publisher)
Created2013
Description
This dissertation investigates the condition of skeletal muscle insulin resistance using bioinformatics and computational biology approaches. Drawing from several studies and numerous data sources, I have attempted to uncover molecular mechanisms at multiple levels. From the detailed atomistic simulations of a single protein, to datamining approaches applied at the systems biology level, I provide new targets to explore for the research community. Furthermore I present a new online web resource that unifies various bioinformatics databases to enable discovery of relevant features in 3D protein structures.
ContributorsMielke, Clinton (Author) / Mandarino, Lawrence (Committee member) / LaBaer, Joshua (Committee member) / Magee, D. Mitchell (Committee member) / Dinu, Valentin (Committee member) / Willis, Wayne (Committee member) / Arizona State University (Publisher)
Created2013
Description
Recombinant protein expression is essential to biotechnology and molecular medicine, but facile methods for obtaining significant quantities of folded and functional protein in mammalian cell culture have been lacking. Here I describe a novel 37-nucleotide in vitro selected sequence that promotes unusually high transgene expression in a vaccinia driven cytoplasmic expression system. Vectors carrying this sequence in a monocistronic reporter plasmid produce >1,000-fold more protein than equivalent vectors with conventional vaccinia promoters. Initial mechanistic studies indicate that high protein expression results from dual activity that impacts both transcription and translation. I suggest that this motif represents a powerful new tool in vaccinia-based protein expression and vaccine development technology.
ContributorsFlores, Julia Anne (Author) / Chaput, John C (Thesis advisor) / Jacobs, Bertram (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2012
Description
The TP53 tumor suppressor gene is the most frequently mutated gene in human cancers. In the highly aggressive triple negative breast cancer (TNBC), TP53 is mutated in 80% of cases. TNBC lacks viable drug targets, resulting in a low prognosis (12.2% 5 year survivability rate). As such, the discovery of druggable targets in TNBC would be beneficial. Mutated p53 protein typically occurs as a missense mutation and often endows cancer cells with gain of function (GOF) properties by dysregulating metabolic pathways. One of these frequently dysregulated pathways is the Hippo/Yes-associated protein-1 (YAP1)/WW Domain Containing Transcription Regulator 1 (TAZ) tumor suppressor pathway. This study therefore analyzed the involvement of the Hippo/YAP1/TAZ pathway in p53-mediated breast cancer cell invasion. From an RNA-seq screen in MCF10A cell lines harboring different TP53 missense mutations, each with a differing invasive phenotype, components of the Hippo pathway were found to correlate with cell invasion. To this end, the active and inactive forms of YAP1 and TAZ were studied. Phosphorylated (inactive) YAP1 and TAZ are retained in the cytoplasm and eventually degraded. Unphosphorylated (active) YAP1 and TAZ translocate to the nucleus to activate TEAD-family transcription factors, inducing cell survival and proliferation genes leading to increased cell invasion. Using quantitative western blot analysis, it was found that inactive TAZ expression was lower in the most invasive cell lines and higher in the least invasive cell lines (p = 0.003). Moreover, the ratio of inactive TAZ protein to total TAZ protein was also shown to be predominantly lower in the invasive cell lines compared to the non-invasive lines (p = 0.04). Finally, active TAZ expression was primarily higher in p53-mutant invasive cell lines and lower in non-invasive p53 mutant cells. Additionally, although YAP1 and TAZ are thought to be functionally redundant, the pattern seen in TAZ was not seen in the YAP1 protein. Taken together, the results demonstrated here suggest that TAZ holds a more dominant role in governing TNBC cell invasion compared to YAP1 and further highlights TAZ as a potential therapeutic target in TNBC.
ContributorsGrief, Dustin (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2022
Description
Glioblastoma (GBM), the most common and aggressive primary brain tumor affecting adults, is characterized by an aberrant yet druggable epigenetic landscape. The Histone Deacetylases (HDACs), a major family of epigenetic regulators, favor transcriptional repression by mediating chromatin compaction and are frequently overexpressed in human cancers, including GBM. Hence, over the last decade there has been considerable interest in using HDAC inhibitors (HDACi) for the treatment of malignant primary brain tumors. However, to date most HDACi tested in clinical trials have failed to provide significant therapeutic benefit to patients with GBM. This is because current HDACi have poor or unknown pharmacokinetic profiles, lack selectivity towards the different HDAC isoforms, and have narrow therapeutic windows. Isoform selectivity for HDACi is important given that broad inhibition of all HDACs results in widespread toxicity across different organs. Moreover, the functional roles of individual HDAC isoforms in GBM are still not well understood. Here, I demonstrate that HDAC1 expression increases with brain tumor grade and is correlated with decreased survival in GBM. I find that HDAC1 is the essential HDAC isoform in glioma stem cells and its loss is not compensated for by its paralogue HDAC2 or other members of the HDAC family. Loss of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner and leads to significant suppression of tumor growth in vivo. While no HDAC isoform-selective inhibitors are currently available, the second-generation HDACi quisinostat harbors high specificity for HDAC1. I show that quisinostat exhibits potent growth inhibition in multiple patient-derived glioma stem cells. Using a pharmacokinetics- and pharmacodynamics-driven approach, I demonstrate that quisinostat is a brain-penetrant molecule that reduces tumor burden in flank and orthotopic models of GBM and significantly extends survival both alone and in combination with radiotherapy. The work presented in this thesis thereby unveils the non-redundant functions of HDAC1 in therapy- resistant glioma stem cells and identifies a brain-penetrant HDACi with higher selectivity towards HDAC1 as a potent radiosensitizer in preclinical models of GBM. Together, these results provide a rationale for developing quisinostat as a potential adjuvant therapy for the treatment of GBM.
