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- Genre: Masters Thesis
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
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
University-level sustainability education in Western academia attempts to focus on eliminating future harm to people and the planet. However, Western academia as an institution upholds systems of oppression and reproduces settler colonialism. This reproduction is antithetical to sustainability goals as it continues patterns of Indigenous erasure and extractive relationships to the Land that perpetuate violence towards people and the planet. Sustainability programs, however, offer several frameworks, including resilience, that facilitate critical interrogations of social-ecological systems. In this thesis, I apply the notion of resilience to the perpetuation of settler colonialism within university-level sustainability education. Specifically, I ask: How is settler colonialism resilient in university-level sustainability education? How are, or could, sustainability programs in Western academic settings address settler colonialism? Through a series of conversational interviews with faculty and leadership from Arizona State University School of Sustainability, I analyzed how university-level sustainability education is both challenging and shaped by settler colonialism. These interviews focused on faculty perspectives on the topic and related issues; the interviews were analyzed using thematic coding in NVivo software. The results of this project highlight that many faculty members are already concerned with and focused on challenging settler colonialism, but that settler colonialism remains resilient in this system due to feedback loops at the personal level and reinforcing mechanisms at the institutional level. This research analyzes these feedback loops and reinforcing mechanisms, among others, and supports the call for anti-colonial and decolonial reconstruction of curriculum, as well as a focus on relationship building, shifting of mindset, and school-wide education on topics of white supremacy, settler colonialism, and systems of oppression in general.
ContributorsBills, Haven (Author) / Klinsky, Sonja (Thesis advisor) / Goebel, Janna (Committee member) / Schoon, Michael (Committee member) / Arizona State University (Publisher)
Created2022
Description
MicroRNAs (miRNAs) are 17-22 nucleotide non-coding RNAs that regulate gene expression by targeting non-complementary elements in the 3’ untranslated regions (3’UTRs) of mRNAs. miRNAs, which form complex networks of interaction that differ by tissue and developmental stage, display conservation in their function across metazoan species. Yet much remains unknown regarding their biogenesis, localization, strand selection, and their absolute abundance due to the difficulty of detecting and amplifying such small molecules. Here, I used an updated HT qPCR-based methodology to follow miRNA expression of 5p and 3p strands for all 190 C. elegans miRNAs described in miRBase throughout all six developmental stages in triplicates (total of 9,708 experiments), and studied their expression levels, tissue localization, and the rules underlying miRNA strand selection. My study validated previous findings and identified novel, conserved patterns of miRNA strand expression throughout C. elegans development, which at times correlate with previously observed developmental phenotypes. Additionally, my results highlighted novel structural principles underlying strand selection, which can be applied to higher metazoans.
Though optimized for use in C. elegans, this method can be easily adapted to other eukaryotic systems, allowing for more scalable quantitative investigation of miRNA biology and/or miRNA diagnostics.
ContributorsMeadows, Dalton Alexander (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Murugan, Vel (Committee member) / Wilson-Rawls, Jeanne (Committee member) / Arizona State University (Publisher)
Created2023
Description
Wildlife rehabilitation as a practice in the United States exists in a complicated ethical landscape. The Wildlife Rehabilitator's Code of Ethics exists to guide the profession and states that rehabilitators must respect the wildness and maintain the dignity of an animal in their care. This thesis explores the question: How do the attitudes and actions of wildlife rehabilitators exemplify the ways in which they understand and enact respect for an animal’s dignity and wildness while in their care? Additionally, in what circumstances do rehabilitators align and diverge from each other in their interpretation and demonstration of this respect? These questions were answered through a literature review, interviews with rehabilitators, and site visits to wildlife rehabilitation centers in the Phoenix metropolitan area. My results suggest that rehabilitators are aligned in their understanding of respect for wildness and dignity as it applies to the animals in their care that are actively undergoing rehabilitation. Rehabilitators achieved consensus on the idea that they should interact with the animals as little as possible while providing their medically necessary care. Rehabilitators began to diverge when considering the animals in their sanctuary spaces. Specifically, they varied in their perception of wildness in sanctuary animals, which informed how some saw their responsibilities to the animals. Lesser perceived wildness correlated to increased acceptance of forming affectionate relationships with the sanctuary animals, and even feelings of obligation to form these relationships. Based on my research, I argue that the Wildlife Rehabilitator's Code of Ethics should be revised to reflect the specific boundary that wildlife rehabilitators identified in the rehabilitation space and provide substantive guidance as to what respecting wildness and dignity means in this field.
ContributorsBernat, Isabella Elyse (Author) / Minteer, Ben (Thesis advisor) / Ellison, Karin (Committee member) / Schoon, Michael (Committee member) / Arizona State University (Publisher)
Created2023
Description
Patients with malignant brain tumors have a median survival of approximately 15 months following diagnosis, regardless of currently available treatments which include surgery followed by radiation and chemotherapy. Improvement in the survival of brain cancer patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is the metabolic dysregulation that is a hallmark of cancer cells. It has therefore been postulated that one approach to treating brain tumors may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. It has been shown that this metabolic therapy enhances survival and potentiates standard therapy in mouse models of malignant gliomas, yet the anti-tumor mechanisms are not fully understood.
The current study reports that KetoCal® (KC; 4:1 fat:protein/carbohydrates), fed ad libitum, alters hypoxia, angiogenic, and inflammatory pathways in a mouse model of glioma. Tumors from animals maintained on KC showed reduced expression of the hypoxia marker carbonic anhydrase 9 (CA IX), a reduction in hypoxia inducible factor 1-alpha (HIF-1α) and decreased activation of nuclear factor kappa B (NF-κB). Animals maintained on KC also showed a reduction in expression of vascular endothelial growth factor receptor 2 (VEGFR2) and decreased microvasculature in their tumors. Further, peritumoral edema was significantly reduced in animals fed the KC and protein analysis showed significantly altered expression of the tight junction protein zona occludens-1 (ZO-1) and the water channeling protein aquaporin-4 (AQP4), both of which have been implicated in malignant processes in glioma, including the formation of peritumoral edema in patients. Taken together the data suggests that KC alters multiple processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use.
The current study reports that KetoCal® (KC; 4:1 fat:protein/carbohydrates), fed ad libitum, alters hypoxia, angiogenic, and inflammatory pathways in a mouse model of glioma. Tumors from animals maintained on KC showed reduced expression of the hypoxia marker carbonic anhydrase 9 (CA IX), a reduction in hypoxia inducible factor 1-alpha (HIF-1α) and decreased activation of nuclear factor kappa B (NF-κB). Animals maintained on KC also showed a reduction in expression of vascular endothelial growth factor receptor 2 (VEGFR2) and decreased microvasculature in their tumors. Further, peritumoral edema was significantly reduced in animals fed the KC and protein analysis showed significantly altered expression of the tight junction protein zona occludens-1 (ZO-1) and the water channeling protein aquaporin-4 (AQP4), both of which have been implicated in malignant processes in glioma, including the formation of peritumoral edema in patients. Taken together the data suggests that KC alters multiple processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use.
ContributorsWoolf, Eric C (Author) / Scheck, Adrienne C (Thesis advisor) / Lake, Douglas F (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2014