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The advent of CRISPR/Cas9 revolutionized the field of genetic engineering and gave rise to the development of new gene editing tools including prime editing. Prime editing is a versatile gene editing method that mediates precise insertions and deletions and can perform all 12 types of point mutations. In turn, prime editing represents great promise in the design of new gene therapies and disease models where editing was previously not possible using current gene editing techniques. Despite advancements in genome modification technologies, parallel enrichment strategies of edited cells remain lagging behind in development. To this end, this project aimed to enhance prime editing using transient reporter for editing enrichment (TREE) technology to develop a method for the rapid generation of clonal isogenic cell lines for disease modeling. TREE uses an engineered BFP variant that upon a C-to-T conversion will convert to GFP after target modification. Using flow cytometry, this BFP-to-GFP conversion assay enables the isolation of edited cell populations via a fluorescent reporter of editing. Prime induced nucleotide engineering using a transient reporter for editing enrichment (PINE-TREE), pairs prime editing with TREE technology to efficiently enrich for prime edited cells. This investigation revealed PINE-TREE as an efficient editing and enrichment method compared to a conventional reporter of transfection (RoT) enrichment strategy. Here, PINE-TREE exhibited a significant increase in editing efficiencies of single nucleotide conversions, small insertions, and small deletions in multiple human cell types. Additionally, PINE-TREE demonstrated improved clonal cell editing efficiency in human induced pluripotent stem cells (hiPSCs). Most notably, PINE-TREE efficiently generated clonal isogenic hiPSCs harboring a mutation in the APOE gene for in vitro modeling of Alzheimer’s Disease. Collectively, results gathered from this study exhibited PINE-TREE as a valuable new tool in genetic engineering to accelerate the generation of clonal isogenic cell lines for applications in developmental biology, disease modeling, and drug screening.
ContributorsKostes, William Warner (Author) / Brafman, David (Thesis advisor) / Jacobs, Bertram (Committee member) / Lapinaite, Audrone (Committee member) / Tian, Xiaojun (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2022
Description
This dissertation research project developed as an urgent response to physical inactivity, which has resulted in increased rates of obesity, diabetes, and metabolic disease worldwide. Incorporating enough daily physical activity (PA) is challenging for most people. This research aims to modulate the brain's reward systems to increase motivation for PA and, thus, slow the rapid increase in sedentary lifestyles. Transcranial direct current stimulation (tDCS) involves brain neuromodulation by facilitating or inhibiting spontaneous neural activity. tDCS applied to the dorsolateral prefrontal cortex (DLPFC) increases dopamine release in the striatum, an area of the brain involved in the reward–motivation pathways. I propose that a repeated intervention, consisting of tDCS applied to the DLPFC followed by a short walking exercise stimulus, enhances motivation for PA and daily PA levels in healthy adults. Results showed that using tDCS followed by short-duration walking exercise may enhance daily PA levels in low-physically active participants but may not have similar effects on those with higher levels of daily PA. Moreover, there was a significant effect on increasing intrinsic motivation for PA in males, but there were no sex-related differences in PA. These effects were not observed during a 2-week follow-up period of the study after the intervention was discontinued. Further research is needed to confirm and continue exploring the effects of tDCS on motivation for PA in larger cohorts of sedentary populations. This novel research will lead to a cascade of new evidence-based technological applications that increase PA by employing approaches rooted in biology.