ContributorsLo Cascio, Costanza (Author) / LaBaer, Joshua (Thesis advisor) / Mehta, Shwetal (Committee member) / Mirzadeh, Zaman (Committee member) / Mangone, Marco (Committee member) / Paek, Andrew (Committee member) / Arizona State University (Publisher)
Created2022
Description
ABSTRACT Over the past several decades, the dilemma of free-roaming horses in the U.S. has proven to be one of the most divisive issues in management of public lands. According to federal land management agencies, without population regulation, horses can increase at the rate of 15-20% a year on arid rangelands with inadequate numbers of natural, large predators. Horses compete for valuable forage and water resources alongside cattle and native wildlife in delicate riparian areas highly susceptible to the negative ecological effects of soil compaction and overgrazing. Most U.S. management policies, therefore, call for increased removal of free-roaming horses as they are categorized as “un-authorized livestock” or "non-native" species. Wild horse advocates, however, continue to petition for improvement in animal welfare and expansion of the horses’ territory. With heightened social conflict spurred by animal rights and ecological concerns, not to mention the often-stark differences over what really “belongs” on the landscape, the success of appropriate management strategies hinges on managing agencies’ preparedness and ability to respond in a timely and inclusive manner. A critical element of the management context is the public’s views toward the wild horse and the science used to manage them. Synthesizing the vast literature in the history and philosophy of wildlife management in the American West, and utilizing an ethnographic and case study approach, my research examines the range of stakeholder concerns and analyzes the factors that have led to the disconnect between public values of wild horses and public policy for the management of the federally protected free-roaming horses in Arizona’s Apache-Sitgreaves National Forests.
ContributorsMurphree, Julie Joan (Author) / Minteer, Ben A. (Thesis advisor) / Schoon, Michael (Thesis advisor) / Bradshaw, Karen (Committee member) / Chew, Matthew (Committee member) / Arizona State University (Publisher)
Created2022
Description
Transient Receptor Potential Vanilloid-1 (TRPV1) is an integral membrane polymodal cation channel involved in various essential biological functions, including thermosensing, thermoregulation, and nociception. Discrete TRPV1 activation modes such as ligand, heat, and proton have been challenging to disentangle. However, dissecting the polymodal nature of TRPV1 is essential for therapeutic development. The human TRPV1 (hTRPV1) voltage-sensing like domain (VSLD; transmembrane helices S1-S4) contains the canonical vanilloid ligand binding site and significantly contributes to thermosensing. Nuclear magnetic resonance (NMR)-detected studies probe the role of the hTRPV1-VSLD in TRPV1 polymodal function. The hTRPV1-VSLD is identified as an allosteric hub for all three primary TRPV1 activation modes and demonstrates plasticity in chemical ligand modulation. The presented results underscore molecular features in the VSLD that dictate TRPV1 function, highlighting important considerations for future therapeutic design.
ContributorsOwens, Aerial M. (Author) / Van Horn, Wade D. (Thesis advisor) / Levitus, Marcia (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, declared in March 2020 resulted in an unprecedented scientific effort that led to the deployment in less than a year of several vaccines to prevent severe disease, hospitalizations, and death from coronavirus disease 2019 (COVID-19). Most vaccine models focus on the production of neutralizing antibodies against the spike (S) to prevent infection. As the virus evolves, new variants emerge that evade neutralizing antibodies produced by natural infection and vaccination, while memory T cell responses are long-lasting and resilient to most of the changes found in variants of concern (VOC). Several lines of evidence support the study of T cell-mediated immunity in SARS-CoV-2 infections. First, T cell reactivity against SARS-CoV-2 is found in both (cluster of differentiation) CD4+ and CD8+ T cell compartments in asymptomatic, mild, and severe recovered COVID-19 patients. Second, an early and stronger CD8+ T cell response correlates with less severe COVID-19 disease [1-4]. Third, both CD4+ and CD8+ T cells that are reactive to SARS-CoV-2 viral antigens are found in healthy unexposed individuals suggesting that cross-reactive and conserved epitopes may be protective against infection.