ContributorsRuiz Tejada, Anaissa (Author) / Katsanos, Christos (Thesis advisor) / Neisewander, Janet (Committee member) / Sadleir, Rosalind (Committee member) / Buman, Matthew (Committee member) / Arizona State University (Publisher)
Created2023
Description
The relationship between sleep and physical activity is an area of growing scientific interest, particularly in the context of older adults. The importance of examining long sleep duration and its influence on physical activity in this demographic becomes increasingly relevant given rising healthcare costs. This dissertation aims to investigate this intricate relationship via secondary analysis by examining the effects of moderate time-in-bed (TIB) restriction (60 minutes per night)) on various intensities of physical activity (sedentary, light, moderate, vigorous, moderate-vigorous physical activity) in older adults classified as long sleepers and average duration sleepers. It was hypothesized that moderate TIB restriction would result in differential changes in physical activity levels across various intensities, with long sleepers exhibiting increased physical activity and average sleepers displaying decreased activity, potentially influenced by alterations in TST (total sleep time) and SE (sleep efficiency). Utilizing a randomized controlled trial design, this study examined the effect of treatment changes in objectively measures activity (waist actigraphy) and subjects physical activity levels as measured by the Godin Leisure-Time Exercise Questionnaire . Eligible participants were long sleepers (sleeping > 9 hours per night) and average sleepers (sleeping 7-9 hours per night). Both types of sleepers were either randomized to TIB restriction or asked to maintain their average sleep patterns. Mean TIB restriction compared with baseline was 39.5 minutes in average sleepers and 52.9 minutes in long sleepers randomized to TIB restriction . Contrary to the original hypothesis, no significant effect of TIB restriction was observed across all physical activity levels in either long sleepers or average sleepers. However, a notable association was found between increased sleep efficiency (+0.09% [SD = ± 4.64%]) and light physical activity (±31 minutes [SD = ± 104.81, R=0.445, P < 0.007]) in long sleepers undergoing TIB restriction. While this study presents several methodological limitations, including its nature as a secondary analysis and the less-than-intended achievement of TIB restriction, it adds a valuable layer to the existing body of research on sleep and physical activity in older adults. The findings suggest that moderate TIB restriction may not be sufficiently impactful to change behavior in physical activity levels, thus highlighting the need for more nuanced, targeted research in this domain.
ContributorsPerry, Christopher (Author) / Youngstedt, Shawn D (Thesis advisor) / Petrov, Megan (Committee member) / Swan, Pamela (Committee member) / Buman, Matthew (Committee member) / Ringenbach, Shannon (Committee member) / Arizona State University (Publisher)
Created2023
Description
The purpose of this investigation was to evaluate the influence of tap water safety perceptions on plain water intake (PWI) and hydration status in US Latinx adults. Participants (n=492; age, 28±7 y; 37.4% female) completed an Adapted Survey of Water Issues in Arizona and household watersecurity experience-based scales. A sub-sample (n=55; age, 33±14 y; body mass index, 27.77±6.60 kg·m2) completed dietary recalls on two weekdays and one weekend day via Automated Self-Administered 24-hour Dietary Assessment Tool to determine average PWI and total water intake (TWI). A 24-h urine sample was collected on one recall day and analyzed for urine osmolality (UOsm). Binary logistic regression determined odds ratios (OR) for the odds of perceiving tap water to be unsafe. Hierarchical linear regression was employed with 24-h UOsm and PWI as primary outcomes for the sub-sample. Overall, 51.2% of all participants and 52.7% of the sub-sample mistrust their tap water safety. The odds of mistrusting tap water were significantly greater (P<0.05) for each additional favorable perception of bottled over tap water (OR=1.94, 95% CI=1.50, 2.50), each additional negative home tap water experience (OR=1.32, 95% CI=1.12, 1.56), each additional use of alternatives and/or modifications to home tap water (OR=1.25, 95% CI=1.04, 1.51), and decreased water quality and acceptability (OR=1.21, 95% CI=1.01, 1.45). The odds of mistrusting tap water were significantly lower (P<0.05) for those whose primary source of drinking water is the public supply (municipal) (OR=0.07, 95% CI=0.01, 0.63) and for those with decreased water access (OR=0.56, 95% CI=0.48, 0.66). There were no differences (n=55, P>0.05) in TWI (2,678±1,139 mL), PWI (1,357±971), or 24-h UOsm (460±234 mosm·kg-1). Tap water safety perceptions did not significantly explain variance in PWI or 24-h UOsm (P > 0.05). In conclusion, Latinx mistrust in tap water safety is prevalent. Mistrust appears to be influenced by organoleptic perceptions and to lead to reliance on alternatives to the home drinking water system. Perceptions of tap water safety do not appear to be related to PWI, TWI, or hydration status in Latinx adults.
ContributorsColburn, Abigail (Author) / Kavouras, Stavros (Thesis advisor) / Buman, Matthew (Committee member) / Ohri-Vachaspati, Punam (Committee member) / Vega-Lopez, Sonia (Committee member) / Wutich, Amber (Committee member) / Arizona State University (Publisher)
Created2022
Description
Ecology has been an actively studied topic recently, along with the rapid development of human microbiota-based technology. Scientists have made remarkable progress using bioinformatics tools to identify species and analyze composition. However, a thorough understanding of interspecies interactions of microbial ecosystems is still lacking, which has been a significant obstacle in the further development of related technologies. In this work, a genetic circuit design principle with synthetic biology approaches is developed to form two-strain microbial consortia with different inter-strain interactions. The microbial systems are well-defined and inducible. Co-culture experiment results show that our microbial consortia behave consistently with previous ecological knowledge and thus serves as excellent model systems to simulate ecosystems with similar interactions. Colony patterns also emerge when co-culturing multiple species on solid media. With the engineered microbial consortia, image-processing based methods were developed to quantify the shape of co-culture colonies and distinguish microbial consortia with different interactions. Factors that affect the population ratios were identified through induction and variations in the inoculation process. Further time-lapse experiments revealed the basic rules of colony growth, composition variation, patterning, and how spatial factors impact the co-culture colony.
ContributorsChen, Xingwen (Author) / Wang, Xiao (Thesis advisor) / Kuang, Yang (Committee member) / Tian, Xiaojun (Committee member) / Brafman, David (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2022
Description
Annually, approximately 1.7 million people suffer a traumatic brain injury (TBI) in the United States. After initial insult, a TBI persists as a series of molecular and cellular events that lead to cognitive and motor deficits which have no treatment. In addition, the injured brain activates the regenerative niches of the adult brain presumably to reduce damage. The subventricular zone (SVZ) niche contains neural progenitor cells (NPCs) that generate astrocytes, oligodendrocyte, and neuroblasts. Following TBI, the injury microenvironment secretes signaling molecules like stromal cell derived factor-1a (SDF-1a). SDF-1a gradients from the injury contribute to the redirection of neuroblasts from the SVZ towards the lesion which may differentiate into neurons and integrate into existing circuitry. This repair mechanism is transient and does not lead to complete recovery of damaged tissue. Further, the mechanism by which SDF-1a gradients reach SVZ cells is not fully understood. To prolong NPC recruitment to the injured brain, exogenous SDF-1a delivery strategies have been employed. Increases in cell recruitment following stroke, spinal cord injury, and TBI have been demonstrated following SDF-1a delivery. Exogenous delivery of SDF-1a is limited by its 28-minute half-life and clearance from the injury microenvironment. Biomaterials-based delivery improves stability of molecules like SDF-1a and offer control of its release.
This dissertation investigates SDF-1a delivery strategies for neural regeneration in three ways: 1) elucidating the mechanisms of spatiotemporal SDF-1a signaling across the brain, 2) developing a tunable biomaterials system for SDF-1a delivery to the brain, 3) investigating SDF-1a delivery on SVZ-derived cell migration following TBI. Using in vitro, in vivo, and in silico analyses, autocrine/paracrine signaling was necessary to produce SDF-1a gradients in the brain. Native cell types engaged in autocrine/paracrine signaling. A microfluidics device generated injectable hyaluronic-based microgels that released SDF-1a peptide via enzymatic cleavage. Microgels (±SDF-1a peptide) were injected 7 days post-TBI in a mouse model and evaluated for NPC migration 7 days later using immunohistochemistry. Initial staining suggested complex presence of astrocytes, NPCs, and neuroblasts throughout the frontoparietal cortex.
Advancement of chemokine delivery was demonstrated by uncovering endogenous chemokine propagation in the brain, generating new approaches to maximize chemokine-based neural regeneration.