The current study is focused on the T cell-mediated response, with special attention to conserved, non-spike-cross-reactive epitopes that may be protective against SARS-CoV-2. The first chapter reviews the importance of epitope prediction in understanding the T cell-mediated responses to a pathogen. The second chapter centers on the validation of SARS-CoV-2 CD8+ T cell predicted peptides to find conserved, immunodominant, and immunoprevalent epitopes that can be incorporated into the next generation of vaccines against severe COVID-19 disease. The third chapter explores pre-existing immunity to SARS-CoV-2 in a pre-pandemic cohort and finds two highly immunogenic epitopes that are conserved among human common cold coronaviruses (HCoVs). To end, the fourth chapter explores the concept of T cell receptor (TCR) cross-reactivity by isolating SARS-CoV-2-reactive TCRs to elucidate the mechanisms of cross-reactivity to SARS-CoV-2 and other human coronaviruses (HCoVs).
ContributorsCarmona, Jacqueline (Author) / Anderson, Karen S (Thesis advisor) / Lake, Douglas (Thesis advisor) / Maley, Carlo (Committee member) / Mangone, Marco (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
Communications around sustainability have been found to be incongruent with eliciting the transformative change required to address global climate change and its' repercussions. Recent research has been exploring storytelling in sustainability, specifically with an emphasis on reflexive and emancipatory methods. These methods encourage embracing and contextualizing complexity and intend to target entire cognitive hierarchies. This study explores the possibility of using emancipatory and reflexive storytelling as a tool to change attitudes pertaining to the Valley Metro Light Rail, an example of a complex sustainability mitigation effort. I explore this in four steps: 1) Conducted a pre-survey to gauge preexisting attitudes and predispositions; 2) Provided a narrative that uses storytelling methodologies of reflexivity and emancipation through a story about the light rail; 3) Conducted a post-survey to gauge attitude shift resulting from the narrative intervention; 4) Facilitated a focus group discussion to examine impact qualitatively. These steps intended to provide an answer to the question: How does emancipatory and reflexive storytelling impact affective, cognitive and conative attitudes regarding local alternative transportation? By using tripartite attitude model, qualitative and quantitative analysis this paper determines that reflexive and emancipatory storytelling impacts attitudinal structures. The impact is marginal in the survey response, though the shift indicated a narrowing of participant responses towards one another, indicative of participants subscribing to emancipation and reflexivity of their held attitudes. From the group discussion, it was evident from qualitative responses that participants engaged in emancipating themselves from their held attitudes and reflected upon them. In doing so they engaged in collaboration to make suggestions and suggest actions to help those with experiences that differed from their own. Though this research doesn’t provide conclusive evidence, it opens the door for future research to assess these methodologies as a tool to elicit shared values, beliefs and norms, which are necessary for collective action leading to transformative change in response to global climate change.
ContributorsSwanson, Jake Ryan (Author) / Roseland, Mark (Thesis advisor) / Larson, Kelli (Committee member) / Calhoun, Craig (Committee member) / Schoon, Michael (Committee member) / Arizona State University (Publisher)
Created2023
Description
Understanding the dynamic interactions between humans and wildlife is essential to establishing sustainable wildlife-based ecotourism (WBE). Animal behavior exists within a complex feedback loop that affects overall ecosystem function, tourist satisfaction, and socioeconomics of local communities. However, the specific value that animal behavior plays in provisioning ecosystem services has not been thoroughly evaluated. People enjoy activities that facilitate intimate contact with animals, and there are many perceived benefits associated with these experiences, such as encouraging pro-environmental attitudes that can lead to greater motivation for conservation. There is extensive research on the effects that unregulated tourism activity can have on wildlife behavior, which include implications for population health and survival. Prior to COVID-19, WBE was developing rapidly on a global scale, and the pause in activity caused by the pandemic gave natural systems the chance to recover from environmental damage from over-tourism and provided insights into how tourism could be less impactful in the future. Until now it has been undetermined how changes in animal behavior can alter the relationships and socioeconomics of this multidimensional system. This dissertation provides a thorough exploration of the behavioral, ecological, and economic parameters required to model biosocial interactions and feedbacks within the whale watching system in Las Perlas Archipelago, Panama. Through observational data collected in the field, this project assessed how unmanaged whale watching activity is affecting the behavior of Humpback whales in the area as well as the socioeconomic and conservation contributions of the industry. Additionally, it is necessary to consider what a sustainable form of wildlife tourism might be, and whether the incorporation of technology will help enhance visitor experience while reducing negative impacts on wildlife. To better ascertain whether this concept of this integration would be favorably viewed, a sample of individuals was surveyed about their experiences about using technology to enhance their interactions with nature. This research highlights the need for more deliberate identification and incorporation of the perceptions of all stakeholders (wildlife included) to develop a less-impactful WBE industry that provides people with opportunities to establish meaningful relationships with nature that motivate them to help meet the conservation challenges of today.
ContributorsSurrey, Katie (Author) / Gerber, Leah (Thesis advisor) / Guzman, Hector (Committee member) / Minteer, Ben (Committee member) / Schoon, Michael (Committee member) / Arizona State University (Publisher)
Created2023