ContributorsHickey, Kassondra (Author) / Stabenfeldt, Sarah E (Thesis advisor) / Holloway, Julianne (Committee member) / Caplan, Michael (Committee member) / Brafman, David (Committee member) / Newbern, Jason (Committee member) / Arizona State University (Publisher)
Created2021
Description
The RNA editing enzyme adenosine deaminase acting on double stranded RNA 2 (ADAR2) converts adenosine into inosine in regions of double stranded RNA. Here, it was discovered that this critical function of ADAR2 was dysfunctional in amyotrophic lateral sclerosis (ALS) mediated by the C9orf72 hexanucleotide repeat expansion, the most common genetic abnormality associated with ALS. Typically a nuclear protein, ADAR2 was localized in cytoplasmic accumulations in postmortem tissue from C9orf72 ALS patients. The mislocalization of ADAR2 was confirmed using immunostaining in a C9orf72 mouse model and motor neurons differentiated from C9orf72 patient induced pluripotent stem cells. Notably, the cytoplasmic accumulation of ADAR2 coexisted in neurons with cytoplasmic accumulations of TAR DNA binding protein 43 (TDP-43). Interestingly, ADAR2 overexpression in mammalian cell lines induced nuclear depletion and cytoplasmic accumulation of TDP-43, reflective of the pathology observed in ALS patients. The mislocalization of TDP-43 was dependent on the catalytic activity of ADAR2 and the ability of TDP-43 to bind directly to inosine containing RNA. In addition, TDP-43 nuclear export was significantly elevated in cells with increased RNA editing. Together these results describe a novel cellular mechanism by which alterations in RNA editing drive the nuclear export of TDP-43 leading to its cytoplasmic mislocalization. Considering the contribution of cytoplasmic TDP-43 to the pathogenesis of ALS, these findings represent a novel understanding of how the formation of pathogenic cytoplasmic TDP-43 accumulations may be initiated. Further research exploring this mechanism will provide insights into opportunities for novel therapeutic interventions.
ContributorsMoore, Stephen Philip (Author) / Sattler, Rita (Thesis advisor) / Zarnescu, Daniela (Committee member) / Brafman, David (Committee member) / Van Keuren-Jensen, Kendall (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2021
Description
The fast pace of global urbanization makes cities the hotspots of population density and anthropogenic activities, leading to intensive emissions of heat and carbon dioxide (CO2), a primary greenhouse gas. Urban climate scientists have been actively seeking effective mitigation strategies over the past decades, aiming to improve the environmental quality for urban dwellers. Prior studies have identified the role of urban green spaces in the relief of urban heat stress. Yet little effort was devoted to quantify their contribution to local and regional CO2 budget. In fact, urban biogenic CO2 fluxes from photosynthesis and respiration are influenced by the microclimate in the built environment and are sensitive to anthropogenic disturbance. The high complexity of the urban ecosystem leads to an outstanding challenge for numerical urban models to disentangling and quantifying the interplay between heat and carbon dynamics.This dissertation aims to advance the simulation of thermal and carbon dynamics in urban land surface models, and to investigate the role of urban greening practices and urban system design in mitigating heat and CO2 emissions. The biogenic CO2 exchange in cities is parameterized by incorporating plant physiological functions into an advanced single-layer urban canopy model in the built environment. The simulation result replicates the microclimate and CO2 flux patterns measured from an eddy covariance system over a residential neighborhood in Phoenix, Arizona with satisfactory accuracy. Moreover, the model decomposes the total CO2 flux from observation and identifies the significant CO2 efflux from soil respiration. The model is then applied to quantify the impact of urban greening practices on heat and biogenic CO2 exchange over designed scenarios. The result shows the use of urban greenery is effective in mitigating both urban heat and carbon emissions, providing environmental co-benefit in cities. Furthermore, to seek the optimal urban system design in terms of thermal comfort and CO2 reduction, a multi-objective optimization algorithm is applied to the machine learning surrogates of the physical urban land surface model. There are manifest trade-offs among ameliorating diverse urban environmental indicators despite the co-benefit from urban greening. The findings of this dissertation, along with its implications on urban planning and landscaping management, would promote sustainable urban development strategies for achieving optimal environmental quality for policy makers, urban residents, and practitioners.
ContributorsLi, Peiyuan (Author) / Wang, Zhihua (Thesis advisor) / Vivoni, Enrique (Committee member) / Huang, Huei-Ping (Committee member) / Myint, Soe (Committee member) / Xu, Tianfang (Committee member) / Arizona State University (Publisher)
Created2021
Description
High fiber diets have been associated with improved cardiometabolic health with specific efforts to lower circulating levels of low-density lipoprotein (LDL cholesterol). Whole grain and grain-based foods are major contributors of dietary fiber in the American diet, of which wheat has been extensively studied. Corn, however, has not been well studied for its cholesterol-lowering properties. Further, the mechanisms by which grains improve cardiometabolic health require further exploration with regard to the human microbiome. The objective of this single-blind randomized controlled, crossover trial was to assess the impact of three different corn flours (whole grain, refined, and bran-enhanced refined flour mixture) on serum LDL cholesterol and the gut microbiota diversity and composition. Twenty-three participants were recruited, between the ages of 18-70 with hypercholesterolemia (Male = 10, Female = 13, LDL >120 mg/dL) who were not taking any cholesterol-lowering medications. Participants consumed each flour mixture for 4 weeks prepared as muffins and pita breads. At the beginning and end of each 4-week period serum for cholesterol assessment, anthropometrics, and stool samples were obtained. Serum cholesterol was assessed using a clinical analyzer. Stool samples were processed, and microbial DNA extracted and sequenced based on the 16S rRNA gene. A generalized linear model demonstrated a significant treatment effect (p=0.016) on LDL cholesterol and explained a majority of the variance (R-squared= 0.89). Post hoc tests revealed bran-enhanced refined flour had a significant effect on cholesterol in comparison to whole grain flour (p=0.001). No statistically significant differences were observed for gut microbial community composition (Jaccard and weighted Unifrac) after corn consumption. However, relative abundance analysis (LEfSE) identified Mycobacterium celatum (p=0.048 FDR=0.975) as a potential marker of post-corn consumption with this microbe being differentially less abundant following bran-enhanced flour treatment. These data suggest that corn flour consumption may be beneficial for individuals with hypercholesterolemia but the role of gut microbiota in this relationship requires further exploration, especially given the small sample size. Further research and analysis of a fully powered cohort is needed to more accurately describe the associations and potential mechanisms of corn-derived dietary fiber on circulating LDL cholesterol and the gut microbiota.
ContributorsWilson, Shannon L (Author) / Whisner, Corrie M (Thesis advisor) / Sears, Dorothy (Committee member) / Buman, Matthew (Committee member) / Dickinson, Jared (Committee member) / Zhu, Qiyun (Committee member) / Arizona State University (Publisher)
Created2022
Description
The GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9orf72 gene is the most common genetic abnormality associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastatingly progressive neurodegenerative diseases. The discovery of this genetic link confirmed that ALS and FTD reside along a spectrum with clinical and pathological commonalities. Historically understood as diseases resulting in neuronal death, the role of non-neuronal cells like astrocytes is still wholly unresolved. With evidence of cortical neurodegeneration leading to cognitive impairments in C9orf72-ALS/FTD, there is a need to investigate the role of cortical astrocytes in this disease spectrum. Here, a patient-derived induced pluripotent stem cell (iPSC) cortical astrocyte model was developed to investigate consequences of C9orf72-HRE pathogenic features in this cell type. Although there were no significant C9orf72-HRE pathogenic features in cortical astrocytes, transcriptomic, proteomic and phosphoproteomic profiles elucidated global disease-related phenotypes. Specifically, aberrant expression of astrocytic-synapse proteins and secreted factors were identified. SPARCL1, a pro-synaptogenic secreted astrocyte factor was found to be selectively decreased in C9orf72-ALS/FTD iPSC-cortical astrocytes. This finding was further validated in human tissue analyses, indicating that cortical astrocytes in C9orf72-ALS/FTD exhibit a reactive transformation that is characterized by a decrease in SPARCL1 expression. Considering the evidence for substantial astrogliosis and synaptic failure leading to cognitive impairments in C9orf72-ALS/FTD, these findings represent a novel understanding of how cortical astrocytes may contribute to the cortical neurodegeneration in this disease spectrum.
ContributorsBustos, Lynette (Author) / Sattler, Rita (Thesis advisor) / Newbern, Jason (Committee member) / Zarnescu, Daniela (Committee member) / Brafman, David (Committee member) / Mehta, Shwetal (Committee member) / Arizona State University (Publisher)
Created2023